Dairy, Hormones and human health by Pedro Bastos

Pedro Carrera Bastos

MILK, DAIRY AND HUMAN HEALTH

Los Angeles, August 2011

HISTORY OF MILK & DAIRY"

Research Reports in Clinical Cardiology 2011:2submit your manuscript | www.dovepress.com

Dovepress

Dovepress

16

Carrera-Bastos et al

African and Bedouin pastoralists. Today, ALP occurs in about 35% of the world’s population.60

The impetus for these genetic changes was not to increase longevity and resistance to chronic degenerative diseases but rather to increase the probability of survival and reproductive success.27,61,62 Occasionally, muta-tions that had positive survival and reproductive value sometimes also caused adverse health effects in the postreproductive years.4,27,61,62 Furthermore, single gene mutations, although relevant for physicians when treat-ing an individual patient, are imperfect models to prevent chronic degenerative diseases whose clinical symptoms normally affect the postreproductive years and involve numerous genes.61

Importantly, 11,000 years represent approximately 366 human generations,63 which comprise only 0.5% of the history of the genus Homo (Table 1).14,63–65 Indeed, the Industrial Revolution and the Modern Age, which mark the beginning of the western lifestyle, represent only seven and four human generations, respectively (Table 1),14,63–65 and were marked by rapid, radical, and still ongoing changes in lifestyle and diet,14,65 coupled with improved public health measures that greatly reduced mortality in the prereproduc-tive years (and hence largely eliminated impaired reproduc-tive fitness as a selection pressure).62,66 As such, it is highly unlikely that genetic adaptations that allow us to thrive on a western diet and lifestyle have occurred.

Health status of preagriculture traditional populationsThe idea that modern Homo sapiens are still adapted to an ancestral environment is reinforced by data showing that hunter–gatherers, and other populations minimally affected by modern habits, exhibit superior health markers, body composition, and physical fitness compared with industrial-ized populations, including:1. Low blood pressure in hunter–gatherers and horticultural-

ists (Table 2)26,67–69 when compared with current optimal values defined by health institutions ( 120 mm Hg

and 80 mm Hg for systolic blood pressure and diastolic blood pressure, respectively)70

2. Lack of association between blood pressure and age in hunter–gatherers ( Table 3)69 and horticulturalists68 com-pared with in North Americans and Swedes26,68,70

3. Persisting excellent insulin sensitivity among middle-aged and older individuals in non-westernized tra-ditional populations that maintain their ancestral lifestyle26,71–81

4. Lower fasting plasma insulin concentrations and higher insulin sensitivity (measured by the Homeostatic Model Assessment [HOMA] index) in the horticulturalists of Kitava (Papua New Guinea) compared with in healthy Swedes (Figures 1 and 2, respectively)74

5. Lower fasting plasma leptin in the horticulturalists of Kitava and the Ache hunter–gatherer Indians of Paraguay compared with in healthy Swedes82 (Figure 3) and North American male distance runners83 (Figure 4), respectively

6. Lower body mass index (BMI) in hunter–gatherers, tradi-tional pastoralists, and horticulturalists26 compared with in westerners.26,84 For instance, as observed by Lindeberg,26 in Kitava, 87% of men and 93% of women aged 40–60 years had a BMI below 22 kg/m2 and not a single individual in this age group was overweight or obese26

7. Lower waist (cm)/height (m) ratio in the horticul-turalists of Kitava compared with in healthy Swedes (Figure 5)82

8. Lower tricipital skinfold (mm) in hunter–gatherers com-pared with in healthy Americans67 (Figure 6)

Table 1 Historical milestones in human generations14,63–65

Historical milestones Generations % total

Homo habilis 76,667 100.0Homo erectus 60,000 78.2Modern Homo sapiens 6666 8.7Neolithic Revolution 366 0.48Industrial Revolution 7 0.009Food industry (junk food) and physical inactivity (Modern Age)

4 0.005

Table 2 Systolic blood pressure (SBP) and diastolic blood pressure (DBP) at age 40–60 years in hunter–gatherers and horticulturalists (mm Hg)26,67–69

Population Men Women

SBP DBP SBP DBP

Bushmen 108 63 118 71Yonomamo 104 65 102 63Xingu 107 68 102 66Kitava 113 71 121 71

Table 3 Systolic blood pressure and diastolic blood pressure in Yanomamo Indians (mm Hg)69

Age (years) Men Women

0–9 93/59 96/6210–19 108/67 105/6520–29 108/69 100/6330–39 106/69 100/6340–49 107/67 98/6250 100/64 106/64

Carrera-Bastos P, Fontes-Villalba M, O'Keefe JH, Lindeberg S, Cordain L. Res Rep Clin Cardiol 2011;2:15-35.

EVIDENCE FOR THE USE OF DAIRY IN THE MIDDLE EAST & EUROPE

66 100 133 167 200 233 267 300 333

Hum

an

Gen

erat

ions

Pres

ent

33

First evidence of dairying in the Middle East (Turkey) 4

First evidence of dairying in Europe (Romania) 5

Domes@ca@on of sheeps, goats and caBle (Middle East) 1-‐3

1 - Hiendleder S, et al. Proc Biol Sci. 2002 May 7;269(1494):893-904 2 - Luikart G, et al. Proc Natl Acad Sci U S A. 2001 May 8;98(10):5927-32 3 - Loftus RT, et al. Mol Ecol. 1999 Dec;8(12):2015-22 4 - Evershed RP et al. Nature. 2008 Sep 25;455(7212):528-31. 5 - Craig OE, et al. Antiquity 2005; 79:882-894 6 - Copley MS et al. Proc Natl Acad Sci U S A. 2003 Feb 18;100(4):1524-9

First evidence of dairying in North. Eur (UK) 6

frequency of Somali people living in Ethiopia does notprevent them from drinking more than 500 ml of milkper day without any obvious discomfort [16]. Thisinter-individual variation of the amount of lactose tol-erated by lactase non-persistent people may be a resultof variation in the composition of the gut flora (par-ticularly the presence of lactic acid bacteria) [16,43].Also, fermented dairy products (i.e. yoghurt orcheese) contain less lactose, allowing consumptionby non-persistent individuals without any of theexpected symptoms [43].

Two contrasting theories have been proposed toexplain the co-distribution of LPand dairying practices.The culture-historical hypothesis [11,12,44] argues LPdeveloped, and was consequently selected, after milkproduction and dairy consumption spread. The oppos-ing hypothesis (mentioned in McCracken [12] as thereverse-cause hypothesis) proposes that only populationswhose frequency of LP was high enough adopteddairying. In other words, human groups were differen-tiated with regards to LP frequency by a processunrelated to milk consumption—through genetic drift[45]—before the invention of dairying. This viewspecifically assumes that drinking milk would not

necessarily have conferred any selective advantage.The culture-historical hypothesis is better supportedby recent findings from archaeological research (see§1c for details). In particular, organic residues pre-served in archaeological pottery provided evidence forthe use of milk after 8500 BP in the western part ofTurkey [40], a region where LP frequency is low today(figure 1a). It suggests that domestic animals weremilked before LP arose or was present at appreciablefrequencies.

(c) The advantage of being lactase persistentSeveral explanations have been proposed for how andwhy LP may have been selected. For example, it maysimply be that milk is a good source of calories, orspecifically an important source of protein and fat.The milk production of a prehistoric cow has beenestimated to range between 400 and 600 kg per wean-ing period. Even when the milk necessary for theraising of the calves is subtracted, some 150–250 kgremains [46]. This is almost equivalent to the caloriegain from the meat of a whole cow. Hence, over theyears, milking may have resulted in a greater energy

(b)

(a)

0° 45° E

30° S

30° N

60° N

0°

90° E 135° E 180° E

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

0° 45° E

30° S

30° N

60° N

0°

90° E 135° E 180° E

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

Figure 1. Interpolated maps of the distribution of LP and the 213910*T allele in the ‘old world’. (a) LP phenotype distri-bution. Data points (dots) were taken from the literature (see text and [14] for details). (b) Distribution of the allele213910*T, associated to LP. Dots represent sample data taken from a previous review [14,26–30]; crosses represent datafor new locations not previously tested and diamonds correspond to locations where additional data have been added.Regularly updated frequency data are available at http://www.ucl.ac.uk//mace-lab/GLAD/ website.

Evolution of lactase persistence P. Gerbault et al. 865

Phil. Trans. R. Soc. B (2011)

on February 18, 2011rstb.royalsocietypublishing.orgDownloaded from

ALP IN EUROPE"

Gerbault P, et al. Philos Trans R Soc Lond B Biol Sci. 2011 Mar 27;366(1566):863-77.

Predicted Old World LP phenotype frequencies based on -13,910 C>T allele frequency data only

Dates of origin: between 2188 and 20650 BP and between 7450 and 12300 BP

ARTICLE

Independent Introduction of Two Lactase-PersistenceAlleles into Human Populations ReflectsDifferent History of Adaptation to Milk Culture

Nabil Sabri Enattah,1,2 Tine G.K. Jensen,3 Mette Nielsen,3 Rikke Lewinski,3 Mikko Kuokkanen,1,2

Heli Rasinpera,1,2 Hatem El-Shanti,4 Jeong Kee Seo,5 Michael Alifrangis,6 Insaf F. Khalil,6

Abdrazak Natah,7 Ahmed Ali,9 Sirajedin Natah,8 David Comas,10 S. Qasim Mehdi,11

Leif Groop,12 Else Marie Vestergaard,13 Faiqa Imtiaz,14 Mohamed S. Rashed,15

Brian Meyer,14 Jesper Troelsen,3 and Leena Peltonen1,2,*

The T!13910 variant located in the enhancer element of the lactase (LCT) gene correlates perfectly with lactase persistence (LP) in Eur-

asian populations whereas the variant is almost nonexistent among Sub-Saharan African populations, showing high prevalence of

LP. Here, we report identification of two new mutations among Saudis, also known for the high prevalence of LP. We confirmed the ab-

sence of the European T!13910 and established two new mutations found as a compound allele: T/G!13915 within the !13910 enhancer

region and a synonymous SNP in the exon 17 of theMCM6 gene T/C!3712, !3712 bp from the LCT gene. The compound allele is driven

to a high prevalence amongMiddle East population(s). Our functional analyses in vitro showed that both SNPs of the compound allele,

located 10 kb apart, are required for the enhancer effect, most probably mediated through the binding of the hepatic nuclear factor 1 a

(HNF1a). High selection coefficient (s) ~0.04 for LP phenotype was found for both T!13910 and the compound allele. The European

T!13910 and the earlier identified East African G!13907 LP allele share the same ancestral background and most likely the same history,

probably related to the same cattle domestication event. In contrast, the compound Arab allele shows a different, highly divergent

ancestral haplotype, suggesting that these two major global LP alleles have arisen independently, the latter perhaps in response to

camel milk consumption. These results support the convergent evolution of the LP in diverse populations, most probably reflecting

different histories of adaptation to milk culture.

Introduction

The dairy culture was initiated some 10,000 years ago inthe Middle East with the domestication of sheep, goat,and cattle.1–4 Lactase activity of intestinal cells, responsiblefor the digestion of the milk sugar, lactose, declines afterweaning for most humans.5 However, in multiple globalsubpopulations, a genetic capacity of adult humans to di-gest milk sugar has evolved that results in the continuingexpression of lactase by intestinal cells, a condition knownas lactase persistence (LP) (MIM 223000).5 We and othershave shown that a single allele, carrying the T!13910 vari-ant 14 kb upstream of lactase (LCT) gene, fully correlateswith LP in many global populations.5–7 Functional evi-dence for the C/T!13910 variant in the regulation of lactaseactivity in intestinal cells has also emerged, lending addi-tional support for this variant being the true causativeone.8–10 Functional studies in vitro have further shown

that the LP trait-related T!13910 allele binds Oct-1 tran-scription factor more strongly than does the C!13910 allele.It has been further demonstrated that a wider DNA regionencompassing the C/T!13910 variant contains an enhancerelement with binding sites for several transcription fact-ors such as Oct-1 and GATA-6 (region from !13909to !13934), HNF4a and Fox/HNF3a (region !13857to !13817), and Cdx-2 (region !14022 to !14032). Allthese factors probably contribute to the regulation of thelactase gene in intestinal cells.10 Furthermore, the expres-sion of Oct-1 has been shown to drive the reporter geneexpression from both T!13910 and C!13910 variant/LCTpromoter constructs only when it is coexpressed withHNF1a, suggesting that the !13910 enhancer effect is me-diated through HNF1a bound to the proximal promoter ofthe LCT gene.Recently published data indicated the lack of the T!13910

variant among Sub-Saharan African populations, known to

1Department of Molecular Medicine, National Public Health Institute, Biomedicum Helsinki, 00251 Helsinki, Finland, and Department of Medical Genet-ics, University of Helsinki, Biomedicum Helsinki, 00251 Helsinki, Finland; 2Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1HH, UK, and TheBroad Institute, MIT, Cambridge, MA 02141-2023, USA; 3Department of Cellular and Molecular Medicine, Panum Institute, University of Copenhagen,2200 Copenhagen, Denmark; 4Division of Medical Genetics, University of Iowa Hospital & Clinics, 2615 JCP, 200 Hawkins Drive, Iowa City, IA 52242,USA; 5Department of Pediatrics, Seoul National University College of Medicine, Clinical Research Institute, Seoul National University Hospital, Seoul110-774, Korea; 6Centre for Medical Parasitology, Department of International Health, Immunology and Microbiology, CSS, University of Copenhagen,1014 Copenhagen, Denmark; 7Program of Natural Sciences, Faculty of Science, 8Department of Physiology, Biophysics and Medicine, GastrointestinalDivision, Health Sciences Center, University of Calgary, Alberta T2N 1N4, Canada; 9Department of Anatomy, Institute of Clinical Medicine, Universityof Helsinki, 00014 Helsinki, Finland; 10Department of Evolutionary Biology, Faculty of Health and Life Sciences, University of Pompeu Fabra, 08003Barcelona, Spain; 11Institute of Biotechnology and Genetic Engineering (KIBGE), University of Karachi, 75270 Karachi, Pakistan; 12Department of Endo-crinology, Malmo University Hospital, S-205 02 Malmo, Sweden; 13Department of Clinical Biochemistry, Aarhus University Hospital, DK 8000 Aarhus,Denmark; 14Arabian Diagnostics Laboratory, 15National Newborn Screening Laboratory, Research Centre, King Faisal Specialist Hospital & Research Centre,Riyadh 11211, Saudi Arabia*Correspondence: [email protected] 10.1016/j.ajhg.2007.09.012. ª2008 by The American Society of Human Genetics. All rights reserved.

The American Journal of Human Genetics 82, 57–72, January 2008 57

Domestication of the Arabian camel: 6000 BP Origin of G-13915 allele in the Arabian Pensinsula: 4000 BP

Enattah NS, et al. Am J Hum Genet. 2008 Jan;82(1):57-72

Tishkoff SA, et al. Nat Genet. 2007 Jan;39(1):31-40

Spread of Pastoralism south of the Sahara: 4,500 BP Spread of Pastoralism into Northern Tanzania: 3,300 BP

Convergent adaptation of human lactase persistencein Africa and EuropeSarah A Tishkoff1,9, Floyd A Reed1,9, Alessia Ranciaro1,2, Benjamin F Voight3, Courtney C Babbitt4,Jesse S Silverman4, Kweli Powell1, Holly M Mortensen1, Jibril B Hirbo1, Maha Osman5, Muntaser Ibrahim5,Sabah A Omar6, Godfrey Lema7, Thomas B Nyambo7, Jilur Ghori8, Suzannah Bumpstead8,Jonathan K Pritchard3, Gregory A Wray4 & Panos Deloukas8

A SNP in the gene encoding lactase (LCT) (C/T-13910) is associated with the ability to digest milk as adults (lactase persistence)in Europeans, but the genetic basis of lactase persistence in Africans was previously unknown. We conducted a genotype-phenotype association study in 470 Tanzanians, Kenyans and Sudanese and identified three SNPs (G/C-14010, T/G-13915 andC/G-13907) that are associated with lactase persistence and that have derived alleles that significantly enhance transcriptionfrom the LCT promoter in vitro. These SNPs originated on different haplotype backgrounds from the European C/T-13910 SNPand from each other. Genotyping across a 3-Mb region demonstrated haplotype homozygosity extending 42.0 Mb onchromosomes carrying C-14010, consistent with a selective sweep over the past B7,000 years. These data provide a markedexample of convergent evolution due to strong selective pressure resulting from shared cultural traits—animal domesticationand adult milk consumption.

In most humans, the ability to digest lactose, the main carbohydratepresent in milk, declines rapidly after weaning because of decreasinglevels of the enzyme lactase-phlorizin hydrolase (LPH). LPH ispredominantly expressed in the small intestine, where it hydrolyzeslactose into glucose and galactose, sugars that are easily absorbed intothe bloodstream1. However, some individuals, particularly descen-dants from populations that have traditionally practiced cattle domes-tication, maintain the ability to digest milk and other dairy productsinto adulthood. These individuals have the ‘lactase persistence’ trait.The frequency of lactase persistence is high in northern Europeanpopulations (490% in Swedes and Danes), decreases in frequencyacross southern Europe and the Middle East (B50% in Spanish,French and pastoralist Arab populations) and is low in non-pastoralistAsian and African populations (B1% in Chinese, B5%–20% inWest African agriculturalists)1–3. Notably, lactase persistence is com-mon in pastoralist populations from Africa (B90% in Tutsi, B50%in Fulani)1,3.Lactase persistence is inherited as a dominant mendelian trait in

Europeans1,2,4. Adult expression of the gene encoding LPH (LCT),located on 2q21, is thought to be regulated by cis-acting elements5

(Fig. 1). A linkage disequilibrium (LD) and haplotype analysis ofFinnish pedigrees identified two single SNPs associated with the

lactase persistence trait: C/T-13910 and G/A-22018, located B14 kbandB22 kb upstream of LCT, respectively, within introns 9 and 13 ofthe adjacent minichromosome maintenance 6 (MCM6) gene4 (Fig. 1).The T-13910 and A-22018 alleles were 100% and 97% associated withlactase persistence, respectively, in the Finnish study4, and the T-13910allele is B86%–98% associated with lactase persistence in otherEuropean populations6–8. Although these alleles could simply be inLD with an unknown regulatory mutation6, several additional lines ofevidence, including mRNA transcription studies in intestinal biopsysamples9 and reporter gene assays driven by the LCT promoterin vitro10–12, suggest that the C/T-13910 SNP regulates LCT transcrip-tion in Europeans.It is hypothesized that natural selection has had a major role in

determining the frequencies of lactase persistence in different humanpopulations since the development of cattle domestication in theMiddle East and North Africa B7,500–9,000 years ago2,3,6,13–18. Aregion of extensive LD spanning 41 Mb has been observed onEuropean chromosomes with the T-13910 allele, consistent with recentpositive selection6,14,16–18. Based on the breakdown of LD on chromo-somes with the T-13910 allele, it is estimated14 that this allele arosewithin the past B2,000–20,000 years within Europeans, probably inresponse to strong selection for the ability to digest milk as adults.

Received 18 August; accepted 20 November; published online 10 December 2006; doi:10.1038/ng1946

1Department of Biology, University of Maryland, College Park, Maryland 20742, USA. 2Department of Biology, University of Ferrara, 44100 Ferrara, Italy. 3Departmentof Human Genetics, University of Chicago, Chicago, Illinois 60637, USA. 4Institute for Genome Sciences & Policy and Department of Biology, Duke University,Durham, North Carolina 27708, USA. 5Department of Molecular Biology, Institute of Endemic Diseases, University of Khartoum, 15-13 Khartoum, Sudan. 6KenyaMedical Research Institute, Centre for Biotechnology Research and Development, 54840-00200 Nairobi, Kenya. 7Department of Biochemistry, Muhimbili UniversityCollege of Health Sciences, Dar es Salaam, Tanzania. 8Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK. 9Theseauthors contributed equally to this work. Correspondence should be addressed to S.A.T. ([email protected]).

NATURE GENETICS VOLUME 39 [ NUMBER 1 [ JANUARY 2007 31

ART I C LES

©20

07 N

atur

e Pu

blis

hing

Gro

up h

ttp://

ww

w.n

atur

e.co

m/n

atur

egen

etic

s

accounted for by the genotypes in the pooled sample, suggesting thatthere are environmental and/or measurement factors, and possiblyunidentified genetic factors, influencing the LTT phenotype in thisdata set.

Frequency of G/C-14010, T/G-13915 and C/G-13907 in AfricansGenotype frequencies for G/C-14010, T/G-13915 and C/G-13907 areshown in Figure 2b, whereas Supplementary Table 1 gives allelefrequencies for these SNPs as well as the European lactase persistence–associated SNPs C/T-13910 and G/A-22018. The T-13910 allele isabsent in all of the African populations tested, and we observed theA-22018 allele in a single heterozygous Akie individual from Tanzania.The C-14010 allele is common in Nilo-Saharan populations fromTanzania (39%) and Kenya (32%) and in Afro-Asiatic populationsfrom Tanzania (46%) but is at lower frequency in the Sandawe (13%)and Afro-Asiatic Kenyan (18%) populations and is absent in the Nilo-Saharan Sudanese and Hadza populations (Fig. 2b and Supplemen-tary Table 1). The C-13907 and G-13915 alleles are at Z5%frequency only in the Afro-Asiatic Beja populations (21% and 12%,respectively) and in the Afro-Asiatic Kenyan populations (5% and9%, respectively).

C-14010, G-13915 and G-13907 affect expression in vitroIn order to test whether the C-14010, G-13915 and G-13907 variantsaffect expression from the LCT core promoter, we transfected thehuman intestinal cell line Caco-2 with luciferase expression vectorsdriven by the basal 3-kb promoter alone or by the promoter fused to

one of five haplotypes of the 2-kb MCM6 intron 13 region:one haplotype with ancestral alleles at the three candidate SNPs(G-14010, T-13915, C-13907), two haplotypes that differed only atthe derived C-14010 or G-13915 alleles, one haplotype that differed atthe derived G-13907 allele as well as at a linked T-13495 allele and onehaplotype that has the ancestral lactase persistence–associated alleles,with a T at position -13945 (to control for the effect of this variant).Differences in luciferase expression between the basal 3-kb LCTcore promoter and the promoter plus any of the five MCM6 intronsequence constructs were highly significant (paired t test, P o 0.001),resulting in more than a twenty-fold increase in expression over thecore promoter alone (Fig. 5).Notably, we also observed differences in expression between the five

MCM6 intron 13 haplotypes that were functionally tested using thedual-luciferase reporter assay (Fig. 5). The C-14010–, G-13915– andG-13907–derived haplotypes consistently drove higher expression(from B18%–30%) than the haplotypes with the ancestral alleles.There was no statistically significant difference in expression betweenthe constructs with the C-14010, G-13907/T-13495 and G-13915 alleles.

Evidence for positive selection of the C-14010 alleleIf a mutation provides a large enough benefit to its carriers (in thiscase, the ability to digest milk as adults), resulting in more viableoffspring, it is expected to rise rapidly to high frequency in thepopulation, together with linked variants (that is, genetic hitchhik-ing)25. Under neutrality, one expects common mutations to be olderand to have lower levels of LD with flanking markers. In contrast, one

Kenya

Tanzania

Sudan

-13907 -13915 -14010 Mean (variance)

Genotype Phenotype

Beja(Afro-Asiatic)

n = 17

Sandawe(Khoisan)

n = 31

CC-TT-GG 234CC-TT-GC 144CC-TT-CC 44CC-GT-GG 11CC-GT-GC 2CC-GG-GG 2

GC-TT-GG 8

GC-TT-GC 3

GC-GG-GG 1

GG-TT-GG 1

Kenya

Sudan

LNPLIPLP

Phenotype

Beja(Afro-Asiatic)

n = 17

Afro-Asiaticn = 64

Nilo-Saharann = 26

Tanzania

Hadza(Khoisan)

n = 18

Sandawe(Khoisan)

n = 30

Niger-Kordofaniann = 61

Nilo-Saharann = 126

Nilo-Saharann = 45

Afro-Asiaticn = 81

Count

1.39 (0.94)2.04 (1.40)

(1.67)2.453.03 (3.41)3.86 (1.84)

(0.27)2.70

3.96 (1.69)

2.37 (1.22)

(NA)2.90

(NA)4.59

Nilo-Saharann = 27

Hadza(Khoisan)

n = 18

Niger-Kordofaniann = 61

Afro-Asiaticn = 99

Nilo-Saharann = 47

Nilo-Saharann = 128

Afro-Asiaticn = 64

a b

Figure 2 Map of phenotype and genotype proportions for each population group considered in this study. (a) Pie charts representing the proportion of eachphenotype by geographic region. LP indicates lactase persistence, LIP indicates lactase intermediate persistence and LNP indicates lactase non-persistence.Phenotypes were binned using an LTT test according to the rise in blood glucose after digestion of 50 g lactose: lactase persistence, 41.7 mM; LIP,between 1.1 mM and 1.7 mM; LNP, o1.1 mM. (b) Proportion of compound genotypes for G/C-13907, T/G-13915 and C/G-14010 in each region. The piecharts are in the approximate geographic location of the sampled individuals.

NATURE GENETICS VOLUME 39 [ NUMBER 1 [ JANUARY 2007 33

ART I C LES

©20

07 N

atur

e Pu

blis

hing

Gro

up h

ttp://

ww

w.n

atur

e.co

m/n

atur

egen

etic

s

ALP in Africa"

000

Various SNPs associated with LP in Sub-Saharan Africans arose

3000-7000 BP

Tishkoff SA, et al. Nat Genet. 2007 Jan;39(1):31-40

The most widely consumed milk in US and Europe is cow’s milk. "

!!"

Park YW, Haenlein GFW. Handbook of milk of non-bovine mammals. Blackwell Publishing, 2006

MILK PRODUCTION (Europe)

Milk" 1980" 2001"Sheep" 1.9%" 1.3%"

Goat" 1.0%" 1.1%"

Bufalo" 0.1%" 0.1%"

Cow" 97.2%" 97.5%"


Cow" 97.2%" 97.5%"

MILK PRODUCTION PER COW (USA)

http://quickstats.nass.usda.gov/results/79C63180-8FF4-3A0F-9CD6-D064D945F49E

http://quickstats.nass.usda.gov/results/B04AD392-D39A-3C2F-A060-89A54CF5BDE1

0!

5000!

10000!

15000!

20000!

25000!

1950 2010

Lbs

RBGH INCREASES MILK PRODUCTION"

""

Bauman DE, Eppard PJ, DeGeeter MJ, Lanza GM. J Dairy Sci. 1985 Jun;68(6):1352-62

Hare E, Norman HD, Wright JR. J Dairy Sci 2006; 89: 365-370.

INDUSTRIAL MODEL OF DAIRYING"

Change in nutrition

reduced the

average age at

first lactation"

MILK, HORMONES & HUMAN HEALTH October, 2006

2

Then, the nomad separates the calf from the mother and binds the

calf to the fence and begins to milk the cow for human use.

Afterward the nomad allows the calf to suckle milk again until it is

satiated. I was told that the cow pictured here was 14 years old and

had given birth to 10 calves.

The volume of milk that was collected from this cow was 2 or 3

liters at most. The volume of this container is about 20 liters.

Nomadic milking

��

� �

��

Mongolian cows feed only on grass and they do not secrete milk

during the latter half of pregnancy. They get pregnant by natural

mating in July or August, and give birth to calves in April or May.

The Mongolian nomads milk their cows for only 5 or 6 months

from May to October, obtaining, at best, 5 liters of milk per day

per cow.

These are pictures of a modern dairy farm in Japan.

MILK, HORMONES & HUMAN HEALTH October, 2006

2

Then, the nomad separates the calf from the mother and binds the

calf to the fence and begins to milk the cow for human use.

Afterward the nomad allows the calf to suckle milk again until it is

satiated. I was told that the cow pictured here was 14 years old and

had given birth to 10 calves.

The volume of milk that was collected from this cow was 2 or 3

liters at most. The volume of this container is about 20 liters.

Nomadic milking

��

� �

��

Mongolian cows feed only on grass and they do not secrete milk

during the latter half of pregnancy. They get pregnant by natural

mating in July or August, and give birth to calves in April or May.

The Mongolian nomads milk their cows for only 5 or 6 months

from May to October, obtaining, at best, 5 liters of milk per day

per cow.

These are pictures of a modern dairy farm in Japan.

Sato A. Health Effects of Cow Milk. Symposium on Milk, Hormones and Human Health, Harvard, Boston, Oct. 2006 www.milksymposium.ca.org

Health Effects of Cow Milk Akio Sato & Ganmaa Davasaambuu

3

• First pregnancy: 14 months after birth

• Length of pregnancy: 280 days

• Colostrum is given to the calf (5 days)�

• Milking for humans (300 days)

• Artificial insemination 2-3 months after

parturition (during the period of milking)

• Milking is stopped for 60 days (dry period)

before next parturition.

• slaughtered after 4-6 “parturition + milking”

Dairy farming in Japan

I don't know whether modern dairy farming practices in Japan are

the same as in other developed countries. In Japan the cow is

impregnated by artificial insemination 12 to 14 months after birth.

The length of pregnancy is 280 days, the same as in humans.

After giving birth, the cow secrets colostrum. Soon after birth the

calf is separated from its mother and the colostrum is milked by

machine and given to the calf in a feeding bottle for five days.

Modern milking

*Artificial insemination Pregnancy �� Dry

*

� � � � � � � ��

��#�� "��& �� #��

� �

�� " !" ��& �$� �$� $� ��! ��# � % �� " !" ��& �$� �$� $�

Nomadic milking

From the sixth day after delivery, the cow is milked every day for

300 days. Two or three months after delivery, the cow is

inseminated artificially and gets pregnant while it is proficient in

milk secretion. Milking is stopped for 60 days just before the next

delivery, which is referred to as the "dry period." The cow is

slaughtered after giving birth to 4 or 6 calves. Modern cows are

milked for 7 or 8 months while they are pregnant, including the

latter half of pregnancy up to the dry period, which is four months

longer into pregnancy than Mongolian cows are milked. One final

comment on Japanese dairy practices, the use of recombinant

bovine growth hormone is legal in Japan as it is in the US.

However, it is said that dairy farmers in Japan do not use this

hormone. I do not know why nor whether this is true.

Estrone sulfate in milk whey

Heap and Hamon (1979)

��1,000 pg/mlPregnancy 220-240 day

��151 pg/mlPregnancy 41-60 day

��30 pg/mlNon-pregnant cow

I would now like to draw your attention to the female sex

hormones that have been measured in the whey fraction of milk.

The most abundant hormone in milk is estrone sulfate. According

to a 1979 study by Heap and Hamon milk from non-pregnant cows

contains 30 picograms per milliliter estrone sulfate. The

concentration rises with pregnancy progression. It is 150

picograms in the first or second month of pregnancy, and in the

last stage of pregnancy it rises to as high as 1,000 picograms per

milliliter.

Milk contains a considerable

amount of estrone sulfate.

The estrone has an oral activity?

Estrone sulfate is marketed

under the name of premarin (=

pregnant mare), which is widely

used for hormonal replacement

therapy (HRT).

Estrone sulfate has a high oral estrogenic activity. Once in the

body it is converted to estrone or estradiol. That estrone sulfate

has a high oral activity can be understood by the fact that

Premarin, which is marketed for hormone replacement therapy

contains naturally occurring conjugated estrogens derived from

pregnant mares.

Health Effects of Cow Milk Akio Sato & Ganmaa Davasaambuu

3

• First pregnancy: 14 months after birth

• Length of pregnancy: 280 days

• Colostrum is given to the calf (5 days)�

• Milking for humans (300 days)

• Artificial insemination 2-3 months after

parturition (during the period of milking)

• Milking is stopped for 60 days (dry period)

before next parturition.

• slaughtered after 4-6 “parturition + milking”

Dairy farming in Japan

I don't know whether modern dairy farming practices in Japan are

the same as in other developed countries. In Japan the cow is

impregnated by artificial insemination 12 to 14 months after birth.

The length of pregnancy is 280 days, the same as in humans.

After giving birth, the cow secrets colostrum. Soon after birth the

calf is separated from its mother and the colostrum is milked by

machine and given to the calf in a feeding bottle for five days.

Modern milking

*Artificial insemination Pregnancy �� Dry

*

� � � � � � � ��

��#�� "��& �� #��

� �

�� " !" ��& �$� �$� $� ��! ��# � % �� " !" ��& �$� �$� $�

Nomadic milking

From the sixth day after delivery, the cow is milked every day for

300 days. Two or three months after delivery, the cow is

inseminated artificially and gets pregnant while it is proficient in

milk secretion. Milking is stopped for 60 days just before the next

delivery, which is referred to as the "dry period." The cow is

slaughtered after giving birth to 4 or 6 calves. Modern cows are

milked for 7 or 8 months while they are pregnant, including the

latter half of pregnancy up to the dry period, which is four months

longer into pregnancy than Mongolian cows are milked. One final

comment on Japanese dairy practices, the use of recombinant

bovine growth hormone is legal in Japan as it is in the US.

However, it is said that dairy farmers in Japan do not use this

hormone. I do not know why nor whether this is true.

Estrone sulfate in milk whey

Heap and Hamon (1979)

��1,000 pg/mlPregnancy 220-240 day

��151 pg/mlPregnancy 41-60 day

��30 pg/mlNon-pregnant cow

I would now like to draw your attention to the female sex

hormones that have been measured in the whey fraction of milk.

The most abundant hormone in milk is estrone sulfate. According

to a 1979 study by Heap and Hamon milk from non-pregnant cows

contains 30 picograms per milliliter estrone sulfate. The

concentration rises with pregnancy progression. It is 150

picograms in the first or second month of pregnancy, and in the

last stage of pregnancy it rises to as high as 1,000 picograms per

milliliter.

Milk contains a considerable

amount of estrone sulfate.

The estrone has an oral activity?

Estrone sulfate is marketed

under the name of premarin (=

pregnant mare), which is widely

used for hormonal replacement

therapy (HRT).

Estrone sulfate has a high oral estrogenic activity. Once in the

body it is converted to estrone or estradiol. That estrone sulfate

has a high oral activity can be understood by the fact that

Premarin, which is marketed for hormone replacement therapy

contains naturally occurring conjugated estrogens derived from

pregnant mares.

“3 Cups per day of fat-free or low-fat milk and milk products”

2005 DIETARY GUIDELINES ADVISORY COMMITTEE !Janet King, PhD, RD (Chair)"

Children’s Hospital Oakland Research Institute, Oakland, CA!"Lawrence J. Appel, MD, MPH"Johns Hopkins Medical Institutions, Baltimore, MD!"Yvonne L. Bronner, ScD, RD, LD"Morgan State University, Baltimore, MD!"Benjamin Caballero, MD, PhD"Johns Hopkins Bloomberg School of Public Health, Baltimore, MD!"Carlos A. Camargo, MD, DrPH"Harvard University, Boston, MA!"Fergus M. Clydesdale, PhD,"University of Massachusetts, !Amherst, Amherst, MA!"

Vay Liang W. Go, MD University of California at Los Angeles, Los Angeles, CA Penny M. Kris-Etherton, PhD, RD Penn State University, University Park, PA Joanne R. Lupton, PhD Texas A&M University, College Station, TX Theresa A. Nicklas, DrPH, MPH, LN Baylor College of Medicine, Houston, TX Russell R. Pate, PhD University of South Carolina, Columbia, SC F. Xavier Pi-Sunyer, MD, MPH Columbia University, New York, NY Connie M. Weaver, PhD Purdue University, West Lafayette, IN

Camargo C. 2005 US Dietary Guidelines: Milk and Milk Products. Symposium on Milk, Hormones and Human Health, Harvard, Boston, Oct. 2006 www.milksymposium.ca.org

WHY 3 CUPS OF MILK? "

Carlos Camargo, MD, DrPH Harvard University, Boston, MA

Camargo C. 2005 US Dietary Guidelines: Milk and Milk Products. Symposium on Milk, Hormones and Human Health, Harvard, Boston, Oct. 2006 www.milksymposium.ca.org

“... the focus was, of course, the low calcium intake of Americans.” “... So in a world where you are trying to increase calcium intake and simultaneously increase potassium and, perhaps Vitamin D; milk looks quite attractive.”

MILK, CALCIUM & FRACTURES

20 Bischoff-Ferrari HA, Dawson-Hughes B, Baron JA, et al. Am J Clin Nutr. 2007 Dec;86(6):1780-900

Total n: 252,841

ü Prospective Studies do not show association between Calcium intake & Hip fracture risk in Men and Women

ü RCTs observe a slight increase in hip fracture risk with Ca monotherapy

. .

Bolland MJ, et al. BMJ. 2008 Feb 2;336(7638):262-6

Bischoff-Ferrari HA et al. J Bone Mineral Res 2011; online Oct 2010, DOI 10.1002/jbmr.279"

Pooled analysis for categories of milk intake and hip fracture risk in women from prospective cohort studies (6 studies, 195 102 women, 3574 fractures). "

RR/+1glass!

MILK AND FRACTURES!No association in observational studies among adults

MAJOR CONSTITUENT’S OF COW’S MILK"

! Water! Fat! Protein! Lactose! Ash!Average! 86,6%! 4,1%! 3,6%! 5,0%! 0,7%!

Chandan RC. Milk composition, physical and processing characteristics. In Hui YH, Chandan RC, Clark S, et al. Handbook of Food Products Manufacturing – Health, Meat, Milk, Poultry, Seafood, and Vegetables.

John Wiley & Sons, 2007, pps 347-377

WHAT IS MILK?"



Milk is a complex fluid, containing more than 100,000 separate molecules, the levels of which vary with the species!



WHAT IS MILK?"!!!From a physiological standpoint, milk is:!!"“A unique biological secretion of the mammary gland endowed by nature to fulfil the entire nutritional needs of the neonate”."

PURPOSE OF MILK



HOW DOES MILK ACHIEVE THIS PURPOSE?

GI & INSULIN

Last AR, Wilson SA. Am Fam Physician 2006;73:1942-8

Gannon MC, Nuttall FQ, Krezowski PA, Billington CJ, Parker S. Diabetologia. 1986 Nov;29(11):784-91.

MILK’S INSULINOTROPIC RESPONSE

Food" GI" II"

Whole Milk" 30" 90"Fermented Milk" 15" 98"Lactose! 68! 50!Refined Wheat Bread! 100! 100!

Ostman EM, et al. Am J Clin Nutr 2001;74:96 –100.

Liljeberg Elmstahl H & Bjorck I. Eur J Clin Nutr 2001; 55:994–999.

Milk (200 ml) + low GI CHO has an insulin response

~ white bread

WHOLE MILK VS SKIM MILK

Glucose SKIM MILK WHOLE MILK

Hoyt G et al. Br J Nutr. 2005 Feb;93(2):175-7

Ostman EM, et al. Am J Clin Nutr 2001;74:96 –100 Hoyt G et al. Br J Nutr. 2005 Feb;93(2):175-7

Consumption of milk

induces a reactive hypoglycaemia

similar to high glycaemic load carbohydrates

IS THIS BENEFICIAL?

Whey Proteins

GLP-1 GIP

Insulin

Nilsson M, Holst JJ, Björck IM. Am J Clin Nutr. 2007 Apr;85(4):996-1004.

Frid AH, Nilsson M, Holst JJ, Björck IM. Am J Clin Nutr. 2005 Jul;82(1):69-75.

Pratley RE, Gilbert M. Rev Diabet Stud. 2008 Summer;5(2):73-94

Insulin Resistance???

Rizza RA, et al. Diabetologia. 1985 Feb;28(2):70-5 Del Prato S, et al. Diabetologia. 1994 Oct;37(10):1025-35. Flores-Riveros JR, McLenithan JC, Ezaki O, Lane MD. Proc Natl Acad Sci 1993;90:512–6.

Hyperinsulinemia

EPIDEMIOLOGY

Positive Studies •  Pereira MA et al. JAMA 2002;287:2081-9

•  Azadbakht L et al. Am J Clin Nutr. 2005 Sep;82(3):523-30

•  Liu S et al. Diabetes Care 2005;28: 2926-32.

•  Elwood PC et al. J Epidemiol Community

Health. 2007 Aug;61(8): 695-8.

•  Hirschler V, et al. J Pediatr. 2009 Jan;154(1):101-5

•  Fumeron F, et al. Diabetes Care. 2011 Apr;34(4):813-7

Negative Studies

•  Sahi T et al. Am J Clin Nutr. 1977;30:476-81

•  Lau C et al. Diabetes Care. 2005 Jun;28(6):1397-403.

•  Lawlor DA et al. Diabet Med. 2005 Jun;22(6):808-11

•  lmon R, et al. Eur J Nutr. 2010 Apr;49(3):141-6

No association

•  Ma B et al. Am J Epidemiol. 2006 Sep 1;164(5):449-58.

•  Snijder MB et al. Am J Clin Nutr. 2007 Apr;85(4):989-95

INTERVENTION STUDIES

Hoppe C, et al. Eur J Clin Nutr. 2005 Mar;59(3):393-8

ü  N= 24 boys (8 years old)

ü  Consumed 53 g/d of Protein (1,5 lt of milk or 250 g meat) for 7 days

Hoppe C, et al. Eur J Clin Nutr. 2005 Mar;59(3):393-8

0,00

20,00

40,00

60,00

80,00

100,00

120,00

%

Insulin HOMA

MILK

Meat

MILK

Meat

Hoppe C, Mølgaard C, Dalum C, Vaag A, Michaelsen KF. Eur J Clin Nutr. 2009 Sep;63(9):1076-83


0

5

10

15

20

25

Insulin HOMA

Whey

Casein

%

RESULTS

Ecuadorian cohortThe study subjects were 99 individuals with GHRD who have beenfollowed by one of the authors (J.G.-A.) at the Institute of Endocrinol-ogy, Metabolism and Reproduction (IEMYR) in Ecuador since 1988(Fig. 1, A and B). Of these, nine subjects died during the course ofmonitoring. The age distribution of the 90 living GHRD subjectsand the control Ecuador population is shown in Fig. 1C (35). Using aquestionnaire (table S1), we collected mortality data for 53 additionalGHRD subjects who died before 1988 and obtained information on ill-nesses and cause of death for 1606 unaffected first- to fourth-degree rel-atives of the GHRD subjects. The GHRD cohort was identified on the

basis of the severe short stature of the subjects (Fig. 1, A and B) (36–38)and confirmed by genotyping (Fig. 1D). Most GHRD subjects in thiscohort were homozygous for an A-to-G splice sitemutation at position180 in exon 6 of the GHR gene (Fig. 1D). This mutation, termed E180,results in a protein that lacks eight amino acids in its extracellular do-main and is possiblymisfolded and degraded (39). TwoGHRD subjectswere homozygous for the R43X mutation, which results in a truncatedGHRprotein as a result of a premature stop codon (Fig. 1D) (40), and twoGHRD subjects were E180/R43X heterozygotes (Fig. 1D).

To confirm IGF deficiency in this cohort, we measured IGF-1 andIGF-2 concentrations (Fig. 1E) in 13 relatives and 16 GHRD subjectsranging in age from 20 to 50 years, including those whose serum waslater used for in vitro studies. Serum IGF-1 ranged from 29 to 310 ng/ml

(mean, 144) among relatives, but was!20 ng/ml in all GHRD subjects (Fig.1E). Serum IGF-2 ranged from 341 to735 ng/ml (mean, 473) among relatives,but was below 164 ng/ml in all GHRDsubjects (Fig. 1E). There was no overlapin the range of IGF-1 and IGF-2 serumvalues between GHRD subjects and rela-tives (P < 0.0001) (Fig. 1E).

The GHRD cohort shows high mortal-ity from common diseases of childhood(Fig. 1F) (41). Because of this, we consi-dered only individuals who survived toat least age 10 for further analysis of dis-eases in this cohort. Of the 30 deaths amongGHRD subjects (data from both monitor-ing and surveys) older than 10, 9 were dueto age-related diseases (8 from cardiac dis-ease, 1 stroke) and 21 were due to non–age-related causes. Compared to their relatives,GHRD subjects died muchmore frequentlyfrom accidents, alcohol-related causes, andconvulsive disorders (Fig. 2A).

Cancer was not a cause of death inGHRD subjects of any age group; how-ever, it accounted for 20% of deaths in therelatives (Fig. 2, A and B). Stomach can-cer was the predominant cause of cancer-relatedmortality in the relatives (Fig. 2C),which is consistent with the high incidenceof this cancer in Ecuador (42). Amongdeaths in each age group, the proportionfrom cancer was lower in the GHRD sub-jects than in relatives (based on the exacthypergeometricdistributionas implementedin StatXact 7, P = 0.003). Cancer accountedfor 17% of all diseases in the relatives (Fig.2D). Of all the GHRD subjects monitoredsince 1988, only one was diagnosed withcancer, a papillary serous epithelial tumorof the ovary in 2008. After treatment, shehas remained cancer-free.

We did not observe any mortality ormorbidity due to type 2 diabetes in theGHRD cohort, although diabetes was

Fig. 1. Ecuadorian cohort. (A and B) Several members of the GHRD cohort with J.G.-A. in (A) 1988 and (B)2009. (C) Agedistribution for 90 livingGHRD subjects and the Ecuadorian (Control) population. (D) Genotypesof the GHRD cohort. All GHRD subjects were identified on the basis of their short stature and very low serumIGF-1 levels. Undetermined, subjects whose genotypes have not been confirmed. (E) Serum IGF-1 and IGF-2levels in 13 unaffected relatives and 16 GHRD subjects. ***P < 0.0001. (F) Survival of the GHRD cohort.

R E S EARCH ART I C L E

www.ScienceTranslationalMedicine.org 16 February 2011 Vol 3 Issue 70 70ra13 2

on

Febr

uary

20,

201

1st

m.s

cien

cem

ag.o

rgD

ownl

oade

d fro

m

AG ING

Growth Hormone Receptor Deficiency Is Associatedwith a Major Reduction in Pro-Aging Signaling,Cancer, and Diabetes in HumansJaime Guevara-Aguirre,1*† Priya Balasubramanian,2,3* Marco Guevara-Aguirre,1 Min Wei,3

Federica Madia,3 Chia-Wei Cheng,3 David Hwang,4 Alejandro Martin-Montalvo,5,6

Jannette Saavedra,1 Sue Ingles,7 Rafael de Cabo,5 Pinchas Cohen,4 Valter D. Longo2,3,8†

Mutations in growth signaling pathways extend life span, as well as protect against age-dependent DNA dam-age in yeast and decrease insulin resistance and cancer in mice. To test their effect in humans, we monitoredfor 22 years Ecuadorian individuals who carry mutations in the growth hormone receptor (GHR) gene that leadto severe GHR and IGF-1 (insulin-like growth factor–1) deficiencies. We combined this information with surveysto identify the cause and age of death for individuals in this community who died before this period. The in-dividuals with GHR deficiency exhibited only one nonlethal malignancy and no cases of diabetes, in contrast toa prevalence of 17% for cancer and 5% for diabetes in control subjects. A possible explanation for the very lowincidence of cancer was suggested by in vitro studies: Serum from subjects with GHR deficiency reduced DNAbreaks but increased apoptosis in human mammary epithelial cells treated with hydrogen peroxide. Serumfrom GHR-deficient subjects also caused reduced expression of RAS, PKA (protein kinase A), and TOR (target ofrapamycin) and up-regulation of SOD2 (superoxide dismutase 2) in treated cells, changes that promote cellular pro-tection and life-span extension in model organisms. We also observed reduced insulin concentrations (1.4 mU/mlversus 4.4 mU/ml in unaffected relatives) and a very low HOMA-IR (homeostatic model assessment–insulinresistance) index (0.34 versus 0.96 in unaffected relatives) in individuals with GHR deficiency, indicating higher in-sulin sensitivity, which could explain the absence of diabetes in these subjects. These results provide evidence for arole of evolutionarily conserved pathways in the control of aging and disease burden in humans.

INTRODUCTIONReduced activity of growth hormone (GH) and insulin-like growthfactor–1 (IGF-1) signaling proteins or of their orthologs in nonhumanorganisms and the activation of stress resistance transcription factorsand antioxidant enzymes contribute to extended life span and protec-tion against age-dependent damage or diseases (1–16). Pathways thatregulate growth and metabolism also promote aging and genomic in-stability, a correspondence that is conserved in simple eukaryotes andmammals (7). In yeast, life span–extending mutations in genes such asSCH9, the homolog of mammalian S6K (S6 kinase), protect againstage-dependent genomic instability (17–19). Similarly, mutations inthe insulin/IGF-1–like signaling pathway increase life span and reduceabnormal cellular proliferation in worms, and mice deficient in GHand IGF-1 are not only long-lived but also show delayed occurrence

of age-dependent mutations and neoplastic disease (20–23). Amongthe frequently detected mutations in human cancers are those that ac-tivate two major signaling proteins downstream of the IGF-1 receptor(IGF-1R)—Ras and Akt—and those in the IGF-1R itself (24, 25). Thisis in agreement with a potential role for the IGF-1 signaling pathway inpromoting age-dependent mutations that lead to the activation ofproto-oncogenes and for oncogenes in exacerbating the generation ofadditional mutations and changes required for cancer progression (26). Ithas been proposed that the growth-promoting and antiapoptoticfunctions of the IGF-1 pathway underlie its putative role in cancerdevelopment and progression (27). This link is supported by somepopulation studies but not others, which instead indicate a modest as-sociation between high IGF-1 concentrations and increased risk ofcertain cancers (27, 28).

GH may also promote insulin resistance. For example, age-dependentinsulin resistance is reduced in GH- and GH receptor (GHR)–deficientmice (29–32), and GH replacement therapy can exacerbate insulin re-sistance in GH-deficient individuals, apparently because it causes aswitch from glucose metabolism to lipolysis (33).

Here, we have monitored an Ecuadorian cohort with GHR defi-ciency (GHRD), which results in IGF-1 deficiency, for 22 years andinvestigated the effect of these deficiencies on cellular responses tostress and on markers of cancer and diabetes. We show that thefundamental link between pro-growth pathways, oxidative stress,age-dependent genomic instability, and cellular damage observed inyeast (2, 15, 17–19), worms, and mice (5, 6, 20–23, 34) is conservedin humans.

1Institute of Endocrinology, Metabolism and Reproduction, Quito, Ecuador. 2Depart-ment of Molecular and Computational Biology, University of Southern California, LosAngeles, CA 90089, USA. 3Andrus Gerontology Center, University of SouthernCalifornia, Los Angeles, CA 90089, USA. 4Division of Pediatric Endocrinology, DavidGeffen School of Medicine, University of California, Los Angeles, CA 90095, USA.5Laboratory of Experimental Gerontology, National Institute on Aging, Baltimore, MD21224, USA. 6Centro Andaluz de Biología del Desarrollo, Universidad Pablo de Olavide–Consejo Superior de Investigaciones Cientificas and Centre for Biomedical Researchon Rare Diseases, ISCIII, E-41013 Sevilla, Spain. 7Division of Epidemiology, Keck Schoolof Medicine, University of Southern California, Los Angeles, CA 90089, USA. 8NorrisCancer Center, University of Southern California, Los Angeles, CA 90089, USA.*These authors contributed equally to this work.†To whom correspondence should be addressed. E-mail: [email protected] (J.G.-A.); [email protected] (V.D.L.).


www.ScienceTranslationalMedicine.org 16 February 2011 Vol 3 Issue 70 70ra13 1 o

n Fe

brua

ry 2

0, 2

011

stm

.sci

ence

mag

.org

Dow

nloa

ded

from

Guevara-Aguirre J, et al. Sci Transl Med 2011, 3:1-9.

responsible for 5% of the deaths and 6%of all diseases in the relatives (Fig. 2, A andD), in agreement with the 5% prevalence ofdiabetes in Ecuador (Fig. 2E) (43). We es-timated the prevalence of diabetes in theGHRD cohort as 0/90 = 0%, with 95% exactClopper-Pearson confidence interval: 0 to4%. To test whether the diabetes prevalencein the GHRD cohort was different from thegeneral population prevalence of 5%, weperformed an exact test of the null hypoth-esis that P = 0.05, based on the binomialdistribution, with the type I error rate, a =0.05. The P value was 0.02, indicating thatthe prevalence in the GHRD cohort is lessthan 5%. This is a particularly striking resultconsidering the elevated prevalence of obe-sity among these GHRD individuals (21%in GHRD subjects versus 13.4% in Ecuador)(Fig. 2E) (42). To investigate the mecha-nisms that could be responsible for the ob-served lack of diabetes in theGHRDcohort,we measured fasting glucose and insulinconcentrations in 13 relatives and 16 GHRDsubjects consisting of both male and femalesubjects between the ages of 20 and 50.Weobserved no significant difference in fast-ing glucose concentrations between them(Fig. 2F). However, the average insulin con-centration in the GHRD group was about athird of that in the relatives (Fig. 2G, P <0.05), and the HOMA-IR (homeostaticmodel assessment–insulin resistance) index(44) indicated that GHRD subjects (HOMA-IR = 0.34) were muchmore insulin-sensitivethan their relatives (HOMA-IR = 0.96) (Fig.2H, P < 0.05). These results are consistentwith the finding that GHRDmice and otherGH-deficient mouse models have lowserum insulin concentrations and areinsulin-sensitive (29–32).

Although GHRD subjects may have ele-vated cardiac disease mortality (Fig. 2A),the mortality from vascular diseases (com-bining cardiac disease and stroke) appears tobe similar to that of their relatives (33% ofdeaths in relatives versus 30% of deaths inGHRDsubjects) (Fig. 2A). Inagreementwithstudies of a human population with isolatedGH deficiency (45), our data suggest thatGHRD does not increase overall vasculardisease mortality (Fig. 2A).

Effect of reduced IGF-1 signalingon DNA damage and apoptosis ofdamaged cellsOur studies in Saccharomyces cerevisiae in-dicate that homologs of mammalian growth

Fig. 2. Diseases and mortality in the Ecuadorian cohort. (A) Causes of death in unaffected relativesand GHRD subjects. (B) Percentage of cancers per age group in unaffected relatives and GHRDsubjects. (C) Percent distribution of cancer deaths in the unaffected relatives. (D) Percentage of cancerand type 2 diabetes in unaffected relatives and GHRD subjects. Data are shown as a percentage of alldiagnosed/reported diseases. ^, one case of cancer; #, no case of diabetes has been recorded. (E)Percent prevalence of obesity and type 2 diabetes in Ecuador (Control) and GHRD subjects. #, no caseof diabetes has been recorded in the GHRDs. Obesity prevalence in Ecuador is based on WHO reportsfor 2010 (42), whereas that for GHRD subjects was defined as body mass index (BMI) > 30 kg/m2.Prevalence of type 2 diabetes in Ecuador was obtained from (43). (F to H) Fasting glucose (F), fastinginsulin (G), and insulin resistance (H) as indicated by HOMA-IR in relatives and GHRD subjects. Datarepresent means ± SEM for 13 control and 16 GHRD samples. *P < 0.05.



on

Febr

uary

20,

201

1st

m.s

cien

cem

ag.o

rgD

ownl

oade

d fro

m

AG ING












on

Febr

uary

20,

201

1st

m.s

cien

cem

ag.o

rgD

ownl

oade

d fro

m



AG ING












n Fe

brua

ry 2

0, 2

011

stm

.sci

ence

mag

.org

Dow

nloa

ded

from

0 Cases

Collectively, these data imply that there is an increasedrisk of pancreatitis in patients treated with either ex-enatide or sitagliptin vs the other therapies.

Pancreatic cancer. Because pancreatitis is a knownrisk factor for pancreatic cancer,17 we evaluated the reportedrates of pancreatic cancer with exenatide and sitagliptincompared to control events relative to rosiglitazone.

The reported event rate for pancreatic cancer was 2.9-fold greater in patients treated with exenatide comparedto other therapies (P ! 9 " 10#5). The reported event ratefor pancreatic cancer was 2.7-fold greater with sitagliptinthan other therapies (P ! .008).

Thyroid cancer. Because thyroid tumors were re-ported to be increased in rodents treated with liraglutidein a filing to the FDA,20 we examined the frequency ofreported adverse events of thyroid cancer with the GLP-1mimetic therapies vs rosiglitazone. The reported event ratefor thyroid cancer in patients treated with GLP-1 mimetictherapy was increased and reached statistical significance inthe exenatide group (OR ! 4.73; P ! 4 " 10#3), but not inthe sitagliptin group (OR ! 1.48; P ! .65).

All other cancers. There has been a suggestionthat DPP-4 inhibition may lead to impaired immunefunction and increased risk for cancers.18,19 Therefore, wealso examined the reported event rate for all other cancers(excluding pancreas and thyroid) associated with sitaglip-tin, exenatide, or the control therapies. Neither sitagliptinor exenatide were associated with a higher reported rate ofother cancers. The risk for cancer increases with age butage was not different between the individuals in whomcancer (mean age, 61 years other therapies, 61 years ex-enatide, 64 years sitagliptin) or a control event (mean age,62 years other therapies, 60 years exenatide, 63 years

sitagliptin) was reported for the drugs included in thisanalysis.

DiscussionWe report a $6-fold increased reported adverse

event rate for pancreatitis with either of the first twoGLP-1#based drugs available on the market in the UnitedStates, exenatide and sitagliptin, in this analysis of theFDA AERS database.

Analysis of the FDA AERS database is not the idealmechanism to compare adverse event rates between drugs.Limitations of the FDA AERS database, including incom-plete data and reporting biases, are well-known.29 How-ever, AERS has proven effective in similar earlier evalua-tions at detecting unintended drug side effects.30 –32 Thisanalysis was undertaken notwithstanding these limita-tions, given the paucity of safety data available for thisclass of drugs, which is gaining a rapid increase in usagefor a common disease. Randomized, controlled clinicaltrials remain the gold standard for such assessment.Those trials are typically powered for efficacy end pointsrelated to the relative attributes of the new drugs inaccomplishing expected goals, such as glycemic controlcompared to previously available drugs. They do not nec-essarily accumulate sufficient data (in either patient num-bers or follow-up) on infrequent or longer-term conse-quences of the drugs (eg, cancers). The primary goal ofthis study was to examine the FDA database as method-ically as possible to establish whether there are sufficientgrounds for concern that would indicate the need forstudies that specifically examine the signals that arise in aprospective manner.

The approach we have taken should be robust against arange of potential reporting biases. In particular, if thetest drugs have an overall increased reporting rate forevents, the OR will be unaffected. Similarly, if the testevents have an overall increased reporting rate, the ORwill be unaffected. However, the approach has significantweaknesses. The analysis is retrospective. Potential con-founders that influenced the choice of drug therapy fortype 2 diabetes could introduce bias. For example if cig-arette smokers were to be more likely treated with GLP-1based therapy than other therapies for type 2 diabetes, abias in favor of pancreatitis or pancreatic cancer would beintroduced. Since cigarette smoking is not reported in theFDA data base we cannot exclude an unexpected bias infavor of diabetes treatment choice in this regard. Moregenerally, the odds ratios reported here will be upwardlybiased if patients who are at higher risk for pancreatitis,pancreatic and/or thyroid cancer received exenatide or stigi-platin, either as a first line therapy, or subsequent to a poorresponse to the therapy of first choice. There are plausiblescenarios where this might happen, but we are unable tothoroughly determine the extent of this bias based on thisretrospective study.

Also, although the controls (drugs and events) wereprospectively defined, the analysis makes certain assump-

Figure 1. Odds ratio of test vs control events for exenatide, sitagliptin,and other therapies. The odds ratio of an adverse report of pancreatitis,pancreatic and thyroid cancer, or any cancer associated with exenatideand/or sitagliptin therapy vs other therapies.

CLIN

ICA

LPA

NCR

EAS

July 2011 GLP-1–BASED THERAPY AND PANCREATITIS 153

Elashoff M, et al. Gastroenterology. 2011 Jul;141(1):150-6.

HORMONES IN MILK

MILK & IGFS

DOES IT MATTER???

Gauthier SF, Pouliot Y, Maubois JL. Lait 2006; 86: 99-125. Baumrucker CR, Erondu NE. J Mammary Gland Biol Neoplasia 2000; 5: 53-64. Collier RJ, Miller MA, Hildebrandt JR, et al. J Dairy Sci 1991; 74:2905-2911.

Published assays relate exclusively to the IGF-I content in the whey fraction of

milk "!!

Underestimate the total IGF-I content in milk"


0 2 4 6 8 10 12 14 16

IGF-‐1 IGF-‐1 / IGBP-‐3

Whey

Casein

CASEIN PER LITER


0

5

10

15

20

25

Grs

COW’S MILK

HUMAN MILK

Xian CJ, Shoubridge CA, Read LC. J Endocrinol. 1995 Aug;146(2):215-25.

•  Casein and, to a lesser degree,

BSA & Lactoferrin

prevent IGF-1

Proteolysis in rats

RBGH INCREASES MILK IGF-1"""

Faulkner A. J Dairy Res. 1999 May;66(2):207-14.

Sheffield LG. J Dairy Sci 1997; 80: 2020-2024. Liebe A, Schams D. J Dairy Res 1998; 65: 93-100. Bruckmaier RM, Ontsouka CE, Blum JW. Vet Med-Czech 2004; 49: 283-290. Shuster DE, Kehrli ME, Baumrucker CR. Proc Soc Exp Biol Med 1995; 210:140–149.

Mastitis increases

IGF-1

concentrations

in Milk"

MILK INCREASES IGF-1 IN HUMANS

Estudo de intervenção

N= 82 moças caucasianas com 12 anos

Duração: 18 meses

Hoppe C, Mølgaard C, Michaelsen KF. Annu Rev Nutr. 2006;26:131-73. Rogers IS, et al. Cancer Epidemiol Biomarkers Prev 2005; 14: 204-212. Hoppe C, et al. Am J Clin Nutr 2004; 80: 447-452. Hoppe C, et al. Eur J Clin Nutr 2004; 58: 1211-1216.

20-30% increase in

Children



Duração: 18 meses

Norat T, et al. Eur J Clin Nutr 2007; 61: 91-98. Ma J, et al. J Natl Cancer Inst 2001, 93:1330-1336. Morimoto LM, et al. Cancro Causes Control 2005; 16: 917-927. Giovannucci E, et al. Cancer Epidemiol Biomarkers Prev 2003, 12:84-89 Holmes MD, et al. Cancer Epidemiol Biomarkers Prev 2002; 11: 852-861.

10-20% Increase in Adults

MILK INCREASES IGF-1 IN HUMANS

MILK INCREASES IGF-1/IGFBP-3 IN HUMANS



Duração: 18 meses

Rich-Edwards JW, Ganmaa D, Pollak MN, et al. Milk consumption and the prepubertal somatotropic axis. Nutr J. 2007 Sep 27;6:28.

Hoppe C, Molgaard C, Juul A, et al. High intakes of skimmed milk, but not meat, increase serum IGF-I and IGFBP-3 in eight-year-old boys. Eur J Clin Nutr. 2004 Sep;58(9):1211-6

Gunnell D, Oliver SE, Peters TJ, et al. Are diet prostate cancer associations mediated by the IGF axis?. A cross-sectional analysis of diet, IGF-I and IGFBP-3 in healthy middle-aged men. Br. J Cancer 2003; 88: 1682-1686.


0 2 4 6 8 10 12 14 16

IGF-‐1 IGF-‐1 / IGBP-‐3

Whey

Casein

ü Standard Method: 71,7ºC for 15’’"

ü UHT: " 130º-150ºC for 1-5’’ "

Thomas EL. J Dairy Sci 1981; 64: 1023-1027.

Elliott AJ, et al. J Dairy Res 2005; 72: 442–446.

Kang SH, et al. J Dairy Sci 2006; 89: 402-409.

UHT 130º for 2’’ reduces IGF-1 by 10%

PASTEURIZATION"

Collier RJ, et al. J Dairy Sci 1991; 74:2905-2911

121ºC for 15 min reduces IGF-1 to virtually undetected levels

Yogurt: "ü 85ºC for 30 min"ü 95ºC for 10 min"

"

http://www.milkfacts.info/Milk%20Processing/Milk%20Processing%20Page.htm

75º for 15 min decreases IGF-1 by 45% Kang SH, et al. J Dairy Sci 2006; 89: 402-409.

PASTEURIZATION"

FERMENTATION"!!

!!!

!!!!!!

"""""Reduces IGF-1 (measured in whey) by 20%"

""

""!

Kang SH, et al. J Dairy Sci 2006; 89: 402-409.

Melnik BC, Schmitz G. Exp Dermatol. 2009 Oct;18(10):833-41

Wilkinson SB, et al. Am J Clin Nutr. 2007 Apr;85(4):1031-40

Cordain L. et al. Comparative Biochemistry and Physiology Part A 2003; 136: 95–112

Elevated plasma IGF-1/ IGFBP-3 ratio represents a risk factor for

certain epithelial cell cancers"

REVIEW

Emerging role of insulin-like growth factor receptor inhibitors in oncology:early clinical trial results and future directions

A Gualberto1,2 and M Pollak3

1Clinical Department, Pfizer Oncology, New London, CT, USA; 2Department of Pathology, Brown University Alpert School ofMedicine, Providence, RI, USA and 3Department of Oncology, McGill University, Montreal, Quebec, Canada

Preclinical evidence that targeting the insulin-like growthfactor receptor (IGF-IR) is effective in cancer treatmenthas been accumulating for almost two decades. Efforts todevelop drugs began in the late 1990s, and initial datafrom clinical trials were reported in 2006. The biologicalrationale for IGF-IR targeting has potential relevanceto many tumor types, and early results have justifiedexpanded programs to evaluate IGF-IR-targeting agentsin many areas of clinical need. More than two dozen drugcandidates have been developed and clinical trials areunderway for at least 12 of these. Early clinical trialsreveal an acceptable safety profile together with pharma-codynamic evidence that the receptor can be successfullytargeted. It is premature to draw conclusions regardingefficacy, but well-documented instances of single-agentactivity were noted during phase I evaluations, andrecent evidence from a phase II study suggests thatco-administration of an anti-IGF-IR antibody withchemotherapy for non-small-cell lung cancer improvesobjective response rate and progression-free survival. Withmore than 70 trials involving a variety of drug candidatesunderway, the IGF-IR is becoming one of the mostintensively investigated molecular targets in oncology.Early results justify the continuation of ongoing researchacross a broad range of cancer indications.Oncogene advance online publication, 6 July 2009;doi:10.1038/onc.2009.172

Keywords: insulin-like growth factor-I receptor inhibi-tors; clinical trials; targeted therapies

Introduction

Research on insulin-like growth factors (IGFs) and theirreceptors in cancer biology has been ongoing for over20 years (Myal et al., 1984; Pollak et al., 1987; Arteagaet al., 1989). Model systems provided evidence forimportant roles in neoplasia. Examples include genetic

alterations that reduce ligand levels (Pollak et al., 2001,2004; Baserga et al., 2003; Majeed et al., 2005; Hartoget al., 2007; Sachdev and Yee, 2007; Samani et al., 2007;Chitnis et al., 2008; Weroha and Haluska, 2008; Yuenand Macaulay, 2008; Pollak, 2008a) as well as knock-down methods (Wu et al., 2003). One important themethat emerged from this work was the notion thatmultiple oncogenes require the presence of the insulin-like growth factor receptor (IGF-IR) to achieve cellulartransformation (Sell et al., 1993; Martin et al., 2006);another was that that IGF-I signaling confers resistanceto many antineoplastic therapies (Wiseman et al., 1993;Lu et al., 2001). Interest in IGF-IR targeting increasedwith the publication of evidence linking IGF-I signalingwith the onset of neoplasia. Examples include evidencefor associations between circulating IGF-I and cancerrisk (Chan et al., 1998), between IGF-I and mammo-graphic breast density (Diorio et al., 2005), betweengrowth rate in adolescence (which is IGF-I mediated)and cancer risk (Ahlgren et al., 2004), and theobservation that IGF-II overexpression was among themost common molecular derangements in colorectalcancer (Zhang et al., 1997).

Earlier review articles (Baserga et al., 2003; Pollaket al., 2004; Hartog et al., 2007; Sachdev and Yee, 2007;Samani et al., 2007; Chitnis et al., 2008; Weroha andHaluska, 2008; Yuen and Macaulay, 2008; Pollak,2008a) have summarized IGF-IR biology, its relevanceto neoplasia and the preclinical evaluation of drugcandidates targeting the IGF-IR. Recent studies haveprovided further evidence that IGF-IR inhibition can beuseful in attenuating the malignant behavior of cancersin which KRAS (Klinakis et al., 2009) or EGF receptorfamily members (Buck et al., 2008; Huang et al., 2009)play important roles in pathophysiology.

Close to 30 drug candidates targeting the IGF-IR arebeing investigated (Baserga et al., 2003; Pollak et al.,2004; Yee, 2006; Hartog et al., 2007; Sachdev and Yee,2007; Samani et al., 2007; Tao et al., 2007; Chitnis et al.,2008; Feng and Dimitrov, 2008; Rodon et al., 2008;Yuen and Macaulay, 2008; Pollak, 2008a, b). Here, wefocus specifically on the available clinical trial andtranslational research data concerning 12 of thesecompounds currently in clinical trials. The large numberof trials planned and in progress provide importantopportunities for the incorporation of ‘companion’studies that will provide serum and tissue samples forReceived 11 March 2009; revised 14 May 2009; accepted 23 May 2009

Correspondence: Dr M Pollak, Segal Cancer Centre, McGillUniversity, 3755, Chemin de la Cote Sainte-Catherine, Room E-763,Montreal, Quebec, Canada H3T 1E2.E-mail: [email protected]

Oncogene (2009), 1–13& 2009 Macmillan Publishers Limited All rights reserved 0950-9232/09 $32.00

www.nature.com/onc

Archives of Physiology and Biochemistry, 2009; 115(2): 58–71

R E V I E W A R T I C L E

!e insulin-like growth factor-I receptor as an oncogene

Haim Werner1, and Ilan Bruchim2

1Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, and 2Gynecologic Oncology Unit, Department of Obstetrics and Gynecology, Meir Medical Center, Kfar Saba 44281, a!liated with the Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel

Address for Correspondence: Haim Werner, Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel. Tel: 972-3-6408542. Fax: 972-3-6406087. E-mail: [email protected]

(Received 23 December 2008; revised 18 January 2009; accepted 27 January 2009)

Introduction

!e availability of highly sophisticated technolo-gies, including “classical” genomic and proteomic approaches as well as novel microarray-based, high-throughput platforms with speci"c aims (e.g. micro-RNA detection, DNA methylation identi"cation, single nucleotide polymorphism analysis, etc.), is having a huge impact in all areas of medicine, including the "eld of cancer research. Speci"cally, these techniques provide us with the opportunity to investigate physi-ological and pathological processes at various levels of regulation, and to tackle biological questions in an integrative and unbiased fashion. !e insulin-like growth factors (IGFs) constitute a group of cellular and secreted factors with important roles in multiple biological systems. Since their discovery in the mid-1950s, IGFs have attracted the attention of develop-mental biologists, endocrinologists, oncologists, and others (LeRoith et al., 2001a; Salmon & Daughaday, 1957). In recent years, the vast amount of informa-tion generated by experimentalists, clinicians, and

epidemiologists led to the development of molecular tools aimed at targeting the IGF axis as a clinically relevant therapeutic target in cancer and, potentially, other conditions (Hartog et al., 2007; Riedemann & Macaulay, 2006). !e aim of this review article is to summarize evidence accumulated over the last 20 years, which identi"ed the IGF-I receptor (IGF-IR) as a potential cellular oncogene. In addition, vari-ous targeted therapies and several speci"c cancers in which the IGF axis is involved will be discussed in more detail.

!e IGF system: Ligands, receptors and binding proteins

!e IGF ligands comprise IGF-I, IGF-II, insulin, and a number of non-classical ligands whose biological role is still controversial (Werner & LeRoith, 2000)(Table 1). At the cellular level, IGF-I is a progression factor that is required by the cell to advance through the various phases of the cell cycle. !e concentration

ISSN 1381-3455 print/ISSN 1744-4160 online © 2009 Informa UK LtdDOI: 10.1080/13813450902783106

AbstractThe insulin-like growth factor-I receptor (IGF-IR) mediates the biological actions of both IGF-I and IGF-II. The IGF-IR is expressed in most transformed cells, where it displays potent antiapoptotic, cell-survival, and transforming activities. IGF-IR expression is a fundamental prerequisite for the acquisition of a malignant phenotype, as suggested by the !nding that IGF-IR-null cells (derived from IGF-IR knock-out embryos) are unable to undergo transformation when exposed to cellular or viral oncogenes. This review article will focus on the underlying molecular mechanisms that are responsible for the normal, physiological control of IGF-IR gene expression, as well as the cellular pathways that underlie its aberrant expression in cancer. Examples from the clinics will be presented, including a description of how the IGF system is involved in breast, prostate, pediatric, and gynecological cancers. Finally, current attempts to target the IGF-IR as a therapeutic approach will be described.

Keywords: Insulin-like growth factor-I (IGF-I); IGF-I receptor; targeted therapies; gene expression; cancer

http://www.informapharmascience.com/arp

Downloaded At: 12:53 14 May 2009

AG ING












on

Febr

uary

20,

201

1st

m.s

cien

cem

ag.o

rgD

ownl

oade

d fro

m


RESULTS

Ecuadorian cohortThe study subjects were 99 individuals with GHRD who have beenfollowed by one of the authors (J.G.-A.) at the Institute of Endocrinol-ogy, Metabolism and Reproduction (IEMYR) in Ecuador since 1988(Fig. 1, A and B). Of these, nine subjects died during the course ofmonitoring. The age distribution of the 90 living GHRD subjectsand the control Ecuador population is shown in Fig. 1C (35). Using aquestionnaire (table S1), we collected mortality data for 53 additionalGHRD subjects who died before 1988 and obtained information on ill-nesses and cause of death for 1606 unaffected first- to fourth-degree rel-atives of the GHRD subjects. The GHRD cohort was identified on the

basis of the severe short stature of the subjects (Fig. 1, A and B) (36–38)and confirmed by genotyping (Fig. 1D). Most GHRD subjects in thiscohort were homozygous for an A-to-G splice sitemutation at position180 in exon 6 of the GHR gene (Fig. 1D). This mutation, termed E180,results in a protein that lacks eight amino acids in its extracellular do-main and is possiblymisfolded and degraded (39). TwoGHRD subjectswere homozygous for the R43X mutation, which results in a truncatedGHRprotein as a result of a premature stop codon (Fig. 1D) (40), and twoGHRD subjects were E180/R43X heterozygotes (Fig. 1D).

To confirm IGF deficiency in this cohort, we measured IGF-1 andIGF-2 concentrations (Fig. 1E) in 13 relatives and 16 GHRD subjectsranging in age from 20 to 50 years, including those whose serum waslater used for in vitro studies. Serum IGF-1 ranged from 29 to 310 ng/ml

(mean, 144) among relatives, but was!20 ng/ml in all GHRD subjects (Fig.1E). Serum IGF-2 ranged from 341 to735 ng/ml (mean, 473) among relatives,but was below 164 ng/ml in all GHRDsubjects (Fig. 1E). There was no overlapin the range of IGF-1 and IGF-2 serumvalues between GHRD subjects and rela-tives (P < 0.0001) (Fig. 1E).

The GHRD cohort shows high mortal-ity from common diseases of childhood(Fig. 1F) (41). Because of this, we consi-dered only individuals who survived toat least age 10 for further analysis of dis-eases in this cohort. Of the 30 deaths amongGHRD subjects (data from both monitor-ing and surveys) older than 10, 9 were dueto age-related diseases (8 from cardiac dis-ease, 1 stroke) and 21 were due to non–age-related causes. Compared to their relatives,GHRD subjects died muchmore frequentlyfrom accidents, alcohol-related causes, andconvulsive disorders (Fig. 2A).

Cancer was not a cause of death inGHRD subjects of any age group; how-ever, it accounted for 20% of deaths in therelatives (Fig. 2, A and B). Stomach can-cer was the predominant cause of cancer-relatedmortality in the relatives (Fig. 2C),which is consistent with the high incidenceof this cancer in Ecuador (42). Amongdeaths in each age group, the proportionfrom cancer was lower in the GHRD sub-jects than in relatives (based on the exacthypergeometricdistributionas implementedin StatXact 7, P = 0.003). Cancer accountedfor 17% of all diseases in the relatives (Fig.2D). Of all the GHRD subjects monitoredsince 1988, only one was diagnosed withcancer, a papillary serous epithelial tumorof the ovary in 2008. After treatment, shehas remained cancer-free.

We did not observe any mortality ormorbidity due to type 2 diabetes in theGHRD cohort, although diabetes was

Fig. 1. Ecuadorian cohort. (A and B) Several members of the GHRD cohort with J.G.-A. in (A) 1988 and (B)2009. (C) Agedistribution for 90 livingGHRD subjects and the Ecuadorian (Control) population. (D) Genotypesof the GHRD cohort. All GHRD subjects were identified on the basis of their short stature and very low serumIGF-1 levels. Undetermined, subjects whose genotypes have not been confirmed. (E) Serum IGF-1 and IGF-2levels in 13 unaffected relatives and 16 GHRD subjects. ***P < 0.0001. (F) Survival of the GHRD cohort.



on

Febr

uary

20,

201

1st

m.s

cien

cem

ag.o

rgD

ownl

oade

d fro

m

AG ING












on

Febr

uary

20,

201

1st

m.s

cien

cem

ag.o

rgD

ownl

oade

d fro

m


responsible for 5% of the deaths and 6%of all diseases in the relatives (Fig. 2, A andD), in agreement with the 5% prevalence ofdiabetes in Ecuador (Fig. 2E) (43). We es-timated the prevalence of diabetes in theGHRD cohort as 0/90 = 0%, with 95% exactClopper-Pearson confidence interval: 0 to4%. To test whether the diabetes prevalencein the GHRD cohort was different from thegeneral population prevalence of 5%, weperformed an exact test of the null hypoth-esis that P = 0.05, based on the binomialdistribution, with the type I error rate, a =0.05. The P value was 0.02, indicating thatthe prevalence in the GHRD cohort is lessthan 5%. This is a particularly striking resultconsidering the elevated prevalence of obe-sity among these GHRD individuals (21%in GHRD subjects versus 13.4% in Ecuador)(Fig. 2E) (42). To investigate the mecha-nisms that could be responsible for the ob-served lack of diabetes in theGHRDcohort,we measured fasting glucose and insulinconcentrations in 13 relatives and 16 GHRDsubjects consisting of both male and femalesubjects between the ages of 20 and 50.Weobserved no significant difference in fast-ing glucose concentrations between them(Fig. 2F). However, the average insulin con-centration in the GHRD group was about athird of that in the relatives (Fig. 2G, P <0.05), and the HOMA-IR (homeostaticmodel assessment–insulin resistance) index(44) indicated that GHRD subjects (HOMA-IR = 0.34) were muchmore insulin-sensitivethan their relatives (HOMA-IR = 0.96) (Fig.2H, P < 0.05). These results are consistentwith the finding that GHRDmice and otherGH-deficient mouse models have lowserum insulin concentrations and areinsulin-sensitive (29–32).

Although GHRD subjects may have ele-vated cardiac disease mortality (Fig. 2A),the mortality from vascular diseases (com-bining cardiac disease and stroke) appears tobe similar to that of their relatives (33% ofdeaths in relatives versus 30% of deaths inGHRDsubjects) (Fig. 2A). Inagreementwithstudies of a human population with isolatedGH deficiency (45), our data suggest thatGHRD does not increase overall vasculardisease mortality (Fig. 2A).

Effect of reduced IGF-1 signalingon DNA damage and apoptosis ofdamaged cellsOur studies in Saccharomyces cerevisiae in-dicate that homologs of mammalian growth

Fig. 2. Diseases and mortality in the Ecuadorian cohort. (A) Causes of death in unaffected relativesand GHRD subjects. (B) Percentage of cancers per age group in unaffected relatives and GHRDsubjects. (C) Percent distribution of cancer deaths in the unaffected relatives. (D) Percentage of cancerand type 2 diabetes in unaffected relatives and GHRD subjects. Data are shown as a percentage of alldiagnosed/reported diseases. ^, one case of cancer; #, no case of diabetes has been recorded. (E)Percent prevalence of obesity and type 2 diabetes in Ecuador (Control) and GHRD subjects. #, no caseof diabetes has been recorded in the GHRDs. Obesity prevalence in Ecuador is based on WHO reportsfor 2010 (42), whereas that for GHRD subjects was defined as body mass index (BMI) > 30 kg/m2.Prevalence of type 2 diabetes in Ecuador was obtained from (43). (F to H) Fasting glucose (F), fastinginsulin (G), and insulin resistance (H) as indicated by HOMA-IR in relatives and GHRD subjects. Datarepresent means ± SEM for 13 control and 16 GHRD samples. *P < 0.05.



n Fe

brua

ry 2

0, 2

011

stm

.sci

ence

mag

.org

Dow

nloa

ded

from

0 Cases

1 Case

CLINICAL STUDY

Congenital IGF1 deficiency tends to confer protection againstpost-natal development of malignanciesRachel Steuerman1, Orit Shevah1 and Zvi Laron1,21Endocrinology and Diabetes Research Unit, Schneider Children’s Medical Center, 14 Kaplan Street, Petah Tikva 49202, Israel and 2WHO CollaboratingCenter for the Study of Diabetes in Youth, and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel

(Correspondence should be addressed to Z Laron at Endocrinology and Diabetes Research Unit, Schneider Children’s Medical Center;Email: [email protected])

Abstract

Objective: To investigate whether congenital IGF1 deficiency confers protection against development ofmalignancies, by comparing the prevalence of malignancies in patients with congenital (secondary)deficiency of IGF1 with the prevalence of cancer in their family members.Method: Only patients with an ascertained diagnosis of either Laron syndrome (LS), congenital IGHD,congenital multiple pituitary hormone deficiency (cMPHD) including GH or GHRHR defect wereincluded in this study. In addition to our own patients, we performed a worldwide survey and collecteddata on a total of 538 patients, 752 of their first-degree family members, of which 274 were siblingsand 131 were further family members.Results: We found that none of the 230 LS patients developed cancer and that only 1 out of 116patients with congenital IGHD, also suffering from xeroderma pigmentosum, had a malignancy. Out of79 patients with GHRHR defects and out of 113 patients with congenital MPHD, we found threepatients with cancer in each group.Among the first-degree family members (most heterozygotes) of LS, IGHD and MPHD, we found30 cases of cancer and 1 suspected. In addition, 31 malignancies were reported among 131further relatives.Conclusions: Our findings bear heavily on the relationship between GH/IGF1 and cancer. Homozygouspatients with congenital IGF1 deficiency and insensitivity to GH such as LS seem protected from futurecancer development, even if treated by IGF1. Patients with congenital IGHD also seem protected.

European Journal of Endocrinology 164 485–489

Introduction

Linked to their proliferative, differentiation and apoptoticproperties, both GH and insulin-like growth factor 1(IGF1) have been identified as risk factors for certainmalignancies (1–5), even in the pediatric age group andyoung adults (6–8). There is also evidence that mosttumors and transformed cells display increased IGF1receptor (IGF-1R) concentration and high IGF1RmRNA,causing enhanced IGF1 binding (9, 10), leading to theaxiom that overexpression of IGF1R is a pre-requirementfor acquisition or progress of malignant tumors (11).Further evidence for the link between GH, IGF1 andcancer is the successful use of GH and IGF-1R-blockingagents in the treatment of malignancy (12). The abovefindings led us to investigate the reverse situation, namelywhether congenital defects of GH and IGF1 action woulddiminish or prevent the development of cancer.

In 2007, we reported preliminary data on 169patients with Laron syndrome (LS) and on 250 of theirfirst- and second-degree relatives and found that none ofthe homozygous LS patients had developed cancer,

whereas 24% of their heterozygous family membershad (13). We also showed that 35 patients withcongenital (c) IGHD and 18 with GHRH receptor (R)mutation reported no malignancies. In contradiction,9–24% of their family members had a history of cancer.

The aim of the present study was to extend ourfindings by enlarging the number of patients with LSand cIGHD and to collect data on patients with GHRHRmutations and congenital multiple pituitary hormonedeficiency (cMPHD), including GH.

Methods

This retrospective study was approved by the ethicscommittee. The following procedures were employed tocollect the data:

a. We updated data from our medical charts of LSpatients, which had increased to 67 and, 28 patientswere with cIGHD.

b. Sent questionnaires to authors of published reportson patients with the diagnoses mentioned.

European Journal of Endocrinology (2011) 164 485–489 ISSN 0804–4643

q 2011 European Society of Endocrinology DOI: 10.1530/EJE-10-0859Online version via www.eje-online.org

cohort) and probably a large percentage of those withGHRHR mutations.

In total, the patients were significantly younger thantheir first-degree relatives (P!0.001), though thedifferences in the GHRHR defect and the cMPHD groupswere not significant. Also, there was no significant agedifference when we compared the age of patients withthat of their siblings. It is worth mentioning that thepatients’ age range was wide, reaching 85 years, andthat 25% of the patients with recorded age were above30 years of age.

Table 2 shows the prevalence of malignancies inthe patients and their relatives in each diagnostic group.It is seen that none of the230 LS patients had developed amalignancy, despite the fact that 66 had been or are stilltreated by IGF1 and two had received hGH as well. Thedifference between the prevalence of malignancies in allthe first-degree relatives and that in the siblings alone,with that in the patients was significant (P!0.001,PZ0.005, respectively). Out of the 116 patients withcIGHD, only one boy suffers from xeroderma pigmento-sum was reported to have a basal cell carcinoma (BCC)diagnosed at the age of 15 years. Fifty-nine of cIGHDpatients had been treated with hGH, including the boywho developed BCC. Out of the 79 patients with GHRHRmutations (12 previously treated with hGH), three hadcancer (one of them previously treated with hGH) andso had three out of 113 patients with cMPHD. In thelatter group, 97 had been previously treated with hGH,in addition to other hormone replacement therapy, andamong them, two had developed malignancies.

Out of 752 first-degree relatives, 26 reported 30instances of cancer (PZ0.019) and out of 131 further

relatives, 30 reported 31 instances of cancer(P!0.001). Nine malignancies were reported in eightof the siblings (PZ0.165). In Table 3, it is evident thatthe majority are lung, breast and prostate cancer,followed by colon and gastric cancer.

Discussion

In this attempt of a worldwide survey of the prevalenceof cancer in patients with secondary congenital IGF1deficiency, we confirmed and enlarged our preliminaryobservation that patients with LS, having bothcongenital GH and IGF1 deficiency and insensitivity toendogenous and exogenous GH, seem protected fromthe development of cancer (13). That observation wasalso confirmed by findings in the large Ecuadoriancohort of LS patients (15). Considering that the lattercohort accounts for 100 living patients, it is obviousthat over 300 patients with LS, the majority now beingadults, have not developed cancer.

In contradistinction, 15 of their 218 first-degreerelatives in the present survey and 24 out of 131 furtherrelatives (proven or supposed heterozygotes for thedisease) reported 43 instances of cancer. The differencebetween presence or absence of malignancy betweenpatients with LS and their first-degree relatives andsiblings was statistically significant (P!0.001,PZ0.005, respectively). In the Ecuadorian cohort, 80out of 1032 relatives died of cancer (15).

Only one boy out of 116 patients with congenitalIGHD presented a BCC of the cheek. This patient alsosuffered from xeroderma pigmentosum, a genetic defect

Table 2 The prevalence of malignancy in the four diagnostic groups.

Diagnostic groups

Laron sydrome cIGHD GHRHR defects cMPHD Total

PatientsTotal number (n) 230 116 79 113 538Number of malignancies 0 1b 3c 3d 7Prevalence of malignancy (%) 0.0 0.9 3.8 2.7 1.3

First-degree relativesTotal number (n) 218 203 150 181 752Number of malignancies 18 (15) 7 (6) 0 5C1a (6) 30 (26C1)a

Prevalence of malignancy (%) 8.3 3.4 0.0 2.8 4.0Further relativesTotal number (n) 113 13 4 1 131Number of malignancies 25 (24) 4 (4) 1 (1) 1 (1) 31 (30)Prevalence of malignancy (%) 22.1 30.8 25 – 23.7

Siblings onlyTotal number (n) 86 96 6 86 274Number of malignancies 5 (4) 2 (2) 0 2 (2) 9 (80)Prevalence of malignancy (%) 5.8 2.1 0.0 2.3 3.3

P valuesPatients versus first-degree relatives !0.001 0.43 0.04 1 0.019Patients versus further relatives !0.001 !0.001 0.182 – !0.001Patients versus siblings 0.005 0.59 1 1 !0.165

Number in parentheses indicate number of individuals. aIndicates one suspected malignancy; bincludes one male basal cell carcinoma diagnosed at 15 years;cincludes one male Hodgkins lymphoma diagnosed at 43 years and two male skin cancers (one diagnosed at 37 years and the other unknown); dincludes twofemale thyroid carcinoma diagnosed at 10.5 and 52 years and one male planocellular carcinoma of the nasal septum diagnosed at 66 years of age.

GH/IGF1 deficiency and cancer 487EUROPEAN JOURNAL OF ENDOCRINOLOGY (2011) 164

www.eje-online.org

LS – secondary congenital deficiency of IGF1 and primary GH insensitivity, due to defects in the GH receptor.

cIGHD – Primary congenital isolated hGH deficiency

GHRHReceptor (R) defect – secondary hGH deficiency.

cMPHD – Congenital multiple pituitary hormone deficiency, including hGH

Prevalence of malignancy in the four diagnostic groups

BETA-CELLULIN

BETA-CELLULIN IN DAIRY

Bastian SE, et al. Measurement of betacellulin levels in bovine serum, colostrum and milk. J Endocrinol. 2001 Jan;168(1):203-12.

TGF-α: Transforming Growth Factor Alpha HB-EGF: Heparin Binding EGF EPR: Epiregulin AR: Amphiregulin (NRG1, NRG2, NRG3, NRG4): Neuregulins 1, 2, 3 and 4

ErB1 – EGF-R

Cordain L. Dietary implications for the development of acne: a shifting paradigm.

In: U.S. Dermatology Review II 2006, (Ed.,Bedlow, J). Touch Briefings Publications, London, 2006.

Cordain L. Dietary implications for the development of acne: a shifting paradigm.

In: U.S. Dermatology Review II 2006, (Ed.,Bedlow, J). Touch Briefings Publications, London, 2006.

EGF in saliva: 0.0512 ng/ ml

Total Saliva Secretion: 691 ml/24 hours

EGF in 24 hour Saliva: 35.3 ng

BTC per liter of Bovine Milk:

1930 ng

Cordain L. U.S. Dermatology Review II 2006, (Ed.,Bedlow, J). Touch Briefings Publications, London, 2006

EGF upregulates its own receptor Increased signalling

ü Breast

ü Colon

ü Prostate

ü Ovaries

ü Lung

Nanda R. Rev Recent Clin Trials. 2007 May;2(2):111-6. Gravis G, Bladou F, Salem N, et al. Ann Oncol. 2008 May 7. Palayekar MJ, Herzog TJ. Int J Gynecol Cancer. 2007 Dec 5. Mándoky L, et al. Anticancer Res. 2004 Jul-Aug;24(4):2219-24.

Cohen D, et al. Clin Genitourin Cancer. 2007 Mar;5(4):264-70. Davies DE, Chamberlin SG. Biochem Pharmacol. 1996 May 3;51(9):1101-10. Seiwert TY, Cohen E. Angiogenesis Oncol 2005;1:7-10 Henson ES, Gibson SB. Cell Signal. 2006 Dec;18(12):2089-97.

ü  Pancreas

ü  Stomach

ü  Testicle

ü Kidney

ü Head & Neck

EGF-R OVEREXPRESSION OCURS IN VARIOUS TYPES OF CANCER

SOME EFFECTS OF BETA-CELLULIN & IGF-1

Milk e aumento da estatura

MILK INCREASES LINEAR GROWTH

Epidemiological Studies

Hoppe et al. Am J Clin Nutr 2004;80:447-52

Paganus et al. Acta Paediatr 1992;81:518-21

Stallings et al. JPGN 1994;18:440-5

Isolauri et al. J Pediatr 1998;132:1004-9

Black et al. Am J Clin Nutr 2002;76:675-80

Annu. Rev. Nutr. 2006. 26:131–73

13 Jun 2006 13:48 AR ANRV282-NU26-06.tex XMLPublishSM(2004/02/24) P1: OKZ

140 HOPPE ! MØLGAARD ! MICHAELSEN

to schoolchildren was examined (164). In a very simple and elegant experiment,Boyd Orr compared the effects of supplementing the diet of children at schoolwith (a) whole milk, (b) skimmed milk, or (c) biscuits that contained the sameamount of energy as the other supplements. In addition, a control group did notreceive any supplementation. The intervention was conducted in three age groupsof children: 5–6, 8–9, and 13–14 years.

Irrespective of the age group, compared with those in the control group, childrenwho received either whole or skim milk grew 20% more in height during sevenmonths. Children who received the biscuits did not grow any differently from thosein the control group (Figure 4). In a follow-up study on these children, Leighton& Clark (126) found that those who continued to receive milk supplements for asecond year continued to grow at the faster rate, and those children who stoppedreceiving milk supplements after a year returned to the slower growth rate. Becauseinformation was not provided, it is not possible to evaluate the degree to which thechildren participating in the study were poorly nourished at the onset of the study.However, because this study was conducted in the 1920s, it is plausible that somedegree of malnutrition was present.

More remarkable results than those of the Boyd Orr study were found with asimilar supplementation program in New Guinea. It was conducted among 7- to13-year-old Bundi children who had a very-low-protein diet and the majority of

Height increase

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

6 y group 9 y group 13 y group

whole milk skimmed milk biscuits control

Inch

es

Figure 4 Height increases during a seven-month period in the classical Boyd Orr studyfrom 1928. The effects of four different interventions given together with the school lunchwere examined. Children were given whole milk, skimmed milk, biscuits that contained thesame energy as the other diets, or they did not receive any supplements. Created from (164).

Ann

u. R

ev. N

utr.

2006

.26:

131-

173.

Dow

nloa

ded

from

ww

w.a

nnua

lrevi

ews.o

rgby

Lun

d U

nive

rsity

Lib

rarie

s, H

ead

Offi

ce o

n 07

/25/

11. F

or p

erso

nal u

se o

nly.

Boyd Orr Study (1928)

Nilo,c Pastoralists Country Height

Dinka1 Sudan 181.9 cm Shilluk1 Sudan 182.6 cm

Maasai2 Tanzania & Kenya 173.5 cm

1 - Roberts DF, Bainbridge DR. Am J Phys Anthropol 1963; 21 (3): 341–370 2 - Mann GV. Am J Clin Nutr. 1974 May;27(5):464-9

Green J. Lancet Oncol. 2011 Jul 20

Wiley AS. Cow’s Milk Consumption and Health – An Evolutionary Perspective. In Trevathan WR, Smith EO, McKenna JJ. Evolutionary

Medicine and Health – New Perspectives. Oxford University Press, 2008.

Milk promotes linear growth & may promote Premature Puberty

Insulin IGF-1

Akt

Akt P

FoxO1

P FoxO1

14-3-3 protein

Proteasome Low nuclear level of FoxO1

PI3K P

P

P

AR

PP FoxO1

Androgen receptor transactivation

Keratinocyte proliferation

Increased seba-ceous lipogenesis

Inflammation NFκB upregulation

Increased insulin/IGF-1 signaling reduces nuclear levels of FoxO1, the key regulator in acne pathogenesis!

P

Melnik BC. Br J Dermatol. 2010 Jun;162(6):1398-400. Melnik BC. J Dtsch Dermatol Ges. 2010 Feb;8(2):105-14.

ESTROGENS IN MILK"

Farlow DW, Xu X, Veenstra TD. J Chromatogr B Analyt Technol Biomed Life Sci. 2009 May 1;877(13):1327-34

Controlo

Sulfato de Estrona no lactosoro

0 100 200 300 400 500 600 700 800 900

1000

Vaca no embarazada

Vaca Embarazada (41-60 dias)

Vaca Embarazada (220-240 dias)

ESTRONE SULFATE (WHEY) pg/ml

Sato A. Health Effects of Cow Milk. Symposium on Milk, Hormones and Human Health, Harvard, Boston, Oct. 2006 www.milksymposium.ca.org

Non Pregnant Cow!

Pregnant Cow!(41-60 days)!

Pregnant Cow!(220-240 days)!

ESTROGENS IN MILK

Farlow DW, Xu X, Veenstra TD. J Chromatogr B 2009 May 1;877(13):1327-34

Highly

Reactive

Catechol

Estrogens

Farlow DW, Xu X, Veenstra TD. J Chromatogr B 2009 May 1;877(13):1327-34

ESTROGENS IN MILK

DOES IT MATTER???

Ganmaa D, Tezuka H, Enkhmaa D, et al. Int J Cancer 2006; 118: 2363-2365. Ganmaa D, Sato A. Med Hypotheses 2005; 65: 1028-37.

Commercial milk is

uterotrophic in rats

Qin LQ, Xu JY, Wang PY, et al. Int J Cancer 2004; 110: 491-496. Qin LQ, Xu JY, Tezuka H, et al. Cancer Detect Prev 2007; 31: 339-343. Ma DF, Katoh R, Zhou H, et al. Acta Histochem Cytochem 2007; 40: 61-67. Zhou H, Qin LQ, Tang FL, et al. Food Chem Toxicol 2007; 45: 1868-1872.

DMBA = 7,12-dimethylbenz(a) anthracene

Commercial whole, low, and nonfat milk promote growth of!DMBA-induced mammary tumors in rats.!

Comparison between basal excretion volumes (basal) and maximum excretion volumes (peak) of (a) urine estrone, (b) estradiol, (c) estriol and (d) pregnanediol before and after intake of cow milk in prepubertal children (n = 6).

the hormones was detected at 1 h in three children, at 3 h in threechildren, and at 4 h in one child. The net increase in volume ofurine excretion of E2 at 4 h after the intake of milk ranged from39 to 109 ng. Urinary basal and peak excretion volumes of E1,E2, E3 and pregnanediol before and after milk intake are shownin Figure 5. Urinary excretion of these hormones significantlyincreased after intake (P < 0.02).

Women

In four out of five women, ovulation occurred during milk intakein the second menstrual cycle, and the timing of ovulation wassimilar among the three menstrual cycles. In these four women,the third menstruation and ovulation occurred regularly. In onewoman, however, aged 36 years who had a menstrual cycle of

37–39 days, ovulation did not occur during the intake of milk.She ovulated 7 days after stopping milk intake.

Discussion

Toxicological and epidemiological studies have indicated that E2could be categorized as a carcinogen.14 Milk is considered to bea rich source of estrogens. Indeed, E2 concentration is higher inmammary drainage than in the peripheral circulation in high-yielding cows.12 Pregnant cows are under the control of relativelyhigh levels of estrogens, and milk produced from pregnant cowscontains correspondingly high concentrations of estrogens.Estrogen concentration in milk has been measured since the1970s, mainly as an indicator of pregnancy.15,16 Concentration ofE1 sulfate increases from 30 pg/mL in non-pregnant cows to

7

6

5

4

1

100(a) (b)

(c) (d)

(ng/

h)(n

g/h)

(ng/

h)(µ

g/h)

80

60

40

20

0

100

Before 1 h

80

60

40

20

02 h 3 h 4 h

120

100

60

40

20

0

Before 1 h0

2 h 3 h 4 h

80

2

3

Fig. 4 Changes in mean urine excretion volume of (a) estrone, (b) estriol, (c) estradiol and (d) pregnanediol after intake of cow milk inprepubertal children (mean 1 SE, n = 6).

140

(a) (b) (c) (d)

(ng/

h)

120

100

80

60

40

20

Basal Peak Basal Peak Basal Peak Basal Peak

140

(ng/

h)120

100

80

60

40

20

140

(ng/

h)

120

100

80

60

40

20

7

(µg/

h)

6

5

4

3

2

1

P<0.02 P<0.02 P<0.02 P<0.02

Fig. 5 Comparison between basal excretion volumes (basal) and maximum excretion volumes (peak) of (a) urine estrone, (b) estradiol, (c)estriol and (d) pregnanediol before and after intake of cow milk in prepubertal children (n = 6).

36 K Maruyama et al.

© 2010 Japan Pediatric Society

Maruyama K, Oshima T, Ohyama K. Pediatr Int. 2010 Feb;52(1):33-8.

0

20

40

60

80

100

120

ng

E2 Production rate ng/day

Net increase of E2 excretion ng/4h

E2 PRODUCTION RATE VS EXCRETION IN PREPUBERTAL BOYS


performed to examine the difference of hormone concentrationsbefore and after the intake of milk. P < 0.05 was taken assignificant.

Approval for the present study was obtained from the ethicscommittee of the University of Yamanashi School of Medicine.

Results

Men

Serum basal and peak concentrations of E1, E2 and progesteroneduring examination of the milk intake are shown in Figure 1.Serum E1 concentration was significantly increased and peaked30–60 min after the intake of milk (mean 1 SE, before and peak:102.3 1 10.3 pg/mL and 128.9 1 11.8 pg/mL, P < 0.02). SerumE2 concentration was unchanged during the 2 h examination(before and peak: 31 1 4 pg/mL and 32 1 4 pg/mL, NS). Serumprogesterone concentration significantly increased and peaked30–60 min after the intake of milk (mean 1 SE, before and peak:0.66 1 0.08 ng/mL and 0.75 1 0.10 ng/mL, P < 0.02).

Serum basal and nadir concentrations of LH, FSH and test-osterone before and after milk intake are shown in Figure 2.Serum LH and FSH concentration gradually decreased in six outof seven men, and reached a nadir 60–120 min after the intake of

milk (before and lowest point: LH, 3.29 1 0.49 mIU/mL and 2.481 0.43 mIU/mL, P < 0.05; FSH, 3.43 1 0.17 mIU/mL and 3.19 10.15 mIU/mL, P < 0.02). Serum testosterone concentrationsdecreased considerably 120 min after intake in all subjects(before and lowest point: 6.04 1 0.38 ng/mL and 4.94 1 0.13 ng/mL, P < 0.02).

As shown in Figure 3, the volume per hour of urinary excre-tion of E1, E2, E3 and pregnanediol significantly increased afterthe intake of milk in all subjects (P < 0.02). Urine E1 excretionincreased 1 h after intake, and reached a peak 4 h after intake infive out of seven men. Urine E2 excretion increased 1 h after theintake in six out of seven men. Peak excretion of E2 in urine wasdetected after 1 h in three subjects, and after 4 h in another threesubjects. Urine E3 excretion also increased after 1 h in six out ofseven men, and reached peak levels after 4 h in five men. Urinepreganediol excretion peaked after 1 h in two out of seven men,and after 4 h in another four men.

Prepubertal children

Changes in urinary excretion volumes of E1, E2, E3 and preg-nanediol are shown in Figure 4. Urinary excretion patterns weresimilar among these four hormones. Peak excretion volume of

(a) (b) (c)

(ng/

mL

)150

100

50

Basal Peak

(pg/

mL

)

40

30

10

Basal Peak

(ng/

mL

)

Basal Peak

20

0.8

0.6

0.2

0.4

P<0.02 P<0.02NS

Fig. 1 Comparison between basal levels and peak levels of (a)serum estrone (E1), (b) estradiol (E2) and (c) progesterone beforeand after intake of cow milk in men (n = 7).

1000

(a) (b) (c) (d)

(ng/

h)

900

800

700

600

500


300

(ng/

h)

250

200

150

100

50

(ng/

h)

120

100

80

60

40

20

(mg/

h)

20

15

10

5

P<0.02

P<0.02

P<0.02 P<0.02

Fig. 3 Comparison between basal excretion volumes (basal) and maximum excretion volumes (peak) of (a) urine estrone, (b) estradiol, (c)estriol and (d) pregnanediol before and after intake of cow milk in men (n = 7).

(a) (b) (c)

(mIU

/mL

)

4

2

1

Basal Nadir Basal Nadir Basal Nadir

(mIU

/mL

)

4

3

1

(ng/

mL

)

2

8

6

2

4

3

P<0.05 P<0.02 P<0.02

Fig. 2 Comparison between basal and nadir levels of (a) serumluteinizing hormone, (b) follicle-stimulating hormone and (c) test-osterone before and after intake of cow milk in men (n = 7).

Exposure to estrogen from pregnant cow milk 35




Results

Men







(a) (b) (c)

(ng/

mL

)

150

100

50

Basal Peak

(pg/

mL

)

40

30

10

Basal Peak

(ng/

mL

)

Basal Peak

20

0.8

0.6

0.2

0.4

P<0.02 P<0.02NS


1000

(a) (b) (c) (d)(n

g/h)

900

800

700

600

500


300

(ng/

h)

250

200

150

100

50

(ng/

h)

120

100

80

60

40

20

(mg/

h)

20

15

10

5

P<0.02

P<0.02

P<0.02 P<0.02


(a) (b) (c)

(mIU

/mL

)

4

2

1


(mIU

/mL

)

4

3

1

(ng/

mL

)

2

8

6

2

4

3

P<0.05 P<0.02 P<0.02




Comparison between basal excretion volumes (basal) and maximum excretion volumes (peak) of (a) urine estrone, (b) estradiol, (c) estriol and (d) pregnanediol before and after

intake of cow milk in men (n = 7).




Results

Men







(a) (b) (c)

(ng/

mL

)

150

100

50

Basal Peak

(pg/

mL

)

40

30

10

Basal Peak

(ng/

mL

)

Basal Peak

20

0.8

0.6

0.2

0.4

P<0.02 P<0.02NS


1000

(a) (b) (c) (d)

(ng/

h)

900

800

700

600

500


300

(ng/

h)

250

200

150

100

50

(ng/

h)

120

100

80

60

40

20

(mg/

h)

20

15

10

5

P<0.02

P<0.02

P<0.02 P<0.02


(a) (b) (c)

(mIU

/mL

)

4

2

1


(mIU

/mL

)

4

3

1

(ng/

mL

)

2

8

6

2

4

3

P<0.05 P<0.02 P<0.02





Comparison between basal and nadir levels of (a) serum luteinizing hormone, (b) follicle-stimulating hormone and (c) testosterone before and after intake of cow

milk in men (n = 7).

.

1.  Schairer C, Hill D, Sturgeon SR et al. Cancer Epidemiol Biomarkers Prev 2005; 14: 1660-1665. 2.  Hankinson SE, Eliassen AH. J Steroid Biochem Mol Biol 2007; 106: 24-30. 3.  Eliassen AH, Missmer SA, Tworoger SS, et al. J Nat Cancer Inst 2006; 98: 1406-1415. 4.  Cavalieri E, Chakravarti D, Guttenplan J, et al. Biochim Biophys Acta 2006; 1766: 63-78. 5.  Persson I. J Steroid Biochem Mol Biol 2000;74:357–64. 6.  Yager JD, Davidson NE. N Engl J Med 2006; 354: 270-82. 7.  Ganmaa D, Sato A. Med Hypotheses 2005; 65: 1028-37 8.  Qin LQ, Wang PY, Kaneko T, et al. Med Hypotheses. 2004;62(1):133-42. 9.  Shantakumar S, Terry MB, Paykin A, et al. Am J Epidemiol. 2007;165: 1187-1198. 10.  Kahlenborn C. Modugno F, Potter DM, et al. Mayo Clin Proc. 2006;81: 1290-1302. 11.  Chlebowski RT, Hendrix SL, Langer RD, et al. JAMA. 2003 Jun 25;289(24):3243-53. 12.  Rossouw JE, Anderson GL, Prentice RL, et al. JAMA. 2002 Jul 17;288(3):321-33 13.  Beral V; Million Women Study Collaborators. Lancet. 2003 Aug 9;362(9382):419-27. 14.  Chen WY, Manson JE, Hankinson SE, et al. Arch Intern Med 2006; 166: 1027-1032.

Estrogens are implicated in Prostate, Ovarian, Uterine & Breast cancer

DHT PRECURSORS IN MILK

Milk Milk

Milk

Danby FW. Clinics in Dermatology 2010; 28: 598–604

1.  BPH

2.  Acne

3.  Male Pattern Baldness

DHT

ARE COWS ON A QUEST FOR REVENGE?? "

CANCER & MILK

Epidemiology

PROSTATE CANCER

24 Nutrition and Cancer 2004

Table 1. Characteristics of Case-Control Studies Related to Milk Consumption and Prostate Cancer Risk from Published Studies

ReferenceCountry(area)

Number ofCases/Controls

Type ofControl Quantile Items

OR(95% CI)a Match or Adjustment

Mishina et al. (9)b Japan 99/99 Population 2 Milk 1.18(0.67–2.07)c

Age, residence

Talamini e al. (10)b Italy 166/202 Hospital 2 Milk and dairyproducts

2.46(1.29–4.69)

Age, marital status,occupation, BMI

Ohno et al. (11) Japan 100/100 Hospital 2 Eggs and milks 0.80(0.45–1.40)

Age, date of admission

Mettlin et al. (12)b New York 371/371 Hospital 4 Whole milk, 2%milk, skim milk

2.49(1.27–4.87)

Age, residence

La Vecchia et al. (13) Italy 96/292 Hospital 3 Milk, cheese,butter

5.0(1.5–16.6)

Age, residence,education, BMI

Talamini et al.(14)b Italy 271/685 Hospital 3 Milk, cheese,butter

1.58(1.06–2.36)

Age, residence,education, BMI

De Stefani et al. (15)b Uruguay 156/302 Hospital 3 Milk 1.7(1.1–2.9)

Age, residence,education, tobacco,beer

Ewings and Bowie (16)b UnitedKingdom

159/325 Hospital 4 Milk 0.95(0.50–1.83)

Age

Chan et al. (17)b Sweden 526/536 Population 4 Dairy 1.49(1.01–2.19)

Age, family history,energy

Jain ets al. (18)b Canada 617/636 Population 4 Milk, yogurt,cheese

1.47(1.11–1.94)

Age, energy, vasectomy,marital status,education, BMI

Hayes et al. (19) United States 932 /1201 Population 4 Dairy 1.2(0.9–1.5)

Age, study site, race

Tzonou et al. (20) Greece 320/246 Hospital 5 Milk and dairyproducts

1.55(0.92–2.62)

Age, height, Queteletindex, education,energy

Deneo-Pellegrini et al. (21)b Uruguay 175/233 Hospital 4 Dairy food 0.8(0.4–1.6)

Age, residence,education, familyhistory, BMI

Bosetti et al. (22)b Greece 320/246 Hospital 3 Milk and dairyproducts, butter

1.34(0.85–2.09)

Age, height, education,energy

Berndt et al. (23)b United States 69/385 Hospital 3 Milk 1.26(0.57–2.79)

Age, energy

a: OR and CI compared the highest with the lowest quantile of consumption and reflected the greatest degree of control for confounders. They were the resultsfrom the first food item in the Items column.

b: Studies included in the calculation of meta-analysis.c: OR and 95% CI were calculated from the numbers of cases and controls using a standard method.

Figure 1. Meta-analysis schematic concerning the OR of prostate cancer associated with milk consumption in 11 case-control studies calculated usingBiostat™ software (7). Horizontal bars represent the 95% CI. The combined OR in the random-effects model was 1.68 (CI = 1.34–2.12).

Dow

nloa

ded

by [L

und

Uni

vers

ity L

ibra

ries]

at 0

8:35

25

July

201

1

META-ANALYSIS SCHEMATIC CONCERNING THE OR OF PROSTATE CANCER ASSOCIATED WITH MILK CONSUMPTION IN 11 CASE-CONTROL STUDIES

Horizontal bars represent the 95% CI. The combined OR in the random-effects model was 1.68 (CI = 1.34–2.12).

Qin LQ, et al. Nutr Cancer. 2004;48(1):22-7

./01234%/5% 678%936:/234%*32;81% )2<6:6068=%>/4?%@"=%9/?%AB=%C8;8DE81%"=%AFFG% &'()*+,-% !""B

;368K/1I% [%%%%%(3E48% A%\?% %V:K7%%%% P81<0<% 4/N%%%%%%% J3:1I% R1/J0;6% :263T8% N3<%%3<%</;:368J%N:67%3%!AM% :2;183<8% :2% 1:<T% 5/1% 6/634%R1/<6368% %;32;81%[''%l%!?!Ac%@GM%*)%l%!?F%6/%!?A\%32J%3%BBM%:2;183<8%:2%1:<T%5/1%3JP32;8J%R1/<6368%;32;81%[''%l%!?BBc%@GM%*)%l%!?F%6/%!?$\?%W/1%%7:K7%%%%%P81<0<%4/N%%%%%%;34;:0D%:263T8=%678%R//48J%18436:P8%1:<T<%5/1%6/634%%:!C!)!;?!)!=>!)!=P!)!=C!<%%32J%3JP32;8J% %:!C!)!;?!)!=P!)!=C!<%%R1/<6368%;32;81%N818%!?B$%[@GM%*)%l%!?F%6/%!?$\%32J%!?^#%[@GM%*)%l%F?"%6/%B?B\=%18<R8;Q6:P84I?%(7818%N3<%<636:<6:;344I%<:K2:!%;326%78681/K828:6I%3D/2K%678%;34;:0D%<60J:8<%[%Q%%68<6]%%/%%5/1%6/634%32J%3JP32;8J%R1/<6368%;32;81%l%?F"%32J%?F!B=%18<R8;6:P84I\=%E06%2/6%3D/2K%678%J3:1I%R1/J0;6%<60JQ:8<%[%Q%%68<6]%%/%o?!%5/1%6/634%32J%3JP32;8J%R1/<6368%;32;81\?%9/28%/5%678% <60JI% ;7313;681:<6:;<=% :2;40J:2K% 678%D832% 3K8%/5% <0Eh8;6<% 36%E3<84:28=%678%J0136:/2%/5%5/44/NQ0R=%678%4/;36:/2%/5%678%<60JI=%/1%678% I831% 678% <60JI% N3<% R0E4:<78J=% 73J% <636:<6:;344I% <:K2:!%;326%%8558;6<%/2%678%R//48J%18436:P8%1:<T<%5/1%R1/<6368%;32;81%3<</;:368J%N:67%J3:1I%R1/J0;6%/1%;34;:0D%:263T8%[%/%o?FG%5/1%344\?%%%%(78%1:<T%/5%R1/<6368%;32;81%:2;183<8J%N:67%:2;183<:2K%:263T8%/5%

J3:1I%5//J<%32J%/5%;34;:0D%[%/%%6182J%%l%?FA@%32J%?F!^=%18<R8;6:P84I\%[%%%%%(3E48%B%\?%Y782%32%:2681;8R6%681D%N3<%:261/J0;8J%:26/%678%3234IQ<8<=%7/N8P81=%678%;/85!%;:826%5/1%J3:1I%:263T8%32J%R1/<6368%;32;81%J8;183<8J%[51/D%F?F#$%6/%F?FGGc%%/%%6182J%%l%?!!\?%&467/0K7%678%;/85Q!%;:826% 5/1% ;34;:0D% :263T8% 32J% R1/<6368% ;32;81% :2;183<8J% [51/D%F?F!%6/%F?F!B%5/1%83;7%!FF%DK%/5%:263T8\=%678%<632J31J%811/1%34</%:2;183<8J%[51/D%F?FFA%6/%F?FF"\=%32J%<636:<6:;34%<:K2:!%;32;8%N3<%4/<6%[%/%%6182J%%l%?!G@\?%)2%E/67%3234I<8<=%678%:2681;8R6<%[F?FB%5/1%J3:1I%R1/J0;6<%32J%%�%F?FB%5/1%;34;:0D\%N818%2/6%<636:<6:;344I%<:K2:!%;326%[%/%o?^% 5/1% E/67\?%Y782% 344% <60J:8<% N818% :2;40J8J% :2% 678% J/<8%X%%18<R/2<8%3234I<8<=%678%;/85!%;:826%5/1%J3:1I%R1/J0;6<%:2%18436:/2%6/%R1/<6368% ;32;81% J8;183<8J% <4:K764I% [51/D% F?F#$% 6/% F?F^@\% E06%%18D3:28J% <636:<6:;344I% <:K2:!%;326% [%/%%6182J%% l% ?FA"\=% N78183<% 678%%;/85!%;:826% 5/1% ;34;:0D% :2;183<8J% [51/D%F?F!% 6/% F?F!#% 5/1% 83;7%!FF%DK%/5%:263T8\%32J%<636:<6:;34%<:K2:!%;32;8%J:J%2/6%;732K8%51/D%678%:2:6:34%18<046<%[%/%%6182J%%l%?F!B\?%(78%3<</;:36:/2%E86N882%J3:1I%:263T8%32J%678%1:<T%/5%R1/<6368%;32;81%J8;183<8J%<4:K764I%N782%N8%3JJ8J%:;8%;183D%:263T8%6/%6/634%J3:1I%5//J<%[;/85!%;:826%l%F?FG@=%%/%%6182J%%l%?FA@\%32J%N782%N8%:2;40J8J%/24I%<60J:8<%R815/1D8J%:2%678%L2:68J%-6368<%[;/85!%;:826%l%F?FGB=%%/%%6182J%%l%?!$G\?%%%

%%%!c*D@!M@!%%%%'8436:P8%1:<T<%/5%R1/<6368%;32;81%;/DR31:2K%678%7:K78<6%N:67%678%4/N8<6%;34;:0D%:263T8%;368K/1:8<?%%"$$5'.C!>$)15%%l%R//48J%18436:P8%1:<T<%5/1%344%<:U%<60J:8<?%%"$$5'.!J%%l%R//48J%18436:P8%1:<T%:2;40J:2K%678%<60JI%EI%_31/2%86%34?%%:!>;!<0%%%%%"$$5'.!M!%l%R//48J%18436:P8%1:<T%5/1%<60J:8<%N:67%P34:J368J%5//J%518k082;I%k08<6:/223:18<%32J%8281KI%3Jh0<6D826%[%%%%l%^\%%:!C!)!K!)!=P!)!=C!<%?%%%%%0$)%%:2J:;368%R/:26%8<6:D368<%5/1%18436:P8%1:<T<% [''\c% '##$#! G1#%% :2J:;368% @GM% ;/2!%J82;8% :2681P34<% [*)<\c% .*16$3.%%:2J:;368%''<%32J%@GM%*)<%51/D%R//48J%3234I<8<?%%

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

!!0!7,4a,,7`_!!

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s:2%86%34?%%:!=J!<%%

18R/168J%3%;/DE:28J%/JJ<%136:/%/5%R1/<6368%;32;81%/5%!?#$%5/1%D82%:2%678%7:K78<6%D:4T%:263T8%;368K/1I%P81<0<%D82%:2%678%4/N8<6%:263T8%;368K/1I=%N7:;7%:<%K183681%6732%678%R//48J%18436:P8%1:<T%/5%R1/<6368%;32;81% [!?!!\% 5/1% <0Eh8;6<% :2% 678% 7:K78<6% J3:1I% :263T8% ;368K/1I%%;/DR318J%N:67%67/<8%:2%678%4/N8<6%;368K/1I%6736%N8%18R/16%7818?%(7:<%J:558182;8%:2%1:<T%8<6:D368<%D3I=%:2%R316=%18"%8;6%18;344%E:3<=%3%;/DD/2%R1/E48D%:2%;3<8%X%;/261/4%<60J:8<%6736%;32%483J%6/%32%/P81Q8<6:D36:/2%/5% 678%3<</;:36:/2%E86N882%3%J:8631I%P31:3E48%32J%678%1:<T% /5% ;32;81% %:!?K!<%?% W016781D/18=% 8:K76% /5% 678% !!% <60J:8<% :2% 678%D863Q3234I<:<%EI%s:2%86%34?%%:!=J!<%%0<8J%7/<R:634QE3<8J%;/261/4%<0EQh8;6<c%;3<8%X%;/261/4%<60J:8<%6736%0<8%7/<R:634QE3<8J%;/261/4%<0Eh8;6<%73P8%E882%<7/N2%6/% 18R/16%K183681%/JJ<%136:/<% 6732%8:6781%;3<8%X%;/261/4% <60J:8<% 0<:2K% R/R0436:/2QE3<8J% ;/261/4% <0Eh8;6<% /1% R1/Q<R8;6:P8%<60J:8<%%:!@J!)!@;!<%?%&2/6781%R/<<:E48%183</2%5/1%678%J:558182;8%:2%1:<T%8<6:D368<%:<%6736%678%R1/<R8;6:P8%32J%;3<8%X%%;/261/4%<60J:8<%73J%J:5581826%8UR/<018%48P84<%5/1%678%7:K78<6%:263T8%;368K/1:8<?%

%%%!c*D@!J@! !%%%'8436:P8%1:<T<%/5%R1/<6368%;32;81%;/DR31:2K%678%7:K78<6%N:67%678%4/N8<6%J3:1I% R1/J0;6% :263T8% ;368K/1:8<?%%% %"$$5'.C!>$)15%% l% R//48J% 18436:P8% 1:<T<% 5/1% 344% !F%<60J:8<?% %"$$5'.!J%%l%pooled relative risk for studies%N:67%P34:J368J%5//J%518k082;I%k08<6:/223:18<%[%%%%l%^\% %:!K!)!=P!!A!!=C!<%?% %"$$5'.!M%%l%R//48J%18436:P8%1:<T%5/1%<60J:8<%N:67%P34:J368J%5//J%518k082;I%k08<6:/223:18<%32J%8281KI%3Jh0<6D826%[%%%%l%B\%%:!K!)!=B!)!=C!<%?%0$)%%:2J:;368%R/:26%8<6:D368<%5/1%18436:P8%1:<T<%[''\c%%'##$#!G1#%!%:2J:;368%@GM%;/2!%J82;8%:2681P34<%[*)<\c%.*16$3.%%:2J:;368%''<%32J%@GM%*)<%51/D%R//48J%3234I<8<?%%%%

at Lund University Library on July 25, 2011

jnci.oxfordjournals.orgD

ownloaded from

Gao X, LaValley MP, Tucker KL. J Natl Cancer Inst. 2005 Dec 7;97(23):1768-77.

1.  Raimondi S, Mabrouk JB, Shatenstein B, Maisonneuve P, Ghadirian P. Diet and prostate cancer risk with specific focus on dairy products and dietary calcium: a case-control study. Prostate. 2010 Jul 1;70(10):1054-65.

2.  Butler LM, Wong AS, Koh WP, Wang R, Yuan JM, Yu MC. Calcium intake increases risk of prostate cancer among Singapore Chinese. Cancer Res. 2010 Jun 15;70(12):4941-8

3.  Kurahashi N, Inoue M, Iwasaki M, et al. Dairy product, saturated fatty acid, and calcium intake and prostate cancer in a prospective cohort of Japanese men. Cancer Epidemiol Biomarkers Prev. 2008 Apr;17(4):930-7.

4.  Ahn J, Albanes D, Peters U, et al. Dairy products, calcium intake, and risk of prostate cancer in the prostate, lung, colorectal, and ovarian cancer screening trial. Cancer Epidemiol Biomarkers Prev. 2007 Dec;16(12):2623-30.

5.  Mitrou PN, Albanes D, Weinstein SJ et al. A prospective study of dietary calcium, dairy products and prostate cancer risk (Finland). Int J Cancer; 120(11):2466-73, 2007.

6.  Rohrmann S, Platz EA, Kavanaugh CJ, et al. Meat and dairy consumption and subsequent risk of prostate cancer in a US cohort study. Cancer Causes Control. 2007 Feb;18(1):41-50.

7.  Gao X, LaValley MP, Tucker KL. Prospective studies of dairy product and calcium intakes and prostate cancer risk: a meta-analysis. J Natl Cancer Inst. 2005 Dec 7;97(23):1768-77.

MILK, CA & PROSTATE CANCER

BREAST CANCER

INCONCLUSIVE EVIDENCE

Controlo

•  Missmer SA, Smith-Warner SA, Spiegelman D, et al. Meat and dairy food consumption and breast cancer: a pooled analysis of cohort studies. Int J Epidemiol 2002;31: 78-85. •  Moorman PG, Terry PD. Consumption of dairy products and the risk of breast cancer: a review of the literature. Am J Clin Nutr 2004; 80: 5-14. •  Michels KB, Mohllajee AP, Roset-Bahmanyar E, Beehler GP, Moysich KB. Diet and breast cancer, a review of the prospective observational studies. Cancer 2007; 109(12 Suppl): 2712–2149. •  Cho E, Spiegelman D, Hunter DJ, Chen WY, Stampfer MJ, Colditz GA, Willett WC. Premenopausal fat intake and risk of breast cancer. J Natl Cancer Inst 2003; 95: 1079-1085. •  Shin M, Holmes MD, Hankinson SE, Wu K, Colditz GA, Willett WC. Intake of dairy products, calcium and vitamin D and risk of breast cancer. J Natl Cancer Inst 2002; 94: 1301-1311.

OVARIAN CANCER

Controlo

Genkinger JM, Hunter DJ, Spiegelman D, et al. Cancer Epidemiol Biomarkers Prev. 2006 Feb;15(2):364-72.

Modest elevation in the risk of ovarian cancer was seen for lactose intake ~ to 3+ servings of milk per day

COLON CANCER

further quantified the association between milk and dairyproduct intakes and colorectal cancer risk by conducting linearand nonlinear dose–response analyses, and in addition, we alsoconducted detailed subgroup analyses to identify potentialsources of heterogeneity. In the most recent report from theWCRF/AICR, it was stated that milk intake probably protectsagainst colorectal cancer risk, while there was limited suggestiveevidence that cheese increases risk. No judgment was providedfor total dairy product intake, but our results suggest aprotective effect. However, the result for total dairy productintake may largely be driven by an effect of milk intake, as milkaccounts for a large part of total dairy intakes in mostpopulations. The difference between our results and the resultsfrom the report, with regard to cheese intake, may partly be dueto two additional studies having been published after the reportwas completed [24, 26], which found inverse associations, thusdriving the overall result toward the null. These additionalstudies may also have adjusted for more dietary confoundersthan several older studies. The inverse associations with milkand total dairy products were restricted to colon cancer, incontrast to the results from the pooled analysis where aninverse association with milk intake was also observed for rectalcancer [55]. However, fewer studies were included in our rectalcancer analysis compared with the pooled analysis, thus we mayhave had limited statistical power in this subgroup analysis.Our meta-analysis may have several limitations that need to

be addressed. Because of the observational nature of the data,it is possible that the observed inverse association between

milk and total dairy product intakes and colorectal cancer riskcould be due to unmeasured or residual confounding. Higherintake of milk and dairy products may be associated withother health behaviors including higher levels of physicalactivity, lower prevalence of smoking and overweight/obesityand lower intakes of alcohol and red and processed meat [27,29], although it is also possible that different types of dairyproducts may be differentially associated with some of theseconfounders [24]. However, many of the studies adjusted forknown confounding factors such as age, body mass index,smoking, alcohol, red and processed meat and energy intake.In addition, the results were generally similar in the subgroupanalyses when we stratified the results according to adjust-ment for confounding factors or other study characteristics,with no heterogeneity between subgroups for total dairyproducts and milk. Only in the analysis of cheese was theresome indication of heterogeneity between studies with andwithout adjustment for red meat with an inverse associationin studies that adjusted for red meat, while studies withoutadjustment for red meat showed a nonsignificant positiveassociation, suggesting potential confounding. Nevertheless,because of the few studies in these subgroups any futurestudies could clarify this finding.Although publication bias can be a problem in meta-analyses

of published literature, we found no evidence of such bias inthis analysis. The few studies that were excluded from the dose–response analysis are not likely to have altered the results,

.4.6

.81

1.2

Est

imat

ed R

R

0 100 200 300 400 500 600 700 800

Total milk (g/day)

Best fitting fractional polynomial95% confidence interval

B

A

.4.6

.81

1.2

Est

imat

ed R

R

0 200 400 600 800 1000 1200

Total dairy products (g/day)

Best fitting fractional polynomial95% confidence interval

Figure 3. Total dairy products and total milk and colorectal cancer,

nonlinear dose–response. RR, relative risk. Relative Risk

.25 .5 .75 1 2

Study Relative Risk (95% CI)

Kampman, 1994 0.92 ( 0.73, 1.17)

Jarvinen, 2001 0.91 ( 0.76, 1.11)

McCullough, 2003 0.94 ( 0.83, 1.05)

Sanjoaquin, 2004 1.00 ( 0.72, 1.39)

Kesse, 2005 0.78 ( 0.57, 1.08)

Lin, 2005 1.03 ( 0.80, 1.34)

Larsson, 2006 0.81 ( 0.72, 0.91)

Park, 2007 0.91 ( 0.83, 0.99)

Lee, 2009 0.81 ( 0.59, 1.11)

Overall 0.90 ( 0.85, 0.94)

B

Relative Risk

.25 .5 .75 1 2

Study Relative Risk (95% CI)

Kampman, 1994 0.86 ( 0.57, 1.29)

Martinez, 1996 0.89 ( 0.56, 1.42)

Jarvinen, 2001 0.72 ( 0.33, 1.57)

McCullough, 2003 0.96 ( 0.78, 1.18)

Sanjoaquin, 2004 1.10 ( 0.65, 1.87)

Kesse, 2005 0.54 ( 0.33, 0.89)

Lin, 2005 1.12 ( 0.72, 1.74)

Larsson, 2006 0.67 ( 0.51, 0.87)

Park, 2007 0.81 ( 0.70, 0.95)

Lee, 2009 0.80 ( 0.50, 1.20)

Overall 0.83 ( 0.74, 0.93)

A

Figure 4. Milk and colorectal cancer. CI, confidence interval.

review Annals of Oncology

6 | Aune et al.

at Lund University Library on July 25, 2011

annonc.oxfordjournals.orgD

ownloaded from

Aune D, et al. Ann Oncol. 2011 May 26

This meta-analysis shows that

milk and total dairy products, but not cheese or

other dairy products, are associated with a

reduction in colorectal cancer

risk.

OTHER TYPES OF CANCER Stang A, BAUMGARDT-ELMS, C. et al. Adolescent milk fat and galactose

consumption and testicular germ cell cancer. Cancer Epidemiol Biomarkers Prev; 15(11):2189-95, 2006.

Ganmaa D, Li XM, Qin LQ, et al. The experience of Japan as a clue to the

etiology of testicular and prostatic cancers. Med Hypotheses. 2003 May;60(5):724-30.

Bravi F, Bosetti C, Scotti L, et al. Food groups and renal cell

carcinoma : a case-control study from Italy. Int J Cancer. 2007 Feb 1;120(3):681-5.

Peters ES, Luckett BG, Applebaum KM, Marsit CJ, McClean MD, Kelsey KT.

Dairy products, leanness, and head and neck squamous cell carcinoma . Head Neck. 2008 Sep;30(9):1193-205.

MILK & ACNE (Epidemiology)

Dose Extra Batata Doce

Controlo

Adebamowo CA, Spiegelman D, Danby FW, et al High school dietary dairy intake and teenage acne. J Am Acad Dermatol; 52(2):207-14, 2005.

Adebamowo CA, Spiegelman D, Berkey CS, et al. Milk consumption and acne in adolescent girls. Dermatol Online J; 12(4):1, 2006. Adebamowo CA, Spiegelman D, Berkey CS, et al. Milk consumption and acne in teenaged boys. J Am Acad Dermatol. 2008 May;58(5):787-93

Thank you very much!

[email protected]

Dairy, Hormones and human health by Pedro Bastos

Business

Transcript of Dairy, Hormones and human health by Pedro Bastos