Association of Peripheral Blood Mitochondrial DNA Content With Type 2 Diabetic

8/18/2019 Association of Peripheral Blood Mitochondrial DNA Content With Type 2 Diabetic

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Journal of Applied Sciences Research, 8(10): 4958-4963, 2012

ISSN 1819-544XThis is a refereed journal and all articles are professionally screened and reviewed

ORIGINAL ARTICLES

Corresponding Author: Hala M. Raslan, Molecular Genetic and Enzymology department, National Research Center.

Association of Peripheral Blood Mitochondrial DNA Content with Type 2 Diabetic

Patients

1Wahiba A. Zarouk,

2Hala M. Raslan,

2Omnia Moguib,

1Ahmed I. Abdelneam,

1Ahmed F. Abd

El Razeek,1Maged M. Mahmoud,

1Mai E. Zekrie.

1 Molecular Genetic and Enzymology department and

2 Internal Medicine department; National Research

Center.

ABSTRACT

Aim: Mitochondrial DNA (mtDNA) content is essential for maintaining normal mitochondrial function, and

the mitochondrial function is critical for the production and the release of insulin in type 2 diabetes mellitus. We

investigated whether peripheral blood mtDNA content was reduced in type 2 diabetes. Methods: Fifteen

Egyptian Type 2 diabetes (T2DM) and twelve normal subjects were enrolled in this study. The quantity ofrelative mtDNA content was measured by a real-time PCR and corrected by simultaneous measurement of the

nuclear DNA. An assay based on real-time quantitative PCR was used for both nuclear DNA (nDNA) and

mtDNA quantification using SYBR green as a fluorescent dye (Invitrogen, Buenos Aires, Argentina). The copy

number of mtDNA and nDNA was calculated from the C T number and by use of the standard curve Plasma

glucose was analysed using automated autoanalyzer. Results: A significant difference in the mtDNA/nDNA

ratio among the control subjects and patients was reported; since results from this study detected reduced

mtDNA content in type 2 diabetes patients (13.36 +/- 6.13) compared to healthy individuals (109.15 +/- 49.4).

Conclusion: Our results demonstrated that lower peripheral blood mtDNA content is associated with type 2diabetes.

Key words: Mitochondrial DNA content; peripheral blood; real-time quantitative polymerase chain reaction;

Type 2 diabetes mellitus.

Introduction

In human cells, mitochondria are the only organelles that contain extrachromosomal DNA. Mitochondria

are essential organelles that primarily function to support aerobic respiration and to provide energy substrates,and their function is intimately related to insulin secretion and possibly insulin action ( Gerbitz et al 1995, Velho

et al 1996, Lee et al 2005, Ritz and Berrut 2005).

Each human cell contains hundreds of mitochondria (Approximately 300-2000 mitochondria are present in

each cell) each with 2 to 10 copies of mitochondrial DNA (mtDNA), which is small, circular double-stranded

DNA of 16,569 bp. Mitochondrial DNA contains 37 genes, which encode 2 ribosomal RNAs (rRNAs), 22

transfer RNAs (tRNAs), and 13 polypeptides. All of the encoded polypeptides are components of the respiratorychain/oxidative phosphorylation system (Anderson et al 1981 , Calvo etal 2006 and Falkenberg et al 2007).

Mitochondrial genetics is unique in many ways. First, mtDNA is more susceptible to oxidative damage and

has a higher mutation rate than nuclear DNA due to a lack of protective histones, lack of an efficient DNArepair system, and its close proximity to reactive oxygen species (ROS) in mitochondria (Croteau and Bohr

1997). Specifically, mtDNA is physically associated with the inner mitochondrial membrane, where highly

mutagenic oxygen radicals are generated as by-products of oxidative phosphorylation (Allen and Raven

1996,Hsin-Chen and Yau-Huei 2005). Second, cells contain hundreds or thousands of mitochondria; since

mtDNA is present in multiple copies, pathologic mutations or variants in mtDNA often result in heteroplasmy,

the coexistence of both wild-type and mutated mtDNA. Virtually every human organ system can be affected,

and many of the pathogenic mtDNA mutations are heteroplasmic. However, most mtDNA SNPs are

homoplasmic and are often related to age, neurodegeneration, or metabolic disorders. Some SNPs in controlregions are also associated with mitochondrial content (Brownlee, 2001 Wong 2007; Lu et al 2010;).

Mitochondria are not only the power house responsible for ATP production via the Krebs cycle and oxidative

phosphorylation to keep cell alive but also responsible for production of about 85% intracellular ROS during the

electron transport to promote cell differentiation or induce apoptosis . The dual roles of mitochondria are

responsible for life and death of the cell (Frisard and Ravussin 2006). The number of mitochondria represents


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not only the capacity of ATP production but also reflects the oxidative stress to which the cell is exposed.

Metabolic regulation is largely dependent on mitochondria (Yakes and Van Houten 1997 and Lee et al 2007).

Approximately 0.5–1.5% of all diabetic patients exhibit pathogenic mtDNA mutations such as duplications

, point mutations , and large-scale deletions (Kadowaki et al 1994, Silva et al 2000 and Maassen et al 2007) .

For many years researchers have argued that type 2 diabetes is a problem of insulin sensitivity, focusing on the

insulin receptors in the peripheral tissue. Other researchers have focused on the dysfunction of the beta-cells,

similar to type 1 diabetes, as the primary dysfunction in type 2. Today it is recognized that both factorscontribute to the disease. Beta-cell malfunction can be traced to various levels of qualitative and quantitative

mitochondrial dysfunction (Ehses et al 2007, Patti and Corvera 2010)). Little attention has been paid to the

quantitative aspects of mtDNA in diabetes and there is as yet no convincing evidence whether the reduction of

mtDNA copy number causes enough disturbance in the glucose metabolism in peripheral tissues such as liver

cells to participate in the development of diabetes. Although the depletion of mtDNA could impair

mitochondrial and pancreatic β-cell function, mitochondrial function can be measured only in the tissues of patients or animals and cannot be measured easily in noninvasive samples or in nondiseased subjects. It has

previously been shown in humans that the mtDNA content in blood cells is partially heritable (Curran et al 2007

and Xing etal 2008). It is believed, therefore, that the peripheral blood mitochondrial DNA (pb-mtDNA) content

could provide an alternative index mitochondrial dysfunction. The aim of the current study was to elucidate the

association of mtDNA content with type 2 diabetes in Egyptian cases

Methods and Procedures:

Subjects and methods:

Fifteen type 2 diabetes patients more than 25 years were recruited from outpatient clinic of Medical

Services Unit at National Research Center and twelve healthy subjects; age and sex matched. Written informed

consent was obtained from all the subjects who participated in this study. All patients and controls weresubjected to detailed history and thorough physical examination. We excluded patients with secondary diabetes,

liver or kidney disease, major organ failure ,auto-immune diseases, or malignancy anywhere in the body.None

of the patients had diabetic complications. Controls did not have any abnormalities regarding their physical

examination and with negative family history of diabetes.

Biochemical measurement:

Serum glucose was detected for all subjects using automated autoanalyzer Cx5 ( Beckman ,USA).

Quantification of mitochondrial DNA using quantitative real-time polymerase chain reaction:

Nucleic acids were extracted from white blood cells from a blood sample using a standard method as

described previously ( Miller et al 1988 and Kawasaki 1990). An assay based on real-time quantitative PCR was

used for both nuclear DNA (nDNA) and mtDNA quantification using SYBR green as a fluorescent dye

(Invitrogen, Buenos Aires, Argentina)).

The primer sequences for mtDNA, mtF3212 (5'-CACCCAAGAACAGGGTTTGT-3') and mtR3319 (5'-

TGGCCATGGGTATGTT-GTTAA-3') and those for nDNA for loading normalization, 18S rRNA gene18S1546F (5'-TAGAGGGACAAGTGGCGTTC-3') and 18S1650R (5'-CGCTGAGCCAGTCA-GTGT3') were

reported (Bai et al 2004).

The PCR profile was 1 cycle of 95 °C for 2 min, followed by 35 cycles (95 °C 15 s and 60 °C 1 min). Real-time quantitative PCR was carried out in a Bio-Rad iCycler (Bio-Rad Laboratories, Hercules, CA). The

calculation of DNA copy number involved extrapolation from the fluorescence readings in the mode of

background subtracted form the Bio-Rad iCycler (Bio-Rad Laboratories) according to Rutledge. Specificity ofamplification and the absence of primer dimers was confirmed by melting curve analysis at the end of each run.

The two-target amplicon sequence (mtDNA and nDNA) was visualized in agarose 2% and purified using

Qiagen Qiaex II, Gel extraction Kit and dilutions of purified amplicons were used as the standard curve. The

copy number of mtDNA and nDNA was calculated from the CT number and by use of the standard curve

according to WONG et al. (Rutledge 2004).

Results:

An assay based on real-time quantitative PCR was used for both nuclear DNA (nDNA) and mtDNA

quantification using SYBR green as a fluorescent dye (Invitrogen, Buenos Aires, Argentina)). The copy numberof mtDNA and nDNA was calculated from the CT number and by use of the standard curve according to Wong


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et al 2004. Amplification occur successfully in patients and controls ; where diagram appear show number of

cycle which occur in amplification .Also the apparatus calculate melting curve to ensure the amplification and

absence of primer dimmers (melting curve appear in all cases). A significant differences in the mtDNA/nDNA

ratio among the control subjects and patients was reported; since results from this study detected reduced

mtDNA content in type 2 diabetes patients (13.36 +/- 6.13) compared to healthy individuals (109.15 +/- 49.4)

(p 0.001).

Discussion:

Metabolic regulation is largely dependent on mitochondria, which play an important role in energy

homeostasis by metabolizing nutrients and producing ATP and heat. The number and size of mitochondria are

correlated with mitochondrial oxidative capacity. Decreased mitochondrial activity might be caused, at least

partly, by reduced content of mtDNA. Expression of nuclear-encoded genes involved in oxidative

phosphorylation is downregulated in insulin-resistant skeletal muscle (Mootha et al 2003). Thus, some

investigators have speculated that mtDNA copy number might be a surrogate marker of mitochondrial function(Cho et al 2007). There is as yet no convincing evidence whether the reduction of mtDNA copy number causes

enough disturbance in the glucose metabolism in peripheral tissues such as liver cells to participate in the

development of diabetes.

In this study, comparing mtDNA/nDNA ratio in control subjects and patients; revealed a significant

decrease of mtDNA content in type 2 diabetes patients compared to healthy individualsThe association of mtDNA content with type 2 diabetes is not confirmed in all studies and showed

conflicting results; evidence is accumulating that mtDNA content is associated with type 2 diabetes. However,

there is debate about whether mitochondrial dysfunction is primary or secondary to type 2 diabetes. Several

lines of evidence support the idea that the alterations of mtDNA quantity may cause type 2 diabetes as one of

the mitochondrial diseases. First, pb-mtDNA content decreased in the offspring of type 2 diabetic patients and,

furthermore, that the pb-mtDNA content is correlated with insulin sensitivity. These findings suggest that thequantitative mtDNA status might be an early genetic marker for type 2 diabetes and possibly for insulin

resistance syndrome ( Song et al 2001). Elegant studies by Petersen et al ( 2004) have found that impaired

mitochondrial function in skeletal muscle of offspring of patients with type 2 diabetes is coupled with an

increase in intramyocellular lipid and insulin resistance. Thus PBMC mtDNA depletion may indicate a greater

depletion of islet or muscle mtDNA content and a greater bioenergetic defect affecting islet function and insulin

action, thereby leading to earlier onset of overt diabetes. Second, early studies with the Goto-Kakizaki rat, a

genetic animal model of type 2 diabetes with impaired insulin secretion, found that the mitochondria of beta-cells were decreased in volume, while the islet tissue had an increased number of mitochondria per unit area, but

a decrease in mtDNA copies (Serradas et al 1995) . The decrease in mtDNA (approximately 50 percent per

mitochondrion) was not found to be associated with any major deletions or mutations. The decrease in mtDNA

was observed in the adult rat tissue (four-month old) but not in fetal tissue. These results suggest a connection

between glucose-stimulated insulin secretion (GSIS) and the somatic progression of mtDNA depletion. This

study shows a correlation between mitochondrial function and type 2 diabetes. The data seem to suggest that

metabolic dysfunction inside the mitochondria produces increased ROS, leading to decreased mtDNA, resulting

in decreased insulin secretion (Alán et al 2011). Third, another example of exogenously induced oxidative

damage to mtDNA can be illustrated with streptozotocin, one of the diabetogenic agents to create diabetes in

test animals; reduced the levels of mtDNA content and its transcripts in pancreatic islets. Streptozotocinincreases ROS causing damage to mtDNA and suppression of mitochondrial transcription. Hydroxyl radicals

have been shown to attack nucleosides of DNA, producing oxidized products such as 8-hydroxy-2’-

deoxyguanosine (8-OHdG). This ROS attack, although known to damage nuclear DNA, can attack mtDNA atrates 3-23 fold higher (Pettepher et al 1991, Moreira et al 1991, Mecocci et al 1994 and Hamilton et al 2001).

Fourth, mtDNA depletion inhibited glucose-stimulated increases of the intracellular free Ca2+

concentration and

insulin secretion in mouse insulinoma cells, which led to glucose intolerance and hyperglycemia ( Soejima et al 1996 ). Finally, a more direct cause-and-effect relationship between mitochondrial dysfunction and human

diseases was demonstrated in the report that antiviral agents used to treat patients with AIDS could damage

mitochondrial function by inhibiting the mitochondrial DNA (mtDNA) polymerase γ, resulting inhyperlactatemia, lipodystrophy (ie, the accumulation of visceral fat, breast adiposity, and cervical fat pads), and

insulin resistance (Davis et al 2002).

In a follow-up study, Morino et al., 2005 proposed that patients with type 2 DM have decreased copies of

mtDNA in insulin-resistant target tissues, such as skeletal muscle and adipose tissue; contributed to the

decreased mitochondrial activity. Other studies argue against previous observations where low mtDNA content

in blood was associated with type 2 diabetes, triglyceride storage, glucose homeostasis, insulin sensitivity, and

insulin secretion (Lee et al 1998, Park et al 1999, Park et al 2001, Song et al 2001 and Weng et al 2009) ; Singhet al., 2007 indicated that reduced mitochondrial DNA content in peripheral blood is not a risk factor for the


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development of type 2 diabetes in the offspring of patients with early onset type 2 diabetes. Reiling and his

colleagues., 2010; confirmed that mtDNA content has a heritability of 35% in Dutch twins, but could not find

evidence for an association of mtDNA content in blood with prevalent or incident T2D and related traits.

Furthermore they were the first to show that the decline in mtDNA content might be male specific. The

observed gender effect on mtDNA content, also observed by Xing et al. (2008), is probably caused by this

gender-specific correlation between mtDNA content and aging. One might speculate that overall mitochondrial

fitness is better retained in females, which might explain the observed difference in life span between males andfemales. However,this hypothesis needs further investigation.- A major difference between studies is the

difference in ethnicity ( Reiling et al 2010) . The centerpiece of the pathophysiologic mechanism of metabolic

syndrome is insulin resistance; it is becoming evident that mitochondrial dysfunction is closely related to insulin

resistance and metabolic syndrome. The underlying mechanism of mitochondrial dysfunction is very complex .

The mechanisms of disease progression and complications in diabetic patients are probably multifactorial; one

factor may be hyperglycemia-induced overproduction of reactive oxygen species (ROS) by mitochondria.

Increased oxidative stress is known to cause mitochondrial damage, dysregulation of mitochondrial function,

and altered mitochondrial biogenesis (Lee et al., 2000, 2002 and 2010).

It is possible that the decreased glucose uptake reduces the glucose supply for enzymes involved in glucose

metabolic pathways. These enzymes may be inactivated or down regulated. Park et al 2001 suggested that the

depletion of mtDNA decreased glucose utilization by suppressing glucose metabolism in addition to reducing

glucose uptake . Furthermore, the attenuated activities of glycolytic enzymes could consequently reduce ATP

production, which may aggravate ATP depletion in a vicious cycle. Also, depletion of mtDNA significantlyattenuated the expression of all types of glucose transporters that are encoded by nuclear DNA .

The mechanisms that control mtDNA content are yet to be fully elucidated. There is evidence that PBMC

mtDNA content has a large genetic determinant (Curran et al 2007) and also evidence suggesting a role for

mitochondria and mtDNA content in both insulin secretion and insulin action. Thus rodent models of diabetes

show reduced mtDNA content in islet cells (Serradas et al 1995) and those with genetic disruption of

mitochondrial transcription factor A in pancreatic beta cells exhibit depleted mtDNA and early diabetes (Silva et

al 2000). Additionally, there is increasing evidence that the monocyte–macrophage lineage plays an independent

and pathogenic role in the development of type 2 diabetes. Furthermore, increased monocyte-derived

macrophage infiltration of islet cells is seen in type 2 diabetes and thought to play a role in causing islet

pathology in that condition. Such evidence supports a direct role of monocytes in the development of glucose

intolerance. In this context, rather than being simply a surrogate for determining mitochondrial action or number

in alternative tissues, reduced mtDNA in monocytes could imply a direct impact on the development and timing

of type 2 diabetes( Ehses et al 2007). The mtDNA molecule is particularly susceptible to damage and defectivereplication by virtue of continued exposure to reactive oxygen species (ROS) and a lack of DNA repair

mechanisms. Furthermore, it has been shown that mtDNA harbouring deleterious mutations are preferentially

clonally amplified as a compensatory response to energy deficiency by making more mitochondria and mtDNA

(Wallace 2005). However, with time, as defective mitochondria accumulate, bioenergetic and replicative

function declines. Therefore, in addition to the possibility of lower mtDNA content predisposing to early-onset

disease, it is equally possible that patients with early-onset disease, exposed to the ravages of longer diabetes

duration, accumulate defects in the mitochondrial genome with resultant defective replication, further depleting

mtDNA content (Wong et al 2009).

Although our study sample was relatively small as compared with other epidemiological and association

studies, the result of this study supports the hypothesis that the mtDNA content in blood is associated with type2 diabetes, and confirm previous observations, where low mtDNA content in blood was associated with type 2

diabetes.

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Association of Peripheral Blood Mitochondrial DNA Content With Type 2 Diabetic

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