Analysis of Metabolism Research

Presented by: Cheryl Gregory

Linh Pho

Kari E. Wong, Frances L. Szeto, Wenshuo Zhang, Honggang Ye, Juan Kong, Zhongyi Zhang, Xiao Jian Sun, and Yan Chun Li

22 January 2009

Data suggests that vit D

� Introduction

� Materials & Methods

� Results� Results◦ Graphs & Diagrams

� Conclusion

� Further Research

� What is the definition of obesity?◦ The Excess of Body Fat

� What is the prevalence of obesity in the United States?◦ In 2008, the obesity prevalence increased to 30%

� What are the associated health � What are the associated health problems?◦ Gangreen◦ Type II Diabetes ◦ Cancer◦ Cardiovascular Disease◦ Hypertension

http://science.kukuchew.com/wp-content/uploads/2009/02/obesity_surgery.jpg

Metabolic Syndrome

ØEnergy Metabolism: the use of fuels to create energy to be used for work.

ØVitamin DVitamin DVitamin DVitamin D----Receptor (VDR) knockout mice used to show Receptor (VDR) knockout mice used to show Receptor (VDR) knockout mice used to show Receptor (VDR) knockout mice used to show Vitamin D, 1alpha,25Vitamin D, 1alpha,25Vitamin D, 1alpha,25Vitamin D, 1alpha,25----dihydroxyvitamin Ddihydroxyvitamin Ddihydroxyvitamin Ddihydroxyvitamin D----3, involvement in 3, involvement in 3, involvement in 3, involvement in energy metabolism energy metabolism energy metabolism energy metabolism

ØVDR-null mice had decreased adiposity and lower plasma lipid levels

ØVDR-null mice had increased Uncoupling Protein expressionØUCPs separate the process of oxidative phosphorylation from ATP production

ØOverall, VDR knockout mice had higher energy expenditure

Examples of fuels: Use more E to create the same amount of ATP

� Animal Treatment◦ Created Vitamin D Receptor (VDR) knockout mice◦ Placed the mice on two diets: High Fat & High Calcium� High Fat (HF)

� Mice were place on HCa for 4 months, then switch to HF dietMice were weighed and monitored weekly� Mice were weighed and monitored weekly

� End, animals were killed: plasma and adipose tissue were harvested

� High Calcium (HCa)� To normalize the plasma calcium level� Used as a control

High Ca diet b/c on regular rodent chow mice were Ca deficient, b/c VDR is involved in Ca Homeostasis High Ca diet b/c on regular rodent chow mice were Ca deficient, b/c VDR is involved in Ca Homeostasis This controlled for any differences that may have been caused by Ca deficiency.

� Assessed Plasma Parameters Using Test Kits◦ Used to determine levels of:� Triglycerides� Cholesterol� Adipokine� Adipokine� Thyroid Stimulating Hormone

Triglycerides: the form of fatty acid found in blood??? Triglyceride levels help indicate the amount of B-oxidation going on (Check this) Cholesterol: a steroid. Helps indicate the level of overall fat Adipokines: the “hormones” of adipose tissue. Higher secretion of adipokines signal lower body fat TSH: known to stimulate metabolism. Increased levels would suggest increased metabolism. Triglycerides: the form of fatty acid found in blood??? Triglyceride levels help indicate the amount of B-oxidation going on (Check this) Cholesterol: a steroid. Helps indicate the level of overall fat Adipokines: the “hormones” of adipose tissue. Higher secretion of adipokines signal lower body fat TSH: known to stimulate metabolism. Increased levels would suggest increased metabolism.

� Observed the Histology ◦ Cultured White Adipose Tissue (WAT)� Storage depot of the body� Stores excess energy in triglyceride form� Releases signals indicating the body’s energy state

◦ Cultured Brown Adipose Tissue (BAT)� Regulates thermogenesis� Expresses uncoupling protein

� Separate oxidative phosphorylation from ATP production

◦ Adipose tissues were:� Processed and Stained

The signal of leptin, etc Separate ATP production from the electron transport chain

� Assayed Fatty Acid B-oxidation◦ Isolated white adipose tissue from male mice ◦ Measure the B-oxidation using tritiated palmitate

� Analyzed Northern Blot of total RNA� Analyzed Northern Blot of total RNA◦ Show the total cellular RNA extraction

� Performed RT-PCR◦ Strands of DNA extracted from the mice◦ To analyze protein and hormone mRNA levels

Carnitine palmitoyltransferase – is protein that transport fatty acid to the midtochonrial matrix

Weight DifferenceWeight DifferenceWeight DifferenceWeight Difference

•VDR (-/-) mice weighed sig. less than WT on both high-Ca++ and high-fat diet

•Gender difference: Male mice more protected against weight gain

MALE FEMALE

•VDR-null mice had a decrease in adiposity•Lower # of adipocytes•Smaller size

There was discrepency btwn in vitro and in vivo. This may be because VDR expression follows a specific temporal pattern during adipocyte differentiation. What would this change??? VDR-null adipocytes appear to be smaller under high fat diet suggesting they store less lipids in the fat tiss. This might be due to a defect in adipogenesis see paragraph on pg 826

ADIPOSE DIFFERENCE

•VDR-null mice on the high-fat diet had smaller adipocytes & less BAT

•The BAT accumulated fewer lipids & had better cell morphology

C. White Adipose Tissue

& had better cell morphology

•VDR-null had less adipose tiss. in all fat depots

•More dramatic difference between males

D. Brown Adipose Tissue

These slides are a cut of adipose tissue that are stain. Point out what is C and what is D.

MALE MICE ONLY

Fatty acid synthase higher in VDR-null mice on HCa diet but diff. not as big on HF diet

ENZYME LEVELS

diff. not as big on HF diet

Malonyl-CoA Dehydrogenase and Stearoyl CoA-Desaturase-1 were not significantly different

LPL lower in mutant mice on HF diet

PPAR-gamma higher in mutant mice on HCa diet

Male only due to extremes differences in weight LPL – Lipoprotein lipase – responsible for the breakdown of plasma triglycerides PPAR Gamma – gene responsible for the expression for some adipocytic gene

Fatty Acid Synthase

Lipoprotein Lipase

PPAR - Gamma

Northern mouse There are high fatty acid PPAR – GAMMA – Peroxisome Proliferator activated receptor

ADIPOKINE LEVELS

Leptin: lower in VDR-null mice on HCa diet and much lower in HF diet

Adiponectin: On the HF diet, mRNA expression was reduced in WT mice

Resistin: no difference between mice on HCa or HFResistin: no difference between mice on HCa or HF

Consistent w/ idea that leptin levels reflect fat mass w/in the body Leptin: reflect the fat mass Adiponectin: anti hyperglycemic adipokine. Increased levels indicate lower body weight. Also, increases b-oxidation and decreases triglyceride levels. Resisten: it’s role is controversial, but it maybe involved in the inflammatory response or in the energy metabolism. Levels increase with increase in the central obesity

PLASMA LIPID LEVELS

Female: No difference in plasma triglycerides, cholesterol, free-fatty acids on both diets

Male: VDR-null mice had lower triglycerides and cholesterol

Triglyceride

Male: VDR-null mice had lower triglycerides and cholesterol on both diets

Top Image: hubpages.com/hub/Triglycerides-can-kill

Bottom Image: www.3dchem.com/molecules.asp?ID=92

(NOT DUE TO: food intake, increased physical activity, decreased intestinal absorption, or increased thyroid stimulating hormone levels)

Cholesterol

To find the cause of lower lipid levels we monitored food intake and found all mice ate the same amount. Then, we investigated intestinal absorption of lipids by gavaging mice with olive oil and testing the plasma triglyceride levels over time. We found no difference in absorption. Wasn’t TSH, It was fatty acid B oxidation

B-Oxidation

•Carnitine palmitolytransferase II (CPTII) protein is responsible for transporting fatty acids into the mitochondrial matrix

• CPTII expression higher in the WAT of VDR-null miceCPTII mRNA increased too

•Increased rate of B-oxidation in adipocytes helps explain increased level of lipids in VDR-null mice

•Suggests more fatty acids are oxidized, which leads to higher basal energy expenditure

Treated WAT with tritiated palmitate then measure the production of 3H2O to measure the level of B-oxidation. CPT – partially explain the plasma lipid

ØUCP expression in BAT was increased in VDR-null mice, the difference was higher when mice where on the HF diet

ØThe expression of the B-adrenergic receptor 3 was unchanged B/c AdrB3 is known to directly regulate UCPsSuggests Vit D directly regulates UCPs

ØDouble Check: treated VDR-null and WT mice with 1,25(OH)2D3As expected, found no effect on VDR-null cells

UNCOUPLING PROTEINS

As expected, found no effect on VDR-null cells

ØVDR transcript was missing in VDR-null mice à Vit D directly targets fat

VDR regulates vitamin D’s affect on UCPs G = Hca H=HF Found ucp high look at b adren = regualt ucp – bc difference in ADR = no difference is directyl To double check this finding we treated it with ADRB3

High Calcium Diet High Fat Diet

To validate out finding here is a chart to show our finding.

� VDR-Knockout Phenotype:◦ Lower fat mass & lower plasma lipid levels

� Vit D-Receptor is involved in Energy Metabolism◦ VDR-null mice had higher energy expenditure as ◦ VDR-null mice had higher energy expenditure as shown by the indirect calorimetric measurement

� Possible Mechanism for this involvement:◦ Vit D directly downregulates UCP expression in BAT

� This Alteration of E Metabolism explains:◦ The lower adiposity and lower plasma lipid profile

� Studies should look at VDR in other body tissues to further understand its role in total body energy metabolism.

� Also, look at the difference btwn in vivo and in vitro

� By better understanding total body energy metabolism, we may be one step closer to helping understand and treat obesity.

� May eventually, lead to a way to alter energy metabolism

Talk about the in vivo and in vitro consistent with in vivo finding Thx the contribuitor

hubpages.com/hub/Triglycerides-can-kill

http://science.kukuchew.com/wp-content/uploads/2009/02/obesity_surgery.jpg

http://ajpendo.physiology.org/cgi/content/full/296/4/E820

http://www.cdc.gov/obesity/data/trends.html

Wong, Kari. E. “Involvement of the vitamin D receptor in energy metabolism: regulation of uncoupling proteins.” Division of Biological Sciences. Dept.

of Medicine Metabolism and Nutrition. Chicago, Illinois. 22 Jan. 2009

www.3dchem.com/molecules.asp?ID=92

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