Roles of Androgen Receptor in lipid and glucose homeostasis -- Using the ARKO mice model

1. Roles of Androgen Receptor in lipid and glucose homeostasis -- Using the ARKO mice model

2. AR - The Nuclear Hormone Receptor Superfamily Known Receptors Classical receptors GR cortisolMR aldosteroneAR testosterone, DHTPR α,β progesteroneER α,β estradiolVDR 1,25-(OH)2 vit D3TR α,β triiodothyronineEcR 20-OH ecdysone Orphan Receptors Vertebrate DrosophilaTR-2 α,β DHR78NGFI-B α,β,γ DHR38ROR α,β,γ DHR3Rev-erb α,β E75, E78SF-1 α,β FTZ-F1 α,β COUP α,β,γ svpHNF-4 α, β HNF-4Tlx α,β tll No known homologsERR α,β,γ knirpsDAX-1 knirps-relatedSHP egonGCNF DHR96 EX-OrphansRAR α β γ all-trans retinoic acidRXR α,β,γ 9-cis retinoic acidPPAR α,β,γ fatty acids, eicosanoidsLXR α,β oxy-sterolsFXR α,β bile acidsBXR α,β benzoates Nearly EX-orphans (natural ligands?)CAR androstanes, xenobioticsSXR/PXR steroids, xenobiotics

3. Target Organ: Testosterone to DHT OH CH3 O OH CH3 CH3 CH3 CH3 CH3 CH3 CH3 O H H HO O O Cholesterol Androstenedione Testosterone Dihydro-testosterone (DHT) Adrenal Glands: Secrete Androstenedione Converting Enzyme: 5-alpha-Reductase Production of Androgens Testes: Secrete Testosterone

4. Converted to Organs Behavior and Reproduction • Hair • Muscle • Brain • Testes • Sex Accessory • Tissues (Prostate) • Aggression • Libido • Spermatogenesis • Potency Effects of Testosterone on the Body Testosterone in the blood DHT in the target organ

5. CBP/p300 RNA Polymerase p160 Gene Transcription (PSA) DNA TATA Box ARE Promoter DHT and AR AR with DHT Ligand (Dimerizes) Testosterone 5a-R DHT Activated AR Enters Nucleus and Binds to DNA AR (Inactive) Expression of Genes Androgen Response Element (ARE)

6. The Worsening Epidemic of Obesity and Diabetes in the USA 1999-2000 NHANES Data (JAMA, Oct 2002) • 31% obese (BMI 30), increase from 23% • 65% overweight (BMI 25), increase from 56% • 4.7% extremely obese (BMI 40), increase from 2.9% • No physical activity in 27%! • No regular activity in additional 28% • Each 1-kg increase in weight =4.5%–9% increase in risk of diabetes The prevalence of obesity in the United States and other “Westernized” countries has reached epidemic proportions. Diabetes was the sixth leading cause of death listed on U.S. death certificates in 2002. NHANES=National Health and Nutrition Examination Survey.

7. Receptor + postreceptor defects Pathophysiology of Type 2 Diabetes Glucose Insulin resistance Liver Increased glucose production Peripheral Tissues (Muscle and Adipose) Pancreas Impaired insulin secretion Diabetes (1996) 45:1661-1669.

8. Progression of Insulin Resistance Time Insulin resistance Insulin production Glucose level Non- diabetes Pre- diabetes Type 2 diabetes Type 2 Diabetes BASICS, International Diabetes Center, 2000.

9. Hypothesis for characterizing male general ARKO mice Obesity in men is associated with a decline in total plasma testosterone and free testosterone which parallels body fat mass. Testosterone also regulates insulin sensitivity, increase lipolysis, and affects body composition. In male the effects of obesity on metabolism variables could involve the presence of lower androgens, which may carry an independent risk factor of diabetes and cardiovascular disease.

10. General ARKO mice (T-AR-/y) Male T-AR-/ymice develop obesity at advanced age

11. Hyperlipidemia +/y +/+ - /y TABLE Weight and blood parameters of 35-wk-old AR , AR , and AR mice +/y +/+ - /y AR (9) AR (10) AR (9) Body weight (g) 33.15 ± 1.2 25.81 ± 0.44 39.06 ± 2.7 * Gonadal fat/body (%) 2.21 ± 0.14 0.34 ± 0.08 3.29 ± 0.23 * Serum TG (mmol/l) 0.30 ± 0.04 0.32 ± 0.08 0.67 ± 0.1 ** Serum FFA (mmol/l) 0.25 ± 0.02 0.25 ± 0.02 0.71 ± 0.04 ***

12. Male T-AR-/y mice are insulin resistant

13. Male T-AR-/y mice are hyperleptinemia

14. Male T-AR-/ymice have reduced PI3k activity and ectopic TG accumulation both in skeletal muscle and liver

15. Loss of AR altered the expression of lipid metabolism genes. P P A R g * * * C / E B P a * a P 2 * S R E B P 1c * * * P P A R a / M u * * * ( 0 . 2 0 f o l d ) P P A R a / L * ( 0 . 1 3 f o l d ) 0 1 2 3 4 - / y F o l d c h a n g e ( ) A R

16. Summary of general ARKO mice. • Poor development of genital organs and low level of testosterone. • Develop obesity in advanced age. • Hyperglycemia and hyperinsulinemia. • Abnormal lipid and adipokine profiles. • Progressively develop insulin and leptin resistance. • Ectopic deposition of triglyceride in liver and skeletal muscle. • Increased lipid synthesis in adipose and reduced beta oxidation in both liver and skeletal muscle. • Yeh et al. PNAS 99, 13498 2002 • Lin et al. Diabetes 54, 1717 2005

17. Androgen receptor roles in glucose and lipid homeostasis Hypothalamus AR Central leptin and insulin resistance Increased leptin levels Steatosis and reduced oxidation Fat AR Steatosis and reduced oxidation Liver AR Muscle AR Increased fat amount and size of adipocytes Increased gluconeogenesis Increased FFA releasing Reduced glucose uptake Insulin resistance status Hyperglycemia, hyperinsulinemia and abnormal lipid profiles

18. Tissue specific ARKO mice

19. Hepatic ARKO Hepatic ARKO mice (H-AR-/y) • Similar body weight compared to wild-type mice • Gained more weight upon HFD feeding.

20. Hepatic ARKO H-AR-/y mice exhibit elevated fasted and fed blood glucose levels and increased serum insulin levels upon HFD feeding.

21. Hepatic ARKO Upon HFD feeding, H-AR-/ymice are insulin resistance assayed by intraperitoneal glucose tolerance test (IGTT).

22. Hepatic ARKO H-AR -/ymice have reduced hepatic PI3-K activity upon insulin stimulation.

23. Hepatic ARKO HFD feeding induced hepatic steatosis in H-AR-/ymice. At advanced age, H-AR-/y mice spontaneously develop hepatic steatosis.

24. Hepatic ARKO Increased gluconeogenesis and lipid synthesis and reduced beta oxidation occurred in H-AR-/ymice, upon HFD feeding.

25. Hepatic ARKO Liver AR regulates glucose and lipid metabolism

26. Hypo ARKO Hypothalamus specific ARKO mice (Hypo-AR-/y) Significant increased body weight in Hypo-AR-/y mice. Hypo-AR-/y Hypo-AR-/y AR+/y AR+/y

27. Hypo ARKO Significant increased serum insulin level in Hypo-AR-/y mice. Hypo-AR-/y AR+/y Hypo-AR-/y AR+/y

28. Hypo ARKO Hypo-AR-/y mice are mild insulin resistant. IGTT ITT

29. Hypo ARKO Hypo-AR-/y Hypo-AR-/y Hypo-AR-/y Hypo-AR-/y mice have increased serum levels of NEFA, Leptin. AR+/y AR+/y AR+/y

30. Hypo ARKO Lep Lep Hypo-AR-/y mice develop leptin resistance

31. HARKO project Androgen receptor roles in glucose and lipid homeostasis Hypothalamus AR Central leptin and insulin resistance Increased leptin levels Steatosis and reduced oxidation Fat AR Steatosis and reduced oxidation Liver AR Muscle AR Increased fat amount and size of adipocytes Increased gluconeogenesis Increased FFA releasing Reduced glucose uptake Insulin resistance status Increased body weight Hyperinsulinemia and abnormal lipid profiles

32. Summary of tissue specific ARKO mice

33. Future Directions Adipose specific ARKO mice • Is there a metabolic phenotype in AARKO mice fed HFD? • Determine the mechanism for reduced size of testis in AARKO mice. Hypothalamus or brain specific ARKO mice • Determine the mechanism for the central AR regulated insulin and leptin sensitivity. Double tissue specific ARKO mice • Determine the whether double tissues specific ARKO mice recapitulate phenotypes observed in general ARKO mice at advanced age.

34. Selective Androgen Receptor Modulator (SARM) A small molecule that binds to and selectively modulates androgen receptors depending on tissue type. In men, SARMs may be able to: * Stimulate testosterone's beneficial action in bone, liver, muscle, and brain * Block testosterone's harmful action in the prostate and skin * Either cross or not cross into the central nervous system to affect lipids Testosterone SARMs

35. Acknowledgements URMC Chang Lab Dr. Hung-Yun Lin Dr. Yei-Tsung Chen Dr. Ruey-Shen Wang Dr. Qingquan Xu URMC Yeh Lab Dr. Shuyuan Yeh URMC Dr. Janet Sparks Dr. Robert Mooney Dept. of Pathology and Lab Medicine