Insulin (+), insulin-induced AKT activity determined from muscle collected from mice 20 min post insulin injection. A. Images of representative western blot results. B. Densitometric quantifica- tion of the ratio of pAKT vs. total AKT before and after insulin injection. The value of WT mice treated with insulin and mutants with and without insulin treatment is normalized to that of WT without insulin treatment. C. The ratio of pAKT before and after insulin injection for each genotype. The basal pAKT was elevated but the net increase of pAKT following insulin injection was attenuated in SERT-deficient muscle. *, p,0.05, ***, p,0.001 The number of mice analyzed is indicated in parentheses. (TIF) Figure S5 Quantification of plasma corticosterone levels in 3- and 6-month old mice. Mice under normal environment References 1. Cassidy F, Ahearn E, Carroll BJ (1999) Elevated frequency of diabetes mellitus in hospitalized manic-depressive patients. Am J Psychiatry 156: 1417–1420. 2. Lustman PJ, Penckofer SM, Clouse RE (2008) Recent advances in understand- ing depression in adults with diabetes. Curr Psychiatry Rep 10: 495–502. 3. Veenstra-VanderWeele J, Anderson GM, Cook EH, Jr. (2000) Pharmacogenet- ics and the serotonin system: initial studies and future directions. Eur J Pharmacol 410: 165–181. 4. Saller CF, Stricker EM (1976) Hyperphagia and increased growth in rats after intraventricular injection of 5,7-dihydroxytryptamine. Science 192: 385–387. 5. Breisch ST, Zemlan FP, Hoebel BG (1976) Hyperphagia and obesity following serotonin depletion by intraventricular p-chlorophenylalanine. Science 192: 382–385. 6. Fletcher PJ, Paterson IA (1989) A comparison of the effects of tryptamine and 5- hydroxytryptamine on feeding following injection into the paraventricular nucleus of the hypothalamus. Pharmacol Biochem Behav 32: 907–911. 7. Tecott LH, Sun LM, Akana SF, Strack AM, Lowenstein DH, et al. (1995) Eating disorder and epilepsy in mice lacking 5-HT2c serotonin receptors. Nature 374: 542–546. 8. Nonogaki K, Strack AM, Dallman MF, Tecott LH (1998) Leptin-independent hyperphagia and type 2 diabetes in mice with a mutated serotonin 5-HT2C receptor gene. Nat Med 4: 1152–1156. 9. Heisler LK, Jobst EE, Sutton GM, Zhou L, Borok E, et al. (2006) Serotonin reciprocally regulates melanocortin neurons to modulate food intake. Neuron 51: 239–249. 10. Norris SL, Zhang X, Avenell A, Gregg E, Schmid CH, et al. (2004) Efficacy of pharmacotherapy for weight loss in adults with type 2 diabetes mellitus: a meta- analysis. Arch Intern Med 164: 1395–1404. 11. Gray DS, Fujioka K, Devine W, Bray GA (1992) Fluoxetine treatment of the obese diabetic. Int J Obes Relat Metab Disord 16: 193–198. 12. Fava M, Judge R, Hoog SL, Nilsson ME, Koke SC (2000) Fluoxetine versus sertraline and paroxetine in major depressive disorder: changes in weight with long-term treatment. J Clin Psychiatry 61: 863–867. 13. Raeder MB, Bjelland I, Emil Vollset S, Steen VM (2006) Obesity, dyslipidemia, and diabetes with selective serotonin reuptake inhibitors: the Hordaland Health Study. J Clin Psychiatry 67: 1974–1982. 14. Yamada J, Sugimoto Y, Inoue K (1999) Selective serotonin reuptake inhibitors fluoxetine and fluvoxamine induce hyperglycemia by different mechanisms. Eur J Pharmacol 382: 211–215. 15. Briscoe VJ, Ertl AC, Tate DB, Dawling S, Davis SN (2008) Effects of a selective serotonin reuptake inhibitor, fluoxetine, on counterregulatory responses to hypoglycemia in healthy individuals. Diabetes 57: 2453–2460. 16. Homberg JR, la Fleur SE, Cuppen E (2010) Serotonin transporter deficiency increases abdominal fat in female, but not male rats. Obesity (Silver Spring) 18: 137–145. 17. Kim H, Toyofuku Y, Lynn FC, Chak E, Uchida T, et al. (2010) Serotonin regulates pancreatic beta cell mass during pregnancy. Nat Med 16: 804–808. 18. Murphy DL, Lesch KP (2008) Targeting the murine serotonin transporter: insights into human neurobiology. Nat Rev Neurosci 9: 85–96. 19. Lam DD, Heisler LK (2007) Serotonin and energy balance: molecular mechanisms and implications for type 2 diabetes. Expert reviews in molecular medicine 9: 1–24. 20. Bengel D, Murphy DL, Andrews AM, Wichems CH, Feltner D, et al. (1998) Altered brain serotonin homeostasis and locomotor insensitivity to 3, 4- methylenedioxymethamphetamine (‘‘Ecstasy’’) in serotonin transporter-deficient mice. Mol Pharmacol 53: 649–655. 21. Stunes AK, Reseland JE, Hauso O, Kidd M, Tommeras K, et al. (2011) Adipocytes express a functional system for serotonin synthesis, reuptake and receptor activation. Diabetes Obes Metab 13: 551–558. 22. Chang AS, Chang SM, Starnes DM, Schroeter S, Bauman AL, et al. (1996) Cloning and expression of the mouse serotonin transporter. Brain Res Mol Brain Res 43: 185–192. Distinct SERT Function in Feeding and Obesity were analyzed. The number of mice analyzed is indicated in parentheses. (TIF) Acknowledgments We thank S. B. Horwitz, C. Chow and D. Cai for providing us antibodies, and the histotechnology and comparative pathology facility at the Albert Einstein Cancer Center for assistance during histological analyses. Author Contributions Conceived and designed the experiments: XC KJM MDG GJS JYS. Performed the experiments: XC GJS. Analyzed the data: XC KJM MDG GJS JYS. Contributed reagents/materials/analysis tools: KJM MDG GJS JYS. Wrote the paper: XC KJM MDG GJS JYS. 23. Ruddell RG, Oakley F, Hussain Z, Yeung I, Bryan-Lluka LJ, et al. (2006) A role for serotonin (5-HT) in hepatic stellate cell function and liver fibrosis. Am J Pathol 169: 861–876. 24. Mathews TA, Fedele DE, Coppelli FM, Avila AM, Murphy DL, et al. (2004) Gene dose-dependent alterations in extraneuronal serotonin but not dopamine in mice with reduced serotonin transporter expression. J Neurosci Methods 140: 169–181. 25. Halaas JL, Gajiwala KS, Maffei M, Cohen SL, Chait BT, et al. (1995) Weight- reducing effects of the plasma protein encoded by the obese gene. Science 269: 543–546. 26. Elwing JE, Lustman PJ, Wang HL, Clouse RE (2006) Depression, anxiety, and nonalcoholic steatohepatitis. Psychosom Med 68: 563–569. 27. Feldstein AE (2010) Novel insights into the pathophysiology of nonalcoholic fatty liver disease. Semin Liver Dis 30: 391–401. 28. Rhodes CJ (2005) Type 2 diabetes-a matter of beta-cell life and death? Science 307: 380–384. 29. Taniguchi CM, Emanuelli B, Kahn CR (2006) Critical nodes in signalling pathways: insights into insulin action. Nat Rev Mol Cell Biol 7: 85–96. 30. Cho H, Mu J, Kim JK, Thorvaldsen JL, Chu Q, et al. (2001) Insulin resistance and a diabetes mellitus-like syndrome in mice lacking the protein kinase Akt2 (PKB beta). Science 292: 1728–1731. 31. George S, Rochford JJ, Wolfrum C, Gray SL, Schinner S, et al. (2004) A family with severe insulin resistance and diabetes due to a mutation in AKT2. Science 304: 1325–1328. 32. Liu HY, Hong T, Wen GB, Han J, Zuo D, et al. (2009) Increased basal level of Akt-dependent insulin signaling may be responsible for the development of insulin resistance. Am J Physiol Endocrinol Metab 297: E898–906. 33. Storgaard H, Song XM, Jensen CB, Madsbad S, Bjornholm M, et al. (2001) Insulin signal transduction in skeletal muscle from glucose-intolerant relatives of type 2 diabetic patients [corrected]. Diabetes 50: 2770–2778. 34. Liu Z, Cao W (2009) p38 mitogen-activated protein kinase: a critical node linking insulin resistance and cardiovascular diseases in type 2 diabetes mellitus. Endocr Metab Immune Disord Drug Targets 9: 38–46. 35. Um SH, D’Alessio D, Thomas G (2006) Nutrient overload, insulin resistance, and ribosomal protein S6 kinase 1, S6K1. Cell Metab 3: 393–402. 36. Sabio G, Davis RJ (2010) cJun NH2-terminal kinase 1 (JNK1): roles in metabolic regulation of insulin resistance. Trends Biochem Sci 35: 490–496. 37. Aguirre V, Uchida T, Yenush L, Davis R, White MF (2000) The c-Jun NH(2)- terminal kinase promotes insulin resistance during association with insulin receptor substrate-1 and phosphorylation of Ser(307). J Biol Chem 275: 9047–9054. 38. Lee YH, Giraud J, Davis RJ, White MF (2003) c-Jun N-terminal kinase (JNK) mediates feedback inhibition of the insulin signaling cascade. J Biol Chem 278: 2896–2902. 39. Tamguney T, Stokoe D (2007) J Cell Sci 120: 4071–4079. 40. Wijesekara N, Konrad D, Eweida M, Jefferies C, Liadis N, et al. (2005) Muscle- specific Pten deletion protects against insulin resistance and diabetes. Mol Cell Biol 25: 1135–1145. 41. Kurlawalla-Martinez C, Stiles B, Wang Y, Devaskar SU, Kahn BB, et al. (2005) Insulin hypersensitivity and resistance to streptozotocin-induced diabetes in mice lacking PTEN in adipose tissue. Mol Cell Biol 25: 2498–2510. 42. Karsenty G (2006) Convergence between bone and energy homeostases: leptin regulation of bone mass. Cell Metab 4: 341–348. 43. Warden SJ, Robling AG, Sanders MS, Bliziotes MM, Turner CH (2005) Inhibition of the serotonin (5-hydroxytryptamine) transporter reduces bone accrual during growth. Endocrinology 146: 685–693. 44. Roberge C, Carpentier AC, Langlois MF, Baillargeon JP, Ardilouze JL, et al. (2007) Adrenocortical dysregulation as a major player in insulin resistance and onset of obesity. American journal of physiology Endocrinology and metabolism 293: E1465–1478. PLoS ONE | www.plosone.org 12 March 2012 | Volume 7 | Issue 3 | e32511