Diabetes Volume 64, May 2015 1591 Junjie Yu, Fei Xiao, Yajie Guo, Jiali Deng, Bin Liu, Qian Zhang, Kai Li, Chunxia Wang, Shanghai Chen, and Feifan Guo Hepatic Phosphoserine Aminotransferase 1 Regulates Insulin Sensitivity in Mice via Tribbles Homolog 3 Diabetes 2015;64:1591–1602 | DOI: 10.2337/db14-1368 Phosphoserine aminotransferase 1 (PSAT1) is an en- particularly branched-chain amino acids, are closely re- zyme participating in serine synthesis. A role of PSAT1 in lated to the development and prediction of insulin resis- the regulation of insulin sensitivity, however, is un- tance in both human and animal models (5–7). Other known. In this study, we showed that hepatic PSAT1 studies have shown that, however, nonessential amino expression and liver serine levels are reduced in genet- acids might also be related to the development of insulin ically engineered leptin receptor–deficient (db/db) mice resistance. For example, high serum glycine levels are as- – and high-fat diet (HFD) induced diabetic mice. Addition- sociated with decreased risk of T2D in humans (8), and ally, overexpression of PSAT1 by adenovirus expressing glutamine supplementation attenuates high-fat diet METABOLISM PSAT1 improved insulin signaling and insulin sensitivity (HFD)-induced insulin resistance in rats (9). Despite these in vitro and in vivo under normal conditions. Opposite studies, the role that nonessential amino acids and the effects were observed when PSAT1 was knocked down enzymes related to their synthesis play in the regulation by adenovirus expressing small hairpin RNA specific for of insulin sensitivity remains unclear. PSAT1 (Ad-shPSAT1). Importantly, overexpression of fi Phosphoserine aminotransferase 1 (PSAT1), an enzyme PSAT1 also signi cantly ameliorated insulin resistance fi fi in diabetic mice. In addition, PSAT1 inhibited the expres- involved in serine biosynthesis that was rst puri ed from sion of hepatic tribbles homolog 3 (TRB3) in vitro and in the brains of sheep, is expressed at high levels in many vivo, and adenoviruses expressing small hairpin RNA tissues, including liver (10). Three steps are involved in against TRB3-mediated inhibition of TRB3 reversed serine synthesis (11), and PSAT1 is involved in the second the attenuated insulin sensitivity in Ad-shPSAT1 mice. step catalyzing the 3-phosphohydroxypyruvate to form Interestingly, we found that serine mediates PSAT1 reg- L-phosphoserine (11,12). Serine contributes to the regula- ulation of TRB3 expression and insulin signaling in vitro. tion of several physiological processes (13–16), and, as one These results identify a novel function for hepatic PSAT1 of a key enzymes in serine synthesis, PSAT1 has some in regulating insulin sensitivity and provide important important metabolic functions (17–19). For example, insights in targeting PSAT1 for treating insulin resis- PSAT1 deficiency results in intractable seizures and tance and type 2 diabetes. Our results also suggest that acquired microcephaly (17), while increased expression of nonessential amino acid serine may play an important PSAT1 is associated with the development of colorectal role in regulating insulin sensitivity. cancer (18). Several lines of evidence indicate that PSAT1 has a role in Insulin resistance, in which insulin fails to effectively controlling blood glucose. For example, PSAT1 regulates activate its downstream signaling pathways, is a common serine synthesis (19), and serine reportedly regulates gly- feature of type 2 diabetes (T2D) (1). Nutrients are impor- cogensynthesis(16).Inaddition,a-ketoglutaric acid, tant contributing factors to the development of insulin as a by-product of PSAT1 catalyzed reaction (14), stim- resistance in patients with T2D (2–4). Previous studies ulates insulin secretion (20). However, it is currently have demonstrated that essential amino acids, unknown whether PSAT1 participates in the regulation Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences, This article contains Supplementary Data online at http://diabetes Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Grad- .diabetesjournals.org/lookup/suppl/doi:10.2337/db14-1368/-/DC1. uate School of the Chinese Academy of Sciences, Shanghai, China © 2015 by the American Diabetes Association. Readers may use this article as Corresponding author: Feifan Guo, [email protected]. long as the work is properly cited, the use is educational and not for profit, and Received 9 September 2014 and accepted 9 December 2014. the work is not altered. 1592 PSAT1 Regulates Insulin Sensitivity Diabetes Volume 64, May 2015 of insulin sensitivity, another important aspect of main- sacrifice. Eight- to 10-week-old mice were maintained taining glucose homeostasis (1). Previous studies have on a 12-h light/dark cycle at 25°C and provided free shown that PSAT1 expression and serine biosynthesis access to commercial rodent chow and tap water prior are increased under protein-restricted diet in liver of to initiation of the experiments. Food intake and body rats (21), a condition demonstrated to be beneficial weight were measured daily. Animal experiments were for improving insulin sensitivity (22), which suggests conducted in accordance with the guidelines of the that PSAT1 might have a novel function in the regu- Institutional Animal Care and Use Committee of the lation of insulin sensitivity. The aim of our current Institute for Nutritional Sciences, Shanghai Institutes study was to investigate this possibility and elucidate for Biological Sciences, Chinese Academy of Sciences underlying mechanisms. (CAS). In this study, we show that hepatic PSAT1 regulates Primary Mouse Hepatocyte Isolation, Cell Culture, and insulin sensitivity in vitro and in vivo under normal and Treatments insulin-resistant conditions. Furthermore, the effects of Primary mouse hepatocytes were prepared by collagenase PSAT1 were mediated by inhibition of tribbles homolog perfusion as previously described (25). HepG2 and Hep1- 3(TRB3).Inaddition,PSAT1regulatedTRB3expression 6 cells (Cell Centre of Shanghai Institutes for Biological in a serine-dependent manner, and decreased liver Sciences, CAS) were maintained in DMEM containing 25 serine levels might contribute to insulin resistance in mmol/L glucose (Gibco, Invitrogen, Carlsbad, CA), 10% vivo. FBS, and 50 mg/mL penicillin and streptomycin, in an environment with 5% CO2/95% air at 37°C. Control (com- RESEARCH DESIGN AND METHODS plete amino acid), (2) ser (serine-deficient), and 23 ser Animals and Treatment (serine-supplemented) medium were prepared from Male C57BL/6J wild-type (WT) mice were obtained amino acid–free DMEM (Invitrogen) by adding back all from Shanghai Laboratory Animal Co., Ltd. (Shanghai, of the amino acids contained in regular DMEM, without China).Leptinreceptor–deficient (db/db)micewere serine only, or double the serine amount, respectively. kindly provided by Dr. Xiang Gao (Nanjing University). Double-stranded small interfering RNA (siRNA) target- Male C57BL/6J WT mice were fed for 16 weeks an HFD ing human phosphoglycerate dehydrogenase (PGDH) or (Research Diets, Inc., New Brunswick, NJ) or chow diet, L-phosphoserine phosphatase (PSPH) were purchased which has been previously used as control for HFD from GenePharma (Shanghai, China). The siRNA se- (23,24). Adenoviruses were injected in to mice under quence specificforhumanPGDHwas59-GGGAG HFD or chow diet for 14 weeks, and then these mice GAAAUUGCUGUUCATT-39 and for human PSPH was were fed with an HFD or chow diet continuously before 59-GGAGUAUUGUAGAGCAUGUTT-39. HepG2 cells were Figure 1—Hepatic PSAT1 expression is reduced under insulin-resistant conditions. PSAT1 expression was analyzed in the livers, WAT, and muscle, and liver serine levels were measured in wild-type (wt) and db/db mice in A–C or WT mice fed a control diet (2 HFD) or HFD (+ HFD) for 16 weeks in D–F. Data were obtained with mice described above (n =10–14 mice/group) and are presented as means 6 SEMs. Statistical significance was calculated using the two-tailed Student t test for the effects of db/db or HFD mice versus control mice (*P < 0.05). A and D: PSAT1 protein expression (top, Western blot; bottom, quantitative measurement of PSAT1 protein relative to tubulin or actin). B and E: Hepatic Psat1 mRNA level. C and F: Liver serine level. diabetes.diabetesjournals.org Yu and Associates 1593 transfected with siRNA using X-tremeGENE siRNA hairpin RNA sequence for mouse PSAT1 is 59-GCATCAGT Transfection Reagent (RocheDiagnostics,Mannheim, GTGCTCGAAATGA-39 andformouseTRB3is59-GGAA Germany). CCTTCAGAGCGACTTGT-39. High-titer stocks of amplified fi Generation and Administration of Recombinant recombinant adenoviruses were puri ed as previously de- Adenoviruses scribed (7). Viruses were diluted in PBS and administered 7 Recombinant adenovirus expressing human PSAT1 was at a dose of 10 plaque-forming units/well in a 12-well plate 3 8 generated using the AdEasy Adenoviral Vector System or via a tail vein injection using 5 10 plaque-forming (Qbiogene, Irvine, CA) according to the manufacturer’s units per mouse. instructions. Adenovirus expressing scrambled or short Blood Glucose, Serum Insulin, Glucose Tolerance Test, hairpin RNA directed against the coding region for mouse Insulin Tolerance Test, and HOMA of Insulin PSAT1 or TRB3 was generated using the BLOCK-iT Resistance Index Adenoviral RNAi Expression System (Invitrogen) accord- Levels of blood glucose and serum insulin were mea- ing to the manufacturer’s instructions. The scrambled sured using a Glucometer