Hepatic Overexpression of ATP Synthase B Subunit Activates PI3K/Akt Pathway to Ameliorate Hyperglycemia of Diabetic Mice

Diabetes Volume 63, March 2014 947

Chunjiong Wang,1,2 Zhenzhen Chen,1 Sha Li,1 Yuan Zhang,3 Shi Jia,1 Jing Li,1 Yujing Chi,1 Yifei Miao,1 Youfei Guan,1,4 and Jichun Yang1

Hepatic Overexpression of ATP Synthase b Subunit Activates PI3K/Akt Pathway to Ameliorate Hyperglycemia of Diabetic Mice

ATP synthase b subunit (ATPSb) had been exclusion of forkhead box O1 in liver cells. In previously shown to play an important role in conclusion, a decrease in hepatic ATPSb expression controlling ATP synthesis in pancreatic b-cells. This in the liver, leading to the attenuation of ATP-P2 METABOLISM study aimed to investigate the role of ATPSb in receptor-CaM-Akt pathway, may play an important regulation of hepatic ATP content and glucose role in the progression of diabetes. metabolism in diabetic mice. ATPSb expression and Diabetes 2014;63:947–959 | DOI: 10.2337/db13-1096 ATP content were both reduced in the livers of type 1 and type 2 diabetic mice. Hepatic overexpression In past decades, diabetes became one of the major dis- of ATPSb elevated cellular ATP content and eases threatening our health, with an estimated global ameliorated hyperglycemia of streptozocin- prevalence of 6.4% in 2010 (1). The liver is the key tissue induced diabetic mice and db/db mice. ATPSb that releases glucose into the circulation in fasting status, overexpression increased phosphorylated Akt and an increase in hepatic glucose production due to (pAkt) levels and reduced PEPCK and G6pase insulin deﬁciency or insulin resistance is the central expression levels in the livers. Consistently, ATPSb event in the development and progression of type 1 or overexpression repressed hepatic glucose type 2 diabetes (2). Moreover, the liver is also one of the production in db/db mice. In cultured hepatocytes, key tissues for lipid metabolism. ATPSb overexpression increased intracellular and ATP serves as an energy molecule as well as a signal extracellular ATP content, elevated the cytosolic free molecule in many cells (3). We previously showed that calcium level, and activated Akt independent of leucine upregulates the ATP synthase b (ATPSb) subunit insulin. The ATPSb-induced increase in cytosolic to enhance ATP synthesis and insulin secretion in rat free calcium and pAkt levels was attenuated by islets, type 2 diabetic human islets, and Rattus insulin-1 inhibition of P2 receptors. Notably, inhibition of (INS-1) cells. Overexpression or knockdown of ATPSb calmodulin (CaM) completely abolished ATPSb- increases or reduces cellular ATP levels in INS-1 cells induced Akt activation in liver cells. Inhibition of P2 (4,5). Overall, these ﬁndings suggest that ATPSb plays receptors or CaM blocked ATPSb-induced nuclear a crucial role in controlling ATP synthesis.

1Department of Physiology and Pathophysiology, Key Laboratory of Molecular Corresponding author: Jichun Yang, [email protected]. Cardiovascular Science, Peking (Beijing) University Health Science Center, Received 12 July 2013 and accepted 21 November 2013. Beijing, China This article contains Supplementary Data online at http://diabetes 2Department of Physiology and Pathophysiology, Tianjin Medical University, .diabetesjournals.org/lookup/suppl/doi:10.2337/db13-1096/-/DC1. Tianjin, China 3Department of Laboratory Medicine, Peking University Third Hospital, Beijing, C.W., Z.C., and S.L. contributed equally to this work. China © 2014 by the American Diabetes Association. See http://creativecommons 4Shenzhen University Diabetes Center, Shenzhen University Health Science .org/licenses/by-nc-nd/3.0/ for details. Center, Shenzhen, China 948 ATPSb Subunit and Hyperglycemia Diabetes Volume 63, March 2014

ATP content was significantly reduced in the livers of Sigma-Aldrich) freshly dissolved in citrate buffer (pH streptozocin (STZ)-induced type 1 diabetic mice, high- 4.2). Control mice were injected with citrate buffer. Mice fat diet (HFD)-induced type 2 diabetic mice, and with a fasting blood glucose (FBG) level .16.4 mmol/L at a methionine- and choline-deficient diet-induced non- 30 days after the final STZ injection were chosen for alcoholic steatohepatitis rats (6–8). Hepatic ATP content experiments. The mice were treated with Ad-ATPSb or was also decreased in insulin-resistant and type 2 di- Ad-GFP as were the db/db mice. FBG was monitored at abetic patients (9,10). A decrease in the hepatic ATP-to- 3 days after the viral injection. On the fourth day, the fed adenosine 5-monophosphate (AMP) ratio stimulates animals were killed for experimental analysis. Serum was food intake by signals via the vagus nerve to the brain, collected for determination of insulin, triglyceride, and which may lead to obesity and insulin resistance in total cholesterol levels, and the liver was taken for bio- humans (11). In obese healthy subjects, hepatic ATP chemical analysis. All procedures were approved by the content was inversely related to BMI, decreasing steadily Peking University Health Science Center Institutional with increasing BMI (12). Hepatic knockdown of PEPCK, Animal Care and Use Committee. one of the key gluconeogenic enzymes, ameliorated hy- OGTTs perglycemia and insulin resistance with increased hepatic Mice were fasted for 6 h (8:00 A.M. to 2:00 P.M.) in a cage ATP content in db/db mice (13). Overall, all of these with fresh bedding before OGTT. Mice were orally ad- studies revealed that a decrease in ATP content is associated with an increase in glucose production in the livers ministered with glucose at the dose of 3 g/kg, and blood glucose levels were monitored at 0, 15, 30, 60, 90 and of diabetic animals and humans. Given the crucial role of 120 min after the glucose load using a Freestyle brand ATPSb in controlling mitochondrial ATP synthesis (4,5), glucometer (Roche) by blood collected from the tail (15). we hypothesized that its expression in the liver may be The blood glucose concentrations at 0 min were de- deregulated and associated with increased hepatic glu- termined as FBG. Blood samples were taken at 0, 15, 30, cose production under diabetic conditions. and 60 min from the tail vein for detecting insulin levels. In this study, we report that ATPSb expression is Plasma insulin levels were measured using the Rat/ reduced and correlated with ATP content in the livers of type 1 and type 2 diabetic mice. Hepatic overexpression Mouse Insulin ELISA kit (Millipore) (16). of ATPSb increased cellular ATP content and suppressed Pyruvate Tolerance Tests gluconeogenesis, leading to the amelioration hypergly- The protocol for pyruvate tolerance tests (PTT) was de- cemia of type 1 and type 2 diabetic mice. tailed elsewhere (17). In brief, db/db mice were infected with Ad-ATPSb or Ad-GFP for 7 days, fasted for 16 h, RESEARCH DESIGN AND METHODS and injected intraperitoneally with pyruvate (0.75 mg/kg Construction of Adenovirus Expressing Rattus ATPSb body weight in saline). Blood glucose levels were mea- Adenovirus (Ad) expressing Rattus ATPSb was con- sured from the tail vein at indicated times using a Free- structed using the ATPSb cDNA coding sequence cloned style glucometer. from INS-1 cells in our previous study (4) with a 6xHis Insulin Tolerance Tests tag inserted in the N-terminus. Insulin tolerance tests (ITT) were done with an in- Overexpression of ATPSb in the Livers of Diabetic traperitoneal injection of insulin (3 units/kg body Mice weight) into mice after 6 h of fasting (18). Plasma glucose To assess the effect of an HFD on hepatic ATPSb ex- was measured using blood drawn from the tail vein at 0, pression, 8-week-old C57BL/6 mice were fed on a 45% 15, 30, 60, 90, 120 min after the insulin injection (19). HFD or a control diet (Diet #MD 45% fat and Diet #MD Immunoprecipitation and Immunoblot Assays 10% fat, Medicience Ltd., Yangzhou, China) for 12 weeks The immunoprecipitation was performed using Protein G (14). The mRNA and protein expression of ATPSb or Agarose beads, as described previously (20). Liver protein other genes in the livers was analyzed by real-time PCR (200 mg) was precipitated with anti-ubiquitin or IgG, and and Western blotting assays. separated by 10–12% SDS gel. Immunoblot was per- For overexpression of ATPSb in the liver, 8- to formed, and the membrane was developed with en- 12-week-old female db/db mice on a BKS background hanced chemiluminescence. were chosen for the experiment. As previously described, 1.0 3 109 plaque-forming unit Ad-ATPSb or Ad-green Cell Culture fluorescent protein (GFP) were injected into the db/db Human hepatocellular carcinoma (HepG2) cells were mice via tail vein (15,16). At the third and seventh day purchased from American Type Culture Collection after virus injection, oral glucose tolerance tests (OGTTs) (Rockville, MD) and cultured at 37°C in 5% CO2 and 95% were performed. On the eighth day, the fed animals were air in Dulbecco’s modified Eagle’s medium. The cells were killed for experimental analysis. infected with 20 multiplicity of infection of Ad-GFP or Male C57BL/6 mice (8 weeks old) were injected intra- Ad-ATPSb for 48 h. For insulin stimulation, the cells peritoneally for 7 consecutive days with STZ (50 mg/kg, were serum-starved for 12 h, followed by treatment with diabetes.diabetesjournals.org Wang and Associates 949

100 nmol/L insulin (Novo Nordisk) for 5 min and then antibodies were from Abcam. Anti-ATPSa,6His-tag, lysed in fresh Roth Lysis buffer. For inhibition of phos- G6Pase, ubiquitin, EIF5, and b-actin antibodies were from phatidylinositide 3-kinase (PI3K), the infected cells were Santa Cruz Biotechnology. Ant-ATPSg and ATPSd anti- treated with indicated concentrations of wortmannin bodies were from BioWorld. Anti-COXIV antibodies were (inhibitor of PI3K), PIK75 (inhibitor of p110a), or from Invitrogen (Medical & Biological Laboratories). TGX221 (inhibitor of p110b) for 30 min before being Peroxidase-conjugated secondary antibodies were from lysed. The infected cells were treated with indicated Santa Cruz Biotechnology. Activation of Akt was evaluated concentrations of PPADS (antagonist of P2 receptor), by analyzing phosphorylation at Ser473 site. U73122 (inhibitor of IP3R), and carboxypeptidase Z (CPZ; inhibitor of calmodulin [CaM]), for 30 min to 1 h Quantitative Real-Time PCR Assay before experimental assay. The lysate was centrifuged at Total RNA of tissues and cells were extracted with 13,000 rpm at 4°C for 10 min, and 20–100 mg total RNApure High-purity Total RNA Rapid Extraction Kit proteins underwent Western blotting assay immediately. (BioTeke Corporation). Quantification of target gene expression was performed by quantitative real-time PCR. Primary Hepatocyte Culture The complementary DNA was synthesized with the use Primary mouse hepatocytes were cultured as previously of RevertAid First Strand cDNA Synthesis Kit (Fermentas described (4). In brief, 5- to 6-week-old male C57BL/6 K1622). Target gene mRNA level was normalized to that mice were anesthetized with 10% chloral hydrate, and of b-actin in the same sample, as detailed previously (21). a catheter was placed in the inferior vena cava. The liver Each sample was measured in duplicate or triplicate in was perfused with 1 mL heparin (320 m/mL), 40 mL each experiment. The melting curve for each PCR product ’ solution I (Krebs s solution and 0.1 mmol/L EGTA), and was analyzed to ensure the specificity of the amplifica- ’ 30 mL solution II (Krebs s solution, 2.74 mmol/L CaCl2, tion product. All of the primer sequences for the real- and 0.05% collagenase I), respectively. The perfused liver time PCR assays are listed in Supplementary Table 1. was passed though a 400-mm screening size filter by flushing with RPMI 1640 medium. The hepatocytes were ATP Content Determination collected by centrifuge at 50g for 2 min. Hepatocytes ATP content was assessed by the bioluminescence were resuspended with RPMI 1640 medium and plated in method using ATP-Lite Assay Kit (Vigorous Bio- six-well plates for experiments after three washes with technology Beijing Co., Ltd.). For hepatic ATP content RPMI 1640. determination, 1 mL lysis buffer was added per 100 mg Immunoblotting frozen tissue. For cellular ATP content determination, 300 mL lysis buffer was added to the 35-mm dish. For Cells or liver tissues were lysed in fresh Roth lysis buffer extracellular ATP content determination, HepG2 cell (50 mmol/L HEPES, 150 mmol/L NaCl, 1% Triton X-100, culture medium was collected. The lysate ATP content or 5 mmol/L EDTA, 5 mmol/L EGTA, 20 mmol/L sodium the ATP content in the medium was measured by lumi- pyrophosphate, 20 mmol/L NaF, 0.2 mg/mL phenyl- nometric determination of the luciferin-luciferase re- methylsulfonyl fluoride, 0.01 mg/mL leupeptin, and 0.01 action. For determination of relative ATP level in the mg/mL aprotinin, pH 7.4). The lysate was centrifuged at cells, the ATP content values (nmol) were first normal- 4°C at 13,000 rpm for 10 min. The protein concentration ized to the protein mount (nmol/mg protein) in the same in the supernatant was determined by bicinchoninic acid sample and then normalized to the control values. For assay, and 20 to 200 mg total protein were separated by determination of relative ATP level in the medium, the 12–15% SDS-PAGE. Proteins in the gel were transferred absolute concentration was determined and normalized to the Hybond-C Extra membrane (Amersham Bio- to the control value. sciences) at 120 V for 2 h at 4°C. The membrane was washed once with Tris-buffered saline with Tween (TBST; Cellular Calcium Measurement 1.2 g/L Tris Base, 5.84 g/L NaCl, and 0.1% Tween-20, pH HepG2 cells seeded on coverslips were loaded with 7.5) before blocking in TBST containing 1% BSA at room 1 mmol/L Fura-2 acetoxymethyl ester (Molecular Probes) temperature for 1 h. The membrane was incubated in for 10 min and then imaged under an Olympus IX71 – 1:200 1:1,000 primary antibodies at 4°C overnight. The fluorescence microscope. The emission intensities under fi membrane was washed ve times with TBST and in- 340 and 380 nm illumination were recorded every 1 s, cubated in 1:5,000 peroxidase-conjugated secondary with the ratio of the emission densities (F340-to-F380) antibodies at room temperature for 1 h before washing reflecting the intracellular free calcium concentration (5). as above, and then developed with ECL. After immuno- For basal calcium concentration measurement, infected blotting assays of target proteins, the membrane was HepG2 cells were treated with indicated concentrations stripped with 0.2 N NaOH and reprobed for EIF5 or an- of PPADS and suramin for 1 h. other housekeeping protein as loading control. Anti- ATPSb, phosphorylated (p)Akt, Akt, forkhead box class Mitochondria Isolation O1 (FOXO1), Fas, and sterol responsive element binding Mitochondria were isolated from liver tissue or HepG2 protein (SREBP1) and its mature form of SREBP1 cells using the Mitochondria/Cytosol Fractionation Kit 950 ATPSb Subunit and Hyperglycemia Diabetes Volume 63, March 2014

(Pierce) according to the manufacturer’s protocol. In incubated with Alexa Fluor 594 antibodies (1:200). Images brief, liver tissue or HepG2 cells were homogenized in were obtained using confocal microscopy. Mito-Cyto extraction buffer provided by the kit, and the Statistical Analysis lysate was centrifuged twice at 800g for 5 min to pellet Data are presented as mean 6 SEM. Statistical signifi- the nucleus and cell debris. The supernatant was col- cance of differences between groups was analyzed by lected and centrifuged at 10,000g for 10 min to pellet unpaired Student t test or by one-way ANOVA when mitochondria, which was washed three times with lysis more than two groups were compared. buffer to clean contaminated cytoplasmic proteins (22). Immunofluorescence RESULTS To detect the translocation of FOXO1, HepG2 cells were ATPSb Expression Was Reduced in the Livers of db/db infected with Ad-GFP or Ad-ATPSb for 48 h and treated and HFD-Fed Diabetic Mice with of PPADS (50 mmol/L) and CPZ (100 mmol/L) for 1 h To determine the potential role of ATPSb in the pro- before been fixed with 4% paraformaldehyde for 15 min gression of hepatic insulin resistance and type 2 diabetes, and permeabilized with 0.5% Triton X-100 for 10 min. its expression in the livers of db/db and HFD-fed diabetic Nonspecific binding was blocked in 1% BSA at room mice was analyzed. The mRNA (Fig. 1A)andprotein(Fig.1B) temperature for 10 min, followed by incubation with anti- levels of ATPSb were significantly reduced in the livers FOXO1 antibody overnight at 4°C. The slides were then of db/db mice. In contrast, the mRNA levels of the a, g, d,

Figure 1—ATPSb expression was reduced in the livers of db/db and HFD-fed diabetic mice. A: The mRNA level of ATPSb was reduced in the livers of db/db mice. B: The protein level of ATPSb was reduced in the livers of db/db mice. C: Hepatic ATP content was reduced in db/ db mice. n =5,*P < 0.05 vs. db/m mice. D: The mRNA level of ATPSb was reduced in the livers of mice fed an HFD for 12 weeks. E: The protein level of ATPSb was reduced in the livers of HFD-fed mice. F: Hepatic ATP content was reduced in HFD-fed mice. n =5,*P < 0.05 vs. control mice. NC, normal chow. Con, control groups of cells or animals. diabetes.diabetesjournals.org Wang and Associates 951 and ´ subunits of the F1 domain, and the b, c, and Hepatic Overexpression of ATPSb Ameliorated d subunits of the F0 domain were not significantly dif- Hyperglycemia in db/db Mice ferent between db/db and db/m mouse livers (Supple- The efficacy of Ad-ATPSb treatment on the cellular mentary Fig. 1A). The protein levels of the a and g ATPSb protein level was firstly determined in HepG2 subunits of the F1 domain, and the d subunits of the F0 cells (Supplementary Fig. 2). Then, ATPSb was overex- domain were confirmed to remain unchanged between pressed in the livers of db/db mice via tail vein injection db/db and db/m mouse livers (Supplementary Fig. 1B). of Ad-ATPSb to evaluate its effect on ATP content and Hepatic ATP content was also reduced in db/db mice glucose metabolism. At 3 days after the virus injection, compared with db/m mice (Fig. 1C). The mRNA and Ad-ATPSb–treated mice exhibited significant improve- protein levels of ATPSb and the ATP content were also ment of fasting hyperglycemia and glucose intolerance reduced in the livers of HFD-fed diabetic mice (Fig. 1D–F). compared with Ad-GFP–treated mice (Fig. 2A and B). At Similarly, the mRNA levels of the a, g, d, ´,b,c,andd 7 days after the virus injection, fasting hyperglycemia subunits of ATP synthase were not significantly different and glucose intolerance were markedly attenuated in Ad- between HFD and normal chow mouse livers (Supple- ATPSb–treatedmicecomparedwithAd-GFP–treated mice mentary Fig. 1C). The protein levels of the a, g,and (Fig. 2C). The FBG of mice at 0, 3, and 7 days after the dsubunitswerealsoconfirmed to remain unchanged in viral injection is shown in Fig. 2D. At day 8 after the virus HFD-fed diabetic mouse livers (Supplementary Fig. 1D). injection, the serum insulin levels in Ad-ATPSb–treated

Figure 2—Hepatic overexpression of ATPSb markedly ameliorated fasting hyperglycemia and glucose intolerance in db/db mice. OGTT before (A)orat3(B) and 7 (C) days after tail vein injection of Ad-GFP or Ad-ATPSb is shown. At 3 and 7 days after virus injection, Ad- ATPSb–transduced mice exhibited significant improvement of hyperglycemia and glucose intolerance. D: FBG results before or at 3 and 7 days after viral injection. E: Ad-ATPSb treatment reduced serum insulin levels.n=8–10, *P < 0.05 vs. Ad-GFP group. 952 ATPSb Subunit and Hyperglycemia Diabetes Volume 63, March 2014 mice were significantly lower than those in control mice in the livers of db/db mice (Fig. 3C–E). The mRNA levels (Fig. 2E). ATPSb overexpression also reduced hepatic lipid of PEPCK and G6Pase in the livers were significantly deposition in db/db mice (Supplementary Fig. 3). repressed after ATPSb overexpression (Fig. 3F). In vivo, OGTT, insulin secretion, ITT, and PTT assays indicated ATPSb Overexpression Activated Akt and Repressed there was no significant difference between saline- and Gluconeogenic Genes in the Livers of db/db Mice Ad-GFP–treated mice (Supplementary Figs. 5 and 6). Ad-ATPSb treatment increased the ATPSb protein level Moreover, ATPSb overexpression failed to affect insulin in the livers of db/db mice (Fig. 3A). Moreover, overex- secretion in db/db mice (Supplementary Fig. 5D and E). pressed ATPSb was predominantly located in mitochon- In vitro, Ad-GFP infection also failed to significantly af- dria (Fig. 3B). In contrast, the injection of Ad-ATPSb had fect Akt activation in HepG2 cells (Supplementary Fig. little effect on the mRNA and protein levels of ATPSb in 5F). These findings suggested that the virus itself, at the skeletal muscle and the pancreas, indicating the speci- dose used in the current study, has little effect on glucose ficity of ATPSb overexpression in the liver (Supplemen- metabolism and Akt activation in mouse liver cells. ITT tary Fig. 4). ATPSb overexpression elevated cellular ATP assays indicated that global insulin sensitivity was im- content, increased phosphorylated Akt (pAkt) levels, and proved by hepatic overexpression of ATPSb (Supple- reduced the protein level of gluconeogenic gene G6Pase mentary Fig. 6A and B). The PTT assays revealed that

Figure 3—ATPSb overexpression repressed gluconeogenic genes in the livers of db/db mice. A: Ad-ATPSb treatment increased ATPSb protein level in the livers. B: Ad-ATPSb treatment increased ATPSb protein level in the mitochondria. Mitochondria were isolated from db/db mouse livers, and immunoblotting assays were performed using antibodies against ATPSb or 6xHis tag. Mi, mitochondrial fraction. C: ATPSb overexpression elevated ATP content in the livers. Shown are representative images (D) and quantitative data (E) of ATPSb overexpression on the protein levels of gluconeogenic genes in the livers. F: ATPSb overexpression reduced the mRNA levels of G6Pase and PEPCK in the livers. n =5–8, *P < 0.05 vs. Ad-GFP group. diabetes.diabetesjournals.org Wang and Associates 953 hepatic glucose production was repressed after ATPSb staining and immunoblotting assays indicated that Ad- overexpression (Supplementary Fig. 6C and D). Overall, ATPSb treatment increased the ATPSb protein level in these findings were consistent with the decrease in glu- the mitochondria (Supplementary Figs. 7 and 8). ATPSb coneogenic genes and the attenuation of hyperglycemia overexpression significantly elevated intracellular and in db/db mice after hepatic ATPSb overexpression. extracellular ATP levels in HepG2 cells (Fig. 4A) and in primary cultured mouse hepatocytes (Fig. 4B). In HepG2 ATPSb Activated PI3K-Akt Signaling Pathway Through cells infected by Ad-GFP, insulin potently stimulated Akt the P2 Receptor activation (Fig. 4C). Ad-ATPSb treatment markedly ele- Because Akt plays a crucial role in controlling glucose vated pAkt levels (Fig. 4C) compared with Ad-GFP–treated metabolism, the effect of ATPSb on Akt activation was cells without insulin stimulation. ATPSb overexpression further analyzed in HepG2 cells and primary cultured also augmented insulin-stimulated Akt activation in mouse hepatocytes. In HepG2 cells, immunofluorescence HepG2 cells (Fig. 4C). Similarly, ATPSb also induced Akt

Figure 4—ATPSb overexpression activated Akt via PI3K in liver cells. ATPSb overexpression increased intracellular and extracellular ATP levels in HepG2 cells (A) and in primary cultured mouse hepatocytes (B). C: ATPSb activated Akt independent of insulin in HepG2 cells. The infected cells were serum-starved for 12 h and treated with or without 100 nmol/L insulin (INS) for 5 min before pAkt levels were analyzed. D: ATPSb activated Akt independent of insulin in primary cultured mouse hepatocytes. E: Effect of inhibition of PI3K on ATPSb- induced Akt activation in HepG2 cells. Infected HepG2 cells were treated with wortmannin (1 mmol/L), p110a inhibitor PIK75 (100 nmol/L), or p110b inhibitor TGX221 (100 nmol/L) for 30 min before pAkt levels were analyzed. D, DMSO; W, wortmannin; a, p110a inhibitor PIK75; b, p110b inhibitor TGX221. n =4–5, *P < 0.05 vs. control cells; #P < 0.05 vs. Ad-GFP–infected cells in the presence of insulin. 954 ATPSb Subunit and Hyperglycemia Diabetes Volume 63, March 2014 activation in primary cultured mouse hepatocytes in the Pretreatment with suramin, another P2 receptor antag- absence of insulin stimulation (Fig. 4D). ATPSb-induced onist, also attenuated ATPSb-induced Akt activation Akt activation was completely blocked by the PI3K in- (Supplementary Fig. 9A). Furthermore, inhibition of the hibitor wortmannin in HepG2 cells in the absence of P2 receptor downstream molecule phospholipase C (PLC) insulin stimulation (Fig. 4E). Inhibition of the PI3K using U73122 also attenuated ATPSb-induced Akt acti- p110a catalytic subunit blocked ATPSb-induced Akt ac- vation (Fig. 5B). ATPSb overexpression elevated the cy- tivation by ;70–80%, whereas inhibition of the PI3K tosolic free calcium level, which was inhibited by PPADS p110b catalytic subunit had little effect on ATPSb- and suramin in HepG2 cells (Fig. 5C). Because an increase induced Akt activation in the absence of insulin (Fig. 4E). in cytosolic free calcium level has been shown to activate Overall, these results suggested that ATPSb-induced Akt the PI3K/Akt signaling axis through CaM in several cell activation requires the activity of the PI3K p110a cata- types (25,26), we further analyzed whether ATPSb induced lytic subunit but is independent of insulin. Akt activation by the calcium-CaM signaling pathway. The ATP can be released to function as a signal molecule in results indicated that depletion of extracellular calcium various cell types (23,24). In HepG2 cells, ATPSb-induced partially attenuated (Supplementary Fig. 9B), whereas in- Akt activation was signiﬁcantly attenuated by the ATP hibition of CaM using CPZ completely abolished, ATPSb- receptor P2 receptor antagonist PPADS (Fig. 5A). induced Akt activation in HepG2 cells (Fig. 5D).

Figure 5—ATPSb induced Akt activation by the P2 receptor/calcium/CaM signaling pathway in HepG2 cells. A: Inhibition of P2 receptors attenuated ATPSb-induced Akt activation in HepG2 cells. Infected cells were treated with PPADS (50 mmol/L) for 1 h before pAkt levels were analyzed. B: Inhibition of PLC attenuated ATPSb-induced Akt activation in HepG2 cells. Infected cells were treated with PLC inhibitor U73122 (5 mmol/L) for 1 h before pAkt levels were analyzed. C: Inhibition of P2 receptors attenuated the ATPSb-induced increase in cytosolic free calcium level. Infected cells were treated with PPADS or suramin (50 mmol/L) for 1 h before cellular calcium levels were analyzed. The calcium levels were obtained from more than 300 single cells in at least three independent experiments. D: Inhibition of CaM completely abolished ATPSb-induced Akt activation. Infected cells were treated with the CaM antagonist CPZ (100 mmol/L) for 1 h before pAkt levels were analyzed. n =5,*P < 0.05 vs. control cells; #P < 0.05 vs. Ad-ATPSb–treated cells. diabetes.diabetesjournals.org Wang and Associates 955

Inhibition of P2 Receptor or CaM Blocked ATPSb- insulin resistance, and fatty liver in db/db mice. Hepatic Induced Translocation of FOXO1 overexpression of ATPSb also elevated cellular ATP and Activation of Akt plays a crucial role in suppressing ameliorated hyperglycemia in STZ-induced diabetic mice. gluconeogenesis by phosphorylating and inactivating ATPSb overexpression increased pAkt levels and re- FOXO1, the key transcriptor controlling the transcrip- pressed the expression of gluconeogenic genes in the tion of gluconeogenic genes PEPCK and G6Pase (27). As livers of these diabetic mice. These findings suggested expected, ATPSb overexpression significantly promoted that inhibition of hepatic glucose production would at- nuclear exclusion of FOXO1 with the activation of Akt in tenuate hyperglycemia and global insulin resistance in HepG2 cells (Fig. 6). In support, inhibition of P2 recep- type 2 diabetic mice. Similarly, a previous report showed tors or CaM blocked ATPSb-promoted nuclear exclusion that silencing of hepatic PEPCK suppressed hepatic glu- of FOXO1 with the attenuation of Akt activation (Fig. 6). cose production and improved global insulin resistance in db/db mice (13). In vitro, ATPSb overexpression in- Hepatic Overexpression of ATPSb Ameliorated Hyperglycemia of STZ-Induced Type 1 Diabetic Mice creased intracellular and extracellular ATP levels in HepG2 cells and primary cultured mouse hepatocytes. In In the livers of STZ-induced type 1 diabetic mice, the support of our previous (4,5) and current findings that mRNA and protein levels of ATPSb expression was also ATPSb plays a crucial role in controlling ATP synthesis, significantly reduced with a decrease in ATP content (Fig. hydrogen peroxide induces ATPSb expression and 7A–C). Hepatic overexpression of ATPSb for 3 days ele- increases cellular ATP level in melanocytes (28). Proteo- vated cellular ATP content and ameliorated fasting hy- mic analysis revealed that ATPSb expression was re- perglycemia in STZ-induced type 1 diabetic mice (Fig. duced, with a decrease in ATP content in mouse livers 7C–E). ATPSb overexpression increased pAkt levels and after ischemia-reperfusion treatment (29). No change of reduced mRNA and protein levels of PEKCK and G6Pase other subunits of ATP synthase was found in mouse in the livers of STZ-treated mice, suggesting that hepatic livers injured by ischemia-reperfusion in the same gluconeogenesis was inhibited (Fig. 7F–H). study (29). DISCUSSION Extracellular ATP can activate the PI3K-Akt pathway In the livers of type 1 and type 2 diabetic mice, ATPSb through P2 receptors in several cell types. Ishikawa et al. expression was reduced and positively correlated with (30) found that pannexin 3 releases ATP into extracel- ATP content. Hepatic overexpression of ATPSb elevated lular space, which activates the PI3K/Akt pathway cellular ATP content, suppressed hepatic glucose pro- through P2 receptors to promote the proliferation of duction, and improved hyperglycemia, hyperinsulinemia, osteoblasts. Electrical pulses can stimulate skeletal muscle cells to release ATP, which enhances glucose uptake by activation of the PI3K-Akt pathway through P2 receptors (31). Extracellular ATP also activates the PI3K-Akt pathway to attenuate ischemia-induced apoptosis of human endothelial cells (32). P2 receptors were divided into P2X receptors and P2Y receptors. P2X receptors are ligand-gated ion channels that are permeable for calcium (33), whereas P2Y receptors are G protein–coupled receptors that stimulate PLC to increase IP3, resulting in calcium release from internal storage (23). Liver cells and HepG2 cells have been shown to release ATP. P2 receptors are also constitutively expressed in liver cells and HepG2 cells (24,34). Exposure to exogenous ATP has been reported to elevate the cytosolic free calcium level in HepG2 cells and primary cultured mouse hepatocytes (35). PPADS is a specific inhibitor for the P2X subtype, whereas suramin is considered to be a broad-spectrum P2 receptor inhibitor (36). PPADS and suramin both significantly attenuated the ATPSb-induced increase in the cytosolic free calcium level and Akt activation in HepG2 cells. Depletion of extracellular calcium also attenuated Figure 6—ATPSb promoted nuclear exclusion of FOXO1 in ATPSb-induced Akt activation. Furthermore, U73122, an HepG2 cells. In the absence of insulin, ATPSb overexpression inhibitor of the P2Y receptor downstream molecule PLC, promoted translocation of FOXO1 from nucleus to cytoplasm, also attenuated ATPSb-induced Akt activation in HepG2 which was blocked by the P2 receptor antagonist PPADS or the cells. These results revealed the involvement of both P2X CaM inhibitor CPZ. The images were obtained using confocal microscopy and are the representatives of at least three in- and P2Y receptors in ATPSb-induced increase in cytosolic dependent experiments. free calcium level and Akt activation in liver cells. 956 ATPSb Subunit and Hyperglycemia Diabetes Volume 63, March 2014

Figure 7—Hepatic overexpression of ATPSb ameliorated fasting hyperglycemia in STZ-induced type 1 diabetic mice. The mRNA (A) and protein (B) levels of ATPSb were reduced in the livers STZ-treated mice. n =5,*P < 0.05 vs. control mice. C: Ad-ATPSb treatment elevated ATP content in the livers. D: Ad-ATPSb treatment increased the ATPSb protein level in the livers of STZ-induced diabetic mice. E: Hepatic overexpression of ATPSb ameliorated fasting hyperglycemia of STZ-treated mice. FBG was monitored 3 days after viral injection. F and G: ATPSb overexpression increased pAkt levels and reduced the protein levels of G6Pase and PEPCK in the livers. H: ATPSb overexpression reduced the mRNA levels of G6Pase and PEPCK in the livers. n =9,*P < 0.05, **P < 0.01 vs. control mice; #P < 0.05 vs. Ad-GFP–treated mice.

Cytosolic free calcium level is the crucial second (Supplementary Fig. 3D). FOXO1 is a crucial transcriptor messenger in the ATP/P2 receptor–signaling pathway. An controlling the transcription of gluconeogenic enzymes increased cytosolic free calcium level activates CaM, PEPCK and G6Pase (27). Insulin-stimulated activation of which can activate PI3K by direct association with its p85 Akt can phosphorylate FOXO1 and promote its trans- regulatory subunit, leading to activation of Akt (37). location from the nucleus to the cytoplasm, suppressing Importantly, inhibition of CaM using CPZ completely the transcription of gluconeogenic genes and hepatic abolished ATPSb-induced Akt activation in HepG2 cells. gluconeogenesis (27). ATPSb overexpression promotes ATPSb-induced Akt activation requires the activity of the nuclear exclusion of FOXO1, which can be inhibited by PI3K p110a catalytic subunit but is independent of in- antagonisms of the P2 receptor or CaM. Overall, these sulin signaling. These findings were consistent with re- findings revealed that ATPSb overexpression elevated cent discoveries that p110a predominantly mediated Akt ATP synthesis and secretion in liver cells. Released ATP activation in liver cells. Deletion of p110a blunted Akt activates both P2X and P2Y receptors to increase the activation in response to insulin, whereas deletion of cytosolic free calcium level, which activates the CaM- p110b had little effect on insulin-stimulated Akt activa- PI3K-Akt signaling axis to suppress gluconeogenesis in- tion in the liver (38). Furthermore, ATPSb over- dependent of insulin. However, it should be noted that expression reduced hepatic and serum triglyceride elevated intracellular ATP levels will inhibit the activity content with little effect on liver and serum cholesterol of the ATP-sensitive K+ channel and increase the influx levels in db/db mice. The protein levels of key lipogenic of extracellular calcium through the L-type voltage-gated genes FAS and mature SREBP1 were reduced in the calcium channel (39,40), which may also contribute to livers of db/db mice after ATPSb overexpression ATPSb-induced increase in free cytosolic calcium and Akt diabetes.diabetesjournals.org Wang and Associates 957

HepG2 cells, which was reversed by overexpression of ATPSb (Supplementary Fig. 11). Palmitate has been shown to induce insulin resistance by stimulating reactive oxygen species (ROS) production in hepatocytes (47). We further found that ATPSb overexpression reduced palmitate-induced ROS production in HepG2 cells (Supplementary Fig. 12). ATPSb also tended to reduce ROS production in basal conditions in HepG2 cells (Supplementary Fig. 12). In a lipid stressed condition, an increase in electron transfer on the respiratory chain as well as a decrease in ATPSb expression may contribute to ROS overproduction and oxidative stress, which is one of the main causes of hepatic insulin resistance (47,48). These findings suggest that repression of ATPSb ex- Figure 8—Proposed mechanism of ATPSb in regulation of glu- pression, leading to a decrease in ATP synthesis and cose metabolism in the livers. ATPSb enhances ATP synthesis and a possible increase in ROS production, might be a novel elevates the extracellular ATP level, which activates P2 receptors mechanism for lipid-induced insulin resistance in liver to increase cytosolic free calcium levels. Finally, elevated cytosolic calcium levels activate the PI3K-Akt signaling pathway in a CaM- and muscle cells. dependent manner, leading to the suppression of hepatic gluco- Activation of AMP-activated protein kinase (AMPK) neogenesis. The → represents activation or enhancement, and ⊥ exerts beneficial effects on amelioration of hyperglycemia represents repression. (49). The activity of AMPK is modulated by various fac- tors such as an increase in the AMP-to-ATP ratio, adi- ponectin, and metformin (49). The antidiabetic drug activation. Interestingly, although we found that ATPSb metformin can target mitochondrial complex I to inhibit fi overexpression for 7 days signi cantly reduced the ATP production (50,51), leading to the activation of mRNA level of PEPCK in db/db mouse livers, its protein AMPK (52), which at least partially mediates metformin’s – level remained unchanged (Fig. 3D F, Supplementary hypoglycemic effects. However, some deleterious effects – Fig. 10A C). The decrease in the PEPCK mRNA level was of AMPK activation, including inhibition of insulin se- fi con rmed by two different sets of primers according to cretion (53), induction of pancreatic b-cell, and renal the mRNA sequence of PEPCK (Supplementary Fig. 10C). medullary interstitial cell apoptosis (54,55), have also At 3 days after ATPSb overexpression, the mRNA and fi been reported. Given the role of ATP as the key energy protein levels of PEPCK were both signi cantly reduced storage molecule and an important signaling molecule in the livers of db/db mice (data not shown) and STZ- (30–32), activation of ATPSb-ATP-P2 receptor signaling induced diabetic mice (Fig. 7). A coimmunoprecipitation may have some unique advantages than activation of assay revealed that the ubiquitination of the PEPCK metformin-AMPK signaling in the treatment of type 2 protein was reduced at 7 days after ATPSb over- diabetes. expression, suggesting that the ubiquitin-mediated deg- In summary, the current study demonstrated that radation of PEPCK protein might decrease ATPSb expression is positively correlated with ATP (Supplementary Fig. 10D). A decrease in PEPCK protein content in the livers of diabetic mice. A decrease in degradation might be a protective mechanism against ATPSb expression in the liver, leading to the reduction of persistent suppression of hepatic glucose production ATP content and attenuation of the P2 receptor-PI3K- induced by long-term ATPSb overexpression. Akt signaling pathway, may play an important role in the That excessive accumulation of free fatty acids (FFAs), progression of diabetes. Upregulating hepatic ATPSb in particular, saturated FFAs such as palmitate, plays an might be an attractive method for the treatment of type important role in the progression of insulin resistance in 1 and type 2 diabetes (Fig. 8). various tissues has been intensively studied (41). Højlund et al. (42,43) found that the ATPSb protein level is reduced in skeletal muscle of insulin-resistant and type 2 Funding. This study was supported by grants from the Ministry of Science diabetic patients. In vivo, FFAs induced insulin resistance and Technology (2012CB517504/2011ZX09102), the Natural Science Foun- in skeletal muscle cells with a decrease in ATP generation dation of China (81170791/30870905/81200625/81322011), and the Beijing (44). Rooibos ameliorates palmitate-induced insulin re- Natural Science Foundation (7122107). sistance in C2C12 muscle cells with increased cellular Duality of Interest. No potential conflicts of interest relevant to this ATP levels (45). In support, inhibition of ATP synthase article were reported. using oligomycin reduces cellular ATP levels and induces Author Contributions. C.W., Z.C., S.L., Y.Z., S.J., and Y.M. researched insulin resistance in cultured human myotubes (46). In data and contributed to discussion. C.W. wrote the manuscript. J.L. and Y.C. the current study, we found that palmitate down- reviewed and edited the manuscript. C.W., Y.G., and J.Y. designed the study regulated ATPSb and induced insulin resistance in and revised and edited the manuscript. J.Y. is the guarantor of this work and, 958 ATPSb Subunit and Hyperglycemia Diabetes Volume 63, March 2014

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