Hepatic p38α regulates gluconeogenesis by suppressing AMPK

Research Article

Hepatic p38a regulates gluconeogenesis by suppressing AMPK Yanyan Jing1,2, , Wei Liu1,2, , Hongchao Cao1, Duo Zhang1, Xuan Yao1, Shengjie Zhang1, Hongfeng Xia1, Dan Li3, Yu-cheng Wang4,5, Jun Yan6, Lijian Hui3, Hao Ying1,2,4,⇑ 1

Key Laboratory of Food Safety Research, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai 200031, China; 2Key Laboratory of Food Safety Risk Assessment, Ministry of Health, Beijing 100021, China; 3Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China; 4Clinical Research Center of Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China; 5Department of Nutrition, Shanghai Xuhui Central Hospital, Shanghai 200031, China; 6Model Animal Research Center, and MOE Key Laboratory of Model Animals for Disease Study, Nanjing University, Nanjing 210061, China

Background & Aims: It is proposed that p38 is involved in gluconeogenesis, however, the genetic evidence is lacking and precise mechanisms remain poorly understood. We sought to delineate the role of hepatic p38a in gluconeogenesis during fasting by applying a loss-of-function genetic approach. Methods: We examined fasting glucose levels, performed pyruvate tolerance test, imaged G6Pase promoter activity, as well as determined the expression of gluconeogenic genes in mice with a targeted deletion of p38a in liver. Results were confirmed both in vivo and in vitro by using an adenoviral dominant-negative form of p38a (p38a-AF) and the constitutively active mitogenactivated protein kinase 6, respectively. Adenoviral dominantnegative form of AMP-activated protein kinase a (DN-AMPKa) was employed to test our proposed model. Results: Mice lacking hepatic p38a exhibited reduced fasting glucose level and impaired gluconeogenesis. Interestingly, hepatic deficiency of p38a did not result in an alteration in CREB phosphorylation, but led to an increase in AMPKa phosphorylation. Adenoviral DN-AMPKa could abolish the effect of p38a-AF on gluconeogenesis. Knockdown of up-steam transforming growth factor b-activated kinase 1 decreased the AMPKa phosphorylation induced by p38a-AF, suggesting a negative feedback loop. Consistently, inverse correlations between p38 and AMPKa phosphorylation were observed during fasting and in diabetic mouse models. Importantly, adenoviral p38a-AF treatment ameliorated hyperglycemia in diabetic mice.

Keywords: p38a; Gluconeogenesis; AMPK; TAK1; Energy state. Received 30 May 2014; received in revised form 3 November 2014; accepted 23 December 2014; available online 13 January 2015 ⇑ Corresponding author. Address: INS, SIBS, CAS, 320 Yueyang Road, Shanghai 200031, China. Tel.: +86 21 54920247; fax: +86 21 54920291. E-mail address: [email protected] (H. Ying).   These authors contributed equally to this work. Abbreviations: MAPK, Mitogen-activated protein kinases; AMPK, AMP-activated protein kinase; CREB, cAMP response element-binding protein; TAK1, transforming growth factor b-activated kinase 1; G6Pase, glucose-6phosphatase; PEPCK, phosphoenolpyruvate carboxykinase; PGC-1a, peroxisome proliferators-activated receptor-c coactivator-1a; PTT, pyruvate tolerance test; p38a-AF, dominant-negative form of p38a; MKK6EE, a constitutive active mitogen-activated protein kinase kinase 6; DN-AMPKa, dominant-negative form of AMPKa; HFD, high-fat diet; HFHSD, high-fat high-sucrose diet.

Conclusions: Our study provides evidence that hepatic p38a functions as a negative regulator of AMPK signaling in maintaining gluconeogenesis, dysregulation of this regulatory network contributes to unrestrained gluconeogenesis in diabetes, and hepatic p38a could be a drug target for hyperglycemia. Ó 2015 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Introduction During periods of nutrient deprivation, the hepatic gluconeogenesis is stimulated to maintain blood glucose levels in a normal range, thereby supplying sufficient energy for glucose-dependent tissues. Dysregulation of gluconeogenesis is believed to be one of the major factors that contribute to metabolic disorders such as type 2 diabetes. Thus better understanding of the physiology and physiopathology of hepatic gluconeogenesis will help us to fight diabetes. p38 mitogen-activated protein kinases (MAPKs) are important in converting extracellular stimuli into a wide range of cellular responses. p38 MAPKs have key roles in inflammation, as well as in tissue homeostasis [1,2]. The p38 MAPK family has four members (p38a, b, c, and d), each encoded by individual genes. p38a is the predominant isoform in liver [3]. Activation of p38 has been observed not only in the livers during fasting, but also in the livers of genetic and diet-induced mouse models of obesity and diabetes [4,5]. It has been shown that p38 might play a regulatory role in hepatic gluconeogenesis [6–11]. However, most of these studies were done in vitro and pyridinyl imidazole compounds, such as SB203580, which inhibits both p38a and b isoforms, were applied, or carried out under pathological but not physiological conditions. Moreover, the precise mechanisms underlying the regulation of glucose homeostasis by hepatic p38 under physiological conditions are still not clear. Recently, the physiological importance of hepatic AMP-activated protein kinase (AMPK) in maintaining whole-body glucose homeostasis has been demonstrated [12–14]. AMPK has been shown as a significant inhibitor of gluconeogenesis [15–18]. However, in response to food deprivation, there is a fall in energy

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Research Article state, which results in a net loss of ATP and an elevation in AMP/ ATP ratio. The increased AMP/ATP ratio will directly activate AMPK signaling, which in turn has an inhibitory role in glucose production. Indeed, according to the literature, the conclusion of whether AMPK is activated in the fasted liver is not consistent, probably due to different experimental conditions or methods [19,20]. Additionally, hepatic energy state has been reported to be attenuated in diabetic mice, suggesting that high AMP/ATP levels are part of the metabolic disease milieu. Moreover, hepatic ATP levels have been shown to be reduced in obese humans [21]. Interestingly, we found the phosphorylation of hepatic AMPKa is inhibited in various diabetic models. Thus, it requires intensive investigation to address how the inhibitory role of AMPK on gluconeogenesis is controlled during fasting when the energy state is reduced, and whether the dysregulation of this regulatory network contribute to the unrestrained gluconeogenesis in diabetes. Here we employed a loss-of-function genetic approach to delineate the role of p38a in the liver. We found hepatic deficiency of p38a had no effect on the phosphorylation of cAMP response element-binding protein (CREB) during fasting but led to AMPKa phosphorylation through an upstream kinase, transforming growth factor b-activated kinase 1 (TAK1), via a negative feedback loop. We propose that the acute and constitutive activation of hepatic p38 during early fasting and in diabetic objects, respectively, antagonizes AMPK signaling under a reduced energy state, which results in either well-controlled gluconeogenesis during fasting or constitutively activated gluconeogenesis and hyperglycemia in diabetes. We also demonstrated that repression of hepatic p38 resulted in improved fasting hyperglycemia in diabetic mouse models, indicating that hepatic p38a represents an exciting pharmacological target for the treatment of hyperglycemia.

Materials and methods Animals were maintained and experiments were performed according to protocols approved by the Animal Care and Use Committees of INS, SIBS, CAS. Mice with a conditional p38a allele were generated as we described previously [22]. Mice with a targeted deletion of p38a in liver (LivKO) were generated by crossing the p38a flox/flox mice (Floxed) with transgenic mice expressing Cre-recombinase under the control of the albumin promoter (Alb-cre). Mice had free access to food and were routinely euthanized in a fed state during daytime or in a fasted state as specifically indicated. Other detailed information of materials and methods are provided as Supplementary Materials.

Results Effect of hepatic p38a on gluconeogenesis It has been reported that hepatic p38 is phosphorylated during fasting. Here we showed that the level of phospho-p38 reached a peak about 6–8 h after food deprivation and declined afterwards when fasting was prolonged (Fig. 1A and B). In contrast, hepatic gluconeogenic programme was activated during both short-term and long-term fasting as indicated by an elevation of glucose-6-phosphatase (G6Pase), phosphoenolpyruvate carboxykinase (PEPCK), and peroxisome proliferators-activated receptor-c coactivator-1a (PGC-1a) mRNA levels (Supplementary Fig. 1A). These data indicated that p38 might be critical for glucose homeostasis during short-term fasting. Since p38a is the major p38 isoform in the liver [3], we decided to explore the action of hepatic p38a in this study. 1320

Because p38a whole-body knockout mice are lethal, we generated a liver-specific knockout mouse by crossing p38a flox/flox mice [22] with Alb-Cre transgenic mice. Liver p38a knockout (p38aLivKO) mice were fertile, and appeared indistinguishable from their wild-type littermates (p38aFloxed). There was no significant difference in body weight and food intake between p38aLivKO mice and control mice (Supplementary Fig. 1B). The specificity of p38a knockout was confirmed, and the efficiency of p38a gene deletion in liver was estimated to be greater than 90% (Fig. 1C; Supplementary Fig. 1C). Interestingly, we found that the fasting glucose level was significantly decreased in both male and female fasted p38aLivKO mice (Fig. 1D; Supplementary Fig. 1D). In agreement with the mice phenotype, the hepatic gluconeogenesis was impaired in p38aLivKO mice as assessed by a pyruvate tolerance test (PTT) (Fig. 1E) [23]. In addition, we also found that PEPCK enzyme activity was decreased in the liver of fasted p38aLivKO mice (Supplementary Fig. 1E). To rule out the contribution of any developmental defects caused by p38a deletion, adenovirus-mediated overexpression of a dominantnegative form of p38a (Ad-p38a-AF) was used to suppress the activity of p38a in the liver (Fig. 1F). We found that the fasting glucose level was lower in mice infected with Ad-p38a-AF as compared to that in control group (Fig. 1G). PTT results suggested that the gluconeogenesis was impaired by Ad-p38a-AF (Fig. 1H). In contrast, when p38 signaling was activated by adenovirus for MKK6EE (Ad-MKK6EE), a constitutively active mitogen-activated protein kinase kinase 6 (MKK6) (Fig. 1I; Supplementary Fig. 1F), mice exhibited fasting hyperglycemia (Fig. 1J) and enhanced gluconeogenesis (Fig. 1K). In agreement with the mice phenotype and impaired gluconeogenesis, the mRNA expression of gluconeogenic G6Pase, PEPCK, and PGC-1a could not be elevated by fasting in mice lacking hepatic p38a (Fig. 2A). In contrast, in mice infected with Ad-MKK6EE, the constitutive activation of p38 resulted in the elevation of G6Pase, PEPCK, and PGC-1a mRNA expression even in a fed state (Fig. 2B). Although the further elevation by p38 activation was only obvious for G6Pase and PGC-1a during fasting, the constitutive increased mRNA expression of these three key players could contribute to the fasting hyperglycemia. To rule out the possibility that MKK6EE was able to regulate gluconeogenesis via signaling pathways other than p38a, p38aLivKO mice were infected with Ad-MKK6EE. As expected, the stimulatory effect of MKK6EE on G6Pase, PEPCK, and PGC-1a mRNA expression was lost in mice without hepatic p38a (Supplementary Fig. 2). In addition, the promoter activity of G6Pase was imaged with IVIS Lumina Imaging System in p38aLivKO mice and mice infected with Ad-MKK6EE in the fasted state [24]. As expected, the promoter activity of G6Pase was significantly suppressed in p38aLivKO mice (Fig. 2C). Conversely, the promoter activity of G6Pase was increased in the liver of mice infected with Ad-MKK6EE (Fig. 2D). The transcriptional regulation of gluconeogenic genes by p38a was then studied in vitro by using primary hepatocytes. Consistent with the observation in vivo, the elevation of G6Pase, PEPCK, and PGC-1a mRNA expression by glucagon was significantly repressed in primary hepatocytes derived from p38aLivKO mice (Fig. 2E). Similar results were obtained in wild-type hepatocytes infected with Ad-p38a-AF (Fig. 2F) and p38aFloxed mice-derived hepatocytes infected with Cre-recombinase adenovirus (Ad-Cre) (Fig. 2G). Luciferase assay by

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JOURNAL OF HEPATOLOGY Relative p-p38 level (normalized by p38α)

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min Fig. 1. Hepatic p38a is essential for glucose metabolism during fasting. (A) and (B) Phosphorylation status of hepatic p38 in mice fasted for 6, 8, 12, or 16 h as indicated. Glucose levels and pyruvate tolerance test (PTT) in male p38aLivKO (n = 15) and p38aFloxed mice (n = 18) (D), or mice infected with Ad-p38a-AF (n = 7) (G) or Ad-MKK6EE (n = 4) (J). (E), (H), and (K) The area under curve (AUC) for PTT was determined. Data are mean ± SEM. t test, ⁄p <0.05; ⁄⁄p <0.01. p38a protein levels were measured in the liver, muscle, and white adipose tissues (WAT) of p38aLivKO and p38aFloxed mice (C). The efficiency of the infection of Flag-tagged Ad-p38a-AF (F) and the hepatic activation of p38 in mice infected with Ad-MKK6EE (I) was evaluated by determining the levels of phospho-p38, total p38a and Flag-tagged p38a-AF. Representative data are shown.

using an adenoviral reporter containing G6Pase promoter revealed that glucagon-stimulated reporter activity was inhibited in hepatocytes from p38aLivKO mice, wild-type hepatocytes infected with Ad-p38a-AF, and p38aFloxed mice-derived hepatocytes infected with Ad-Cre, respectively (Fig. 2H). These data suggest that hepatic p38a controls the transcriptional regulation of G6Pase, PEPCK, and PGC-1a in a cell-autonomous manner. Regulation of AMPK by hepatic p38a Previously, p38 was shown to regulate gluconeogenesis through phosphorylation of CREB [7]. To our surprise, the phosphorylation of CREB was slightly increased rather than suppressed in fasted p38aLivKO mice (Fig. 3A). This unexpected finding prompted us

to explore new regulatory network pathways involved. Interestingly, we observed the elevated level of phosphoAMPKa in the liver of both p38aLivKO mice and mice infected with Ad-p38a-AF (Fig. 3A and B). In contrast, the level of phospho-AMPKa was significantly decreased in the liver of mice infected with Ad-MKK6EE (Fig. 3C). We also performed kinase assay to determine the AMPK activity in the liver of p38aLivKO mice and mice infected with Ad-p38a-AF. As expected, the elevation in AMPK activity was observed in these mice, further supporting that AMPK is activated when p38a is absent or repressed (Fig. 3D). Thus, we hypothesized that p38a might affect gluconeogenesis by antagonizing AMPK. To test our hypothesis, we inactivated AMPK signaling by using adenovirus containing a dominant-negative form of

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Fig. 2. Hepatic p38a regulates the expression of gluconeogenic genes. The mRNA expression of gluconeogenic genes in p38aLivKO (fed group, n = 8; fasted group, n = 8) and p38aFloxed mice (fed group, n = 5; fasted group, n = 6) (A) or mice infected with Ad-MKK6EE (fed group, n = 11; fasted group, n = 12) or control virus (fed group, n = 4; fasted group, n = 11) (B). Ad-G6Pase-luc activity imaged and quantified in p38aLivKO and p38aFloxed mice (C) or mice infected with Ad-MKK6EE (D) in the fasted state. (n = 3–6). The mRNA expression of gluconeogenic genes in primary hepatocytes from p38aLivKO (KO) and p38aFloxed (f/f) mice (E) (n = 4) or wild-type primary hepatocytes infected with Ad-p38a-AF (F) (n = 4), or primary p38aFloxed mice hepatocytes (f/f) infected with Ad-Cre or Ad-GFP (G) (n = 4). (H) Ad-G6Pase-luc activity in primary KO and f/f hepatocytes, or p38a-AF infected hepatocytes, and Ad-Cre infected f/f hepatocytes that were exposed to glucagon (GL) or vehicle (VEH) (n = 3–6). Data are mean ± SEM. t test, *p <0.05; **p <0.01; ***p <0.001. (This figure appears in colour on the web.)

AMPKa (Ad-DN-AMPKa). Consistent with the results in Fig. 1G, Ad-p38a-AF infection reduced the fasting glucose level in mice (Fig. 3E). This phenotype induced by Ad-p38a-AF was fully rescued by additional Ad-DN-AMPKa infection (Fig. 3E). Consistent with the finding in vivo, Ad-p38a-AF impaired the promoter activity of G6Pase, while Ad-DN-AMPKa could abolish the inhibitory effect of Ad-p38a-AF in vitro (Fig. 3F). Moreover, Ad-DN-AMPKa could attenuate the repression of G6Pase and PEPCK mRNA expression by Ad-p38a-AF and SB203580 in primary hepatocytes, respectively (Fig. 3G; Supplementary Fig. 3A). Similar results were obtained when Compound C (AMPK inhibitor) was used (Supplementary Fig. 3B). In agreement with these data, Ad-DNAMPKa could diminish the inhibitory effect of Ad-p38a-AF on glucose production in primary hepatocytes (Fig. 3H). These results suggested that p38a is located upstream of AMPK signaling and AMPK mediates the regulatory role of p38a in hepatic glucose metabolism. TAK1 is known to be an upstream kinase of p38 and could be suppressed by p38 through a negative feedback loop [25,26]. Recently, TAK1 has been shown to be an upstream regulator of AMPK [27–29]. We hypothesized that p38 might be able to inhibit

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AMPK activity through repressing TAK1 in hepatocytes. To test our hypothesis, we checked the TAK1 activity in p38aLivKO mice. As we expected, the phosphorylation level of MKK3/6, which are TAK1 downstream kinases, was significantly higher in p38aLivKO mice as compared to p38aFloxed mice (Fig. 3I). Consistently, the hepatic TAK1 activity was increased in the liver of p38aLivKO mice as compared to that in p38aFloxed mice (Fig. 3J). Similar results were obtained in mice infected with Adp38a-AF (Fig. 3K and L). In addition, knockdown of TAK1 in primary hepatocytes and hepatocyte cell line by lentiviral shRNA and siRNA, respectively, could effectively suppress the phosphorylation of AMPKa induced by Ad-p38a-AF (Fig. 3M; Supplementary Fig. 3C). Moreover, knockdown of TAK1 diminished the repressive effect of Ad-p38a-AF on the mRNA expression of gluconeogenic genes (Fig. 3N). Consistently, inhibition of TAK1 attenuated the inhibitory effect of Ad-p38a-AF on glucose production in primary hepatocytes (Fig. 3O). These data indicate that p38a has the capacity to antagonize AMPK signaling through TAK1 via a negative feedback circuit and loss of hepatic p38a results in decreased gluconeogenesis and lowered fasting glucose level (Fig. 3P).

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Fig. 3. AMPK mediates the regulatory role of p38a in hepatic glucose metabolism. Immunoblot of hepatic phospho-AMPKa protein in p38aLivKO and p38aFloxed mice (A) (n = 3), mice infected with Ad-p38a-AF (B) (n = 4) or Ad-MKK6EE (C) (n = 3) in the fasted state. Phosphorylation level of CREB was determined in p38aLivKO mice (A). Relative phospho-AMPKa and phospho-CREB levels were normalized. (D) The AMPK activity in the liver of p38aLivKO mice and mice infected with Ad-p38a-AF was analyzed in vitro, respectively. AMPK activity was represented by relative phospho-ACC1 levels vs. total His-ACC1 levels (n = 3). (E) Fasting glucose levels in mice infected with Ad-p38a-AF or both Ad-p38a-AF and Ad-DN-AMPKa (n = 5–6). Ad-G6Pase-luc activity (F) (n = 3), G6Pase and PEPCK mRNA expression (G) (n = 4), and glucose production (H) (n = 4) in primary hepatocytes infected with Ad-p38a-AF or both Ad-p38a-AF and Ad-DN-AMPKa. Hepatocytes were exposed to glucagon (GL) or vehicle (VEH). Immunoblot of hepatic phospho-MKK3/6 protein (TAK1 downstream kinase) (I) (K) (n = 3–4) and in vitro assay for TAK1 activity (J) (L) (n = 3–4) in p38aLivKO and p38aFloxed mice or mice infected with Ad-p38a-AF in the fasted state. Relative phospho-MKK3/6 levels were normalized. TAK1 activity was represented by relative phospho-MKK3/6 levels vs. total His-MKK6 levels. Effect of TAK1 knockdown on the AMPKa phosphorylation (M), mRNA expression of gluconeogenic genes (N) (n = 3) and glucose production (O) (n = 3) in primary hepatocytes infected with Ad-p38a-AF. The data are representative of at least three experiments. Data are mean ± SEM. t test, ⁄ p <0.05; ⁄⁄p <0.01, ⁄⁄⁄p <0.001. (P) The schematic representation for the negative feedback circuit discovered in this study. (This figure appears in colour on the web.)

Phosphorylation status of p38 and AMPKa in response to metabolic stresses During fasting, reduced energy state and elevated AMP/ATP ratio (Supplementary Fig. 4A) in the liver are supposed to activate AMPK, which has inhibitory effect on gluconeogenesis. In order to turn on gluconeogenesis effectively to fit the whole-body glucose requirement during fasting, AMPK activity should be tightly controlled. Indeed, we observed a reduction of AMPKa phosphorylation during fasting (6–8 h), which was inversely

correlated with p38 phosphorylation (Fig. 4A and B). Since we revealed that p38a is able to suppress AMPKa phosphorylation, we proposed that acute activation of p38a during early fasting maintains AMPK signaling in a less-activated status to facilitate gluconeogenesis. Interestingly, hepatic energy state was also reduced in diabetic db/db, ob/ob, and high-fat diet (HFD)-fed mice (Supplementary Fig. 4B–D). Hepatic phospho-p38 level was increased, while phosphorylation level of AMPKa was decreased in these diabetic mouse models (Fig. 4C; Supplementary Fig. 4E and F), suggesting

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B Relative p-p38 level (normalized by p38α)

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Research Article

Fig. 4. The phosphorylation status of hepatic p38 and under reduced energy state. (A) Immunoblot of phospho-p38 and phospho-AMPKa in the liver of fasted mice. (B) Relative phosphorylation levels of p38 and AMPKa were normalized and plotted against fasting time. (C) Hepatic phospho-p38, phospho-MKK3/6, and phospho-AMPKa protein levels in db/db mice (n = 3). Relative phospho-p38 and phospho-AMPKa levels were quantified and plotted. (D) Schematic representation for the role of hepatic p38a in gluconeogenesis in response to metabolic stress under physiological and pathological condition. (E–H) Fasting blood glucose levels were decreased in db/db mice infected with Ad-p38a-AF (E) (n = 5–6) or HFHSD-fed mice infected with Ad-p38a-AF (G) (n = 8–9) as compared to control mice. Hepatic phospho-p38 and phospho-AMPKa protein levels in db/db (F) and HFHSD-fed (H) mice were determined. (I) Fasting glucose levels in HFHSD-fed p38aLivKO and p38aFloxed mice (n = 14). (J) PTT in HFHSD-fed p38aLivKO and p38aFloxed mice (n = 4–5). The AUC for PTT was determined. (K) Relative mRNA expression of gluconeogenic genes in the liver of HFHSD-fed p38aLivKO and p38aFloxed mice (n = 5–8). Representative results are shown. Data are mean ± SEM. t test, ⁄p <0.05; ⁄⁄p <0.01, ⁄⁄⁄p <0.001. (This figure appears in colour on the web.)

that chronic activation of p38 might contribute to the suppressed AMPK signaling, constitutively activated gluconeogenesis, and hyperglycemia in diabetes. As expected, the phosphorylation level of MKK3/6 was reduced in db/db mice due to the feedback loop reaction (Fig. 4C). To rule out the possibility that p38a affects AMPK action through changing energy state, energy charge was determined in fed and fasted p38aLivKO and p38aFloxed mice. As expected, deficiency in hepatic p38a did not change energy charge (Supplementary Fig. 4G). Taken together, we proposed that hepatic p38a plays a crucial regulatory role in glucose homeostasis both under physiological and pathological conditions (Fig. 4D): 1) during early fasting, acute activation of hepatic p38a by glucagon signaling inhibits the phosphorylation of AMPKa, thereby inhibiting the suppressive effect of AMPK on gluconeogenesis and maintaining the blood glucose level in a proper range; and 2) in diabetic subjects, the chronic activation

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of hepatic p38a suppresses the phosphorylation of AMPKa, resulting in unrestrained gluconeogenesis. Repression of p38a ameliorates hyperglycemia in diabetic mice To determine whether repression of p38a activity in the livers of diabetic mice would elevate the phosphorylation level of AMPKa and reduce blood glucose levels, db/db mice, high-fat high-sucrose diet (HFHSD)-fed mice, and HFD-fed mice were infected with Adp38a-AF through tail vein injection, respectively (Fig. 4E–H; Supplementary Fig. 4H–K). As we expected, the fasting blood glucose levels were significantly reduced in the Ad-p38a-AF-infected db/db mice, HFHSD-fed mice, or HFD-fed mice compared with control mice (Fig. 4E, G, and Supplementary Fig. 4J). Moreover, the phosphorylation level of AMPKa was found to be increased after Ad-p38a-AF infection (Fig. 4F, H; Supplementary Fig. 4K).

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JOURNAL OF HEPATOLOGY In agreement with our proposed model, Ad-p38a-AF infection reduced the gluconeogenesis in HFHSD-fed mice as shown by PTT and qRT-PCR analysis of the mRNA expression of gluconeogenic genes (Supplementary Fig. 4H and I). In addition, we also tested whether mice deficient in hepatic p38a were resistant to streptozotocin (STZ)-induced diabetes and HFHSD-induced hyperglycemia (Fig. 4I–K; Supplementary Fig. 4L and N). As expected, the degree of STZ-induced or HFHSD-induced hyperglycemia was less severe in p38aLivKO mice as compared to the control mice (Fig. 4I; Supplementary Fig. 4L). Consistent with our proposed mechanisms, the elevated levels of phospho-AMPKa were observed in the liver of these mice lacking hepatic p38a (Supplementary Fig. 4M and N). As expected, we observed a decrease of gluconeogenesis in HFHSD-fed p38aLivKO mice as shown by PTT and gluconeogenic gene expression analysis (Fig. 4J and K). These results suggest that inhibition of hepatic p38a could be a new therapeutic approach to treat diabetes by suppressing gluconeogenesis and ameliorating hyperglycemia.

Discussion The prevalence of type 2 diabetes mellitus has reached epidemic proportions. Dysregulation of gluconeogenesis is one of the major contributing factors of type 2 diabetes. Understanding the molecular basis underlying hepatic gluconeogenesis is crucial for better management of type 2 diabetes. At present, it is still unclear how multiple signaling pathways are coordinated to regulate the activity of key transcription factors and co-regulators in gluconeogenesis. In this study, we took advantage of conditional liver-specific p38a knockout mice to delineate the role of hepatic p38a in glucose homeostasis. Our study demonstrates that hepatic p38 is a pivotal regulator of hepatic gluconeogenesis. During early fasting, a fall in energy state is likely to activate AMPK which has adverse effects on glucose production. However, acute activation of p38a by glucagon is able to attenuate AMPKa phosphorylation through TAK1 via a negative feedback loop, thereby promoting gluconeogenesis to ensure a sufficient supply of energy. We propose that hepatic p38a might functions as a gatekeeper of AMPK signaling in response to metabolic stress such as fasting which will lead to a reduced energy state. A previous study demonstrated that p38 stimulates hepatic gluconeogenesis by regulating the phosphorylation of CREB [7]. However, we did not detect any defect in the activation of CREB during fasting in p38aLivKO mice, suggesting that p38 signaling might not be required for the activation of CREB during fasting. This unexpected result prompted us to find other factors mediating the action of p38 in controlling gluconeogenesis. Then we found the phospho-AMPKa levels were abnormally elevated in the liver of p38aLivKO mice, which might contribute to the fasting hypoglycemia. Moreover, we discovered that deficiency of p38 resulted in the constitutive activation of TAK1, which in turn could increase AMPKa phosphorylation. Thus, our study reveals a novel regulatory network of gluconeogenesis, in which p38 signaling crosstalks with AMPK signaling through upstream kinase TAK1. AMPK has been shown as a significant inhibitor of gluconeogenesis. However, in response to food deprivation, there is a fall in energy state, which results in a net loss of ATP and an elevation in AMP/ATP ratio. Since increased AMP/ATP ratio will

directly stimulate AMPK activity, which has adverse effect on glucose production, there must be a sophisticated regulatory network to control AMPK activity in this setting. Here, we provide in vivo and in vitro evidence that p38a is a negative regulator of AMPK signaling and is able to inhibit AMPKa phosphorylation. Our study suggest that during early fasting, in order to ensure a sufficient supply of energy, acute activation of p38a is critical for counteracting the suppressive effect of AMPK on gluconeogenesis. Thus, we propose that hepatic p38a functions as a gatekeeper of AMPK signaling for maintain proper hepatic glucose production in response to metabolic stresses. To be noted, our findings of reduced AMPKa phosphorylation during fasting could also explain why mice lacking hepatic AMPKa exhibit normal fasting glucose level [10]. Since mice lacking hepatic AMPKa and mice with reduced AMPKa phosphorylation due to fasting were compared in the experiment, it would be difficult to detect any changes in fasting glucose level. Hepatic glucose production is an important driver of hyperglycemia. The model we proposed in the current study is able to explain the excessive gluconeogenesis and inactivation of AMPK in diabetic subjects which also face metabolic stress and reduced energy state. Attenuated hepatic energy state has been reported in diabetic mice, suggesting that high AMP/ATP levels are part of the metabolic disease milieu. Moreover, reduced hepatic ATP levels have been observed in obese humans. In this study, we found that AMPKa phosphorylation is inhibited, while p38 signaling is activated in the liver of various diabetic models. We speculate that the inappropriately activated hepatic p38 will suppress hepatic AMPK activity in a cell-autonomous manner and lead to dysregulation of hepatic gluconeogenesis, thereby contributing to the development of type 2 diabetes. In this study, we also provided evidence that inhibition of hepatic p38a could be a new therapeutic approach to manage diabetes by suppressing gluconeogenesis and ameliorating hyperglycemia. Regarding the mechanisms of p38 activation, it is known that glucagon is able to activate p38 in hepatocytes. However the underlying molecular mechanisms remain not completely understood. Glucagon binds to the glucagon receptor, which leads to subsequent activation of the coupled G proteins. Gsa and Gq are two classes of G proteins involved in the signal transduction of the glucagon receptor. The activation of Gsa results in activation of adenylate cyclase, which will increase cAMP levels and subsequently activate protein kinase A (PKA). In contrast, the activation of Gq leads to the activation of phospholipase C (PLC), which will finally cause intracellular calcium release [30]. Additionally, cAMP may increase calcium mobilization through the PKA-dependent phosphorylation of the intracellular calcium channel [31]. Thus, PKA, PLC, and calcium signaling are thought to be major mediators for glucagon action. Indeed, efforts have been made to distinguish which pathway mediates the glucagon-induced p38 activation. One published paper suggested that PKA is not required for p38 activation by glucagon [32]. In contrast, a recent paper indicated that a calcium-sensing enzyme, calcium calmodulin-dependent kinase II (CaMKII), might be involved, but its activation by glucagon requires PKA [10]. Therefore, how glucagon activates calcium mobilization and calcium signaling activates p38 signaling are still not very clear and require further exploration. Regarding the relationship between energy state and p38, we found that p38a deficiency in the liver did not change energy charge, indicating that p38a affects AMPK action not through changing energy state (Supplementary

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Research Article Fig. 4G). However, whether the alteration of energy state affects the p38 signaling is not known and requires further study. In summary, our study using genetically modified animal models strongly suggests that p38 is critical in the control of glucose homeostasis in response to metabolic stress. Our study reveals a novel regulatory network of gluconeogenesis, in which p38a signaling crosstalk with AMPK signaling through upstream kinase TAK1. Our data also suggest that excessive activation of hepatic p38a in obesity and diabetes might contribute to the unrestrained gluconeogenesis and pathogenesis of diabetes, and repression of hepatic p38a might be therapeutic.

Financial support This work was supported by grants from the Ministry of Science and Technology of China (2010CB912500, 2011CB944104, 2010CB945101, and 2012BAK01B00), the National Natural Science Foundation (31371189, 31070679, 31100550, 81172009, 81372168), Shanghai Institutes for Biological Sciences, Chinese Academy of Science – China (SIBS2012004), CAS/SAFEA International Partnership Program for Creative Research Teams, and Xuhui Central Hospital (Shanghai, China).

Conflict of interest The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.

Author contributions Y.J., W.L., L.H., and H.Y. did the literature research and designed the experiments. Y.J., W.L., H.C., S.Z., H.X., and D.L. carried out the experimental work. Y.J., W.L., D.Z., and H.Y. performed data analysis and interpreted the data. X.Y., Y.W., J.Y., L.H., and H.Y. supervised the project and contributed to manuscript preparation and editing. Y.J. and W.L. contributed equally to this work. Acknowledgements We would like to thank Dr. Yi Liu, Dr. Yong Liu, Dr. Jia Li, Dr. Qiwei Zhai, Dr. Baoliang Song, Dr. Huiyong Yin, and Dr. Xiaolong Liu (SIBS, CAS) for the technical support and constructive suggestions. This work was supported by grants from the Ministry of Science and Technology of China (2010CB912500, 2011CB944104, 2010CB945101, and 2012BAK01B00), the National Natural Science Foundation (31371189, 31070679, 31100550, 81172009, 81372168), Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (SIBS2012004), CAS/SAFEA International Partnership Program for Creative Research Teams, and Xuhui Central Hospital (Shanghai, China).

Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jhep.2014.12. 032. 1326

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