O-GlcNAcylation Increases ChREBP Protein Content and Transcriptional Activity in the Liver

International audienceOBJECTIVE Carbohydrate-responsive element–binding protein (ChREBP) is a key transcription factor that mediates the effects of glucose on glycolytic and lipogenic genes in the liver. We have previously reported that liver-specific inhibition of ChREBP prevents hepatic steatosis in ob/ob mice by specifically decreasing lipogenic rates in vivo. To better understand the regulation of ChREBP activity in the liver, we investigated the implication of O-linked β-N-acetylglucosamine (O-GlcNAc or O-GlcNAcylation), an important glucose-dependent posttranslational modification playing multiple roles in transcription, protein stabilization, nuclear localization, and signal transduction. RESEARCH DESIGN AND METHODS O-GlcNAcylation is highly dynamic through the action of two enzymes: the O-GlcNAc transferase (OGT), which transfers the monosaccharide to serine/threonine residues on a target protein, and the O-GlcNAcase (OGA), which hydrolyses the sugar. To modulate ChREBPOG in vitro and in vivo, the OGT and OGA enzymes were overexpressed or inhibited via adenoviral approaches in mouse hepatocytes and in the liver of C57BL/6J or obese db/db mice. RESULTS Our study shows that ChREBP interacts with OGT and is subjected to O-GlcNAcylation in liver cells. O-GlcNAcylation stabilizes the ChREBP protein and increases its transcriptional activity toward its target glycolytic (L-PK) and lipogenic genes (ACC, FAS, and SCD1) when combined with an active glucose flux in vivo. Indeed, OGT overexpression significantly increased ChREBPOG in liver nuclear extracts from fed C57BL/6J mice, leading in turn to enhanced lipogenic gene expression and to excessive hepatic triglyceride deposition. In the livers of hyperglycemic obese db/db mice, ChREBPOG levels were elevated compared with controls. Interestingly, reducing ChREBPOG levels via OGA overexpression decreased lipogenic protein content (ACC, FAS), prevented hepatic steatosis, and improved the lipidic profile of OGA-treated db/db mice. CONCLUSIONS Taken together, our results reveal that O-GlcNAcylation represents an important novel regulation of ChREBP activity in the liver under both physiological and pathophysiological conditions

Cop1 constitutively regulates c-Jun protein stability and functions as a tumor suppressor in mice

Biochemical studies have suggested conflicting roles for the E3 ubiquitin ligase constitutive photomorphogenesis protein 1 (Cop 1; also known as Rfwd2) in tumorigenesis, providing evidence for both the oncoprotein c-Jun and the tumor suppressor p53 as its targets. Here we present what we believe to be the first in vivo investigation of the role of Cop1 in cancer etiology. Using an innovative genetic approach to generate an allelic series of Cop1, we found that Cop1 hypomorphic mice spontaneously developed malignancy at a high frequency in the first year of life and were highly susceptible to radiation-induced lymphomagenesis. Further analysis revealed that c-Jun was a key physiological target for Cop1 and that Cop1 constitutively kept c-Jun at low levels in vivo and thereby modulated c-Jun/AP-1 transcriptional activity. Importantly, Cop1 deficiency stimulated cell proliferation in a c-Jun-dependent manner. Focal deletions of COP1 were observed at significant frequency across several cancer types, and COP1 loss was determined to be one of the mechanisms leading to c-Jun upregulation in human cancer. We therefore conclude that Cop1 is a tumor suppressor that functions, at least in part, by antagonizing c-Jun oncogenic activity. In the absence of evidence for a genetic interaction between Cop1 and p53, our data strongly argue against the use of Cop1-inhibitory drugs for cancer therapy

Adipocyte CREB Promotes Insulin Resistance in Obesity

SummaryIncreases in adiposity trigger metabolic and inflammatory changes that interfere with insulin action in peripheral tissues, culminating in beta cell failure and overt diabetes. We found that the cAMP Response Element Binding protein (CREB) is activated in adipose cells under obese conditions, where it promotes insulin resistance by triggering expression of the transcriptional repressor ATF3 and thereby downregulating expression of the adipokine hormone adiponectin as well as the insulin-sensitive glucose transporter 4 (GLUT4). Transgenic mice expressing a dominant-negative CREB transgene in adipocytes displayed increased whole-body insulin sensitivity in the contexts of diet-induced and genetic obesity, and they were protected from the development of hepatic steatosis and adipose tissue inflammation. These results indicate that adipocyte CREB provides an early signal in the progression to type 2 diabetes

The LKB1-salt-inducible kinase pathway functions as a key gluconeogenic suppressor in the liver

LKB1 is a master kinase that regulates metabolism and growth through adenosine monophosphate-activated protein kinase (AMPK) and 12 other closely related kinases. Liver-specific ablation of LKB1 causes increased glucose production in hepatocytes in vitro and hyperglycaemia in fasting mice in vivo. Here we report that the salt-inducible kinases (SIK1, 2 and 3), members of the AMPK-related kinase family, play a key role as gluconeogenic suppressors downstream of LKB1 in the liver. The selective SIK inhibitor HG-9-91-01 promotes dephosphorylation of transcriptional co-activators CRTC2/3 resulting in enhanced gluconeogenic gene expression and glucose production in hepatocytes, an effect that is abolished when an HG-9-91-01-insensitive mutant SIK is introduced or LKB1 is ablated. Although SIK2 was proposed as a key regulator of insulin-mediated suppression of gluconeogenesis, we provide genetic evidence that liver-specific ablation of SIK2 alone has no effect on gluconeogenesis and insulin does not modulate SIK2 phosphorylation or activity. Collectively, we demonstrate that the LKB1-SIK pathway functions as a key gluconeogenic gatekeeper in the liver

Metformin inhibits hepatic gluconeogenesis in mice independently of the LKB1/AMPK pathway via a decrease in hepatic energy state.

pages 1-15International audienceMetformin is widely used to treat hyperglycemia in individuals with type 2 diabetes. Recently the LKB1/AMP-activated protein kinase (LKB1/AMPK) pathway was proposed to mediate the action of metformin on hepatic gluconeogenesis. However, the molecular mechanism by which this pathway operates had remained elusive. Surprisingly, here we have found that in mice lacking AMPK in the liver, blood glucose levels were comparable to those in wild-type mice, and the hypoglycemic effect of metformin was maintained. Hepatocytes lacking AMPK displayed normal glucose production and gluconeogenic gene expression compared with wild-type hepatocytes. In contrast, gluconeogenesis was upregulated in LKB1-deficient hepatocytes. Metformin decreased expression of the gene encoding the catalytic subunit of glucose-6-phosphatase (G6Pase), while cytosolic phosphoenolpyruvate carboxykinase (Pepck) gene expression was unaffected in wild-type, AMPK-deficient, and LKB1-deficient hepatocytes. Surprisingly, metformin-induced inhibition of glucose production was amplified in both AMPK- and LKB1-deficient compared with wild-type hepatocytes. This inhibition correlated in a dose-dependent manner with a reduction in intracellular ATP content, which is crucial for glucose production. Moreover, metformin-induced inhibition of glucose production was preserved under forced expression of gluconeogenic genes through PPARgamma coactivator 1alpha (PGC-1alpha) overexpression, indicating that metformin suppresses gluconeogenesis via a transcription-independent process. In conclusion, we demonstrate that metformin inhibits hepatic gluconeogenesis in an LKB1- and AMPK-independent manner via a decrease in hepatic energy state

SIK2 regulates CRTCs, HDAC4 and glucose uptake in adipocytes

Salt-inducible kinase 2 (SIK2) is an AMP-activated protein kinase (AMPK) related kinase abundantly expressed in adipose tissue. Our aim was to identify molecular targets and functions of SIK2 in adipocytes, and to address the role of PKA-mediated phosphorylation of SIK2 on Ser358. Modulation of SIK2 in adipocytes resulted in altered phosphorylation of CREB-regulated transcription co-activator 2 (CRTC2), CRTC3 and class IIa histone deacetylase 4 (HDAC4). Furthermore, CRTC2, CRTC3, HDAC4 and protein phosphatase 2A (PP2A) interacted with SIK2, and the binding of CRTCs and PP2A to wild-type but not Ser358Ala SIK2, was reduced by cAMP elevation. Silencing of SIK2 resulted in reduced GLUT4 (also known as SLC2A4) protein levels, whereas cells treated with CRTC2 or HDAC4 siRNA displayed increased levels of GLUT4. Overexpression or pharmacological inhibition of SIK2 resulted in increased and decreased glucose uptake, respectively. We also describe a SIK2–CRTC2–HDAC4 pathway and its regulation in human adipocytes, strengthening the physiological relevance of our findings. Collectively, we demonstrate that SIK2 acts directly on CRTC2, CRTC3 and HDAC4, and that the cAMP–PKA pathway reduces the interaction of SIK2 with CRTCs and PP2A. Downstream, SIK2 increases GLUT4 levels and glucose uptake in adipocytes

Glycerol-3-Phosphate Acyltransferase 1 Deficiency in ob/ob Mice Diminishes Hepatic Steatosis but Does Not Protect Against Insulin Resistance or Obesity

OBJECTIVEHepatic steatosis is strongly associated with insulin resistance, but a causal role has not been established. In ob/ob mice, sterol regulatory element binding protein 1 (SREBP1) mediates the induction of steatosis by upregulating target genes, including glycerol-3-phosphate acyltransferase-1 (Gpat1), which catalyzes the first and committed step in the pathway of glycerolipid synthesis. We asked whether ob/ob mice lacking Gpat1 would have reduced hepatic steatosis and improved insulin sensitivity.RESEARCH DESIGN AND METHODSHepatic lipids, insulin sensitivity, and hepatic insulin signaling were compared in lean (Lep+/?), lean-Gpat1−/−, ob/ob (Lepob/ob), and ob/ob-Gpat1−/− mice.RESULTSCompared with ob/ob mice, the lack of Gpat1 in ob/ob mice reduced hepatic triacylglycerol (TAG) and diacylglycerol (DAG) content 59 and 74%, respectively, but increased acyl-CoA levels. Despite the reduction in hepatic lipids, fasting glucose and insulin concentrations did not improve, and insulin tolerance remained impaired. In both ob/ob and ob/ob-Gpat1−/− mice, insulin resistance was accompanied by elevated hepatic protein kinase C-ε activation and blunted insulin-stimulated Akt activation.CONCLUSIONSThese results suggest that decreasing hepatic steatosis alone does not improve insulin resistance, and that factors other than increased hepatic DAG and TAG contribute to hepatic insulin resistance in this genetically obese model. They also show that the SREBP1-mediated induction of hepatic steatosis in ob/ob mice requires Gpat1

Paradoxical Regulation of Human FGF21 by Both Fasting and Feeding Signals: Is FGF21 a Nutritional Adaptation Factor?

Fibroblast growth factor 21 (FGF21) has recently emerged as a metabolic hormone involved in regulating glucose and lipid metabolism in mouse, but the regulatory mechanisms and actions of FGF21 in humans remain unclear. Here we have investigated the regulatory mechanisms of the human FGF21 gene at the transcriptional level. A deletion study of the human FGF21 promoter (−1672 to +230 bp) revealed two fasting signals, including peroxisome proliferator-activated receptor α (PPARα) and glucagon signals, that independently induced human FGF21 gene transcription in mouse primary hepatocytes. In addition, two feeding signals, glucose and xylitol, also dose-dependently induced human FGF21 gene transcription and mRNA expression in both human HepG2 cells and mouse primary hepatocytes. FGF21 protein expression and secretion were also induced by high glucose stimulation. The human FGF21 promoter (−1672 to +230 bp) was found to have a carbohydrate-responsive element at −380 to −366 bp, which is distinct from the PPAR response element (PPRE). Knock-down of the carbohydrate response element binding protein by RNAi diminished glucose-induced human FGF21 transcription. Moreover, we found that a region from −555 to −443 bp of the human FGF21 promoter region exerts an important role in the activation of basic transcription. In conclusion, human FGF21 gene expression is paradoxically and independently regulated by both fasting and feeding signals. These regulatory mechanisms suggest that human FGF21 is increased with nutritional crisis, including starvation and overfeeding