Insulin resistance per se drives early and reversible dysbiosis-mediated gut barrier impairment and bactericidal dysfunction.

OBJECTIVE: A common feature of metabolic diseases is their association with chronic low-grade inflammation. While enhanced gut permeability and systemic bacterial endotoxin translocation have been suggested as key players of this metaflammation, the mechanistic bases underlying these features upon the diabesity cascade remain partly understood. METHODS: Here, we show in mice that, independently of obesity, the induction of acute and global insulin resistance and associated hyperglycemia, upon treatment with an insulin receptor (IR) antagonist (S961), elicits gut hyperpermeability without triggering systemic inflammatory response. RESULTS: Of note, S961-treated diabetic mice display major defects of gut barrier epithelial functions, such as increased epithelial paracellular permeability and impaired cell-cell junction integrity. We also observed in these mice the early onset of a severe gut dysbiosis, as characterized by the bloom of pro-inflammatory Proteobacteria, and the later collapse of Paneth cells antimicrobial defense. Interestingly, S961 treatment discontinuation is sufficient to promptly restore both the gut microbial balance and the intestinal barrier integrity. Moreover, fecal transplant approaches further confirm that S961-mediated dybiosis contributes at least partly to the disruption of the gut selective epithelial permeability upon diabetic states. CONCLUSIONS: Together, our results highlight that insulin signaling is an indispensable gatekeeper of intestinal barrier integrity, acting as a safeguard against microbial imbalance and acute infections by enteropathogens.

LKB1 as a Gatekeeper of Hepatocyte Proliferation and Genomic Integrity during Liver Regeneration.

Liver kinase B1 (LKB1) is involved in several biological processes and is a key regulator of hepatic metabolism and polarity. Here, we demonstrate that the master kinase LKB1 plays a dual role in liver regeneration, independently of its major target, AMP-activated protein kinase (AMPK). We found that the loss of hepatic Lkb1 expression promoted hepatocyte proliferation acceleration independently of metabolic/energetic balance. LKB1 regulates G0/G1 progression, specifically by controlling epidermal growth factor receptor (EGFR) signaling. Furthermore, later in regeneration, LKB1 controls mitotic fidelity. The deletion of Lkb1 results in major alterations to mitotic spindle formation along the polarity axis. Thus, LKB1 deficiency alters ploidy profile at late stages of regeneration. Our findings highlight the dual role of LKB1 in liver regeneration, as a guardian of hepatocyte proliferation and genomic integrity.

A Specific ChREBP and PPARα Cross-Talk Is Required for the Glucose-Mediated FGF21 Response.

While the physiological benefits of the fibroblast growth factor 21 (FGF21) hepatokine are documented in response to fasting, little information is available on Fgf21 regulation in a glucose-overload context. We report that peroxisome-proliferator-activated receptor α (PPARα), a nuclear receptor of the fasting response, is required with the carbohydrate-sensitive transcription factor carbohydrate-responsive element-binding protein (ChREBP) to balance FGF21 glucose response. Microarray analysis indicated that only a few hepatic genes respond to fasting and glucose similarly to Fgf21. Glucose-challenged Chrebp-/- mice exhibit a marked reduction in FGF21 production, a decrease that was rescued by re-expression of an active ChREBP isoform in the liver of Chrebp-/- mice. Unexpectedly, carbohydrate challenge of hepatic Pparα knockout mice also demonstrated a PPARα-dependent glucose response for Fgf21 that was associated with an increased sucrose preference. This blunted response was due to decreased Fgf21 promoter accessibility and diminished ChREBP binding onto Fgf21 carbohydrate-responsive element (ChoRE) in hepatocytes lacking PPARα. Our study reports that PPARα is required for the ChREBP-induced glucose response of FGF21.

Alternative splicing of hepatitis B virus: A novel virus/host interaction altering liver immunity.

BACKGROUND & AIMS: Hepatitis B virus (HBV) RNA can undergo alternative splicing, but the relevance of this post-transcriptional regulation remains elusive. The mechanism of HBV alternative splicing regulation and its impact on liver pathogenesis were investigated. METHODS: HBV RNA-interacting proteins were identified by RNA pull-down, combined with mass spectrometry analysis. HBV splicing regulation was investigated in chemically and surgically induced liver damage, in whole HBV genome transgenic mice and in hepatoma cells. Viral and endogenous gene expression were quantified by quantitative reverse transcription polymerase chain reaction, Western blot and enzyme-linked immunosorbent assay. Resident liver immune cells were studied by fluorescence-activated cell sorting. RESULTS: HBV pregenomic RNA-interacting proteins were identified and 15% were directly related to the splicing machinery. Expression of these splicing factors was modulated in HBV transgenic mice with liver injuries and contributed to an increase of the HBV spliced RNA encoding for HBV splicing-generated protein (HBSP). HBSP transgenic mice with chemically induced liver fibrosis exhibited attenuated hepatic damage. The protective effect of HBSP resulted from a decrease of inflammatory monocyte/macrophage recruitment through downregulation of C-C motif chemokine ligand 2 (CCL2) expression in hepatocytes. In human hepatoma cells, the ability of HBSP to control CCL2 expression was confirmed and maintained in a whole HBV context. Finally, viral spliced RNA detection related to a decrease of CCL2 expression in the livers of HBV chronic carriers underscored this mechanism. CONCLUSION: The microenvironment, modified by liver injury, increased HBSP RNA expression through splicing factor regulation, which in turn controlled hepatocyte chemokine synthesis. This feedback mechanism provides a novel insight into liver immunopathogenesis during HBV infection. Lay summary: Hepatitis B virus persists for decades in the liver of chronically infected patients. Immune escape is one of the main mechanisms developed by this virus to survive. Our study highlights how the crosstalk between virus and liver infected cells may contribute to this immune escape.

Growth factor receptor binding protein 14 inhibition triggers insulin-induced mouse hepatocyte proliferation and is associated with hepatocellular carcinoma.

Metabolic diseases such as obesity and type 2 diabetes are recognized as independent risk factors for hepatocellular carcinoma (HCC). Hyperinsulinemia, a hallmark of these pathologies, is suspected to be involved in HCC development. The molecular adapter growth factor receptor binding protein 14 (Grb14) is an inhibitor of insulin receptor catalytic activity, highly expressed in the liver. To study its involvement in hepatocyte proliferation, we specifically inhibited its liver expression using a short hairpin RNA strategy in mice. Enhanced insulin signaling upon Grb14 inhibition was accompanied by a transient induction of S-phase entrance by quiescent hepatocytes, indicating that Grb14 is a potent repressor of cell division. The proliferation of Grb14-deficient hepatocytes was cell-autonomous as it was also observed in primary cell cultures. Combined Grb14 down-regulation and insulin signaling blockade using pharmacological approaches as well as genetic mouse models demonstrated that Grb14 inhibition-mediated hepatocyte division involved insulin receptor activation and was mediated by the mechanistic target of rapamycin complex 1-S6K pathway and the transcription factor E2F1. In order to determine a potential dysregulation in GRB14 gene expression in human pathophysiology, a collection of 85 human HCCs was investigated. This revealed a highly significant and frequent decrease in GRB14 expression in hepatic tumors when compared to adjacent nontumoral parenchyma, with 60% of the tumors exhibiting a reduced Grb14 mRNA level. CONCLUSION: Our study establishes Grb14 as a physiological repressor of insulin mitogenic action in the liver and further supports that dysregulation of insulin signaling is associated with HCC. (Hepatology 2017;65:1352-1368).

Novel Grb14-Mediated Cross Talk between Insulin and p62/Nrf2 Pathways Regulates Liver Lipogenesis and Selective Insulin Resistance.

A long-standing paradox in the pathophysiology of metabolic diseases is the selective insulin resistance of the liver. It is characterized by a blunted action of insulin to reduce glucose production, contributing to hyperglycemia, while de novo lipogenesis remains insulin sensitive, participating in turn to hepatic steatosis onset. The underlying molecular bases of this conundrum are not yet fully understood. Here, we established a model of selective insulin resistance in mice by silencing an inhibitor of insulin receptor catalytic activity, the growth factor receptor binding protein 14 (Grb14) in liver. Indeed, Grb14 knockdown enhanced hepatic insulin signaling but also dramatically inhibited de novo fatty acid synthesis. In the liver of obese and insulin-resistant mice, downregulation of Grb14 markedly decreased blood glucose and improved liver steatosis. Mechanistic analyses showed that upon Grb14 knockdown, the release of p62/sqstm1, a partner of Grb14, activated the transcription factor nuclear factor erythroid-2-related factor 2 (Nrf2), which in turn repressed the lipogenic nuclear liver X receptor (LXR). Our study reveals that Grb14 acts as a new signaling node that regulates lipogenesis and modulates insulin sensitivity in the liver by acting at a crossroad between the insulin receptor and the p62-Nrf2-LXR signaling pathways.

The role of chicken ovalbumin upstream promoter transcription factor II in the regulation of hepatic fatty acid oxidation and gluconeogenesis in newborn mice.

Chicken ovalbumin upstream promoter transcription factor II (COUP-TFII) is an orphan nuclear receptor involved in the control of numerous functions in various organs (organogenesis, differentiation, metabolic homeostasis, etc.). The aim of the present work was to characterize the regulation and contribution of COUP-TFII in the control of hepatic fatty acid and glucose metabolisms in newborn mice. Our data show that postnatal increase in COUP-TFII mRNA levels is enhanced by glucagon (via cAMP) and PPARα. To characterize COUP-TFII function in the liver of suckling mice, we used a functional (dominant negative form; COUP-TFII-DN) and a genetic (shRNA) approach. Adenoviral COUP-TFII-DN injection induces a profound hypoglycemia due to the inhibition of gluconeogenesis and fatty acid oxidation secondarily to reduced PEPCK, Gl-6-Pase, CPT I, and mHMG-CoA synthase gene expression. Using the crossover plot technique, we show that gluconeogenesis is inhibited at two different levels: 1) pyruvate carboxylation and 2) trioses phosphate synthesis. This could result from a decreased availability in fatty acid oxidation arising cofactors such as acetyl-CoA and reduced equivalents. Similar results are observed using the shRNA approach. Indeed, when fatty acid oxidation is rescued in response to Wy-14643-induced PPARα target genes (CPT I and mHMG-CoA synthase), blood glucose is normalized in COUP-TFII-DN mice. In conclusion, this work demonstrates that postnatal increase in hepatic COUP-TFII gene expression is involved in the regulation of liver fatty acid oxidation, which in turn sustains an active hepatic gluconeogenesis that is essential to maintain an appropriate blood glucose level required for newborn mice survival.

AMPKα1 controls hepatocyte proliferation independently of energy balance by regulating Cyclin A2 expression.

BACKGROUND: AMP-activated protein kinase (AMPK) is an evolutionarily conserved sensor of cellular energy status that contributes to restoration of energy homeostasis by slowing down ATP-consuming pathways and activating ATP-producing pathways. Unexpectedly, in different systems, AMPK is also required for proper cell division. In the current study, we evaluated the potential effect of the AMPK catalytic subunit, AMPKα1, on hepatocyte proliferation. METHODS: Hepatocyte proliferation was determined in AMPKα1 knockout and wild-type mice in vivo after two thirds partial hepatectomy, and in vitro in primary hepatocyte cultures. The activities of metabolic and cell cycle-related signaling pathways were measured. RESULTS: After partial hepatectomy, hepatocytes proliferated rapidly, correlating with increased AMPK phosphorylation. Deletion of AMPKα1 delayed liver regeneration by impacting on G1/S transition phase. The proliferative defect of AMPKα1-deficient hepatocytes was cell autonomous, and independent of energy balance. The priming phase, lipid droplet accumulation, protein anabolic responses and growth factor activation after partial hepatectomy occurred normally in the absence of AMPKα1 activity. By contrast, mRNA and protein expression of cyclin A2, a key driver of S phase progression, were compromised in the absence of AMPK activity. Importantly, AMPKα1 controlled cyclin A2 transcription mainly through the ATF/CREB element. CONCLUSIONS: Our study highlights a novel role for AMPKα1 as a positive regulator of hepatocyte division occurring independently of energy balance.

Enhancing liver mitochondrial fatty acid oxidation capacity in obese mice improves insulin sensitivity independently of hepatic steatosis.

BACKGROUND & AIMS: Despite major public health concern, therapy for non-alcoholic fatty liver, the liver manifestation of the metabolic syndrome often associated with insulin resistance (IR), remains elusive. Strategies aiming to decrease liver lipogenesis effectively corrected hepatic steatosis and IR in obese animals. However, they also indirectly increased mitochondrial long-chain fatty acid oxidation (mFAO) by decreasing malonyl-CoA, a lipogenic intermediate, which is the allosteric inhibitor of carnitine palmitoyltransferase 1 (CPT1A), the key enzyme of mFAO. We thus addressed whether enhancing hepatic mFAO capacity, through a direct modulation of liver CPT1A/malonyl-CoA partnership, can reverse an already established hepatic steatosis and IR in obese mice. METHODS: Adenovirus-mediated liver expression of a malonyl-CoA-insensitive CPT1A (CPT1mt) in high-fat/high-sucrose (HF/HS) diet-induced or genetically (ob/ob) obese mice was followed by metabolic and physiological investigations. RESULTS: In association with increased hepatic mFAO capacity, liver CPT1mt expression improved glucose tolerance and insulin response to a glucose load in HF/HS and ob/ob mice, showing increased insulin sensitivity, and corrected IR in ob/ob mice. Surprisingly, hepatic steatosis was not affected in CPT1mt-expressing obese mice, indicating a clear dissociation between hepatic steatosis and IR. Moreover, liver CPT1mt expression rescued HF/HS-induced impaired hepatic insulin signaling at the level of IRS-1, IRS-2, Akt, and GSK-3ß, most likely through the observed decrease in the HF/HS-induced accumulation of lipotoxic lipids, oxidative stress, and JNK activation. CONCLUSIONS: Enhancing hepatic mFAO capacity is sufficient to reverse a state of IR and glucose intolerance in obese mice independently of hepatic steatosis.

Diet and gastrointestinal bypass-induced weight loss: the roles of ghrelin and peptide YY.

OBJECTIVE: Bariatric surgery causes durable weight loss. Gut hormones are implicated in obesity pathogenesis, dietary failure, and mediating gastrointestinal bypass (GIBP) surgery weight loss. In mice, we determined the effects of diet-induced obesity (DIO), subsequent dieting, and GIBP surgery on ghrelin, peptide YY (PYY), and glucagon-like peptide-1 (GLP-1). To evaluate PYY's role in mediating weight loss post-GIBP, we undertook GIBP surgery in PyyKO mice. RESEARCH DESIGN AND METHODS: Male C57BL/6 mice randomized to a high-fat diet or control diet were killed at 4-week intervals. DIO mice underwent switch to ad libitum low-fat diet (DIO-switch) or caloric restriction (CR) for 4 weeks before being killed. PyyKO mice and their DIO wild-type (WT) littermates underwent GIBP or sham surgery and were culled 10 days postoperatively. Fasting acyl-ghrelin, total PYY, active GLP-1 concentrations, stomach ghrelin expression, and colonic Pyy and glucagon expression were determined. Fasting and postprandial PYY and GLP-1 concentrations were assessed 30 days postsurgery in GIBP and sham pair-fed (sham.PF) groups. RESULTS: DIO progressively reduced circulating fasting acyl-ghrelin, PYY, and GLP-1 levels. CR and DIO-switch caused weight loss but failed to restore circulating PYY to weight-appropriate levels. After GIBP, WT mice lost weight and exhibited increased circulating fasting PYY and colonic Pyy and glucagon expression. In contrast, the acute effects of GIBP on body weight were lost in PyyKO mice. Fasting PYY and postprandial PYY and GLP-1 levels were increased in GIBP mice compared with sham.PF mice. CONCLUSIONS: PYY plays a key role in mediating the early weight loss observed post-GIBP, whereas relative PYY deficiency during dieting may compromise weight-loss attempts.

Maintenance of red blood cell integrity by AMP-activated protein kinase alpha1 catalytic subunit.

AMP-activated protein kinase (AMPK) plays a pivotal role in regulating cellular energy metabolism. We previously showed that AMPKalpha1-/- mice develop moderate anemia associated with splenomegaly and high reticulocytosis. Here, we report that splenectomy of AMPKalpha1-/- mice worsened anemia supporting evidence that AMPKalpha1-/- mice developed a compensatory response through extramedullary erythropoiesis in the spleen. Transplantation of bone marrow from AMPKalpha1-/- mice into wild-type recipients recapitulated the hematologic phenotype. Further, AMPKalpha1-/- red blood cells (RBC) showed less deformability in response to shear stress limiting their membrane flexibility. Thus, our results highlight the crucial role of AMPK to preserve RBC integrity.

Intestinal gluconeogenesis is a key factor for early metabolic changes after gastric bypass but not after gastric lap-band in mice.

Unlike the adjustable gastric banding procedure (AGB), Roux-en-Y gastric bypass surgery (RYGBP) in humans has an intriguing effect: a rapid and substantial control of type 2 diabetes mellitus (T2DM). We performed gastric lap-band (GLB) and entero-gastro anastomosis (EGA) procedures in C57Bl6 mice that were fed a high-fat diet. The EGA procedure specifically reduced food intake and increased insulin sensitivity as measured by endogenous glucose production. Intestinal gluconeogenesis increased after the EGA procedure, but not after gastric banding. All EGA effects were abolished in GLUT-2 knockout mice and in mice with portal vein denervation. We thus provide mechanistic evidence that the beneficial effects of the EGA procedure on food intake and glucose homeostasis involve intestinal gluconeogenesis and its detection via a GLUT-2 and hepatoportal sensor pathway.

The transcription factor COUP-TFII is negatively regulated by insulin and glucose via Foxo1- and ChREBP-controlled pathways.

COUP-TFII has an important role in regulating metabolism in vivo. We showed this previously by deleting COUP-TFII from pancreatic beta cells in heterozygous mutant mice, which led to abnormal insulin secretion. Here, we report that COUP-TFII expression is reduced in the pancreas and liver of mice refed with a carbohydrate-rich diet and in the pancreas and liver of hyperinsulinemic and hyperglycemic mice. In pancreatic beta cells, COUP-TFII gene expression is repressed by secreted insulin in response to glucose through Foxo1 signaling. Ex vivo COUP-TFII reduces insulin production and secretion. Our results suggest that beta cell insulin secretion is under the control of an autocrine positive feedback loop by alleviating COUP-TFII repression. In hepatocytes, both insulin, through Foxo1, and high glucose concentrations repress COUP-TFII expression. We demonstrate that this negative glucose effect involves ChREBP expression. We propose that COUP-TFII acts in a coordinate fashion to control insulin secretion and glucose metabolism.

The MODY1 gene for hepatocyte nuclear factor 4alpha and a feedback loop control COUP-TFII expression in pancreatic beta cells.

Pancreatic islet beta cell differentiation and function are dependent upon a group of transcription factors that maintain the expression of key genes and suppress others. Knockout mice with the heterozygous deletion of the gene for chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) or the complete disruption of the gene for hepatocyte nuclear factor 4alpha (HNF4alpha) in pancreatic beta cells have similar insulin secretion defects, leading us to hypothesize that there is transcriptional cross talk between these two nuclear receptors. Here, we demonstrate specific HNF4alpha activation of a reporter plasmid containing the COUP-TFII gene promoter region in transfected pancreatic beta cells. The stable association of the endogenous HNF4alpha with a region of the COUP-TFII gene promoter that contains a direct repeat 1 (DR-1) binding site was revealed by chromatin immunoprecipitation. Mutation experiments showed that this DR-1 site is essential for HNF4alpha transactivation of COUP-TFII. The dominant negative suppression of HNF4alpha function decreased endogenous COUP-TFII expression, and the specific inactivation of COUP-TFII by small interfering RNA caused HNF4alpha mRNA levels in 832/13 INS-1 cells to decrease. This positive regulation of HNF4alpha by COUP-TFII was confirmed by the adenovirus-mediated overexpression of human COUP-TFII (hCOUP-TFII), which increased HNF4alpha mRNA levels in 832/13 INS-1 cells and in mouse pancreatic islets. Finally, hCOUP-TFII overexpression showed that there is direct COUP-TFII autorepression, as COUP-TFII occupies the proximal DR-1 binding site of its own gene in vivo. Therefore, COUP-TFII may contribute to the control of insulin secretion through the complex HNF4alpha/maturity-onset diabetes of the young 1 (MODY1) transcription factor network operating in beta cells.

Liver-specific inhibition of ChREBP improves hepatic steatosis and insulin resistance in ob/ob mice.

Obesity is a metabolic disorder often associated with type 2 diabetes, insulin resistance, and hepatic steatosis. Leptin-deficient (ob/ob) mice are a well-characterized mouse model of obesity in which increased hepatic lipogenesis is thought to be responsible for the phenotype of insulin resistance. We have recently demonstrated that carbohydrate responsive element-binding protein (ChREBP) plays a key role in the control of lipogenesis through the transcriptional regulation of lipogenic genes, including acetyl-CoA carboxylase and fatty acid synthase. The present study reveals that ChREBP gene expression and ChREBP nuclear protein content are significantly increased in liver of ob/ob mice. To explore the involvement of ChREBP in the physiopathology of hepatic steatosis and insulin resistance, we have developed an adenovirus-mediated RNA interference technique in which short hairpin RNAs (shRNAs) were used to inhibit ChREBP expression in vivo. Liver-specific inhibition of ChREBP in ob/ob mice markedly improved hepatic steatosis by specifically decreasing lipogenic rates. Correction of hepatic steatosis also led to decreased levels of plasma triglycerides and nonesterified fatty acids. As a consequence, insulin signaling was improved in liver, skeletal muscles, and white adipose tissue, and overall glucose tolerance and insulin sensitivity were restored in ob/ob mice after a 7-day treatment with the recombinant adenovirus expressing shRNA against ChREBP. Taken together, our results demonstrate that ChREBP is central for the regulation of lipogenesis in vivo and plays a determinant role in the development of the hepatic steatosis and of insulin resistance in ob/ob mice.

Hepatic glucokinase is required for the synergistic action of ChREBP and SREBP-1c on glycolytic and lipogenic gene expression.

Hepatic glucokinase (GK) catalyzes the phosphorylation of glucose to glucose 6-phosphate (G6P), a step which is essential for glucose metabolism in liver as well as for the induction of glycolytic and lipogenic genes. The sterol regulatory element-binding protein-1c (SREBP-1c) has emerged as a major mediator of insulin action on hepatic gene expression, but the extent to which its transcriptional effect is caused by an increased glucose metabolism remains unclear. Through the use of hepatic GK knockout mice (hGK-KO) we have shown that the acute stimulation by glucose of l-pyruvate kinase (l-PK), fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC), and Spot 14 genes requires GK expression. To determine whether the effect of SREBP-1c requires GK expression and subsequent glucose metabolism, a transcriptionally active form of SREBP-1c was overexpressed both in vivo and in primary cultures of control and hGK-KO hepatocytes. Our results demonstrate that the synergistic action of SREBP-1c and glucose metabolism via GK is necessary for the maximal induction of l-PK, ACC, FAS, and Spot 14 gene expression. Indeed, in hGK-KO hepatocytes overexpressing SREBP-1c, the effect of glucose on glycolytic and lipogenic genes is lost because of the impaired ability of these hepatocytes to efficiently metabolize glucose, despite a marked increase in low K(m) hexokinase activity. Our studies also reveal that the loss of glucose effect observed in hGK-KO hepatocytes is associated with a decreased in the carbohydrate responsive element-binding protein (ChREBP) gene expression, a transcription factor suggested to mediate glucose signaling in liver. Decreased ChREBP gene expression, achieved using small interfering RNA, results in a loss of glucose effect on endogenous glycolytic (l-PK) and lipogenic (FAS, ACC) gene expression, thereby demonstrating the direct implication of ChREBP in glucose action. Together these results support a model whereby both SREBP-1c and glucose metabolism, acting via ChREBP, are necessary for the dietary induction of glycolytic and lipogenic gene expression in liver.