Liver-specific Insulin Receptor Knockout Research Articles

Chronic hyperinsulinemia promotes human hepatocyte senescence

ObjectiveCellular senescence, an irreversible proliferative cell arrest, is caused by excessive intracellular or extracellular stress/damage. Increased senescent cells have been identified in multiple tissues in different metabolic and other aging-related diseases. Recently, several human and mouse studies emphasized the involvement of senescence in development and progression of NAFLD. Hyperinsulinemia, seen in obesity, metabolic syndrome, and other conditions of insulin resistance, has been linked to senescence in adipocytes and neurons. Here, we investigate the possible direct role of chronic hyperinsulinemia in the development of senescence in human hepatocytes. MethodsUsing fluorescence microscopy, immunoblotting, and gene expression, we tested senescence markers in human hepatocytes subjected to chronic hyperinsulinemia in vitro and validated the data in vivo by using liver-specific insulin receptor knockout (LIRKO) mice. The consequences of hyperinsulinemia were also studied in senescent hepatocytes following doxorubicin as a model of stress-induced senescence. Furthermore, the effects of senolytic agents in insulin- and doxorubicin-treated cells were analyzed. ResultsResults showed that exposing the hepatocytes to prolonged hyperinsulinemia promotes the onset of senescence by increasing the expression of p53 and p21. It also further enhanced the senescent phenotype in already senescent hepatocytes. Addition of insulin signaling pathway inhibitors prevented the increase in cell senescence, supporting the direct contribution of insulin. Furthermore, LIRKO mice, in which insulin signaling in the liver is abolished due to deletion of the insulin receptor gene, showed no differences in senescence compared to their wild-type counterparts despite having marked hyperinsulinemia indicating these are receptor-mediated effects. In contrast, the persistent hyperinsulinemia in LIRKO mice enhanced senescence in white adipose tissue. In vitro, senolytic agents dasatinib and quercetin reduced the prosenescent effects of hyperinsulinemia in hepatocytes. ConclusionOur findings demonstrate a direct link between chronic hyperinsulinemia and hepatocyte senescence. This effect can be blocked by reducing the levels of insulin receptors or administration of senolytic drugs, such as dasatinib and quercetin.

1336-P: FoxO1-ATGL-Sirt1-FoxO1 Feed Forward Signaling Mediates Effects on Hepatic Gene Expression and Glucose Homeostasis in LIRKO Mice

FoxO1 is a major target of insulin action and regulates expression of ATGL and its inhibitor, G0S2, in the liver (Cell Rep 15:349, 2016) . Here, we asked whether ATGL mediates effects of FoxO1 in liver-specific insulin receptor knockout (LIRKO) mice. Studies in LIRKO and IR/FoxO1 double knockout (LIRFKO) mice show ATGL expression is increased and G0S2 is suppressed in a FoxO1-dependent fashion when insulin signaling is disrupted in the liver. ATGL knockdown (KD) reduces high plasma glucose and insulin concentrations, HOMA-IR and fatty acid oxidation, and improves glucose tolerance in LIRKO mice. Hyperinsulinemic-euglycemic clamp studies show ATGL KD improves insulin sensitivity due to effects on both HGP and glucose utilization, reflecting changes in WAT lipolysis, BAT metabolism and hepatic gene expression. The expression of FoxO1-regulated gluconeogenic genes (PEPCK, G6Pase, PCO, PGC-1α) , hepatokines (e.g., follistatin) and IGFBP-1 is increased in LIRKO mice and reduced by ATGL KD, reflecting changes in acetylation of FoxO1 and its co-activator, PGC-1α (decreased in LIRKO, increased by ATGL KD) . Rictor acetylation, which promotes activation of Akt and phosphorylation of FoxO1, also is increased by ATGL KD in LIRKO mice and isolated hepatocytes. Since ATGL promotes activation of Sirt1 (Mol Cell 77:810, 2020) and Sirt1 deacetylates FoxO1, PGC-1α and rictor, these studies suggest FoxO1 promotes increased ATGL and Sirt1 activity, and that Sirt1 activation enhances the effects of FoxO1 on gene expression and glucose homeostasis in a feed forward fashion (FoxO1 -> ATGL -> Sirt1 -> FoxO1) . Targeting the FoxO1/ATGL/Sirt1 pathway may provide a useful strategy for improving glucose homeostasis when insulin signaling is impaired in the liver. Disclosure I.O-sullivan: None. S.Bhattacharyya: None. C.Liew: None. S.M.Lee: None. J.Cordoba-chacon: None. R.J.Perry: None. G.I.Shulman: Advisory Panel; 89bio, Inc., AstraZeneca, Equator Therapeutics, Inc., Janssen Research & Development, LLC, Merck & Co., Inc., Consultant; DiCerna Pharmaceuticals, Inc., Novo Nordisk, Other Relationship; Generian Pharmaceuticals, iMetabolic Biopharma Corporation, Maze Therapeutics, The Liver Company, Stock/Shareholder; Levels Health, Inc. . T.Unterman: None. Funding Department of Veterans Affairs (5I01BX001968)

Severe Hepatic Insulin Resistance Induces Vascular Dysfunction: Improvement by Liver-Specific Insulin Receptor Isoform A Gene Therapy in a Murine Diabetic Model.

Background: Cardiovascular dysfunction is linked to insulin-resistant states. In this paper, we analyzed whether the severe hepatic insulin resistance of an inducible liver-specific insulin receptor knockout (iLIRKO) might generate vascular insulin resistance and dysfunction, and whether insulin receptor (IR) isoforms gene therapy might revert it. Methods: We studied in vivo insulin signaling in aorta artery and heart from iLIRKO. Vascular reactivity and the mRNA levels of genes involved in vascular dysfunction were analyzed in thoracic aorta rings by qRT-PCR. Finally, iLIRKO mice were treated with hepatic-specific gene therapy to analyze vascular dysfunction improvement. Results: Our results suggest that severe hepatic insulin resistance was expanded to cardiovascular tissues. This vascular insulin resistance observed in aorta artery from iLIRKO mice correlated with a reduction in both PI3K/AKT/eNOS and p42/44 MAPK pathways, and it might be implicated in their vascular alterations characterized by endothelial dysfunction, hypercontractility and eNOS/iNOS levels’ imbalance. Finally, regarding long-term hepatic expression of IR isoforms, IRA was more efficient than IRB in the improvement of vascular dysfunction observed in iLIRKO mice. Conclusion: Severe hepatic insulin resistance is sufficient to produce cardiovascular insulin resistance and dysfunction. Long-term hepatic expression of IRA restored the vascular damage observed in iLIRKO mice.

Parental metabolic syndrome epigenetically reprograms offspring hepatic lipid metabolism in mice.

The prevalence of nonalcoholic fatty liver disease (NAFLD) is increasing worldwide. Although gene-environment interactions have been implicated in the etiology of several disorders, the impact of paternal and/or maternal metabolic syndrome on the clinical phenotypes of offspring and the underlying genetic and epigenetic contributors of NAFLD have not been fully explored. To this end, we used the liver-specific insulin receptor knockout (LIRKO) mouse, a unique nondietary model manifesting 3 hallmarks that confer high risk for the development of NAFLD: hyperglycemia, insulin resistance, and dyslipidemia. We report that parental metabolic syndrome epigenetically reprograms members of the TGF-β family, including neuronal regeneration-related protein (NREP) and growth differentiation factor 15 (GDF15). NREP and GDF15 modulate the expression of several genes involved in the regulation of hepatic lipid metabolism. In particular, NREP downregulation increases the protein abundance of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) and ATP-citrate lyase (ACLY) in a TGF-β receptor/PI3K/protein kinase B-dependent manner, to regulate hepatic acetyl-CoA and cholesterol synthesis. Reduced hepatic expression of NREP in patients with NAFLD and substantial correlations between low serum NREP levels and the presence of steatosis and nonalcoholic steatohepatitis highlight the clinical translational relevance of our findings in the context of recent preclinical trials implicating ACLY in NAFLD progression.

Identification of two microRNA nodes as potential cooperative modulators of liver metabolism.

AimHepatic insulin resistance is a hallmark of type 2 diabetes and non‐alcoholic fatty liver disease. Dysregulation of microRNA (miRNA) expression in insulin‐resistant livers might coordinate impaired hepatic metabolic function. Here, we aimed to discover miRNAs and their downstream targets involved in hepatic insulin resistance.MethodsWe determined miRNA expression profiles by small RNA sequencing of two mouse models of impaired hepatic insulin action: high‐fat diet‐induced obesity and liver‐specific insulin receptor knockout. Conversely, we assessed the hepatic miRNA expression profile after treatment with the antidiabetic hormone, fibroblast growth factor 21 (FGF21). Ontology analysis of predicted miRNA gene targets was performed to identify regulated gene pathways. Target enrichment analysis and miRNA mimic overexpression in vitro were used to identify unified protein targets of nodes of regulated miRNAs.ResultsWe identified an array of miRNA species regulated by impaired liver insulin action or after fibroblast growth factor 21 treatment. Ontology analysis of predicted miRNA gene targets identified pathways controlling hepatic energy metabolism and insulin sensitivity. We identified a node of two miRNAs downregulated in the livers of liver‐specific insulin receptor knockout mice, miR‐883b and miR‐205, which positively regulate the expression of transcription factor zinc finger E‐box‐binding homeobox 1 (ZBED1). We found another node of two miRNAs upregulated in the livers of fibroblast growth factor 21‐treated mice, miR‐155‐3p and miR‐1968‐5p, which canonically downregulates the caveola component, polymerase I and transcript release factor (PTRF), a gene previously implicated in hepatic energy metabolism.ConclusionsThis study identifies two nodes of coregulated miRNAs that might coordinately control hepatic energy metabolism in states of insulin resistance.

1874-P: A Novel Role for SerpinB1 in the Regulation of Insulin Signaling in Hepatocytes

Compensatory expansion of β cell mass has been pursued as a potential therapeutic strategy to cure diabetes. We previously identified SerpinB1 as a hepatocyte-derived circulating factor that contributes to adaptive beta cell expansion in the liver-specific insulin receptor knockout (LIRKO) mouse. To understand how SerpinB1 is regulated and secreted, we began by first investigating the mechanism(s) and signaling pathways that modulate the gene and protein expression of SerpinB1 in hepatocytes. Hepatic SerpinB1 expression was elevated in insulin resistant mouse models such as db/db and ob/ob, and circulating interleukin-6 (IL-6) levels were higher in these mice. Indeed, IL-6 induced SerpinB1 expression in HepG2 cells and human primary hepatocytes via the JAK/STAT3 pathway, indicating an important role for this cytokine in inducing the expression of the protease inhibitor in insulin resistant states. Conversely, silencing of SERPINB1 gene in hepatocytes decreased INSR gene and protein expression with a consequent alteration of expression of several downstream signaling genes related to gluconeogenesis and cell proliferation in hepatocytes. Ribosomal protein S3 (RPS3), identified as a binding partner of SerpinB1 in HepG2 cells, exhibited reduced expression in db/db and ob/ob mice liver along with reduced insulin receptor (IR) expression. Silencing of RPS3 gene in HepG2 cells, phenocopied the effects of SerpinB1 gene silencing, and decreased IR protein expression with consequent alterations in downstream signaling proteins. Additional studies are required to examine the precise regulation of IR expression by SerpinB1 and how the latter is secreted from insulin resistant hepatocytes. These results indicate that SerpinB1 expression is upregulated in insulin resistance by IL-6 and the SerpinB1/RPS3 complex plays a role in regulating insulin signaling in hepatocytes. Disclosure K. Orime: None. D.F. De Jesus: None. U. Jhala: None. R. Kulkarni: None.

SAT-150 Forkhead Box Protein O1 (foxo1) Regulates Hepatic Serine Protease Inhibitor B1 (serpinb1) Expression In A Cell Non-autonomous Fashion

Forkhead box O (FoxO) proteins are major targets of insulin action, and FoxO1 mediates insulin’s effects on hepatic glucose metabolism. We previously reported that serine protease inhibitor B1 (serpinB1) is a liver-secreted factor (hepatokine) that promotes adaptive β-cell proliferation in response to insulin resistance in liver-specific insulin receptor knockout (LIRKO) mice. Here, we report that FoxO1 promotes serpinB1 expression in hepatic insulin resistance in a cell non-autonomous manner. Mice lacking both the insulin receptor and FoxO1 in the liver (LIRFKO) exhibited lower β-cell mass than LIRKO mice due to attenuated β-cell proliferation. Hepatic expression of serpinb1mRNA and protein levels were increased in the LIRKO mice, whereas in the LIRFKO mice, these levels returned to those in control mice. Furthermore, liver-specific expression of constitutively active FoxO1 increased hepatic serpinB1 mRNA and protein levels. Conversely, serpinB1 mRNA and protein levels were reduced in FoxOKO mice lacking FoxO proteins in the liver. ChIP experiments indicated that FoxO1 binds to three distinct DNA sites located ~9 kb upstream of the serpinb1 gene in primary mouse hepatocytes and that this binding is enhanced in LIRKO hepatocytes. However, although adenoviral expression of wildtype or constitutively active FoxO1 and insulin treatment regulated other known FoxO1 target genes (IGFBP-1, PEPCK, G6Pase), it did not alter serpinB1 expression in mouse primary hepatocytes. These results indicate that liver FoxO1 promotes serpinB1 expression in hepatic insulin resistance and that cell non-autonomous mechanisms contribute to FoxO1-dependent effects on hepatic serpinB1 expression.

MicroRNA-146a-5p association with the cardiometabolic disease risk factor TMAO.

Trimethylamine-N-oxide (TMAO), a microbial choline metabolism byproduct that is processed in the liver and excreted into circulation, is associated with increased atherosclerotic lesion formation and cardiovascular disease risk. Genetic regulators of TMAO levels are largely unknown. In the present study, we used 288 mice from a genetically heterogeneous mouse population [Diversity Outbred (DO)] to determine hepatic microRNA associations with TMAO in the context of an atherogenic diet. We also validated findings in two additional animal models of atherosclerosis: liver-specific insulin receptor knockout mice fed a chow diet (LIRKO) and African green monkeys fed high-fat/high-cholesterol diet. Small RNA-sequencing analysis in DO mice, LIRKO mice, and African green monkeys identified only one hepatic microRNA (miR-146a-5p) that is aberrantly expressed across all three models. Moreover, miR-146a-5p levels are associated with circulating TMAO after atherogenic diet in each of these models. We also performed high-resolution genetic mapping and identified a novel quantitative trait locus on Chromosome 12 for TMAO levels. This interval includes two genes, Numb and Dlst, which are inversely correlated with both miR-146a and TMAO and are predicted targets of miR-146a. Both of these genes have been validated as direct targets of miR-146a, though in other cellular contexts. This is the first report to our knowledge of a link between miR-146 and TMAO. Our findings suggest that miR-146-5p, as well as one or more genes at the Chromosome 12 QTL (possibly Numb or Dlst), is strongly linked to TMAO levels and likely involved in the control of atherosclerosis.

Forkhead box protein O1 (FoxO1) regulates hepatic serine protease inhibitor B1 (serpinB1) expression in a non-cell-autonomous fashion

FoxO proteins are major targets of insulin action, and FoxO1 mediates the effects of insulin on hepatic glucose metabolism. We reported previously that serpinB1 is a liver-secreted factor (hepatokine) that promotes adaptive β-cell proliferation in response to insulin resistance in the liver-specific insulin receptor knockout (LIRKO) mouse. Here we report that FoxO1 plays a critical role in promoting serpinB1 expression in hepatic insulin resistance in a non-cell-autonomous manner. Mice lacking both the insulin receptor and FoxO1 (LIRFKO) exhibit reduced β-cell mass compared with LIRKO mice because of attenuation of β-cell proliferation. Although hepatic expression of serpinB1 mRNA and protein levels was increased in LIRKO mice, both the mRNA and protein levels returned to control levels in LIRFKO mice. Furthermore, liver-specific expression of constitutively active FoxO1 in transgenic mice induced an increase in hepatic serpinB1 mRNA and protein levels in refed mice. Conversely, serpinB1 mRNA and protein levels were reduced in mice lacking FoxO proteins in the liver. ChIP studies demonstrated that FoxO1 binds to three distinct sites located ∼9 kb upstream of the serpinb1 gene in primary mouse hepatocytes and that this binding is enhanced in hepatocytes from LIRKO mice. However, adenoviral expression of WT or constitutively active FoxO1 and insulin treatment are sufficient to regulate other FoxO1 target genes (IGFBP-1 and PEPCK) but not serpinB1 expression in mouse primary hepatocytes. These results indicate that liver FoxO1 promotes serpinB1 expression in hepatic insulin resistance and that non-cell-autonomous factors contribute to FoxO1-dependent effects on serpinB1 expression in the liver.

NREP Bridges TGF-ß Signaling and Lipid Metabolism in the Epigenetic Reprogramming of NAFLD in the Offspring of Insulin-Resistant Parents

Nonalcoholic fatty liver disease (NAFLD) prevalence is increasing worldwide and few studies have associated maternal diabetes and birth weights with increased risk for NAFLD. We used an unique non-dietary model, manifesting hyperglycemia and hyperinsulinemia-two hallmarks of gestational and type 2 diabetes. We aimed to determine the genetic and epigenetic effects of paternal vs. maternal genetic insulin resistance on the developmental programming in the offspring of the liver-specific insulin receptor knockout (LIRKO) mice. Male control F1 offspring from father LIRKO (FL), mother LIRKO (ML) or control mothers and fathers (C) were weaned on a chow or high-fat-diet (HFD) and followed for 3 months. FL and ML showed impaired growth and body weight composition. FL and ML developed hepatic steatosis compared to C when challenged with HFD and exhibited increased hepatic expression of lipogenesis-associated genes. Hepatic transcriptomic and genome-wide DNA methylation analyses of FL and ML on chow diet presented enriched-pathways associated with TGF-β signaling and lipid synthesis. FL and ML hepatic NREP mRNA levels were decreased 50% (p<0.05) on HFD compared to C. In-vivo and in-vitro modeling of hepatic steatosis in HepG2 and human primary hepatocytes decreased NREP mRNA and protein levels. Knock-down experiments performed in HepG2 cells revealed that NREP acts by regulating triglyceride and cholesterol synthesis transcriptional network via regulation of ATP-citrate lyase (ACL) in a phospho-AKT dependent manner. Finally, reduced NREP mRNA levels in patients with hepatic steatosis by 40% (p<0.05) and recent preclinical trials implicating ACL in NAFLD highlight the translation relevance of our findings. These data suggest that prenatal insulin resistance epigenetically regulates a novel gene, NREP, that has detrimental effects on metabolic adaptation and transcriptional regulation of hepatic metabolism in the development of NAFLD. Disclosure D.F. De Jesus: None. K. Orime: None. C. Wang: None. J. Hu: None. E. Dirice: None. A.M. Silva: None. Y. Tseng: Other Relationship; Self; Chugai Pharmaceutical Co., Ltd.. Research Support; Self; MedImmune. J. Pihlajamaki: None. R. Kulkarni: None.

A Novel Role for SerpinB1, a Protease Inhibitor, in Hepatic Biology

SerpinB1, a protease inhibitor that is secreted from the liver in response to insulin resistance, was recently reported to promote pancreatic β cell proliferation. We focused on investigating the significance of SerpinB1 in the liver to gain insights into its role in regulating whole body metabolism. We determined SerpinB1 gene expression in multiple metabolic tissues harvested under fasting or fed states from ∼34-week-old male Liver-specific insulin receptor knockout (LIRKO) mice and littermate controls. While in controls SerpinB1 expression was reduced by ∼50% after feeding when compared to the fasting state, (P=0.02, n=4-5), the LIRKOs, in contrast, exhibited a 2.3-fold higher (P=0.01, n=5-6) expression in the fed vs. fasting states and the higher levels were consistent with earlier observations in the LIRKO livers. We next took advantage of the availability of liver samples from 18 to 24-week-old male SerpinB1 KO mice and littermate controls and subjected them to RNA-sequencing. As expected, SerpinB1 gene expression was absent in the KO samples, and interestingly, was associated with a significant decrease in gene expression related to cell cycle, NF-kappa B signaling, and inflammatory pathways. Independent validation of these data by RT-qPCR confirmed a decrease in cell cycle-related genes such as Ccna2 (-60%, P=0.04), Ccnb2 (-54%, P=0.04), Ccnd1 (-46%, P=0.03), and Ccne1 (-51%, P=0.07), and the cell proliferation marker, Mki67 (-60%, P=0.00) (n=5). Furthermore, knockdown of SerpinB1 in the human hepatocyte cell line, HepG2, exhibited significantly decreased expression of Ccnb1 (-36%, P=0.01), Ccnb2 (-44%, P=0.00), Ccne1 (-21%, P=0.00), MKi67 (-30%, P=0.00), and MCM2 (-29%, P=0.00) genes, and increased expression of P21 (+165%, P=0.00) (n=3 for control vs. knockdown groups). These novel data implicate endogenous SerpinB1 in cell proliferation in hepatocytes and warrant further investigation of its role in hepatocyte function and the overall impact on glucose homeostasis. Disclosure K. Orime: None. D.F. De Jesus: None. R. Kulkarni: None.

Insulin-Inducible SMILE Inhibits Hepatic Gluconeogenesis.

The role of a glucagon/cAMP-dependent protein kinase-inducible coactivator PGC-1α signaling pathway is well characterized in hepatic gluconeogenesis. However, an opposing protein kinase B (PKB)/Akt-inducible corepressor signaling pathway is unknown. A previous report has demonstrated that small heterodimer partner-interacting leucine zipper protein (SMILE) regulates the nuclear receptors and transcriptional factors that control hepatic gluconeogenesis. Here, we show that hepatic SMILE expression was induced by feeding in normal mice but not in db/db and high-fat diet (HFD)-fed mice. Interestingly, SMILE expression was induced by insulin in mouse primary hepatocyte and liver. Hepatic SMILE expression was not altered by refeeding in liver-specific insulin receptor knockout (LIRKO) or PKB β-deficient (PKBβ(-/-)) mice. At the molecular level, SMILE inhibited hepatocyte nuclear factor 4-mediated transcriptional activity via direct competition with PGC-1α. Moreover, ablation of SMILE augmented gluconeogenesis and increased blood glucose levels in mice. Conversely, overexpression of SMILE reduced hepatic gluconeogenic gene expression and ameliorated hyperglycemia and glucose intolerance in db/db and HFD-fed mice. Therefore, SMILE is an insulin-inducible corepressor that suppresses hepatic gluconeogenesis. Small molecules that enhance SMILE expression would have potential for treating hyperglycemia in diabetes.

FoxO1 integrates direct and indirect effects of insulin on hepatic glucose production and glucose utilization.

FoxO proteins are major targets of insulin action. To better define the role of FoxO1 in mediating insulin effects in the liver, we generated liver-specific insulin receptor knockout (LIRKO) and IR/FoxO1 double knockout (LIRFKO) mice. Here we show that LIRKO mice are severely insulin resistant based on glucose, insulin and C-peptide levels, and glucose and insulin tolerance tests, and genetic deletion of hepatic FoxO1 reverses these effects. (13)C-glucose and insulin clamp studies indicate that regulation of both hepatic glucose production (HGP) and glucose utilization is impaired in LIRKO mice, and these defects are also restored in LIRFKO mice corresponding to changes in gene expression. We conclude that (1) inhibition of FoxO1 is critical for both direct (hepatic) and indirect effects of insulin on HGP and utilization, and (2) extrahepatic effects of insulin are sufficient to maintain normal whole-body and hepatic glucose metabolism when liver FoxO1 activity is disrupted.

PKB/Akt phosphorylation of ERRγ contributes to insulin-mediated inhibition of hepatic gluconeogenesis.

Insulin resistance, a major contributor to the pathogenesis of type 2 diabetes, leads to increased hepatic glucose production (HGP) owing to an impaired ability of insulin to suppress hepatic gluconeogenesis. Nuclear receptor oestrogen-related receptor γ (ERRγ) is a major transcriptional regulator of hepatic gluconeogenesis. In this study, we investigated insulin-dependent post-translational modifications (PTMs) altering the transcriptional activity of ERRγ for the regulation of hepatic gluconeogenesis. We examined insulin-dependent phosphorylation and subcellular localisation of ERRγ in cultured cells and in the liver of C57/BL6, leptin receptor-deficient (db/db), liver-specific insulin receptor knockout (LIRKO) and protein kinase B (PKB) β-deficient (Pkbβ (-/-)) mice. To demonstrate the role of ERRγ in the inhibitory action of insulin on hepatic gluconeogenesis, we carried out an insulin tolerance test in C57/BL6 mice expressing wild-type or phosphorylation-deficient mutant ERRγ. We demonstrated that insulin suppressed the transcriptional activity of ERRγ by promoting PKB/Akt-mediated phosphorylation of ERRγ at S179 and by eliciting translocation of ERRγ from the nucleus to the cytoplasm through interaction with 14-3-3, impairing its ability to promote hepatic gluconeogenesis. In addition, db/db, LIRKO and Pkbβ (-/-) mice displayed enhanced ERRγ transcriptional activity due to a block in PKBβ-mediated ERRγ phosphorylation during refeeding. Finally, the phosphorylation-deficient mutant ERRγ S179A was resistant to the inhibitory action of insulin on HGP. These results suggest that ERRγ is a major contributor to insulin action in maintaining hepatic glucose homeostasis.

Insulin‐dependent and ‐independent regulation of sterol regulatory element‐binding protein‐1c

Insulin‐dependent and ‐independent regulation of sterol regulatory element‐binding protein‐1c

Liver-Derived Systemic Factors Drive β Cell Hyperplasia in Insulin-Resistant States

Integrative organ crosstalk regulates key aspects of energy homeostasis, and its dysregulation may underlie metabolic disorders such as obesity and diabetes. To test the hypothesis that crosstalk between the liver and pancreatic islets modulates β cell growth in response to insulin resistance, we used the liver-specific insulin receptor knockout (LIRKO) mouse, a unique model that exhibits dramatic islet hyperplasia. Using complementary invivo parabiosis and transplantation assays, as well as invitro islet culture approaches, we demonstrate that humoral, nonneural, non-cell-autonomous factor(s) induces β cell proliferation in LIRKO mice. Furthermore, we report that a hepatocyte-derived factor(s) stimulates mouse and human β cell proliferation in exvivo assays, independent of ambient glucose and insulin levels. These data implicate the liver as a critical source of β cell growth factor(s) in insulin-resistant states.

β-Cell Hyperplasia Induced by Hepatic Insulin Resistance

OBJECTIVEType 2 diabetes results from a combination of insulin resistance and impaired insulin secretion. To directly address the effects of hepatic insulin resistance in adult animals, we developed an inducible liver-specific insulin receptor knockout mouse (iLIRKO).RESEARCH DESIGN AND METHODSUsing this approach, we were able to induce variable insulin receptor (IR) deficiency in a tissue-specific manner (liver mosaicism).RESULTSiLIRKO mice presented progressive hepatic and extrahepatic insulin resistance without liver dysfunction. Initially, iLIRKO mice displayed hyperinsulinemia and increased β-cell mass, the extent of which was proportional to the deletion of hepatic IR. Our studies of iLIRKO suggest a cause-and-effect relationship between progressive insulin resistance and the fold increase of plasma insulin levels and β-cell mass. Ultimately, the β-cells failed to secrete sufficient insulin, leading to uncontrolled diabetes. We observed that hepatic IGF-1 expression was enhanced in iLIRKO mice, resulting in an increase of circulating IGF-1. Concurrently, the IR-A isoform was upregulated in hyperplastic β-cells of iLIRKO mice and IGF-1–induced proliferation was higher than in the controls. In mouse β-cell lines, IR-A, but not IR-B, conferred a proliferative capacity in response to insulin or IGF-1, providing a potential explanation for the β-cell hyperplasia induced by liver insulin resistance in iLIRKO mice.CONCLUSIONSOur studies of iLIRKO mice suggest a liver-pancreas endocrine axis in which IGF-1 functions as a liver-derived growth factor to promote compensatory pancreatic islet hyperplasia through IR-A.

High Circulating Leptin Receptors with Normal Leptin Sensitivity in Liver-specific Insulin Receptor Knock-out (LIRKO) Mice

Liver-specific insulin receptor knock-out (LIRKO) mice display hyperinsulinemia, abnormal glucose metabolism, and progressive liver dysfunction. In addition, circulating leptin levels appear to be increased more than 10-fold. However, food intake, body weight, and adipose mass are not significantly altered in LIRKO mice compared with wild-type littermates. Using a ligand immunofunctional assay, we found that the apparent increase in circulating leptin in LIRKO mice is because of an 80-fold increased serum level of soluble leptin receptor. Gene expression analysis by microarray and real time PCR reveals the liver as the source of soluble leptin receptor in LIRKO mice, with an increase in expression of the short (Ob-Ra), long (Ob-Rb), and soluble (Ob-Re) forms of the leptin receptor. Direct control of leptin receptor expression by insulin could also be demonstrated in isolated hepatocytes from normal mice. Despite the markedly increased levels of leptin receptor in their circulation, LIRKO mice exhibit normal or even enhanced leptin sensitivity, as assessed by their physiological and molecular responses to exogenous leptin administration and their lower base-line hypothalamic levels of SOCS3 mRNA. Thus, insulin signaling in the liver plays an important role in control of leptin receptor expression and shedding. In the LIRKO mouse, this is lost, leading to markedly increased leptin receptors into the circulation. These high levels of circulating leptin receptor bind leptin and likely alter its clearance, but do not inhibit leptin action and may actually potentiate leptin action. In this manner, insulin signaling in liver plays an important role in leptin homeostasis and fine modulation of leptin action.

Insulin receptors in beta-cells are critical for islet compensatory growth response to insulin resistance.

Insulin and insulin-like growth factor 1 (IGF1) are ubiquitous growth factors that regulate proliferation in most mammalian tissues including pancreatic islets. To explore the specificity of insulin receptors in compensatory beta-cell growth, we examined two models of insulin resistance. In the first model, we used liver-specific insulin receptor knockout (LIRKO) mice, which exhibit hyperinsulinemia without developing diabetes due to a compensatory increase in beta-cell mass. LIRKO mice, also lacking functional insulin receptors in beta-cells (beta IRKO/LIRKO), exhibited severe glucose intolerance but failed to develop compensatory islet hyperplasia, together leading to early death. In the second model, we examined the relative significance of insulin versus IGF1 receptors in islet growth by feeding high-fat diets to beta IRKO and beta-cell-specific IGF1 receptor knockout (beta IGFRKO) mice. Although both groups on the high-fat diet developed insulin resistance, beta IRKO, but not beta IGFRKO, mice exhibited poor islet growth consistent with insulin-stimulated phosphorylation, nuclear exclusion of FoxO1, and reduced expression of Pdx-1. Together these data provide direct genetic evidence that insulin/FoxO1/Pdx-1 signaling is one pathway that is crucial for islet compensatory growth response to insulin resistance.

Role of Insulin Receptor in the Regulation of Glucose Uptake in Neonatal Hepatocytes

The liver plays a major role in the regulation of glucose homoeostasis. Evidence from liver-specific insulin receptor knockout mice (LIRKO) suggested that insulin's direct and indirect effects on glucose utilization by the liver both require the presence of hepatic insulin receptors (IR). To address this issue, we have generated immortalized neonatal hepatocytes bearing (HIR LoxP) or not (HIR KO) IR. The lack of IR significantly decreased basal glucose uptake in neonatal hepatocytes from 3- to 14-d-old mice, and the expression of glucose transporter 1 (GLUT1), GLUT2, and glucokinase (GK) remained unchanged throughout development. HIR KO reconstituted hepatocytes with IRA but not with IRB isoform and restored basal glucose uptake up to the levels observed in HIR LoxP cells. However, both IR isoforms associated with GLUT1 or GLUT2. Overexpression of IGF-I receptor (IGF-IR) increased basal glucose uptake in neonatal hepatocytes lacking or not IR. This effect was also accompanied by its association with GLUT1 or GLUT2. Exogenous expression of GLUT4 had no effect on basal glucose uptake in neonatal hepatocytes. However, HIR LoxP hepatocytes expressing exogenous GLUT4 increased glucose uptake in the presence of insulin without showing association between GLUT4 and IR. Our data clearly indicate that IR plays a direct role in the regulation of basal glucose uptake/transport by the hepatocytes, and either type A IR or IGF-IR works on glucose uptake as a GLUT1- or GLUT2-associated cotransporter. Thus, IR mediates glucose uptake through its specific association with endogenous, but not with exogenous, glucose transporters in neonatal hepatocytes.