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Nonalcoholic fatty liver disease, recently renamed metabolic dysfunction- associated steatotic liver disease (MASLD), is a progressive metabolic disorder that begins with aberrant triglyceride accumulation in the liver and can lead to cirrhosis and cancer. A common variant in the gene PNPLA3, encoding the protein PNPLA3- I148M, is the strongest known genetic risk factor for MASLD. Despite its discovery 20 y ago, the function of PNPLA3, and now the role of PNPLA3- I148M, remain unclear. In this study, we sought to dissect the biogenesis of PNPLA3 and PNPLA3- I148M and characterize changes induced by endogenous expression of the disease- causing variant. Contrary to bioinformatic predictions and prior studies with overexpressed proteins, we demonstrate here that PNPLA3 and PNPLA3- I148M are not endoplasmic reticulum- resident transmembrane proteins. To identify their intracellular associations, we generated a paired set of isogenic human hepatoma cells expressing PNPLA3 and PNPLA3- I148M at endogenous levels. Both proteins were enriched in lipid droplet, Golgi, and endosomal fractions. Purified PNPLA3 and PNPLA3- I148M proteins associated with phosphoinositides commonly found in these compartments. Despite a similar fractionation pattern as the wild- type variant, PNPLA3- I148M induced morphological changes in the Golgi apparatus, including increased lipid droplet--Golgi contact sites, which were also observed in I148M- expressing primary human patient hepatocytes. In addition to lipid droplet accumulation, PNPLA3- I148M expression caused significant proteomic and transcriptomic changes that resembled all stages of liver disease. Cumulatively, we validate an endogenous human cellular system for investigating PNPLA3- I148M biology and identify the Golgi apparatus as a central hub of PNPLA3- I148M- driven cellular change. [ABSTRACT FROM AUTHOR]

Antagonism of the glucagon receptor (GCGR) is associated with increased circulating levels of glucagon-like peptide-1 (GLP-1). To investigate the contribution of GLP-1 to the antidiabetic actions of GCGR antagonism, we administered an anti-GCGR monoclonal antibody (mAb B) to wild-type mice and GLP-1 receptor knockout (GLP-1R KO) mice. Treatment of wild-type mice with mAb B lowered fasting blood glucose, improved glucose tolerance, and enhanced glucose-stimulated insulin secretion during an intraperitoneal glucose tolerance test (ipGTT). In contrast, treatment of GLP-1R KO mice with mAb B had little efficacy during an ipGTr. Furthermore, pretreatment with the GLP-1R antagonist exendin-(9-39) diminished the antihyperglycemic effects of mAb B in wild-type mice. To determine the mechanism whereby mAb B improves glucose tolerance, we generated a monoclonal antibody that specifically antagonizes the human GLP-1R. Using a human islet transplanted mouse model, we demonstrated that pancreatic islet GLP-1R signaling is required for the full efficacy of the GCGR antagonist. To identify the source of the elevated GLP-1 observed in GCGR mAb-treated mice, we measured active GLP-1 content in pancreas and intestine from db/db mice treated with anti-GCGR mAb for 8 wk. Elevated GLP-1 in GCGR mAb-treated mice was predominantly derived from increased pancreatic GLP-1 synthesis and processing. All together, these data show that pancreatic GLP-1 is a significant contributor to the glucose-lowering effects observed in response to GCGR antagonist treatment. glucagon receptor; glucagon-like peptide-1; glucagon receptor antagonism; monoclonal antibody; type 2 diabetes; glucagon-like peptide-1 signal doi: 10.1152/ajpendo.00102.2010.

There is widespread interest in defining factors and mechanisms that stimulate proliferation of pancreatic islet cells. Wnt signaling is an important regulator of organ growth and cell fates, and genes encoding Wnt-signaling factors are expressed in the pancreas. However, it is unclear whether Wnt signaling regulates pancreatic islet proliferation and differentiation. Here we provide evidence that Wnt signaling stimulates islet [beta] cell proliferation. The addition of purified Wnt3a protein to cultured [beta] cells or islets promoted expression of Pitx2, a direct target of Wnt signaling, and Cyclin D2, an essential regulator of [beta] cell cycle progression, and led to increased [beta] cell proliferation in vitro. Conditional pancreatic [beta] cell expression of activated [beta]-catenin, a crucial Wnt signal transduction protein, produced similar phenotypes in vivo, leading to [beta] cell expansion, increased insulin production and serum levels, and enhanced glucose handling. Conditional [beta] cell expression of Axin, a potent negative regulator of Wnt signaling, led to reduced Pitx2 and Cyclin D2 expression by [beta] cells, resulting in reduced neonatal [beta] cell expansion and mass and impaired glucose tolerance. Thus, Wnt signaling is both necessary and sufficient for islet [beta] cell proliferation, and our study provides previously unrecognized evidence of a mechanism governing endocrine pancreas growth and function. Cyclin D2 | diabetes mellitus | islets of Langerhans | pancreas | Pitx2

The discovery of brown adipose tissue (BAT) as a key regulator of energy expenditure has sparked interest in identifying novel soluble factors capable of activating inducibleBAT(iBAT) to combat obesity. Using a high content cell-based screen, we identified fibroblast growth factor 16 (FGF16) as a potent inducer of several physical and transcriptional characteristics analogous to those of both "classical" BAT and iBAT. Overexpression of Fgf16 in vivo recapitulated several of our in vitro findings, specifically the significant induction of the Ucp1 gene and UCP1 protein expression in inguinal white adipose tissue (iWAT), a common site for emergent active iBAT. Despite significant UCP1 up-regulation in iWAT and dramatic weight loss, the metabolic improvements observed due to Fgf16 overexpression in vivo were not the result of increased energy expenditure, as measured by indirect calorimetric assessment. Instead, a pattern of reduced food and water intake, combined with feces replete with lipid and bile acid, indicated a phenotype more akin to that of starvation and intestinal malabsorption. Gene expression analysis of the liver and ileum indicated alterations in several steps of bile acid metabolism, including hepatic synthesis and reabsorption. Histological analysis of intestinal tissue revealed profound abnormalities in support of this conclusion. The in vivo data, together with FGF receptor binding analysis, indicate that the in vivo outcome observed is the likely result of both direct and indirect mechanisms and probably involves multiple receptors. These results highlight the complexity of FGF signaling in the regulation of various metabolic processes. [ABSTRACT FROM AUTHOR]

Pancreatic amyloid formation by islet amyloid polypeptide (IAPP) is a hallmark pathological feature of type 2 diabetes. IAPP is stored in the secretory granules of pancreatic beta-cells and co-secreted with insulin to maintain glucose homeostasis. IAPP is innocuous under homeostatic conditions but imbalances in production or processing of IAPP may result in homodimer formation leading to the rapid production of cytotoxic oligomers and amyloid fibrils. The consequence is beta-cell dysfunction and the accumulation of proteinaceous plaques in and around pancreatic islets. Beta-site APP-cleaving enzyme 2, BACE2, is an aspartyl protease commonly associated with BACE1, a related homolog responsible for amyloid processing in the brain and strongly implicated in Alzheimer’s disease. Herein, we identify two distinct sites of the mature human IAPP sequence that are susceptible to BACE2-mediated proteolytic activity. The result of proteolysis is modulation of human IAPP fibrillation and human IAPP protein degradation. These results suggest a potential therapeutic role for BACE2 in type 2 diabetes-associated hyperamylinaemia. [ABSTRACT FROM AUTHOR]

Elucidating the role of secreted frizzled-related protein 5 (SFRP5) in metabolism and obesity has been complicated by contradictory findings when knockout mice were used to determine metabolic phenotypes. By overexpressing SFRP5 in obese, prediabetic mice we consistently observed elevated hyperglycemia and glucose intolerance, supporting SFRP5 as a negative regulator of glucose metabolism. Accordingly, Sfrp5 mRNA expression analysis of both epididymal and subcutaneous adipose depots of mice indicated a correlation with obesity. Thus, we generated a monoclonal antibody (mAb) against SFRP5 to ascertain the effect of SFRP5 inhibition in vivo. Congruent with SFRP5 overexpression worsening blood glucose levels and glucose intolerance, anti-SFRP5 mAb therapy improved these phenotypes in vivo. The results from both the overexpression and mAb inhibition studies suggest a role for SFRP5 in glucose metabolism and pancreatic ß-cell function and thus establish the use of an anti-SFRP5 mAb as a potential approach to treat type 2 diabetes. [ABSTRACT FROM AUTHOR]

Antagonism of the glucagon receptor (GCGR) is associated with increased circulating levels of glucagon-like peptide-1 (GLP-1). To investigate the contribution of GLP-1 to the antidiabetic actions of GCGR antagonism, we administered an anti-GCGR monoclonal antibody (mAb B) to wild-type mice and GLP-1 receptor knockout (GLP-1R KO) mice. Treatment of wild-type mice with mAb B lowered fasting blood glucose, improved glucose tolerance, and enhanced glucose-stimulated insulin secretion during an intraperitoneal glucose tolerance test (ipGTT). In contrast, treatment of GLP-1R KO mice with mAb B had little efficacy during an ipGTT. Furthermore, pretreatment with the GLP-1R antagonist exendin-(9-39) diminished the antihyperglycemic effects of mAb B in wild-type mice. To determine the mechanism whereby mAb B improves glucose tolerance, we generated a monoclonal antibody that specifically antagonizes the human GLP-1R. Using a human islet transplanted mouse model, we demonstrated that pancreatic islet GLP-1R signaling is required for the full efficacy of the GCGR antagonist. To identify the source of the elevated GLP-1 observed in GCGR mAb-treated mice, we measured active GLP-1 content in pancreas and intestine from db/db mice treated with anti-GCGR mAb for 8 wk. Elevated GLP-1 in GCGR mAb-treated mice was predominantly derived from increased pancreatic GLP-1 synthesis and processing. All together, these data show that pancreatic GLP-1 is a significant contributor to the glucose-lowering effects observed in response to GCGR antagonist treatment. [ABSTRACT FROM AUTHOR]

Several studies have shown that the adult pancreas possesses a limited potential for beta-cell regeneration upon tissue injury. One of the difficulties in studying beta-cell regeneration has been the lack of a robust, synchronized animal model system that would allow controlled regulation of beta-cell loss and subsequent proliferation in adult pancreas. Here we present a transgenic mouse regeneration model in which the c-Myc transcription factor/mutant estrogen receptor (cMycER(TAM)) fusion protein can be specifically activated in mature beta-cells. We have studied these transgenic mice by immunohistochemical and biochemical methods to assess the ablation and posterior regeneration of beta-cells. Activation of the cMycER(TAM) fusion protein results in synchronous and selective beta-cell apoptosis followed by the onset of acute diabetes. Inactivation of c-Myc leads to gradual regeneration of insulin-expressing cells and reversal of diabetes. Our results demonstrate that the mature pancreas has the ability to fully recover from almost complete ablation of all existing beta-cells. Our results also suggest the regeneration of beta-cells is mediated by replication of beta-cells rather than neogenesis from pancreatic ducts. [ABSTRACT FROM AUTHOR]

The article presents a study which explores how KLF15 (Kruppel-like factor 15) cistromes reveal a hepatocyte pathway governing plasma corticosteroid transport and systemic inflammation. It provide critical mechanistic insight into KLF15 function and identify a hepatocyte-intrinsic transcriptional module that potently regulates systemic corticosteroid transport and inflammation.

Circulating corticosteroids orchestrate stress adaptation, including inhibition of inflammation. While pathways governing corticosteroid biosynthesis and intracellular signaling are well understood, less is known about mechanisms controlling plasma corticosteroid transport. Here, we show that hepatocyte KLF15 (Kruppel-like factor 15) controls plasma corticosteroid transport and inflammatory responses through direct transcriptional activation of Serpina6 , which encodes corticosteroid-binding globulin (CBG). Klf15 -deficient mice have profoundly low CBG, reduced plasma corticosteroid binding capacity, and heightened mortality during inflammatory stress. These defects are completely rescued by reconstituting CBG, supporting that KLF15 works primarily through CBG to control plasma corticosterone homeostasis. To understand transcriptional mechanisms, we generated the first KLF15 cistromes using newly engineered Klf15 3xFLAG mice. Unexpectedly, liver KLF15 is predominantly promoter enriched, including Serpina6 , where it binds a palindromic GC-rich motif, opens chromatin, and transactivates genes with minimal associated direct gene repression. Overall, we provide critical mechanistic insight into KLF15 function and identify a hepatocyte-intrinsic transcriptional module that potently regulates systemic corticosteroid transport and inflammation.

The polarization of tissue resident macrophages toward the alternatively activated, anti-inflammatory M2 phenotype is believed to positively impact obesity and insulin resistance. Here we show that the soluble form of the extracellular domain (ECD) of C-type lectin-like receptor 2, CLEC2, regulates Kupffer cell polarization in the liver and improves glucose and lipid parameters in diabetic animal models. Over-expression of Fc-CLEC2(ECD) in mice via in vivo gene delivery, or injection of recombinant Fc-CLEC2(ECD) protein, results in a reduction of blood glucose and liver triglyceride levels and improves glucose tolerance. Furthermore, Fc-CLEC2(ECD) treatment improves cytokine profiles and increases both the M2 macrophage population and the genes involved in the oxidation of lipid metabolism in the liver. These data reveal a previously unidentified role for CLEC2 as a regulator of macrophage polarity, and establish CLEC2 as a promising therapeutic target for treatment of diabetes and liver disease.