MAF bZIP transcription factor A (MafA) transactivates multiple genes involved in β-cell maturity function. MafA transactivation is increased by recruitment and binding of multiple lysine acetyltransferases, Kat2b and CBP, but mechanisms underlying this phenomenon are unclear. We performed a LC-MS/MS screen and validation experiments that revealed that Kat2b and CBP increased MafA acetylation at lysines (K) 32, 33 and 255. To understand functional consequences of MafA acetylation, we mutated all three to glutamine (3KQ) or arginine (3KR) to mimic constitutively acetylated and deacetylated MafA, respectively, and performed luciferase assays with these mimetics that showed increased transactivation ability at the insulin promoter by 3KQ, independent of stability, DNA binding or MafA localization. Intriguingly, 3KQ MafA also showed retarded mobility on SDS-PAGE, suggesting that acetylation induced another post-translational modification (PTM). In a second LC-MS/MS screen followed by alanine scanning, we identified that acetylation increased MafA phosphorylation at serine (S) 14. Thus, S14A mutation attenuated the effects of acetylation. Simultaneously, MafA acetylation at K32 prevents SUMOylation at the same site; thus, inhibition of SUMOylation increased MafA acetylation and activity. Overall, our findings suggest that acetylation induces a novel PTM switch in MafA - enhancing S14 phosphorylation and blocking K32 SUMOylation, to potentiate transactivation at target promoters. Disclosure M. Chirikjian: None. J.Z. Liang: None. A. Bartolomé: None. R. Soni: None. R. Stein: None. U. Pajvani: None.

ObjectiveNotch signaling, re-activated in β cells from obese mice and causal to β cell dysfunction, is determined in part by transmembrane ligand availability in a neighboring cell. We hypothesized that β cell expression of Jagged1 determines the maladaptive Notch response and resultant insulin secretory defects in obese mice.MethodsWe assessed expression of Notch pathway components in high-fat diet-fed (HFD) or leptin receptor-deficient (db/db) mice, and performed single-cell RNA sequencing (scRNA-Seq) in islets from patients with and without type 2 diabetes (T2D). We generated and performed glucose tolerance testing in inducible, β cell-specific Jagged1 gain-of- and loss-of-function mice. We also tested effects of monoclonal neutralizing antibodies to Jagged1 in glucose-stimulated insulin secretion (GSIS) assays in isolated islets.ResultsJag1 was the only Notch ligand that tracked with increased Notch activity in HFD-fed and db/db mice, as well as in metabolically-inflexible β cells enriched in patients with T2D. Neutralizing antibodies to block Jagged1 in islets isolated from HFD-fed and db/db mice potentiated GSIS ex vivo. To demonstrate if β cell Jagged1 is sufficient to cause glucose tolerance in vivo, we generated inducible β cell-specific Jag1 transgenic (β-Jag1TG) and loss-of-function (iβ-Jag1KO) mice. While forced Jagged1 impaired glucose intolerance due to reduced GSIS, loss of β cell Jagged1 did not protect against HFD-induced insulin secretory defects.ConclusionsJagged1 is increased in islets from obese mice and in patients with T2D, and neutralizing Jagged1 antibodies lead to improved GSIS, suggesting that inhibition of Jagged1-Notch signaling may have therapeutic benefit. However, genetic loss-of-function experiments suggest that β cells are not a likely source of the Jagged1 signal.

Objective: Notch signaling, re-activated in (3 cells from obese mice and causal to (3 cell dysfunction, is determined in part by transmembrane ligand availability in a neighboring cell. We hypothesized that (3 cell expression of Jagged1 determines the maladaptive Notch response and resultant insulin secretory defects in obese mice. Methods: We assessed expression of Notch pathway components in high-fat diet-fed (HFD) or leptin receptor-deficient (db/db) mice, and performed single-cell RNA sequencing (scRNA-Seq) in islets from patients with and without type 2 diabetes (T2D). We generated and performed glucose tolerance testing in inducible, (3 cell-specific Jagged1 gain-of- and loss-of-function mice. We also tested effects of monoclonal neutralizing antibodies to Jagged1 in glucose-stimulated insulin secretion (GSIS) assays in isolated islets. Results: Jag1 was the only Notch ligand that tracked with increased Notch activity in HFD-fed and db/db mice, as well as in metabolicallyinflexible (3 cells enriched in patients with T2D. Neutralizing antibodies to block Jagged1 in islets isolated from HFD-fed and db/db mice potentiated GSIS ex vivo. To demonstrate if (3 cell Jagged1 is sufficient to cause glucose tolerance in vivo, we generated inducible (3 cell-specific Jag1 transgenic ((3-Jag1TG) and loss-of-function (i(3-Jag1KO) mice. While forced Jagged1 impaired glucose intolerance due to reduced GSIS, loss of (3 cell Jagged1 did not protect against HFD-induced insulin secretory defects. Conclusions: Jagged1 is increased in islets from obese mice and in patients with T2D, and neutralizing Jagged1 antibodies lead to improved GSIS, suggesting that inhibition of Jagged1-Notch signaling may have therapeutic benefit. However, genetic loss-of-function experiments suggest that (3 cells are not a likely source of the Jagged1 signal. (c) 2024 The Author(s). Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Patients with nonalcoholic steatohepatitis (NASH) have increased expression of liver monocyte chemoattractant protein-1 (MCP-1), but its cellular source and contribution to various aspects of NASH pathophysiology remain debated. We demonstrated increased liver CCL2 (which encodes MCP-1) expression in patients with NASH, and commensurately, a 100-fold increase in hepatocyte Ccl2 expression in a mouse model of NASH, accompanied by increased liver monocyte-derived macrophage (MoMF) infiltrate and liver fibrosis. To test repercussions of increased hepatocyte-derived MCP-1, we generated hepatocyte-specific Ccl2-knockout mice, which showed reduced liver MoMF infiltrate as well as decreased liver fibrosis. Forced hepatocyte MCP-1 expression provoked the opposite phenotype in chow-fed wild-type mice. Consistent with increased hepatocyte Notch signaling in NASH, we observed a close correlation between markers of Notch activation and CCL2 expression in patients with NASH. We found that an evolutionarily conserved Notch/recombination signal binding protein for immunoglobulin kappa J region binding site in the Ccl2 promoter mediated transactivation of the Ccl2 promoter in NASH diet-fed mice. Increased liver MoMF infiltrate and liver fibrosis seen in opposite gain-of-function mice was ameliorated with concomitant hepatocyte Ccl2 knockout or CCR2 inhibitor treatment. Hepatocyte Notch activation prompts MCP-1-dependent increase in liver MoMF infiltration and fibrosis.

Pancreatic beta cells play a critical role in maintaining glucose homeostasis by serving as the primary source of insulin [...].

The increase of functional β cell mass is paramount to maintain glucose homeostasis in the setting of systemic insulin resistance and/or augmented metabolic load. Understanding compensatory mechanisms that allow β cell mass adaptation may allow discovery of therapeutically actionable control nodes. In this study, we report the rapid and robust β cell hyperplasic effect in a mouse model of overfeeding-induced obesity (OIO) based on direct gastric caloric infusion. By performing RNA sequencing in islets isolated from OIO mice, we identified Sin3a as a novel transcriptional regulator of β cell mass adaptation. β cell-specific Sin3a knockout animals showed profound diabetes, due to defective acquisition of postnatal β cell mass. These findings reveal a novel regulatory pathway in β cell proliferation, and validate OIO as a model for discovery of other mechanistic determinants to β cell adaptation.

Altered islet architecture is associated with β cell dysfunction and type 2 diabetes (T2D) progression, but molecular effectors of islet spatial organization remain mostly unknown. Although Notch signaling is known to regulate pancreatic development, we observed "reactivated" β cell Notch activity in obese mouse models. To test the repercussions and reversibility of Notch effects, we generated doxycycline-dependent, β cell-specific Notch gain-of-function mice. As predicted, we found that Notch activation in postnatal β cells impaired glucose-stimulated insulin secretion and glucose intolerance, but we observed a surprising remnant glucose intolerance after doxycycline withdrawal and cessation of Notch activity, associated with a marked disruption of normal islet architecture. Transcriptomic screening of Notch-active islets revealed increased Ephrin signaling. Commensurately, exposure to Ephrin ligands increased β cell repulsion and impaired murine and human pseudoislet formation. Consistent with our mouse data, Notch and Ephrin signaling were increased in metabolically inflexible β cells in patients with T2D. These studies suggest that β cell Notch/Ephrin signaling can permanently alter islet architecture during a morphogenetic window in early life.

β-cells are insulin-producing cells in the pancreas that maintain euglycemic conditions. Pancreatic β-cell maturity and function are regulated by a variety of transcription factors that enable the adequate expression of the cellular machinery involved in nutrient sensing and commensurate insulin secretion. One of the key factors in this regulation is MAF bZIP transcription factor A (MafA). MafA expression is decreased in type 2 diabetes, contributing to β-cell dysfunction and disease progression. The molecular biology underlying MafA is complex, with numerous transcriptional and post-translational regulatory nodes. Understanding these complexities may uncover potential therapeutic targets to ameliorate β-cell dysfunction. This article will summarize the role of MafA in normal β-cell function and disease, with a special focus on known transcriptional and post-translational regulators of MafA expression.

Pancreatic β cell dysfunction is a central component of diabetes progression. During the last decades, the genetic basis of several monogenic forms of diabetes has been recognized. Genome-wide association studies (GWAS) have also facilitated the identification of common genetic variants associated with an increased risk of diabetes. These studies highlight the importance of impaired β cell function in all forms of diabetes. However, how most of these risk variants confer disease risk, remains unanswered. Understanding the specific contribution of genetic variants and the precise role of their molecular effectors is the next step toward developing treatments that target β cell dysfunction in the era of personalized medicine. Protocols that allow derivation of β cells from pluripotent stem cells, represent a powerful research tool that allows modeling of human development and versatile experimental designs that can be used to shed some light on diabetes pathophysiology. This article reviews different models to study the genetic basis of β cell dysfunction, focusing on the recent advances made possible by stem cell applications in the field of diabetes research.

Beta cells are exquisite sensors of the organism’s nutritional status, and a key metabolic regulator via insulin secretion. Systemic insulin requirements vary during the day, but also through life in response to pathophysiological circumstances such as pregnancy or the insulin resistance associated with obesity. These changes impose a burden on beta cells, which must adapt to conditions of higher metabolic load by increasing insulin synthesis and secretion. This adaptation can be achieved by increases in beta cell mass (hyperplasia and hypertrophy), as well as enhanced function per unit of mass. The key molecular machinery that allows beta cell flexibility is the mechanistic target of rapamycin (MTOR). MTOR, an evolutionarily conserved serine/ threonine kinase, exists in 1 of 2 complexes defined by regulatory and targeting subunits Raptor (defining MTORC1) or Rictor (MTORC2). Of these, MTORC1 has emerged as a key sensor of the energetic and nutritional status of the cell, integrating hormonal and metabolic cues. This balance provides regulation of both anabolic and catabolic processes, such as protein synthesis and degradation, and decision trees involved in cell growth and proliferation (1). Specifically, MTORC1 and its upstream signaling regulators are necessary and sufficient to induce beta cell mass adaptation to stress in rodent models (2, 3). These findings had a translational counterpoint in the known toxic effect of MTORC1 inhibition, commonly used in immunosuppressant regimens following solid organ transplantation, on human islets. MTORC1 inhibition is likely causal to delayed post-transplantation diabetes (4), and may complicate interpretations of success of islet transplantation following the Edmonton Protocol (5). Whether these compelling rodent and pharmacologic data, showing MTORC1 effects on beta cell biology, are also seen in progression of human type 2 diabetes (T2D) was unknown. To address this important question, in this issue of the Journal of Clinical Endocrinology & Metabolism, Ni and colleagues perform histological characterization of MTORC1 activity in beta cells in patients undergoing partial pancreatectomy, presumably for evaluation of a suspicious pancreatic mass (6). After exclusion of patients with a diagnosis of a malignant tumor, the authors enrolled body mass index–matched subjects who were nondiabetic (ND), had impaired fasting glucose (IFG), or had a clinical history of T2D. Although higher than ND subjects, glycated hemoglobin levels were similar and not statistically different in IFG (6.1%) and T2D (6.4%) groups, suggesting the T2D patients were very well controlled. By staining paraffinfixed sections of pancreatic tissue with an antibody corresponding to a downstream readout of MTORC1 activity (phosphorylation of ribosomal protein S6), the authors determined that beta cell MTORC1 activity positively correlated with fasting blood glucose in ND subjects, but not subjects with IFG or T2D. Intriguingly, beta cell MTORC1 activity was significantly increased in patients with IFG (but not T2D) when compared with ND. A stratification of the IFG cases according to their MTORC1 activity found that IFG patients with higher MTORC1 activity showed greater retention of markers of beta cell functional maturity, such as UCN3 and GLUT2, as compared to patients with lower MTORC1 activity. Overall, these results suggest

Increased Jagged1 in pericentral hepatocytes causes liver fibrosis in response to hepatocyte TLR4–NF-κB pathway activation in NASH.

Angiotensin converting enzyme 2 (ACE2) is a key regulator of the renin-angiotensin system, but also the functional receptor of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Based on structural similarity with other γ-secretase (γS) targets, we hypothesized that ACE2 may be affected by γS proteolytic activity. We found that after ectodomain shedding, ACE2 is targeted for intramembrane proteolysis by γS, releasing a soluble ACE2 C-terminal fragment. Consistently, chemical or genetic inhibition of γS results in the accumulation of a membrane-bound fragment of ectodomain-deficient ACE2. Although chemical inhibition of γS does not alter SARS-CoV-2 cell entry, these data point to a novel pathway for cellular ACE2 trafficking.

EIF2AK4, which encodes the amino acid deficiency-sensing protein GCN2, has been implicated as a susceptibility gene for type 2 diabetes in the Japanese population. However, the mechanism by which GCN2 affects glucose homeostasis is unclear. Here, we show that insulin secretion is reduced in individuals harboring the risk allele of EIF2AK4 and that maintenance of GCN2-deficient mice on a high-fat diet results in a loss of pancreatic β cell mass. Our data suggest that GCN2 senses amino acid deficiency in β cells and limits signaling by mechanistic target of rapamycin complex 1 to prevent β cell failure during the consumption of a high-fat diet.

BACKGROUND & AIMS:The hepatocyte Notch pathway is a pathogenic factor in non-alcoholic steatohepatitis (NASH)-associated fibrosis, but its role in hepatocellular carcinoma (HCC) is less well defined. Herein, we aimed to characterize the molecular and clinical features of Notch-active human HCC, and to investigate the mechanisms by which Notch affects NASH-driven HCC. METHODS:Using a 14-gene Notch score, we stratified human HCCs from multiple comprehensively profiled datasets. We performed gene set enrichment analyses to compare Notch-active HCCs with published HCC subtype signatures. Next, we sorted Notch-active hepatocytes from Notch reporter mice for RNA sequencing and characterized Notch-active tumors in an HCC model combining a carcinogen and a NASH-inducing diet. We used genetic mouse models to manipulate hepatocyte Notch to investigate the sufficiency and necessity of Notch in NASH-driven tumorigenesis. RESULTS:Notch-active signatures were found in ~30% of human HCCs that transcriptionally resemble cholangiocarcinoma-like HCC, exhibiting a lack of activating CTNNB1 (β-catenin) mutations and a generally poor prognosis. Endogenous Notch activation in hepatocytes is associated with repressed β-catenin signaling and hepatic metabolic functions, in lieu of increased interactions with the extracellular matrix in NASH. Constitutive hepatocyte Notch activation is sufficient to induce β-catenin-inactive HCC in mice with NASH. Notch and β-catenin show a pattern of mutual exclusivity in carcinogen-induced HCC; in this mouse model, chronic blockade of Notch led to β-catenin-dependent tumor development. CONCLUSIONS:Notch activity characterizes a distinct HCC molecular subtype with unique histology and prognosis. Sustained Notch signaling in chronic liver diseases can drive tumor formation without acquiring specific genomic driver mutations. LAY SUMMARY:The Notch signaling pathway is known to be involved in the pathogenesis of liver fibrosis. However, its role in liver cancer has not been well defined. Herein, we show that Notch activity is increased in a subset of liver cancers and is associated with poor outcomes. We also used a mouse model to show that aberrant Notch activity can drive cancer progression in obese mice.

Energy sensing is indispensable to balance anabolic and catabolic processes for the maintenance of cell viability. Pancreatic β cells are especially relevant because of their involvement in the coordination of insulin secretion when glucose concentration arises in the local milieu. In this work, we uncover the increased susceptibility of pancreatic β cells to cell death in response to different energy stressors. Upon glucose decline, from 25 to 5 mM, caused by stimulation with either 2-deoxyglucose or metformin, only pancreatic β cells showed an increase in cell death. Very interestingly, when we transfected either mouse insulinoma cell or human embryo kidney cells with a phospho-mutant form of B cell lymphoma 2 associated agonist of cell death at serine 155 (BAD S155D), an increase in the pro-survival factor B cell lymphoma 2 was detected in pancreatic β cells and not in human embryonic kidney cells in the presence of the energetic stressors. This data suggests that the protective capacity of this mutant form is only present in cells that present glucokinase. In contrast, upon hyperactivation of mechanistic target of rapamycin complex 1 signaling by knocking-down tuberous sclerosis complex protein, we observed increased susceptibility to cell death in response to energy stress in both pancreatic and non-pancreatic β cells. Therefore, mechanistic target of rapamycin complex 1 signaling presents a dual effect on cell viability. On the one hand, a chronic inhibition of mechanistic target of rapamycin complex 1 activity in response to the energy status is deleterious for pancreatic β cells, being attenuated by the overexpression of B cell lymphoma 2 associated agonist of cell death S155D. On the other hand, mechanistic target of rapamycin complex 1 hyperactivity provokes a susceptibility to energetic stress-induced cell death. Taken together, these results may open potential implications for the use of glucokinase activators or mechanistic target of rapamycin complex 1 modulators for the maintenance of pancreatic β cells for longer periods of time avoiding its loss in different pathologies such as type 2 diabetes mellitus.

Although β cell dysfunction is a well-appreciated contributor to type 2 diabetes (T2D) pathophysiology, determinants of β cell function are incompletely understood. For instance, disrupted islet architecture is associated with T2D progression in humans and rodent models, but the identity of the molecular elements that govern islet architecture remain mostly unknown. Nevertheless, data suggest that the spatial arrangement of the different cell types that form the endocrine islets influences cell-to-cell communication and β cell function. The Notch pathway transduces signals between neighboring cells in pancreas development, then thought to be silenced in the developed endocrine islet. However, recent reports have uncovered a maladaptive Notch response in adult β cells in T2D patients and obese rodent models. To test the repercussions of Notch activity, we created β cell-specific gain-of-function mice that express a constitutively-active Notch intracellular domain (β-NICD). β-NICD mice show a profound disorganization of islet architecture, loss of β cell maturity and marked glucose intolerance. Importantly, we generated a second model that allows doxycycline-dependent expression of NICD (β-TetO-NICD), which recapitulates the phenotypes of β-NICD mice, even when NICD is induced in adulthood. Upon doxycycline withdrawal, β-TetO-NICD mice recover markers of β cell maturity, but disrupted islet architecture and glucose intolerance persist in β-TetO-NICD mice as compared to controls. To assess the molecular mechanisms of these findings, we performed unbiased transcriptomic screening of adult islets, which identified differentially expressed pathways that regulate cell-to-cell communication in different systems. These studies uncover β cell Notch signaling as a novel determinant of islet architecture, and suggest that Notch inhibitors repurposed from oncology pipelines may have potential application to correct obesity-related β cell dysfunction in patients with T2D. Disclosure A. Bartolome: None. U. Pajvani: Research Support; Self; Sanofi. Funding American Diabetes Association (1-17-PMF-025 to A.B.)

Fibrosis is the major determinant of morbidity and mortality in patients with nonalcoholic steatohepatitis (NASH) but has no approved pharmacotherapy in part because of incomplete understanding of its pathogenic mechanisms. Here, we report that hepatocyte Notch activity tracks with disease severity and treatment response in patients with NASH and is similarly increased in a mouse model of diet-induced NASH and liver fibrosis. Hepatocyte-specific Notch loss-of-function mouse models showed attenuated NASH-associated liver fibrosis, demonstrating causality to obesity-induced liver pathology. Conversely, forced activation of hepatocyte Notch induced fibrosis in both chow- and NASH diet-fed mice by increasing Sox9-dependent Osteopontin (Opn) expression and secretion from hepatocytes, which activate resident hepatic stellate cells. In a cross-sectional study, we found that OPN explains the positive correlation between liver Notch activity and fibrosis stage in patients. Further, we developed a Notch inhibitor [Nicastrin antisense oligonucleotide (Ncst ASO)] that reduced fibrosis in NASH diet-fed mice. In summary, these studies demonstrate the pathological role and therapeutic accessibility of the maladaptive hepatocyte Notch response in NASH-associated liver fibrosis.

Weight is defended so that increases or decreases in body mass elicit responses that favor restoration of one's previous weight. While much is known about the signals that respond to weight loss and the central role that leptin plays, the lack of experimental systems studying the overfed state has meant little is known about pathways defending against weight gain. We developed a system to study this physiology and found that overfed mice defend against increased weight gain with graded anorexia but, unlike weight loss, this response is independent of circulating leptin concentration. In overfed mice that are unresponsive to orexigenic stimuli, adipose tissue is transcriptionally and immunologically distinct from fat of ad libitum-fed obese animals. These findings provide evidence that overfeeding-induced obesity alters adipose tissue and central responses in ways that are distinct from ad libitum obesity and activates a non-leptin system to defend against weight gain.