Calorie restriction activates a gastric Notch-FOXO1 pathway to expand ghrelin cells.

Calorie restriction increases lifespan. Among the tissue-specific protective effects of calorie restriction, the impact on the gastrointestinal tract remains unclear. We report increased numbers of chromogranin A-positive (+), including orexigenic ghrelin+ cells, in the stomach of calorie-restricted mice. This effect was accompanied by increased Notch target Hes1 and Notch ligand Jag1 and was reversed by blocking Notch with DAPT, a gamma-secretase inhibitor. Primary cultures and genetically modified reporter mice show that increased endocrine cell abundance is due to altered Lgr5+ stem and Neurog3+ endocrine progenitor cell proliferation. Different from the intestine, calorie restriction decreased gastric Lgr5+ stem cells, while increasing a FOXO1/Neurog3+ subpopulation of endocrine progenitors in a Notch-dependent manner. Further, activation of FOXO1 was sufficient to promote endocrine cell differentiation independent of Notch. The Notch inhibitor PF-03084014 or ghrelin receptor antagonist GHRP-6 reversed the phenotypic effects of calorie restriction in mice. Tirzepatide additionally expanded ghrelin+ cells in mice. In summary, calorie restriction promotes Notch-dependent, FOXO1-regulated gastric endocrine cell differentiation.

Protective hepatocyte signals restrain liver fibrosis in metabolic dysfunction-associated steatohepatitis.

Metabolic dysfunction-associated steatotic liver disease (MASLD) affects nearly 40% of the global adult population and may progress to metabolic dysfunction-associated steatohepatitis (MASH), and MASH-associated liver fibrosis and cirrhosis. Despite numerous studies unraveling the mechanism of hepatic fibrogenesis, there are still no approved antifibrotic therapies. The development of MASLD and liver fibrosis results from complex cell-cell interactions that often initiate within hepatocytes but remain incompletely understood. In this issue of the JCI, Yan and colleagues describe an ATF3/HES1/CEBPA/OPN pathway that links hepatocyte signals to fibrogenic activation of hepatic stellate cells and may provide new perspectives on therapeutic options for MASLD-induced liver fibrosis.

The first MASH drug therapy on the horizon: Current perspectives of resmetirom.

The rising prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD) poses a significant global health challenge, affecting over 30% of adults worldwide. MASLD is linked to increased mortality rates and substantial healthcare costs, primarily driven by its progression to metabolic dysfunction-associated steatohepatitis (MASH), which can lead to severe liver complications including cirrhosis and hepatocellular carcinoma. Despite its growing burden, effective pharmacotherapy for MASLD/MASH has been lacking until the recent conditional approval of resmetirom by the FDA. Resmetirom, a liver-targeted thyroid hormone receptor-ß selective drug, has shown promise in clinical trials for treating non-cirrhotic MASH with moderate to advanced fibrosis. It has demonstrated efficacy in reducing hepatic fat content, improving liver histology (both MASH resolution and fibrosis improvement), and ameliorating biomarkers of liver damage without significant effects on body weight or glucose metabolism. Notably, resmetirom also exhibits favourable effects on circulating lipids, potentially reducing cardiovascular risk in MASLD/MASH patients. The safety profile of resmetirom appears acceptable, with gastrointestinal adverse events being the most common, though generally mild or moderate. However, long-term surveillance is warranted to monitor for potential risks related to thyroid, gonadal, or bone diseases. Clinical implementation of resmetirom faces challenges in patient selection and monitoring treatment response, and will heavily rely on non-invasive tests for liver fibrosis assessment. Nonetheless, resmetirom represents a landmark breakthrough in MASLD/MASH treatment, paving the way for future therapeutic strategies aiming to mitigate the multifaceted risks associated with this complex metabolic liver disease.

ß-cell Jagged1 is sufficient but not necessary for islet Notch activity and insulin secretory defects in obese mice.

OBJECTIVE: Notch signaling, re-activated in ß cells from obese mice and causal to ß cell dysfunction, is determined in part by transmembrane ligand availability in a neighboring cell. We hypothesized that ß 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, ß 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 metabolically-inflexible ß 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 ß cell Jagged1 is sufficient to cause glucose tolerance in vivo, we generated inducible ß cell-specific Jag1 transgenic (ß-Jag1TG) and loss-of-function (iß-Jag1KO) mice. While forced Jagged1 impaired glucose intolerance due to reduced GSIS, loss of ß 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 ß cells are not a likely source of the Jagged1 signal.

Liver insulinization as a driver of triglyceride dysmetabolism.

Metabolic dysfunction-associated fatty liver disease (MAFLD) is an increasingly prevalent fellow traveller with the insulin resistance that underlies type 2 diabetes mellitus. However, the mechanistic connection between MAFLD and impaired insulin action remains unclear. In this Perspective, we review data from humans to elucidate insulin's aetiological role in MAFLD. We focus particularly on the relative preservation of insulin's stimulation of triglyceride (TG) biosynthesis despite its waning ability to curb hepatic glucose production (HGP). To explain this apparent 'selective insulin resistance', we propose that hepatocellular processes that lead to TG accumulation require less insulin signal transduction, or 'insulinization,' than do those that regulate HGP. As such, mounting hyperinsulinaemia that barely compensates for aberrant HGP in insulin-resistant states more than suffices to maintain hepatic TG biosynthesis. Thus, even modestly elevated or context-inappropriate insulin levels, when sustained day and night within a heavily pro-lipogenic metabolic milieu, may translate into substantial cumulative TG biosynthesis in the insulin-resistant state.

Calorie Restriction activates a gastric Notch-FOXO1 pathway to expand Ghrelin cells.

Calorie restriction increases lifespan. While some tissue-specific protective effects of calorie restriction have been described, the impact of calorie restriction on the gastrointestinal tract remains unclear. We found increased abundance of chromogranin A+, including orexigenic ghrelin+, endocrine cells in the stomach of calorie-restricted mice. This effect coincided with increased Notch target Hes1 and Notch ligand Jag1 and was reversed when Notch signaling was blocked using the γ-secretase inhibitor DAPT. Using primary cultures and genetically-modified reporter mice, we determined that increased endocrine cell abundance was due to altered stem and progenitor proliferation. Different from the intestine, calorie restriction decreased gastric Lgr5+ stem cells, while increasing a FOXO1/Neurog3+ subpopulation of endocrine progenitors in a Notch-dependent manner. Further, calorie restriction triggered nuclear localization of FOXO1, which was sufficient to promote endocrine cell differentiation. Taken together, the data indicate that calorie restriction promotes gastric endocrine cell differentiation triggered by active Notch signaling and regulated by FOXO1.

Notch-mediated hepatocyte MCP-1 secretion causes liver fibrosis.

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.

Hepatocyte Kctd17 Inhibition Ameliorates Glucose Intolerance and Hepatic Steatosis Caused by Obesity-induced Chrebp Stabilization.

BACKGROUND & AIMS: Obesity predisposes to type 2 diabetes (T2D) and nonalcoholic fatty liver disease (NAFLD), but underlying mechanisms are incompletely understood. Potassium channel tetramerization domain-containing protein 17 (Kctd17) levels are increased in livers from obese mice and humans. In this study, we investigated the mechanism of increased Kctd17 and whether it is causal to obesity-induced metabolic complications. METHODS: We transduced Rosa26-LSL-Cas9 knockin mice with AAV8-TBG-Cre (Control), AAV8-U6-Kctd17 sgRNA-TBG-Cre (L-Kctd17), AAV8-U6-Oga sgRNA-TBG-Cre (L-Oga), or AAV8-U6-Kctd17/Oga sgRNA-TBG-Cre (DKO). We fed mice a high-fat diet (HFD) and assessed for hepatic glucose and lipid homeostasis. We generated Kctd17, O-GlcNAcase (Oga), or Kctd17/Oga-knockout hepatoma cells by CRISPR-Cas9, and Kctd17-directed antisense oligonucleotide to test therapeutic potential in vivo. We analyzed transcriptomic data from patients with NAFLD. RESULTS: Hepatocyte Kctd17 expression was increased in HFD-fed mice due to increased Srebp1c activity. HFD-fed L-Kctd17 or Kctd17 antisense oligonucleotide-treated mice show improved glucose tolerance and hepatic steatosis, whereas forced Kctd17 expression caused glucose intolerance and hepatic steatosis even in lean mice. Kctd17 induced Oga degradation, resulting in increasing carbohydrate response element-binding protein (Chrebp) protein, so concomitant Oga knockout negated metabolic benefits of hepatocyte Kctd17 deletion. In patients with NAFLD, KCTD17 messenger RNA was positively correlated with expression of Chrebp target and other lipogenic genes. CONCLUSIONS: Srebp1c-induced hepatocyte Kctd17 expression in obesity disrupted glucose and lipid metabolism by stabilizing Chrebp, and may represent a novel therapeutic target for obesity-induced T2D and NAFLD.

PPARγ Acetylation Orchestrates Adipose Plasticity and Metabolic Rhythms.

Systemic glucose metabolism and insulin activity oscillate in response to diurnal rhythms and nutrient availability with the necessary involvement of adipose tissue to maintain metabolic homeostasis. However, the adipose-intrinsic regulatory mechanism remains elusive. Here, the dynamics of PPARγ acetylation in adipose tissue are shown to orchestrate metabolic oscillation in daily rhythms. Acetylation of PPARγ displays a diurnal rhythm in young healthy mice, with the peak at zeitgeber time 0 (ZT0) and the trough at ZT18. This rhythmic pattern is deranged in pathological conditions such as obesity, aging, and circadian disruption. The adipocyte-specific acetylation-mimetic mutation of PPARγ K293Q (aKQ) restrains adipose plasticity during calorie restriction and diet-induced obesity, associated with proteolysis of a core circadian component BMAL1. Consistently, the rhythmicity in glucose tolerance and insulin sensitivity is altered in aKQ and the complementary PPARγ deacetylation-mimetic K268R/K293R (2KR) mouse models. Furthermore, the PPARγ acetylation-sensitive downstream target adipsin is revealed as a novel diurnal factor that destabilizes BMAL1 and mediates metabolic rhythms. These findings collectively signify that PPARγ acetylation is a hinge connecting adipose plasticity and metabolic rhythms, the two determinants of metabolic health.

Opposing roles of hepatic stellate cell subpopulations in hepatocarcinogenesis.

Hepatocellular carcinoma (HCC), the fourth leading cause of cancer mortality worldwide, develops almost exclusively in patients with chronic liver disease and advanced fibrosis1,2. Here we interrogated functions of hepatic stellate cells (HSCs), the main source of liver fibroblasts3, during hepatocarcinogenesis. Genetic depletion, activation or inhibition of HSCs in mouse models of HCC revealed their overall tumour-promoting role. HSCs were enriched in the preneoplastic environment, where they closely interacted with hepatocytes and modulated hepatocarcinogenesis by regulating hepatocyte proliferation and death. Analyses of mouse and human HSC subpopulations by single-cell RNA sequencing together with genetic ablation of subpopulation-enriched mediators revealed dual functions of HSCs in hepatocarcinogenesis. Hepatocyte growth factor, enriched in quiescent and cytokine-producing HSCs, protected against hepatocyte death and HCC development. By contrast, type I collagen, enriched in activated myofibroblastic HSCs, promoted proliferation and tumour development through increased stiffness and TAZ activation in pretumoural hepatocytes and through activation of discoidin domain receptor 1 in established tumours. An increased HSC imbalance between cytokine-producing HSCs and myofibroblastic HSCs during liver disease progression was associated with increased HCC risk in patients. In summary, the dynamic shift in HSC subpopulations and their mediators during chronic liver disease is associated with a switch from HCC protection to HCC promotion.

An Overfeeding-Induced Obesity Mouse Model Reveals Necessity for Sin3a in Postnatal Peak ß-Cell Mass Acquisition.

The increase of functional ß-cell mass is paramount to maintaining glucose homeostasis in the setting of systemic insulin resistance and/or augmented metabolic load. Understanding compensatory mechanisms that allow ß-cell mass adaptation may allow for the discovery of therapeutically actionable control nodes. In this study, we report the rapid and robust ß-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 ß-cell mass adaptation. ß-Cell-specific Sin3a knockout animals showed profound diabetes due to defective acquisition of postnatal ß-cell mass. These findings reveal a novel regulatory pathway in ß-cell proliferation and validate OIO as a model for discovery of other mechanistic determinants of ß-cell adaptation.

MafA Regulation in ß-Cells: From Transcriptional to Post-Translational Mechanisms.

ß-cells are insulin-producing cells in the pancreas that maintain euglycemic conditions. Pancreatic ß-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 ß-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 ß-cell dysfunction. This article will summarize the role of MafA in normal ß-cell function and disease, with a special focus on known transcriptional and post-translational regulators of MafA expression.

Notch-mediated Ephrin signaling disrupts islet architecture and ß cell function.

Altered islet architecture is associated with ß 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" ß cell Notch activity in obese mouse models. To test the repercussions and reversibility of Notch effects, we generated doxycycline-dependent, ß cell-specific Notch gain-of-function mice. As predicted, we found that Notch activation in postnatal ß 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 ß cell repulsion and impaired murine and human pseudoislet formation. Consistent with our mouse data, Notch and Ephrin signaling were increased in metabolically inflexible ß cells in patients with T2D. These studies suggest that ß cell Notch/Ephrin signaling can permanently alter islet architecture during a morphogenetic window in early life.

Acetylation of PPARγ in macrophages promotes visceral fat degeneration in obesity.

Obesity is characterized by chronic, low-grade inflammation, which is driven by macrophage infiltration of adipose tissue. PPARγ is well established to have an anti-inflammatory function in macrophages, but the mechanism that regulates its function in these cells remains to be fully elucidated. PPARγ undergoes post-translational modifications (PTMs), including acetylation, to mediate ligand responses, including on metabolic functions. Here, we report that PPARγ acetylation in macrophages promotes their infiltration into adipose tissue, exacerbating metabolic dysregulation. We generated a mouse line that expresses a macrophage-specific, constitutive acetylation-mimetic form of PPARγ (K293Qflox/flox:LysM-cre, mK293Q) to dissect the role of PPARγ acetylation in macrophages. Upon high-fat diet feeding to stimulate macrophage infiltration into adipose tissue, we assessed the overall metabolic profile and tissue-specific phenotype of the mutant mice, including responses to the PPARγ agonist Rosiglitazone. Macrophage-specific PPARγ K293Q expression promotes proinflammatory macrophage infiltration and fibrosis in epididymal white adipose tissue, but not in subcutaneous or brown adipose tissue, leading to decreased energy expenditure, insulin sensitivity, glucose tolerance, and adipose tissue function. Furthermore, mK293Q mice are resistant to Rosiglitazone-induced improvements in adipose tissue remodeling. Our study reveals that acetylation is a new layer of PPARγ regulation in macrophage activation, and highlights the importance and potential therapeutic implications of such PTMs in regulating metabolism.

TOX4, an insulin receptor-independent regulator of hepatic glucose production, is activated in diabetic liver.

Increased hepatic glucose production (HGP) contributes to hyperglycemia in type 2 diabetes. Hormonal regulation of this process is primarily, but not exclusively, mediated by the AKT-FoxO1 pathway. Here, we show that cAMP and dexamethasone regulate the high-mobility group superfamily member TOX4 to mediate HGP, independent of the insulin receptor/FoxO1 pathway. TOX4 inhibition decreases glucose production in primary hepatocytes and liver and increases glucose tolerance. Combined genetic ablation of TOX4 and FoxO1 in liver has additive effects on glucose tolerance and gluconeogenesis. Moreover, TOX4 ablation fails to reverse the metabolic derangement brought by insulin receptor knockout. TOX4 expression is increased in livers of patients with steatosis and diabetes and in diet-induced obese and db/db mice. In the latter two murine models, knockdown Tox4 decreases glycemia and improves glucose tolerance. We conclude that TOX4 is an insulin receptor-independent regulator of HGP and a candidate contributor to the pathophysiology of diabetes.

TAZ-induced Cybb contributes to liver tumor formation in non-alcoholic steatohepatitis.

BACKGROUND & AIMS: Non-alcoholic steatohepatitis (NASH) is a leading cause of hepatocellular carcinoma (HCC), but mechanisms linking NASH to eventual tumor formation remain poorly understood. Herein, we investigate the role of TAZ/WWTR1, which is induced in hepatocytes in NASH, in the progression of NASH to HCC. METHODS: The roles of hepatocyte TAZ and its downstream targets were investigated in diet-induced and genetic models of NASH-HCC using gene-targeting, adeno-associated virus 8 (AAV8)-H1-mediated gene silencing, or AAV8-TBG-mediated gene expression. The biochemical signature of the newly elucidated pathway was probed in liver specimens from humans with NASH-HCC. RESULTS: When hepatocyte-TAZ was silenced in mice with pre-tumor NASH using AAV8-H1-shTaz (short-hairpin Taz), subsequent HCC tumor development was suppressed. In this setting, the tumor-suppressing effect of shTaz was not dependent of TAZ silencing in the tumors themselves and could be dissociated from the NASH-suppressing effects of shTaz. The mechanism linking pre-tumor hepatocyte-TAZ to eventual tumor formation involved TAZ-mediated induction of the NOX2-encoding gene Cybb, which led to NADPH-mediated oxidative DNA damage. As evidence, DNA damage and tumor formation could be suppressed by treatment of pre-tumor NASH mice with AAV8-H1-shCybb; AAV8-TBG-OGG1, encoding the oxidative DNA-repair enzyme 8-oxoguanine glycosylase; or AAV8-TBG-NHEJ1, encoding the dsDNA repair enzyme non-homologous end-joining factor 1. In surrounding non-tumor tissue from human NASH-HCC livers, there were strong correlations between TAZ, NOX2, and oxidative DNA damage. CONCLUSIONS: TAZ in pre-tumor NASH-hepatocytes, via induction of Cybb and NOX2-mediated DNA damage, contributes to subsequent HCC tumor development. These findings illustrate how NASH provides a unique window into the early molecular events that can lead to tumor formation and suggest that NASH therapies targeting TAZ might also prevent NASH-HCC. LAY SUMMARY: Non-alcoholic steatohepatitis (NASH) is emerging as the leading cause of a type of liver cancer called hepatocellular carcinoma (HCC), but molecular events in pre-tumor NASH hepatocytes leading to HCC remain largely unknown. Our study shows that a protein called TAZ in pre-tumor NASH-hepatocytes promotes damage to the DNA of hepatocytes and thereby contributes to eventual HCC. This study reveals a very early event in HCC that is induced in pre-tumor NASH, and the findings suggest that NASH therapies targeting TAZ might also prevent NASH-HCC.

Tumorigenesis from non-alcoholic steatohepatitis to hepatocellular carcinoma.

Non-alcoholic steatohepatitis (NASH) with metabolic syndrome is increasing to be a main cause of hepatocellular carcinoma (HCC). However, the mechanism of tumorigenesis in NASH induced HCC is still not clear. In this perspective, we will discuss the recent progress that has been made to understand the genetic change and the immune microenvironment of HCC, and the remaining questions. Based on the current study, NASH-HCC is likely to have novel mechanism, which needs more investigation in future.