An indispensable role for dynamin-related protein 1 in beige and brown adipogenesis.

Emerging evidence indicates that proper mitochondrial dynamics are critical for adipocyte differentiation and functional thermogenic capacity. We found that the mitochondrial fission protein dynamin-related protein 1 (DRP1, also known as DNML1) is highly expressed in brown adipose tissue compared to expression in white adipose tissue, and these expression levels increase during brown adipocyte differentiation. Our results reveal that the inhibition of DRP1 using mdivi-1 mitigates beige adipocyte differentiation and differentiation-associated mitochondrial biogenesis. We found that DRP1 is essential for the induction of the early-phase beige adipogenic transcriptional program. Intriguingly, inhibition of DRP1 is dispensable following the induction of beige adipogenesis and adipogenesis-associated mitochondrial biogenesis. Altogether, we demonstrate that DRP1 in preadipocytes plays an essential role in beige and brown adipogenesis.This article has an associated First Person interview with the first author of the paper.

Epigenetically active chromatin in neonatal iWAT reveals GABPα as a potential regulator of beige adipogenesis.

Background: Thermogenic beige adipocytes, which dissipate energy as heat, are found in neonates and adults. Recent studies show that neonatal beige adipocytes are highly plastic and contribute to >50% of beige adipocytes in adults. Neonatal beige adipocytes are distinct from recruited beige adipocytes in that they develop independently of temperature and sympathetic innervation through poorly defined mechanisms. Methods: We characterized the neonatal beige adipocytes in the inguinal white adipose tissue (iWAT) of C57BL6 postnatal day 3 and 20 mice (P3 and P20) by imaging, genome-wide RNA-seq analysis, ChIP-seq analysis, qRT-PCR validation, and biochemical assays. Results: We found an increase in acetylated histone 3 lysine 27 (H3K27ac) on the promoter and enhancer regions of beige-specific gene UCP1 in iWAT of P20 mice. Furthermore, H3K27ac ChIP-seq analysis in the iWAT of P3 and P20 mice revealed strong H3K27ac signals at beige adipocyte-associated genes in the iWAT of P20 mice. The integration of H3K27ac ChIP-seq and RNA-seq analysis in the iWAT of P20 mice reveal epigenetically active signatures of beige adipocytes, including oxidative phosphorylation and mitochondrial metabolism. We identify the enrichment of GA-binding protein alpha (GABPα) binding regions in the epigenetically active chromatin regions of the P20 iWAT, particularly on beige genes, and demonstrate that GABPα is required for beige adipocyte differentiation. Moreover, transcriptomic analysis and glucose oxidation assays revealed increased glycolytic activity in the neonatal iWAT from P20. Conclusions: Our findings demonstrate that epigenetic mechanisms regulate the development of peri-weaning beige adipocytes via GABPα. Further studies to better understand the upstream mechanisms that regulate epigenetic activation of GABPα and characterization of the metabolic identity of neonatal beige adipocytes will help us harness their therapeutic potential in metabolic diseases.

Hepatocyte-specific EGFR deletion promotes fibrosis but has no effect on steatosis in fast food diet model of MASLD.

BACKGROUND & AIMS: MASLD has become the most prevalent chronic liver disorder, with no approved treatment. Our previous work demonstrated the efficacy of a pan-ErbB inhibitor, Canertinib, in reducing steatosis and fibrosis in a murine fast-food diet (FFD) model of MASLD. The current study explores the effects of hepatocyte-specific ErbB1 (i.e. EGFR) deletion in the FFD model. METHODS: EGFRflox/flox mice, treated with AAV8-TBG-CRE to delete EGFR specifically in hepatocytes (EGFR-KO), were fed either a chow-diet or FFD for 2 or 5-months. RESULTS: Hepatocyte-specific EGFR deletion reduced serum triglyceride levels but did not prevent steatosis. Surprisingly, hepatic fibrosis was increased in EGFR-KO mice in the long-term study, which correlated with activation of TGFß1/fibrosis signaling pathways. Further, nuclear levels of some of the major MASLD regulating transcription factors (SREBP1, PPARγ, PPARα, and HNF4α) were altered in FFD-fed EGFR-KO mice. Transcriptomic analysis revealed significant alteration of lipid metabolism pathways in EGFR-KO mice with changes in several relevant genes, including downregulation of fatty-acid synthase and induction of lipolysis gene, Pnpla2, without impacting overall steatosis. Interestingly, EGFR downstream signaling mediators, including AKT, remain activated in EGFR-KO mice, which correlated with increased activity pattern of other receptor tyrosine kinases, including ErbB3/MET, in transcriptomic analysis. Lastly, Canertinib treatment in EGFR-KO mice, which inhibits all ErbB receptors, successfully reduced steatosis, suggesting the compensatory roles of other ErbB receptors in supporting MASLD without EGFR. CONCLUSIONS: Hepatocyte-specific EGFR-KO did not impact steatosis, but enhanced fibrosis in the FFD model of MASLD. Gene-networks associated with lipid metabolism were greatly altered in EGFR-KO, but phenotypic effects might be compensated by alternate signaling-pathways.

Hepatic ketogenesis regulates lipid homeostasis via ACSL1-mediated fatty acid partitioning.

Liver-derived ketone bodies play a crucial role in fasting energy homeostasis by fueling the brain and peripheral tissues. Ketogenesis also acts as a conduit to remove excess acetyl-CoA generated from fatty acid oxidation and protects against diet-induced hepatic steatosis. Surprisingly, no study has examined the role of ketogenesis in fasting-associated hepatocellular lipid metabolism. Ketogenesis is driven by the rate-limiting mitochondrial enzyme 3-hydroxymethylglutaryl CoA synthase (HMGCS2) abundantly expressed in the liver. Here, we show that ketogenic insufficiency via disruption of hepatic HMGCS2 exacerbates liver steatosis in fasted chow and high-fat-fed mice. We found that the hepatic steatosis is driven by increased fatty acid partitioning to the endoplasmic reticulum (ER) for re-esterification via acyl-CoA synthetase long-chain family member 1 (ACSL1). Mechanistically, acetyl-CoA accumulation from impaired hepatic ketogenesis is responsible for the elevated translocation of ACSL1 to the ER. Moreover, we show increased ER-localized ACSL1 and re-esterification of lipids in human NASH displaying impaired hepatic ketogenesis. Finally, we show that L-carnitine, which buffers excess acetyl-CoA, decreases the ER-associated ACSL1 and alleviates hepatic steatosis. Thus, ketogenesis via controlling hepatocellular acetyl-CoA homeostasis regulates lipid partitioning and protects against hepatic steatosis.

Oxidative Stress and Redox Signaling in the Pathophysiology of Liver Diseases.

The increased production of derivatives of molecular oxygen and nitrogen in the form of reactive oxygen species (ROS) and reactive nitrogen species (RNS) lead to molecular damage called oxidative stress. Under normal physiological conditions, the ROS generation is tightly regulated in different cells and cellular compartments. Any disturbance in the balance between the cellular generation of ROS and antioxidant balance leads to oxidative stress. In this article, we discuss the sources of ROS (endogenous and exogenous) and antioxidant mechanisms. We also focus on the pathophysiological significance of oxidative stress in various cell types of the liver. Oxidative stress is implicated in the development and progression of various liver diseases. We narrate the master regulators of ROS-mediated signaling and their contribution to liver diseases. Nonalcoholic fatty liver diseases (NAFLD) are influenced by a "multiple parallel-hit model" in which oxidative stress plays a central role. We highlight the recent findings on the role of oxidative stress in the spectrum of NAFLD, including fibrosis and liver cancer. Finally, we provide a brief overview of oxidative stress biomarkers and their therapeutic applications in various liver-related disorders. Overall, the article sheds light on the significance of oxidative stress in the pathophysiology of the liver. © 2022 American Physiological Society. Compr Physiol 12:3167-3192, 2022.

Emerging Role of Hepatic Ketogenesis in Fatty Liver Disease.

Non-alcoholic fatty liver disease (NAFLD), the most common chronic liver diseases, arise from non-alcoholic fatty liver (NAFL) characterized by excessive fat accumulation as triglycerides. Although NAFL is benign, it could progress to non-alcoholic steatohepatitis (NASH) manifested with inflammation, hepatocyte damage and fibrosis. A subset of NASH patients develops end-stage liver diseases such as cirrhosis and hepatocellular carcinoma. The pathogenesis of NAFLD is highly complex and strongly associated with perturbations in lipid and glucose metabolism. Lipid disposal pathways, in particular, impairment in condensation of acetyl-CoA derived from ß-oxidation into ketogenic pathway strongly influence the hepatic lipid loads and glucose metabolism. Current evidence suggests that ketogenesis dispose up to two-thirds of the lipids entering the liver, and its dysregulation significantly contribute to the NAFLD pathogenesis. Moreover, ketone body administration in mice and humans shows a significant improvement in NAFLD. This review focuses on hepatic ketogenesis and its role in NAFLD pathogenesis. We review the possible mechanisms through which impaired hepatic ketogenesis may promote NAFLD progression. Finally, the review sheds light on the therapeutic implications of a ketogenic diet in NAFLD.

Intestinal HIF-2α Regulates GLP-1 Secretion via Lipid Sensing in L-Cells.

BACKGROUND & AIMS: Compelling evidence shows that glucagon-like peptide-1 (GLP-1) has a profound effect in restoring normoglycemia in type 2 diabetic patients by increasing pancreatic insulin secretion. Although L-cells are the primary source of circulating GLP-1, the current therapies do not target L-cells to increase GLP-1 levels. Our study aimed to determine the molecular underpinnings of GLP-1 secretion as an impetus to identify new interventions to target endogenous L-cells. METHODS: We used genetic mouse models of intestine-specific overexpression of hypoxia-inducible factor (HIF)-1α and HIF-2α (VhlΔIE), conditional overexpression of intestinal HIF-2α (Hif-2αLSL;Vilin-Cre/ERT2), and intestine-specific HIF-2α knockout mice (Hif-2αΔIE) to show that HIF signaling, especially HIF-2α, regulates GLP-1 secretion. RESULTS: Our data show that intestinal HIF signaling improved glucose homeostasis in a GLP-1-dependent manner. Intestinal HIF potentiated GLP-1 secretion via the lipid sensor G-protein-coupled receptor (GPR)40 enriched in L-cells. We show that HIF-2α regulates GPR40 in L-cells and potentiates fatty acid-induced GLP-1 secretion via extracellular regulated kinase (ERK). Using a genetic model of intestine-specific overexpression of HIF-2α, we show that HIF-2α is sufficient to increase GLP-1 levels and attenuate diet-induced metabolic perturbations such as visceral adiposity, glucose intolerance, and hepatic steatosis. Lastly, we show that intestinal HIF-2α signaling acts as a priming mechanism crucial for postprandial lipid-mediated GLP-1 secretion. Thus, disruption of intestinal HIF-2α decreases GLP-1 secretion. CONCLUSIONS: In summary, we show that intestinal HIF signaling, particularly HIF-2α, regulates the lipid sensor GPR40, which is crucial for the lipid-mediated GLP-1 secretion, and suggest that HIF-2α is a potential target to induce endogenous GLP-1 secretion.

Hypoxia via ERK Signaling Inhibits Hepatic PPARα to Promote Fatty Liver.

BACKGROUND & AIMS: Fatty liver or nonalcoholic fatty liver disease (NAFLD) is the most common liver disease associated with comorbidities such as insulin resistance and cardiovascular and metabolic diseases. Chronic activation of hypoxic signaling, in particular, hypoxia-inducible factor (HIF)2α, promotes NAFLD progression by repressing genes involved in fatty acid ß-oxidation through unclear mechanisms. Therefore, we assessed the precise mechanism by which HIF2α promotes fatty liver and its physiological relevance in metabolic homeostasis. METHODS: Primary hepatocytes from VHL (VhlΔHep) and PPARα (Ppara-null) knockout mice that were loaded with fatty acids, murine dietary protocols to induce hepatic steatosis, and fasting-refeeding dietary regimen approaches were used to test our hypothesis. RESULTS: Inhibiting autophagy using chloroquine did not decrease lipid contents in VhlΔHep primary hepatocytes. Inhibition of ERK using MEK inhibitor decreased lipid contents in primary hepatocytes from a genetic model of constitutive HIF activation and primary hepatocytes loaded with free fatty acids. Moreover, MEK-ERK inhibition potentiated ligand-dependent activation of PPARα. We also show that MEK-ERK inhibition improved diet-induced hepatic steatosis, which is associated with the induction of PPARα target genes. During fasting, fatty acid ß-oxidation is induced by PPARα, and refeeding inhibits ß-oxidation. Our data show that ERK is involved in the post-prandial repression of hepatic PPARα signaling. CONCLUSIONS: Overall, our results demonstrate that ERK activated by hypoxia signaling plays a crucial role in fatty acid ß-oxidation genes by repressing hepatocyte PPARα signaling.

Tregs facilitate obesity and insulin resistance via a Blimp-1/IL-10 axis.

Interleukin-10 (IL-10) is a critical cytokine used by immune cells to suppress inflammation. Paradoxically, immune cell-derived IL-10 can drive insulin resistance in obesity by suppressing adipocyte energy expenditure and thermogenesis. However, the source of IL-10 necessary for the suppression of adipocyte thermogenesis is unknown. We show here that CD4+Foxp3+ regulatory T cells (Tregs) are a substantial source of IL-10 and that Treg-derived IL-10 can suppress adipocyte beiging. Unexpectedly, Treg-specific loss of IL-10 resulted in increased insulin sensitivity and reduced obesity in high-fat diet-fed male mice. Mechanistically, we determined that Treg-specific loss of the transcription factor Blimp-1, a driver of IL-10 expression by Tregs, phenocopied the Treg-specific IL-10-deficient mice. Loss of Blimp-1 expression in Tregs resulted in reduced ST2+KLRG1+, IL-10-secreting Tregs, particularly in the white adipose tissue. Blimp-1-deficient mice were protected from glucose intolerance, insulin resistance, and diet-induced obesity, through increased white adipose tissue browning. Taken together, our data show that Blimp-1-regulated IL-10 secretion by Tregs represses white adipose tissue beiging to maintain adipose tissue homeostasis.

Sustained mitochondrial biogenesis is essential to maintain caloric restriction-induced beige adipocytes.

BACKGROUND: Caloric restriction (CR) delays the onset of metabolic and age-related disorders. Recent studies have demonstrated that formation of beige adipocytes induced by CR is strongly associated with extracellular remodeling in adipose tissue, decrease in adipose tissue inflammation, and improved systemic metabolic homeostasis. However, beige adipocytes rapidly transition to white upon CR withdrawal through unclear mechanisms. MATERIALS AND METHODS: Six-week old C57BL6 mice were fed with 40% CR chow diet for 6 weeks. Subsequently, one group of mice was switched back to ad libitum chow diet, which was continued for additional 2 weeks. Adipose tissues were assessed histologically and biochemically for beige adipocytes. RESULTS: Beige adipocytes induced by CR rapidly transition to white adipocytes when CR is withdrawn independent of parkin-mediated mitophagy. We demonstrate that the involution of mitochondria during CR withdrawal is strongly linked with a decrease in mitochondrial biogenesis. We further demonstrate that beige-to-white fat transition upon ß3-AR agonist-withdrawal could be attenuated by CR, partly via maintenance of mitochondrial biogenesis. CONCLUSION: In the model of CR, our study highlights the dominant role of mitochondrial biogenesis in the maintenance of beige adipocytes. We propose that loss of beige adipocytes upon ß3-AR agonist withdrawal could be attenuated by CR.