Search

The obesity epidemic continues to worsen worldwide, driving metabolic and chronic inflammatory diseases. Thiazolidinediones, such as rosiglitazone (Rosi), are PPARγ agonists that promote 'M2-like' adipose tissue macrophage (ATM) polarization and cause insulin sensitization. As ATM-derived small extracellular vesicles (ATM-sEVs) from lean mice are known to increase insulin sensitivity, we assessed the metabolic effects of ATM-sEVs from Rosi-treated obese male mice (Rosi-ATM-sEVs). Here we show that Rosi leads to improved glucose and insulin tolerance, transcriptional repolarization of ATMs and increased sEV secretion. Administration of Rosi-ATM-sEVs rescues obesity-induced glucose intolerance and insulin sensitivity in vivo without the known thiazolidinedione-induced adverse effects of weight gain or haemodilution. Rosi-ATM-sEVs directly increase insulin sensitivity in adipocytes, myotubes and primary mouse and human hepatocytes. Additionally, we demonstrate that the miRNAs within Rosi-ATM-sEVs, primarily miR-690, are responsible for these beneficial metabolic effects. Thus, using ATM-sEVs with specific miRNAs may provide a therapeutic path to induce insulin sensitization.

The mechanisms of hepatic stellate cell (HSC) activation and the development of liver fibrosis are not fully understood. Here, we show that deletion of a nuclear seven transmembrane protein, TM7SF3, accelerates HSC activation in liver organoids, primary human HSCs, and in vivo in metabolic-dysfunction-associated steatohepatitis (MASH) mice, leading to activation of the fibrogenic program and HSC proliferation. Thus, TM7SF3 knockdown promotes alternative splicing of the Hippo pathway transcription factor, TEAD1, by inhibiting the splicing factor heterogeneous nuclear ribonucleoprotein U (hnRNPU). This results in the exclusion of the inhibitory exon 5, generating a more active form of TEAD1 and triggering HSC activation. Furthermore, inhibiting TEAD1 alternative splicing with a specific antisense oligomer (ASO) deactivates HSCs in vitro and reduces MASH diet-induced liver fibrosis. In conclusion, by inhibiting TEAD1 alternative splicing, TM7SF3 plays a pivotal role in mitigating HSC activation and the progression of MASH-related fibrosis.

Hepatocytes have important roles in liver iron homeostasis, abnormalities in which are tightly associated with liver steatosis and fibrosis. Here, we show that non-alcoholic fatty liver disease (NAFLD) and steatohepatitis (NASH) are characterized by iron-deficient hepatocytes and iron overload in hepatic stellate cells (HSCs). Iron deficiency enhances hepatocyte lipogenesis and insulin resistance through HIF2α-ATF4 signaling. Elevated secretion of iron-containing hepatocyte extracellular vesicles (EVs), which are normally cleared by Kupffer cells, accounts for hepatocyte iron deficiency and HSC iron overload in NAFLD/NASH livers. Iron accumulation results in overproduction of reactive oxygen species that promote HSC fibrogenic activation. Conversely, blocking hepatocyte EV secretion or depleting EV iron cargo restores liver iron homeostasis, concomitant with mitigation of NAFLD/NASH-associated liver steatosis and fibrosis. Taken together, these studies show that iron distribution disorders contribute to the development of liver metabolic diseases.

Nonalcoholic steatohepatitis (NASH) is a liver disease associated with significant morbidity. Kupffer cells (KCs) produce endogenous miR-690 and, via exosome secretion, shuttle this miRNA to other liver cells, such as hepatocytes, recruited hepatic macrophages (RHMs), and hepatic stellate cells (HSCs). miR-690 directly inhibits fibrogenesis in HSCs, inflammation in RHMs, and de novo lipogenesis in hepatocytes. When an miR-690 mimic is administered to NASH mice in vivo, all the features of the NASH phenotype are robustly inhibited. During the development of NASH, KCs become miR-690 deficient, and miR-690 levels are markedly lower in mouse and human NASH livers than in controls. KC-specific KO of miR-690 promotes NASH pathogenesis. A primary target of miR-690 is NADK mRNA, and NADK levels are inversely proportional to the cellular miR-690 content. These studies show that KCs play a central role in the etiology of NASH and raise the possibility that miR-690 could emerge as a therapeutic for this condition.

In chronic obesity, hepatocytes become insulin resistant and exert important effects on systemic metabolism. Here we show that in early onset obesity (4 weeks high-fat diet), hepatocytes secrete exosomes that enhance insulin sensitivity both in vitro and in vivo. These beneficial effects were due to exosomal microRNA miR-3075, which is enriched in these hepatocyte exosomes. FA2H is a direct target of miR-3075 and small interfering RNA depletion of FA2H in adipocytes, myocytes and primary hepatocytes leads to increased insulin sensitivity. In chronic obesity (16-18 weeks of a high-fat diet), hepatocyte exosomes promote a state of insulin resistance. These chronic obese hepatocyte exosomes do not directly cause impaired insulin signalling in vitro but do promote proinflammatory activation of macrophages. Taken together, these studies show that in early onset obesity, hepatocytes produce exosomes that express high levels of the insulin-sensitizing miR-3075. In chronic obesity, this compensatory effect is lost and hepatocyte-derived exosomes from chronic obese mice promote insulin resistance.

[Figure: see text].

Insulin resistance is a major pathophysiologic defect in type 2 diabetes and obesity, while anti-inflammatory M2-like macrophages are important in maintaining normal metabolic homeostasis. Here, we show that M2 polarized bone marrow-derived macrophages (BMDMs) secrete miRNA-containing exosomes (Exos), which improve glucose tolerance and insulin sensitivity when given to obese mice. Depletion of their miRNA cargo blocks the ability of M2 BMDM Exos to enhance insulin sensitivity. We found that miR-690 is highly expressed in M2 BMDM Exos and functions as an insulin sensitizer both in vivo and in vitro. Expressing an miR-690 mimic in miRNA-depleted BMDMs generates Exos that recapitulate the effects of M2 BMDM Exos on metabolic phenotypes. Nadk is a bona fide target mRNA of miR-690, and Nadk plays a role in modulating macrophage inflammation and insulin signaling. Taken together, these data suggest miR-690 could be a new therapeutic insulin-sensitizing agent for metabolic disease.

Background & aimsLiver CRIg+ (complement receptor of the immunoglobulin superfamily) macrophages play a critical role in filtering bacteria and their products from circulation. Translocation of microbiota-derived products from an impaired gut barrier contributes to the development of obesity-associated tissue inflammation and insulin resistance. However, the critical role of CRIg+ macrophages in clearing microbiota-derived products from the bloodstream in the context of obesity is largely unknown.MethodsWe performed studies with CRIg-/-, C3-/-, cGAS-/-, and their wild-type littermate mice. The CRIg+ macrophage population and bacterial DNA abundance were examined in both mouse and human liver by either flow cytometric or immunohistochemistry analysis. Gut microbial DNA-containing extracellular vesicles (mEVs) were adoptively transferred into CRIg-/-, C3-/-, or wild-type mice, and tissue inflammation and insulin sensitivity were measured in these mice. After coculture with gut mEVs, cellular insulin responses and cGAS/STING-mediated inflammatory responses were evaluated.ResultsGut mEVs can reach metabolic tissues in obesity. Liver CRIg+ macrophages efficiently clear mEVs from the bloodstream through a C3-dependent opsonization mechanism, whereas obesity elicits a marked reduction in the CRIg+ macrophage population. Depletion of CRIg+ cells results in the spread of mEVs into distant metabolic tissues, subsequently exacerbating tissue inflammation and metabolic disorders. Additionally, in vitro treatment of obese mEVs directly triggers inflammation and insulin resistance of insulin target cells. Depletion of microbial DNA blunts the pathogenic effects of intestinal EVs. Furthermore, the cGAS/STING pathway is crucial for microbial DNA-mediated inflammatory responses.ConclusionsDeficiency of CRIg+ macrophages and leakage of intestinal EVs containing microbial DNA contribute to the development of obesity-associated tissue inflammation and metabolic diseases.

ObjectiveRecent evidence indicates that inhibition of prolyl hydroxylase domain (PHD) proteins can exert beneficial effects to improve metabolic abnormalities in mice and humans. However, the underlying mechanisms are not clearly understood. This study was designed to address this question.MethodsA pan-PHD inhibitor compound was injected into WT and liver-specific hypoxia-inducible factor (HIF)-2α KO mice, after onset of obesity and glucose intolerance, and changes in glucose and glucagon tolerance were measured. Tissue-specific changes in basal glucose flux and insulin sensitivity were also measured by hyperinsulinemic euglycemic clamp studies. Molecular and cellular mechanisms were assessed in normal and type 2 diabetic human hepatocytes, as well as in mouse hepatocytes.ResultsAdministration of a PHD inhibitor compound (PHDi) after the onset of obesity and insulin resistance improved glycemic control by increasing insulin and decreasing glucagon sensitivity in mice, independent of body weight change. Hyperinsulinemic euglycemic clamp studies revealed that these effects of PHDi treatment were mainly due to decreased basal hepatic glucose output and increased liver insulin sensitivity. Hepatocyte-specific deletion of HIF-2α markedly attenuated these effects of PHDi treatment, showing PHDi effects are HIF-2α dependent. At the molecular level, HIF-2α induced increased Irs2 and cyclic AMP-specific phosphodiesterase gene expression, leading to increased and decreased insulin and glucagon signaling, respectively. These effects of PHDi treatment were conserved in human and mouse hepatocytes.ConclusionsOur results elucidate unknown mechanisms for how PHD inhibition improves glycemic control through HIF-2α-dependent regulation of hepatic insulin and glucagon sensitivity.

Insulin resistance is a major factor in obesity-linked type 2 diabetes. PPARγ is a master regulator of adipogenesis, and small molecule agonists, termed thiazolidinediones, are potent therapeutic insulin sensitizers. Here, we studied the role of transcriptional co-activator with PDZ-binding motif (TAZ) as a transcriptional co-repressor of PPARγ. We found that adipocyte-specific TAZ knockout (TAZ AKO) mice demonstrate a constitutively active PPARγ state. Obese TAZ AKO mice show improved glucose tolerance and insulin sensitivity compared to littermate controls. PPARγ response genes are upregulated in adipose tissue from TAZ AKO mice and adipose tissue inflammation was also decreased. In vitro and in vivo mechanistic studies revealed that the TAZ-PPARγ interaction is partially dependent on ERK-mediated Ser112 PPARγ phosphorylation. As adipocyte PPARγ Ser112 phosphorylation is increased in obesity, repression of PPARγ activity by TAZ could contribute to insulin resistance. These results identify TAZ as a new factor in the development of obesity-induced insulin resistance.

Insulin resistance is a key feature of obesity and type 2 diabetes. PU.1 is a master transcription factor predominantly expressed in macrophages but after HFD feeding PU.1 expression is also significantly increased in adipocytes. We generated adipocyte specific PU.1 knockout mice using adiponectin cre to investigate the role of PU.1 in adipocyte biology, insulin and glucose homeostasis. In HFD-fed obese mice systemic glucose tolerance and insulin sensitivity were improved in PU.1 AKO mice and clamp studies indicated improvements in both adipose and liver insulin sensitivity. At the level of adipose tissue, macrophage infiltration and inflammation was decreased and glucose uptake was increased in PU.1 AKO mice compared with controls. While PU.1 deletion in adipocytes did not affect the gene expression of PPARg itself, we observed increased expression of PPARg target genes in eWAT from HFD fed PU.1 AKO mice compared with controls. Furthermore, we observed decreased phosphorylation at serine 273 in PU.1 AKO mice compared with fl/fl controls, indicating that PPARg is more active when PU.1 expression is reduced in adipocytes. Therefore, in obesity the increased expression of PU.1 in adipocytes modifies the adipocyte PPARg cistrome resulting in impaired glucose tolerance and insulin sensitivity.

Serine rich splicing factor 3 (SRSF3) plays a critical role in liver function and its loss promotes chronic liver damage and regeneration. As a consequence, genetic deletion of SRSF3 in hepatocytes caused progressive liver disease and ultimately led to hepatocellular carcinoma. Here we show that SRSF3 is decreased in human liver samples with non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), or cirrhosis that was associated with alterations in RNA splicing of known SRSF3 target genes. Hepatic SRSF3 expression was similarly decreased and RNA splicing dysregulated in mouse models of NAFLD and NASH. We showed that palmitic acid-induced oxidative stress caused conjugation of the ubiquitin like NEDD8 protein to SRSF3 and proteasome mediated degradation. SRSF3 was selectively neddylated at lysine11 and mutation of this residue (SRSF3-K11R) was sufficient to prevent both SRSF3 degradation and alterations in RNA splicing. Finally prevention of SRSF3 degradation in vivo partially protected mice from hepatic steatosis, fibrosis and inflammation. These results highlight a neddylation-dependent mechanism regulating gene expression in the liver that is disrupted in early metabolic liver disease and may contribute to the progression to NASH, cirrhosis and ultimately hepatocellular carcinoma.

Decreased adipose tissue oxygen tension and increased HIF-1α expression can trigger adipose tissue inflammation and dysfunction in obesity. Our current understanding of obesity-associated decreased adipose tissue oxygen tension is mainly focused on changes in oxygen supply and angiogenesis. Here, we demonstrate that increased adipocyte O2 demand, mediated by ANT2 activity, is the dominant cause of adipocyte hypoxia. Deletion of adipocyte Ant2 improves obesity-induced intracellular adipocyte hypoxia by decreasing obesity-induced adipocyte oxygen demand, without effects on mitochondrial number or mass, or oligomycin-sensitive respiration. This led to decreased adipose tissue HIF-1α expression and inflammation with improved glucose tolerance and insulin resistance in both a preventative or therapeutic setting. Our results suggest that ANT2 may be a target for the development of insulin sensitizing drugs and that ANT2 inhibition might have clinical utility.

Proinflammatory hepatic macrophage activation plays a key role in the development of nonalcoholic steatohepatitis (NASH). This involves increased embryonic hepatic Kupffer cell (KC) death, facilitating the replacement of KCs with bone marrow–derived recruited hepatic macrophages (RHMs) that highly express proinflammatory genes. Moreover, phago/efferocytic activity of KCs is diminished in NASH, enhancing liver inflammation. However, the molecular mechanisms underlying these changes in KCs are not known. Here, we show that hypoxia-inducible factor 2α (HIF-2α) mediates NASH-associated decreased KC growth and efferocytosis by enhancing lysosomal stress. At the molecular level, HIF-2α stimulated mammalian target of rapamycin (mTOR)– and extracellular signal–regulated kinase-dependent inhibitory transcription factor EB (TFEB) phosphorylation, leading to decreased lysosomal and phagocytic gene expression. With increased metabolic stress and phago/efferocytic burden in NASH, these changes were sufficient to increase lysosomal stress, causing decreased efferocytosis and lysosomal cell death. Of interest, HIF-2α–dependent TFEB regulation only occurred in KCs but not RHMs. Instead, in RHMs, HIF-2α promoted mitochondrial reactive oxygen species production and proinflammatory activation by increasing ANT2 expression and mitochondrial permeability transition. Consequently, myeloid lineage–specific or KC-specific HIF-2α depletion or the inhibition of mTOR-dependent TFEB inhibition using antisense oligonucleotide treatment protected against the development of NASH in mice. Moreover, treatment with an HIF-2α–specific inhibitor reduced inflammatory and fibrogenic gene expression in human liver spheroids cultured under a NASH-like condition. Together, our results suggest that macrophage subtype–specific effects of HIF-2α collectively contribute to the proinflammatory activation of liver macrophages, leading to the development of NASH. Editor's summary: Abberant hepatic macrophage activity has been implicated in NASH development, but the mechanism of this immune dysregulation is unclear. Jeelani et al. show that a hypoxia-inducible factor, HIF-2α, promotes differential effects on macrophage subpopulations during NASH in obesity. HIF-2α suppressed the growth and function of endogenous liver macrophages (Kupffer cells) but increased the proinflammatory activation of the myeloid-derived macrophages that are subsequently recruited to the liver and that start to replace the Kupffer cells. HIF-2α–specific approaches in a murine NASH model and human organoids suggested that this disease biology may be therapeutically targetable. —Catherine Charneski [ABSTRACT FROM AUTHOR]

The activation of Kupffer cells (KCs) and monocyte-derived recruited macrophages (McMΦs) in the liver contributes to obesity-induced insulin resistance and type 2 diabetes. Mice with diet-induced obesity (DIO mice) treated with chromogranin A peptide catestatin (CST) showed several positive results. These included decreased hepatic/plasma lipids and plasma insulin, diminished expression of gluconeogenic genes, attenuated expression of proinflammatory genes, increased expression of anti-inflammatory genes in McMΦs, and inhibition of the infiltration of McMΦs resulting in improvement of insulin sensitivity. Systemic CST knockout (CST-KO) mice on normal chow diet (NCD) ate more food, gained weight, and displayed elevated blood glucose and insulin levels. Supplementation of CST normalized glucose and insulin levels. To verify that the CST deficiency caused macrophages to be very proinflammatory in CST-KO NCD mice and produced glucose intolerance, we tested the effects of (sorted with FACS) F4/80+Ly6C- cells (representing KCs) and F4/80-Ly6C+ cells (representing McMΦs) on hepatic glucose production (HGP). Both basal HGP and glucagon-induced HGP were markedly increased in hepatocytes cocultured with KCs and McMΦs from NCD-fed CST-KO mice, and the effect was abrogated upon pretreatment of CST-KO macrophages with CST. Thus, we provide a novel mechanism of HGP suppression through CST-mediated inhibition of macrophage infiltration and function.

We have previously reported that the fractalkine (FKN)/CX3CR1 system represents a novel regulatory mechanism for insulin secretion and β cell function. Here, we demonstrate that chronic administration of a long-acting form of FKN, FKN-Fc, can exert durable effects to improve glucose tolerance with increased glucose-stimulated insulin secretion and decreased β cell apoptosis in obese rodent models. Unexpectedly, chronic FKN-Fc administration also led to decreased α cell glucagon secretion. In islet cells, FKN inhibited ATP-sensitive potassium channel conductance by an ERK-dependent mechanism, which triggered β cell action potential (AP) firing and decreased α cell AP amplitude. This results in increased glucose-stimulated insulin secretion and decreased glucagon secretion. Beyond its islet effects, FKN-Fc also exerted peripheral effects to enhance hepatic insulin sensitivity due to inhibition of glucagon action. In hepatocytes, FKN treatment reduced glucagon-stimulated cAMP production and CREB phosphorylation in a pertussis toxin-sensitive manner. Together, these results raise the possibility of use of FKN-based therapy to improve type 2 diabetes by increasing both insulin secretion and insulin sensitivity.

Chromogranin A (CgA) is a prohormone and a granulogenic factor that regulates secretory pathways in neuroendocrine tissues. In β-cells of the endocrine pancreas, CgA is a major cargo in insulin secretory vesicles. The impact of CgA deficiency on the formation and exocytosis of insulin vesicles is yet to be investigated. In addition, no literature exists on the impact of CgA on mitochondrial function in β-cells. Using three different antibodies, we demonstrate that CgA is processed to vasostatin- and catestatin-containing fragments in pancreatic islet cells. CgA deficiency in Chga-KO islets leads to compensatory overexpression of chromogranin B, secretogranin II, SNARE proteins and insulin genes, as well as increased insulin protein content. Ultrastructural studies of pancreatic islets revealed that Chga-KO β-cells contain fewer immature secretory granules than wild-type (WT) control but increased numbers of mature secretory granules and plasma membrane-docked vesicles. Compared to WT control, CgA-deficient β-cells exhibited increases in mitochondrial volume, numerical densities and fusion, as well as increased expression of nuclear encoded genes (Ndufa9, Ndufs8, Cyc1 and Atp5o). These changes in secretory vesicles and the mitochondria likely contribute to the increased glucose-stimulated insulin secretion observed in Chga-KO mice. We conclude that CgA is an important regulator for coordination of mitochondrial dynamics, secretory vesicular quanta and GSIS for optimal secretory functioning of β-cells, suggesting a strong, CgA-dependent positive link between mitochondrial fusion and GSIS.

Tissue inflammation is a key component of obesity-induced insulin resistance, with a variety of immune cell types accumulating in adipose tissue. Here, we have demonstrated increased numbers of B2 lymphocytes in obese adipose tissue and have shown that high-fat diet-induced (HFD-induced) insulin resistance is mitigated in B cell-deficient (Bnull) mice. Adoptive transfer of adipose tissue B2 cells (ATB2) from wild-type HFD donor mice into HFD Bnull recipients completely restored the effect of HFD to induce insulin resistance. Recruitment and activation of ATB2 cells was mediated by signaling through the chemokine leukotriene B4 (LTB4) and its receptor LTB4R1. Furthermore, the adverse effects of ATB2 cells on glucose homeostasis were partially dependent upon T cells and macrophages. These results demonstrate the importance of ATB2 cells in obesity-induced insulin resistance and suggest that inhibition of the LTB4/LTB4R1 axis might be a useful approach for developing insulin-sensitizing therapeutics.

Chromogranin A (CgA) is widely expressed in endocrine and neuroendocrine tissues as well as in the central nervous system. We observed CgA expression (mRNA and protein) in the gastrocnemius (GAS) muscle and found that performance of CgA-deficient Chga-KO mice in treadmill exercise was impaired. Supplementation with CgA in Chga-KO mice restored exercise ability suggesting a novel role for endogenous CgA in skeletal muscle function. Chga-KO mice display (i) lack of exercise-induced stimulation of pAKT, pTBC1D1 and phospho-p38 kinase signaling, (ii) loss of GAS muscle mass, (iii) extensive formation of tubular aggregates (TA), (iv) disorganized cristae architecture in mitochondria, (v) increased expression of the inflammatory cytokines Tnfα, Il6 and Ifnγ, and fibrosis. The impaired maximum running speed and endurance in the treadmill exercise in Chga-KO mice correlated with decreased glucose uptake and glycolysis, defects in glucose oxidation and decreased mitochondrial cytochrome C oxidase activity. The lack of adaptation to endurance training correlated with the lack of stimulation of p38MAPK that is known to mediate the response to tissue damage. As CgA sorts proteins to the regulated secretory pathway, we speculate that lack of CgA could cause misfolding of membrane proteins inducing aggregation of sarcoplasmic reticulum (SR) membranes and formation of tubular aggregates that is observed in Chga-KO mice. In conclusion, CgA deficiency renders the muscle energy deficient, impairs performance in treadmill exercise and prevents regeneration after exercise-induced tissue damage.

The heart possesses a remarkable inherent capability to adapt itself to a wide array of genetic and extrinsic factors to maintain contractile function. Failure to sustain its compensatory responses results in cardiac dysfunction, leading to cardiomyopathy. Diabetic cardiomyopathy (DCM) is characterized by left ventricular hypertrophy and reduced diastolic function, with or without concurrent systolic dysfunction in the absence of hypertension and coronary artery disease. Changes in substrate metabolism, oxidative stress, endoplasmic reticulum stress, formation of extracellular matrix proteins, and advanced glycation end products constitute the early stage in DCM. These early events are followed by steatosis (accumulation of lipid droplets) in cardiomyocytes, which is followed by apoptosis, changes in immune responses with a consequent increase in fibrosis, remodeling of cardiomyocytes, and the resultant decrease in cardiac function. The heart is an omnivore, metabolically flexible, and consumes the highest amount of ATP in the body. Altered myocardial substrate and energy metabolism initiate the development of DCM. Diabetic hearts shift away from the utilization of glucose, rely almost completely on fatty acids (FAs) as the energy source, and become metabolically inflexible. Oxidation of FAs is metabolically inefficient as it consumes more energy. In addition to metabolic inflexibility and energy inefficiency, the diabetic heart suffers from impaired calcium handling with consequent alteration of relaxation-contraction dynamics leading to diastolic and systolic dysfunction. Sarcoplasmic reticulum (SR) plays a key role in excitation-contraction coupling as Ca2+ is transported into the SR by the SERCA2a (sarcoplasmic/endoplasmic reticulum calcium-ATPase 2a) during cardiac relaxation. Diabetic cardiomyocytes display decreased SERCA2a activity and leaky Ca2+ release channel resulting in reduced SR calcium load. The diabetic heart also suffers from marked downregulation of novel cardioprotective microRNAs (miRNAs) discovered recently. Since immune responses and substrate energy metabolism are critically altered in diabetes, the present review will focus on immunometabolism and miRNAs.