Ceramides are fuel gauges on the drive to cardiometabolic disease.

Ceramides are signals of fatty acid excess that accumulate when a cell's energetic needs have been met and its nutrient storage has reached capacity. As these sphingolipids accrue, they alter the metabolism and survival of cells throughout the body including in the heart, liver, blood vessels, skeletal muscle, brain, and kidney. These ceramide actions elicit the tissue dysfunction that underlies cardiometabolic diseases such as diabetes, coronary artery disease, metabolic-associated steatohepatitis, and heart failure. Here, we review the biosynthesis and degradation pathways that maintain ceramide levels in normal physiology and discuss how the loss of ceramide homeostasis drives cardiometabolic pathologies. We highlight signaling nodes that sense small changes in ceramides and in turn reprogram cellular metabolism and stimulate apoptosis. Finally, we evaluate the emerging therapeutic utility of these unique lipids as biomarkers that forecast disease risk and as targets of ceramide-lowering interventions that ameliorate disease.

Lipidomic QTL in Diversity Outbred mice identifies a novel function for α/ß hydrolase domain 2 (Abhd2) as an enzyme that metabolizes phosphatidylcholine and cardiolipin.

We and others have previously shown that genetic association can be used to make causal connections between gene loci and small molecules measured by mass spectrometry in the bloodstream and in tissues. We identified a locus on mouse chromosome 7 where several phospholipids in liver showed strong genetic association to distinct gene loci. In this study, we integrated gene expression data with genetic association data to identify a single gene at the chromosome 7 locus as the driver of the phospholipid phenotypes. The gene encodes α/ß-hydrolase domain 2 (Abhd2), one of 23 members of the ABHD gene family. We validated this observation by measuring lipids in a mouse with a whole-body deletion of Abhd2. The Abhd2KO mice had a significant increase in liver levels of phosphatidylcholine and phosphatidylethanolamine. Unexpectedly, we also found a decrease in two key mitochondrial lipids, cardiolipin and phosphatidylglycerol, in male Abhd2KO mice. These data suggest that Abhd2 plays a role in the synthesis, turnover, or remodeling of liver phospholipids.

Sphingosine-1-phosphate controls endothelial sphingolipid homeostasis via ORMDL.

Disruption of sphingolipid homeostasis and signaling has been implicated in diabetes, cancer, cardiometabolic, and neurodegenerative disorders. Yet, mechanisms governing cellular sensing and regulation of sphingolipid homeostasis remain largely unknown. In yeast, serine palmitoyltransferase, catalyzing the first and rate-limiting step of sphingolipid de novo biosynthesis, is negatively regulated by Orm1 and 2. Lowering sphingolipids triggers Orms phosphorylation, upregulation of serine palmitoyltransferase activity and sphingolipid de novo biosynthesis. However, mammalian orthologs ORMDLs lack the N-terminus hosting the phosphosites. Thus, which sphingolipid(s) are sensed by the cells, and mechanisms of homeostasis remain largely unknown. Here, we identify sphingosine-1-phosphate (S1P) as key sphingolipid sensed by cells via S1PRs to maintain homeostasis. The increase in S1P-S1PR signaling stabilizes ORMDLs, restraining SPT activity. Mechanistically, the hydroxylation of ORMDLs at Pro137 allows a constitutive degradation of ORMDLs via ubiquitin-proteasome pathway, preserving SPT activity. Disrupting S1PR/ORMDL axis results in ceramide accrual, mitochondrial dysfunction, impaired signal transduction, all underlying endothelial dysfunction, early event in the onset of cardio- and cerebrovascular diseases. Our discovery may provide the molecular basis for therapeutic intervention restoring sphingolipid homeostasis.

Endothelial-Specific Reduction in Arf6 Impairs Insulin-Stimulated Vasodilation and Skeletal Muscle Blood Flow Resulting in Systemic Insulin Resistance in Mice.

BACKGROUND: Much of what we know about insulin resistance is based on studies from metabolically active tissues such as the liver, adipose tissue, and skeletal muscle. Emerging evidence suggests that the vascular endothelium plays a crucial role in systemic insulin resistance; however, the underlying mechanisms remain incompletely understood. Arf6 (ADP ribosylation factor 6) is a small GTPase that plays a critical role in endothelial cell function. Here, we tested the hypothesis that the deletion of endothelial Arf6 will result in systemic insulin resistance. METHODS: We used mouse models of constitutive endothelial cell-specific Arf6 deletion (Arf6f/- Tie2Cre+) and tamoxifen-inducible Arf6 knockout (Arf6f/f Cdh5CreER+). Endothelium-dependent vasodilation was assessed using pressure myography. Metabolic function was assessed using a battery of metabolic assessments including glucose and insulin tolerance tests and hyperinsulinemic-euglycemic clamps. We used a fluorescence microsphere-based technique to measure tissue blood flow. Skeletal muscle capillary density was assessed using intravital microscopy. RESULTS: Endothelial Arf6 deletion impaired insulin-stimulated vasodilation in white adipose tissue and skeletal muscle feed arteries. The impairment in vasodilation was primarily due to attenuated insulin-stimulated nitric oxide bioavailability but independent of altered acetylcholine-mediated or sodium nitroprusside-mediated vasodilation. Endothelial cell-specific deletion of Arf6 also resulted in systematic insulin resistance in normal chow-fed mice and glucose intolerance in high-fat diet-fed obese mice. The underlying mechanisms of glucose intolerance were reductions in insulin-stimulated blood flow and glucose uptake in the skeletal muscle and were independent of changes in capillary density or vascular permeability. CONCLUSIONS: Results from this study support the conclusion that endothelial Arf6 signaling is essential for maintaining insulin sensitivity. Reduced expression of endothelial Arf6 impairs insulin-mediated vasodilation and results in systemic insulin resistance. These results have therapeutic implications for diseases that are associated with endothelial cell dysfunction and insulin resistance such as diabetes.

Ceramides Increase Fatty Acid Utilization in Intestinal Progenitors to Enhance Stemness and Increase Tumor Risk.

BACKGROUND & AIMS: Cancers of the alimentary tract, including esophageal adenocarcinomas, colorectal cancers, and cancers of the gastric cardia, are common comorbidities of obesity. Prolonged, excessive delivery of macronutrients to the cells lining the gut can increase one's risk for these cancers by inducing imbalances in the rate of intestinal stem cell proliferation vs differentiation, which can produce polyps and other aberrant growths. We investigated whether ceramides, which are sphingolipids that serve as a signal of nutritional excess, alter stem cell behaviors to influence cancer risk. METHODS: We profiled sphingolipids and sphingolipid-synthesizing enzymes in human adenomas and tumors. Thereafter, we manipulated expression of sphingolipid-producing enzymes, including serine palmitoyltransferase (SPT), in intestinal progenitors of mice, cultured organoids, and Drosophila to discern whether sphingolipids altered stem cell proliferation and metabolism. RESULTS: SPT, which diverts dietary fatty acids and amino acids into the biosynthetic pathway that produces ceramides and other sphingolipids, is a critical modulator of intestinal stem cell homeostasis. SPT and other enzymes in the sphingolipid biosynthesis pathway are up-regulated in human intestinal adenomas. They produce ceramides, which serve as prostemness signals that stimulate peroxisome-proliferator activated receptor-α and induce fatty acid binding protein-1. These actions lead to increased lipid utilization and enhanced proliferation of intestinal progenitors. CONCLUSIONS: Ceramides serve as critical links between dietary macronutrients, epithelial regeneration, and cancer risk.

Glucagon blockade restores functional ß-cell mass in type 1 diabetic mice and enhances function of human islets.

We evaluated the potential for a monoclonal antibody antagonist of the glucagon receptor (Ab-4) to maintain glucose homeostasis in type 1 diabetic rodents. We noted durable and sustained improvements in glycemia which persist long after treatment withdrawal. Ab-4 promoted ß-cell survival and enhanced the recovery of insulin+ islet mass with concomitant increases in circulating insulin and C peptide. In PANIC-ATTAC mice, an inducible model of ß-cell apoptosis which allows for robust assessment of ß-cell regeneration following caspase-8-induced diabetes, Ab-4 drove a 6.7-fold increase in ß-cell mass. Lineage tracing suggests that this restoration of functional insulin-producing cells was at least partially driven by α-cell-to-ß-cell conversion. Following hyperglycemic onset in nonobese diabetic (NOD) mice, Ab-4 treatment promoted improvements in C-peptide levels and insulin+ islet mass was dramatically increased. Lastly, diabetic mice receiving human islet xenografts showed stable improvements in glycemic control and increased human insulin secretion.

Identification of genetic drivers of plasma lipoprotein size in the Diversity Outbred mouse population.

Despite great progress in understanding lipoprotein physiology, there is still much to be learned about the genetic drivers of lipoprotein abundance, composition, and function. We used ion mobility spectrometry to survey 16 plasma lipoprotein subfractions in 500 Diversity Outbred mice maintained on a Western-style diet. We identified 21 quantitative trait loci (QTL) affecting lipoprotein abundance. To refine the QTL and link them to disease risk in humans, we asked if the human homologs of genes located at each QTL were associated with lipid traits in human genome-wide association studies. Integration of mouse QTL with human genome-wide association studies yielded candidate gene drivers for 18 of the 21 QTL. This approach enabled us to nominate the gene encoding the neutral ceramidase, Asah2, as a novel candidate driver at a QTL on chromosome 19 for large HDL particles (HDL-2b). To experimentally validate Asah2, we surveyed lipoproteins in Asah2-/- mice. Compared to wild-type mice, female Asah2-/- mice showed an increase in several lipoproteins, including HDL. Our results provide insights into the genetic regulation of circulating lipoproteins, as well as mechanisms by which lipoprotein subfractions may affect cardiovascular disease risk in humans.

The Lard Works in Mysterious Ways: Ceramides in Nutrition-Linked Chronic Disease.

Diet influences onset, progression, and severity of several chronic diseases, including heart failure, diabetes, steatohepatitis, and a subset of cancers. The prevalence and clinical burden of these obesity-linked diseases has risen over the past two decades. These metabolic disorders are driven by ectopic lipid deposition in tissues not suited for fat storage, leading to lipotoxic disruption of cell function and survival. Sphingolipids such as ceramides are among the most deleterious and bioactive metabolites that accrue, as they participate in selective insulin resistance, dyslipidemia, oxidative stress and apoptosis. This review discusses our current understanding of biochemical pathways controlling ceramide synthesis, production and action; influences of diet on ceramide levels; application of circulating ceramides as clinical biomarkers of metabolic disease; and molecular mechanisms linking ceramides to altered metabolism and survival of cells. Development of nutritional or pharmacological strategies to lower ceramides could have therapeutic value in a wide range of prevalent diseases.

CB1Rs in VMH neurons regulate glucose homeostasis but not body weight.

Cannabinoid 1 receptor (CB1R) inverse agonists reduce body weight and improve several parameters of glucose homeostasis. However, these drugs have also been associated with deleterious side effects. CB1R expression is widespread in the brain and in peripheral tissues, but whether specific sites of expression can mediate the beneficial metabolic effects of CB1R drugs, while avoiding the untoward side effects, remains unclear. Evidence suggests inverse agonists may act on key sites within the central nervous system to improve metabolism. The ventromedial hypothalamus (VMH) is a critical node regulating energy balance and glucose homeostasis. To determine the contributions of CB1Rs expressed in VMH neurons in regulating metabolic homeostasis, we generated mice lacking CB1Rs in the VMH. We found that the deletion of CB1Rs in the VMH did not affect body weight in chow- and high-fat diet-fed male and female mice. We also found that deletion of CB1Rs in the VMH did not alter weight loss responses induced by the CB1R inverse agonist SR141716. However, we did find that CB1Rs of the VMH regulate parameters of glucose homeostasis independent of body weight in diet-induced obese male mice.NEW & NOTEWORTHY Cannabinoid 1 receptors (CB1Rs) regulate metabolic homeostasis, and CB1R inverse agonists reduce body weight and improve parameters of glucose metabolism. However, the cell populations expressing CB1Rs that regulate metabolic homeostasis remain unclear. CB1Rs are highly expressed in the ventromedial hypothalamic nucleus (VMH), which is a crucial node that regulates metabolism. With CRISPR/Cas9, we generated mice lacking CB1Rs specifically in VMH neurons and found that CB1Rs in VMH neurons are essential for the regulation of glucose metabolism independent of body weight regulation.

PPARγ-K107 SUMOylation regulates insulin sensitivity but not adiposity in mice.

The nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) is a master regulator of adipocyte differentiation and is the target for the insulin-sensitizing thiazolidinedione (TZD) drugs used to treat type 2 diabetes. In cell-based in vitro studies, the transcriptional activity of PPARγ is inhibited by covalent attachment of small ubiquitin-related modifier (SUMOylation) at K107 in its N terminus. However, whether this posttranslational modification is relevant in vivo remains unclear. Here, using mice homozygous for a mutation (K107R) that prevents SUMOylation at this position, we demonstrate that PPARγ is SUMOylated at K107 in white adipose tissue. We further show that in the context of diet-induced obesity PPARγ-K107R-mutant mice have enhanced insulin sensitivity without the corresponding increase in adiposity that typically accompanies PPARγ activation by TZDs. Accordingly, the PPARγ-K107R mutation was weaker than TZD treatment in stimulating adipocyte differentiation in vitro. Moreover, we found that both the basal and TZD-dependent transcriptomes of inguinal and epididymal white adipose tissue depots were markedly altered in the K107R-mutant mice. We conclude that PPARγ SUMOylation at K107 is physiologically relevant and may serve as a pharmacologic target for uncoupling PPARγ's beneficial insulin-sensitizing effect from its adverse effect of weight gain.

Depletion of adipocyte sphingosine kinase 1 leads to cell hypertrophy, impaired lipolysis, and nonalcoholic fatty liver disease.

Sphingolipids have become established participants in the pathogenesis of obesity and its associated maladies. Sphingosine kinase 1 (SPHK1), which generates S1P, has been shown to increase in liver and adipose of obese humans and mice and to regulate inflammation in hepatocytes and adipose tissue, insulin resistance, and systemic inflammation in mouse models of obesity. Previous studies by us and others have demonstrated that global sphingosine kinase 1 KO mice are protected from diet-induced obesity, insulin resistance, systemic inflammation, and NAFLD, suggesting that SPHK1 may mediate pathological outcomes of obesity. As adipose tissue dysfunction has gained recognition as a central instigator of obesity-induced metabolic disease, we hypothesized that SPHK1 intrinsic to adipocytes may contribute to HFD-induced metabolic pathology. To test this, we depleted Sphk1 from adipocytes in mice (SK1fatKO) and placed them on a HFD. In contrast to our initial hypothesis, SK1fatKO mice displayed greater weight gain on HFD and exacerbated impairment in glucose clearance. Pro-inflammatory cytokines and neutrophil content of adipose tissue were similar, as were levels of circulating leptin and adiponectin. However, SPHK1-null adipocytes were hypertrophied and had lower basal lipolytic activity. Interestingly, hepatocyte triacylglycerol accumulation and expression of pro-inflammatory cytokines and collagen 1a1 were exacerbated in SK1fatKO mice on a HFD, implicating a specific role for adipocyte SPHK1 in adipocyte function and inter-organ cross-talk that maintains overall metabolic homeostasis in obesity. Thus, SPHK1 serves a previously unidentified essential homeostatic role in adipocytes that protects from obesity-associated pathology. These findings may have implications for pharmacological targeting of the SPHK1/S1P signaling axis.

Liver-specific ceramide reduction alleviates steatosis and insulin resistance in alcohol-fed mice.

Alcohol's impairment of both hepatic lipid metabolism and insulin resistance (IR) are key drivers of alcoholic steatosis, the initial stage of alcoholic liver disease (ALD). Pharmacologic reduction of lipotoxic ceramide prevents alcoholic steatosis and glucose intolerance in mice, but potential off-target effects limit its strategic utility. Here, we employed a hepatic-specific acid ceramidase (ASAH) overexpression model to reduce hepatic ceramides in a Lieber-DeCarli model of experimental alcoholic steatosis. We examined effects of alcohol on hepatic lipid metabolism, body composition, energy homeostasis, and insulin sensitivity as measured by hyperinsulinemic-euglycemic clamp. Our results demonstrate that hepatic ceramide reduction ameliorates the effects of alcohol on hepatic lipid droplet (LD) accumulation by promoting VLDL secretion and lipophagy, the latter of which involves ceramide cross-talk between the lysosomal and LD compartments. We additionally demonstrate that hepatic ceramide reduction prevents alcohol's inhibition of hepatic insulin signaling. These effects on the liver are associated with a reduction in oxidative stress markers and are relevant to humans, as we observe peri- LD ASAH expression in human ALD. Together, our results suggest a potential role for hepatic ceramide inhibition in preventing ALD.

Dapagliflozin suppresses glucagon signaling in rodent models of diabetes.

Sodium-glucose cotransporter 2 (SGLT2) inhibitors are a class of antidiabetic drug used for the treatment of diabetes. These drugs are thought to lower blood glucose by blocking reabsorption of glucose by SGLT2 in the proximal convoluted tubules of the kidney. To investigate the effect of inhibiting SGLT2 on pancreatic hormones, we treated perfused pancreata from rats with chemically induced diabetes with dapagliflozin and measured the response of glucagon secretion by alpha cells in response to elevated glucose. In these type 1 diabetic rats, glucose stimulated glucagon secretion by alpha cells; this was prevented by dapagliflozin. Two models of type 2 diabetes, severely diabetic Zucker rats and db/db mice fed dapagliflozin, showed significant improvement of blood glucose levels and glucose disposal, with reduced evidence of glucagon signaling in the liver, as exemplified by reduced phosphorylation of hepatic cAMP-responsive element binding protein, reduced expression of phosphoenolpyruvate carboxykinase 2, increased hepatic glycogen, and reduced hepatic glucose production. Plasma glucagon levels did not change significantly. However, dapagliflozin treatment reduced the expression of the liver glucagon receptor. Dapagliflozin in rodents appears to lower blood glucose levels in part by suppressing hepatic glucagon signaling through down-regulation of the hepatic glucagon receptor.

Acute loss of adipose tissue-derived adiponectin triggers immediate metabolic deterioration in mice.

AIM/HYPOTHESIS: Adiponectin (APN), a circulating hormone secreted by mature adipocytes, has been extensively studied because it has beneficial metabolic effects. While many studies have focused on the congenital loss of APN and its effects on systemic body glucose and lipid metabolism, little is known about the effects triggered by acute loss of APN in the adult mouse. We anticipated that genetically induced acute depletion of APN in adult mice would have a more profound effect on systemic metabolic health than congenital deletion of Adipoq, the gene encoding APN, with its associated potential for adaptive responses that may mask the phenotypes. METHODS: Mice carrying loxP-flanked regions of Adipoq were generated and bred to the Adipoq (APN) promoter-driven reverse tetracycline-controlled transactivator (rtTA) (APN-rtTA) gene and a tet-responsive Cre line (TRE-Cre) to achieve acute depletion of APN. Upon acute removal of APN in adult mice, systemic glucose and lipid homeostasis were assessed under basal and insulinopenic conditions. RESULTS: The acute depletion of APN results in more severe systemic insulin resistance and hyperlipidaemia than in mice with congenital loss of APN. Furthermore, the acute depletion of APN in adult mice results in a much more dramatic reduction in survival rate, with 50% of inducible knockouts dying in the first 5 days under insulinopenic conditions compared with 0% of congenital Adipoq knockout mice under similar conditions. CONCLUSIONS/INTERPRETATION: Acute systemic removal of APN results in a much more negative metabolic phenotype compared with congenital knockout of Adipoq. Specifically, our data demonstrate that acute depletion of APN is especially detrimental to lipid homeostasis, both under basal and insulinopenic conditions. This suggests that compensatory mechanisms exist in congenital knockout mice that offset some of the metabolic actions covered by APN.

PPARα agonist fenofibrate enhances fatty acid ß-oxidation and attenuates polycystic kidney and liver disease in mice.

Peroxisome proliferator-activated receptor α (PPARα) is a nuclear hormone receptor that promotes fatty acid ß-oxidation (FAO) and oxidative phosphorylation (OXPHOS). We and others have recently shown that PPARα and its target genes are downregulated, and FAO and OXPHOS are impaired in autosomal dominant polycystic kidney disease (ADPKD). However, whether PPARα and FAO/OXPHOS are causally linked to ADPKD progression is not entirely clear. We report that expression of PPARα and FAO/OXPHOS genes is downregulated, and in vivo ß-oxidation rate of 3H-labeled triolein is reduced in Pkd1RC/RC mice, a slowly progressing orthologous model of ADPKD that closely mimics the human ADPKD phenotype. To evaluate the effects of upregulating PPARα, we conducted a 5-mo, randomized, preclinical trial by treating Pkd1RC/RC mice with fenofibrate, a clinically available PPARα agonist. Fenofibrate treatment resulted in increased expression of PPARα and FAO/OXPHOS genes, upregulation of peroxisomal and mitochondrial biogenesis markers, and higher ß-oxidation rates in Pkd1RC/RC kidneys. MRI-assessed total kidney volume and total cyst volume, kidney-weight-to-body-weight ratio, cyst index, and serum creatinine levels were significantly reduced in fenofibrate-treated compared with untreated littermate Pkd1RC/RC mice. Moreover, fenofibrate treatment was associated with reduced kidney cyst proliferation and infiltration by inflammatory cells, including M2-like macrophages. Finally, fenofibrate treatment also reduced bile duct cyst number, cyst proliferation, and liver inflammation and fibrosis. In conclusion, our studies suggest that promoting PPARα activity to enhance mitochondrial metabolism may be a useful therapeutic strategy for ADPKD.

Hepatic ANGPTL3 regulates adipose tissue energy homeostasis.

Angiopoietin-like protein 3 (ANGPTL3) is a circulating inhibitor of lipoprotein and endothelial lipase whose physiological function has remained obscure. Here we show that ANGPTL3 plays a major role in promoting uptake of circulating very low density lipoprotein-triglycerides (VLDL-TGs) into white adipose tissue (WAT) rather than oxidative tissues (skeletal muscle, heart brown adipose tissue) in the fed state. This conclusion emerged from studies of Angptl3(-/-) mice. Whereas feeding increased VLDL-TG uptake into WAT eightfold in wild-type mice, no increase occurred in fed Angptl3(-/-) animals. Despite the reduction in delivery to and retention of TG in WAT, fat mass was largely preserved by a compensatory increase in de novo lipogenesis in Angptl3(-/-) mice. Glucose uptake into WAT was increased 10-fold in KO mice, and tracer studies revealed increased conversion of glucose to fatty acids in WAT but not liver. It is likely that the increased uptake of glucose into WAT explains the increased insulin sensitivity associated with inactivation of ANGPTL3. The beneficial effects of ANGPTL3 deficiency on both glucose and lipoprotein metabolism make it an attractive therapeutic target.

Glucose transporter 4-deficient hearts develop maladaptive hypertrophy in response to physiological or pathological stresses.

Pathological cardiac hypertrophy may be associated with reduced expression of glucose transporter 4 (GLUT4) in contrast to exercise-induced cardiac hypertrophy, where GLUT4 levels are increased. However, mice with cardiac-specific deletion of GLUT4 (G4H-/-) have normal cardiac function in the unstressed state. This study tested the hypothesis that cardiac GLUT4 is required for myocardial adaptations to hemodynamic demands. G4H-/- and control littermates were subjected to either a pathological model of left ventricular pressure overload [transverse aortic constriction (TAC)] or a physiological model of endurance exercise (swim training). As predicted after TAC, G4H-/- mice developed significantly greater hypertrophy and more severe contractile dysfunction. Somewhat surprisingly, after exercise training, G4H-/- mice developed increased fibrosis and apoptosis that was associated with dephosphorylation of the prosurvival kinase Akt in concert with an increase in protein levels of the upstream phosphatase protein phosphatase 2A (PP2A). Exercise has been shown to decrease levels of ceramide; G4H-/- hearts failed to decrease myocardial ceramide in response to exercise. Furthermore, G4H-/- hearts have reduced levels of the transcriptional coactivator peroxisome proliferator-activated receptor-γ coactivator-1, lower carnitine palmitoyl-transferase activity, and reduced hydroxyacyl-CoA dehydrogenase activity. These basal changes may also contribute to the impaired ability of G4H-/- hearts to adapt to hemodynamic stresses. In conclusion, GLUT4 is required for the maintenance of cardiac structure and function in response to physiological or pathological processes that increase energy demands, in part through secondary changes in mitochondrial metabolism and cellular stress survival pathways such as Akt.NEW & NOTEWORTHY Glucose transporter 4 (GLUT4) is required for myocardial adaptations to exercise, and its absence accelerates heart dysfunction after pressure overload. The requirement for GLUT4 may extend beyond glucose uptake to include defects in mitochondrial metabolism and survival signaling pathways that develop in its absence. Therefore, GLUT4 is critical for responses to hemodynamic stresses.

Hyperglycemia in rodent models of type 2 diabetes requires insulin-resistant alpha cells.

To determine the role of glucagon action in diet-induced and genetic type 2 diabetes (T2D), we studied high-fat-diet-induced obese (DIO) and leptin receptor-defective (LepR(-/-)) rodents with and without glucagon receptors (GcgRs). DIO and LepR(-/-),GcgR(+/+) mice both developed hyperinsulinemia, increased liver sterol response element binding protein 1c, and obesity. DIO GcgR(+/+) mice developed mild T2D, whereas LepR(-/-),GcgR(+/+) mice developed severe T2D. High-fat-fed (HFF) glucagon receptor-null mice did not develop hyperinsulinemia, increased liver sterol response element binding protein 1c mRNA, or obesity. Insulin treatment of HFF GcgR(-/-) to simulate HFF-induced hyperinsulinemia caused obesity and mild T2D. LepR(-/-),GcgR(-/-) did not develop hyperinsulinemia or hyperglycemia. Adenoviral delivery of GcgR to GcgR(-/-),LepR(-/-) mice caused the severe hyperinsulinemia and hyperglycemia of LepR(-/-) mice to appear. Spontaneous disappearance of the GcgR transgene abolished the hyperinsulinemia and hyperglycemia. In conclusion, T2D hyperglycemia requires unsuppressible hyperglucagonemia from insulin-resistant α cells and is prevented by glucagon suppression or blockade.

High-Mobility Group Box 1 Disrupts Metabolic Function with Cigarette Smoke Exposure in a Ceramide-Dependent Manner.

We have previously found that cigarette smoke disrupts metabolic function, in part, by increasing muscle ceramide accrual. To further our understanding of this, we sought to determine the role of the cytokine high-mobility group box 1 (HMGB1), which is increased with smoke exposure, in smoke-induced muscle metabolic perturbations. To test this theory, we determined HMGB1 from lungs of human smokers, as well as from lung cells from mice exposed to cigarette smoke. We also treated cells and mice directly with HMGB1, in the presence or absence of myriocin, an inhibitor of serine palmitoyltransferase, the rate-limiting enzyme in ceramide biosynthesis. Outcomes included assessments of insulin resistance and muscle mitochondrial function. HMGB1 was significantly increased in both human lungs and rodent alveolar macrophages. Further testing revealed that HMGB1 treatment elicited a widespread increase in ceramide species and reduction in myotube mitochondrial respiration, an increase in reactive oxygen species, and reduced insulin-stimulated Akt phosphorylation. Inhibition of ceramide biosynthesis with myriocin was protective. In mice, by comparing treatments of HMGB1 injections with or without myriocin, we found that HMGB1 injections resulted in increased muscle ceramides, especially C16 and C24, which were necessary for reduced muscle mitochondrial respiration and compromised insulin and glucose tolerance. In conclusion, HMGB1 may be a necessary intermediate in the ceramide-dependent metabolic consequences of cigarette smoke exposure.