Overproduction of endothelin-1 impairs glucose tolerance but does not promote visceral adipose tissue inflammation or limit metabolic adaptations to exercise.

Endothelin-1 (ET-1) is a potent vasoconstrictor and proinflammatory peptide that is upregulated in obesity. Herein, we tested the hypothesis that ET-1 signaling promotes visceral adipose tissue (AT) inflammation and disrupts glucose homeostasis. We also tested if reduced ET-1 is a required mechanism by which exercise ameliorates AT inflammation and improves glycemic control in obesity. We found that 1) diet-induced obesity, AT inflammation, and glycemic dysregulation were not accompanied by significantly increased levels of ET-1 in AT or circulation in wild-type mice and that endothelial overexpression of ET-1 and consequently increased ET-1 levels did not cause AT inflammation yet impaired glucose tolerance; 2) reduced AT inflammation and improved glucose tolerance with voluntary wheel running was not associated with decreased levels of ET-1 in AT or circulation in obese mice nor did endothelial overexpression of ET-1 impede such exercise-induced metabolic adaptations; 3) chronic pharmacological blockade of ET-1 receptors did not suppress AT inflammation in obese mice but improved glucose tolerance; and 4) in a cohort of human subjects with a wide range of body mass indexes, ET-1 levels in AT, or circulation were not correlated with markers of inflammation in AT. In aggregate, we conclude that ET-1 signaling is not implicated in the development of visceral AT inflammation but promotes glucose intolerance, thus representing an important therapeutic target for glycemic dysregulation in conditions characterized by hyperendothelinemia. Furthermore, we show that the salutary effects of exercise on AT and systemic metabolic function are not contingent on the suppression of ET-1 signaling.

Removal of interscapular brown adipose tissue increases aortic stiffness despite normal systemic glucose metabolism in mice.

Brown adipose tissue (BAT) is considered protective against obesity and related cardiometabolic dysfunction. Indeed, activation of BAT improves glucose homeostasis and attenuates cardiovascular disease development. However, whether a reduction in BAT mass perturbs metabolic function and increases risk for cardiovascular disease remains largely unknown. To address this question, C57BL/6J male mice underwent a sham procedure or surgical bilateral excision of interscapular BAT (iBATx) and were fed a normal chow or a Western diet for 18 wk, creating four groups ( n = 10/group). Mice were housed at 25°C. As expected, the Western diet increased final body weight and adiposity; however, contrary to our hypothesis, iBATx did not potentiate adiposity independent of diet. Furthermore, iBATx did not affect indexes of glycemic control (HbA1c, fasting glucose and insulin, and glucose area under the curve during a glucose tolerance test) and produced minimal-to-no effects on lipid homeostasis. The absence of metabolic disturbances with iBATx was not attributed to regrowth of iBAT or a "browning" or proliferative compensatory response of other BAT depots. Notably, iBATx caused an increase in aortic stiffness in normal chow-fed mice only, which was associated with an increase in aortic uncoupling protein-1. Collectively, we demonstrated that, at 25°C (i.e., limited thermal stress conditions), a substantial reduction in BAT mass via iBATx does not disrupt systemic glucose metabolism, challenging the current dogma that preservation of BAT is obligatory for optimal metabolic function. However, iBATx caused aortic stiffening in lean mice, hence supporting the existence of an interplay between iBAT and aortic stiffness, independent of alterations in glucose homeostasis.

Deletion of UCP1 enhances ex vivo aortic vasomotor function in female but not male mice despite similar susceptibility to metabolic dysfunction.

Females are typically more insulin sensitive than males, which may be partly attributed to greater brown adipose tissue (BAT) activity and uncoupling protein 1 (UCP1) content. Accordingly, we tested the hypothesis that UCP1 deletion would abolish sex differences in insulin sensitivity and that whitening of thoracic periaortic BAT caused by UCP1 loss would be accompanied with impaired thoracic aortic function. Furthermore, because UCP1 exerts antioxidant effects, we examined whether UCP1 deficiency-induced metabolic dysfunction was mediated by oxidative stress. Compared with males, female mice had lower HOMA- and AT-insulin resistance (IR) despite no significant differences in BAT UCP1 content. UCP1 ablation increased HOMA-IR, AT-IR, and whitening of BAT in both sexes. Expression of UCP1 in thoracic aorta was greater in wild-type females compared with males. Importantly, deletion of UCP1 enhanced aortic vasomotor function in females only. UCP1 ablation did not promote oxidative stress in interscapular BAT. Furthermore, daily administration of the free radical scavenger tempol for 8 wk did not abrogate UCP1 deficiency-induced increases in adiposity, hyperinsulinemia, or liver steatosis. Collectively, we report that 1) in normal chow-fed mice housed at 25°C, aortic UCP1 content was greater in females than males and its deletion improved ex vivo aortic vasomotor function in females only; 2) constitutive UCP1 content in BAT was similar between females and males and loss of UCP1 did not abolish sex differences in insulin sensitivity; and 3) the metabolic disruptions caused by UCP1 ablation did not appear to be contingent upon increased oxidative stress in mice under normal dietary conditions.

Loss of 3-O-sulfotransferase enzymes, Hs3st3a1 and Hs3st3b1, reduces kidney and glomerular size and disrupts glomerular architecture.

Heparan sulfate (HS) is an important component of the kidney anionic filtration barrier, the glomerular basement membrane (GBM). HS chains attached to proteoglycan protein cores are modified by sulfotransferases in a highly ordered series of biosynthetic steps resulting in immense structural diversity due to negatively charged sulfate modifications. 3-O-sulfation is the least abundant modification generated by a family of seven isoforms but creates the most highly sulfated HS domains. We analyzed the kidney phenotypes in the Hs3st3a1, Hs3st3b1 and Hs3st6 -knockout (KO) mice, the isoforms enriched in kidney podocytes. Individual KO mice show no overt kidney phenotype, although Hs3st3b1 kidneys were smaller than wildtype (WT). Furthermore, Hs3st3a1-/-; Hs3st3b1-/- double knockout (DKO) kidneys were smaller but also had a reduction in glomerular size relative to wildtype (WT). Mass spectrometry analysis of kidney HS showed reduced 3-O-sulfation in Hs3st3a1-/- and Hs3st3b1-/-, but not in Hs3st6-/- kidneys. Glomerular HS showed reduced HS staining and reduced ligand-and-carbohydrate engagement (LACE) assay, a tool that detects changes in binding of growth factor receptor-ligand complexes to HS. Interestingly, DKO mice have increased levels of blood urea nitrogen, although no differences were detected in urinary levels of albumin, creatinine and nephrin. Finally, transmission electron microscopy showed irregular and thickened GBM and podocyte foot process effacement in the DKO compared to WT. Together, our data suggest that loss of 3-O-HS domains disrupts the kidney glomerular architecture without affecting the glomerular filtration barrier and overall kidney function.

Muscle-Liver Trafficking of BCAA-Derived Nitrogen Underlies Obesity-Related Glycine Depletion.

Glycine levels are inversely associated with branched-chain amino acids (BCAAs) and cardiometabolic disease phenotypes, but biochemical mechanisms that explain these relationships remain uncharted. Metabolites and genes related to BCAA metabolism and nitrogen handling were strongly associated with glycine in correlation analyses. Stable isotope labeling in Zucker fatty rats (ZFRs) shows that glycine acts as a carbon donor for the pyruvate-alanine cycle in a BCAA-regulated manner. Inhibition of the BCAA transaminase (BCAT) enzymes depletes plasma pools of alanine and raises glycine levels. In high-fat-fed ZFRs, dietary glycine supplementation raises urinary acyl-glycine content and lowers circulating triglycerides but also results in accumulation of long-chain acyl-coenzyme As (acyl-CoAs), lower 5' adenosine monophosphate-activated protein kinase (AMPK) phosphorylation in muscle, and no improvement in glucose tolerance. Collectively, these studies frame a mechanism for explaining obesity-related glycine depletion and also provide insight into the impact of glycine supplementation on systemic glucose, lipid, and amino acid metabolism.

Chronic Elevation of Endothelin-1 Alone May Not Be Sufficient to Impair Endothelium-Dependent Relaxation.

Endothelin-1 (ET-1) is a powerful vasoconstrictor peptide considered to be causally implicated in hypertension and the development of cardiovascular disease. Increased ET-1 is commonly associated with reduced NO bioavailability and impaired vascular function; however, whether chronic elevation of ET-1 directly impairs endothelium-dependent relaxation (EDR) remains elusive. Herein, we report that (1) prolonged ET-1 exposure (ie, 48 hours) of naive mouse aortas or cultured endothelial cells did not impair EDR or reduce eNOS (endothelial NO synthase) activity, respectively (P>0.05); (2) mice with endothelial cell-specific ET-1 overexpression did not exhibit impaired EDR or reduced eNOS activity (P>0.05); (3) chronic (8 weeks) pharmacological blockade of ET-1 receptors in obese/hyperlipidemic mice did not improve aortic EDR or increase eNOS activity (P>0.05); and (4) vascular and plasma ET-1 did not inversely correlate with EDR in resistance arteries isolated from human subjects with a wide range of ET-1 levels (r=0.0037 and r=-0.1258, respectively). Furthermore, we report that prolonged ET-1 exposure downregulated vascular UCP-1 (uncoupling protein-1; P<0.05), which may contribute to the preservation of EDR in conditions characterized by hyperendothelinemia. Collectively, our findings demonstrate that chronic elevation of ET-1 alone may not be sufficient to impair EDR.