Role of adropin in arterial stiffening associated with obesity and type 2 diabetes.

Adropin is a peptide largely secreted by the liver and known to regulate energy homeostasis; however, it also exerts cardiovascular effects. Herein, we tested the hypothesis that low circulating levels of adropin in obesity and type 2 diabetes (T2D) contribute to arterial stiffening. In support of this hypothesis, we report that obesity and T2D are associated with reduced levels of adropin (in liver and plasma) and increased arterial stiffness in mice and humans. Establishing causation, we show that mesenteric arteries from adropin knockout mice are also stiffer, relative to arteries from wild-type counterparts, thus recapitulating the stiffening phenotype observed in T2D db/db mice. Given the above, we performed a set of follow-up experiments, in which we found that 1) exposure of endothelial cells or isolated mesenteric arteries from db/db mice to adropin reduces filamentous actin (F-actin) stress fibers and stiffness, 2) adropin-induced reduction of F-actin and stiffness in endothelial cells and db/db mesenteric arteries is abrogated by inhibition of nitric oxide (NO) synthase, and 3) stimulation of smooth muscle cells or db/db mesenteric arteries with a NO mimetic reduces stiffness. Lastly, we demonstrated that in vivo treatment of db/db mice with adropin for 4 wk reduces stiffness in mesenteric arteries. Collectively, these findings indicate that adropin can regulate arterial stiffness, likely via endothelium-derived NO, and thus support the notion that "hypoadropinemia" should be considered as a putative target for the prevention and treatment of arterial stiffening in obesity and T2D.NEW & NOTEWORTHY Arterial stiffening, a characteristic feature of obesity and type 2 diabetes (T2D), contributes to the development and progression of cardiovascular diseases. Herein we establish that adropin is decreased in obese and T2D models and furthermore provide evidence that reduced adropin may directly contribute to arterial stiffening. Collectively, findings from this work support the notion that "hypoadropinemia" should be considered as a putative target for the prevention and treatment of arterial stiffening in obesity and T2D.

Young Women Are Protected Against Vascular Insulin Resistance Induced by Adoption of an Obesogenic Lifestyle.

Vascular insulin resistance is a feature of obesity and type 2 diabetes that contributes to the genesis of vascular disease and glycemic dysregulation. Data from preclinical models indicate that vascular insulin resistance is an early event in the disease course, preceding the development of insulin resistance in metabolically active tissues. Whether this is translatable to humans requires further investigation. To this end, we examined if vascular insulin resistance develops when young healthy individuals (n = 18 men, n = 18 women) transition to an obesogenic lifestyle that would ultimately cause whole-body insulin resistance. Specifically, we hypothesized that short-term (10 days) exposure to reduced ambulatory activity (from >10 000 to <5000 steps/day) and increased consumption of sugar-sweetened beverages (6 cans/day) would be sufficient to prompt vascular insulin resistance. Furthermore, given that incidence of insulin resistance and cardiovascular disease is lower in premenopausal women than in men, we postulated that young females would be protected against vascular insulin resistance. Consistent with this hypothesis, we report that after reduced ambulation and increased ingestion of carbonated beverages high in sugar, young healthy men, but not women, exhibited a blunted leg blood flow response to insulin and suppressed skeletal muscle microvascular perfusion. These findings were associated with a decrease in plasma adropin and nitrite concentrations. This is the first evidence in humans that vascular insulin resistance can be provoked by short-term adverse lifestyle changes. It is also the first documentation of a sexual dimorphism in the development of vascular insulin resistance in association with changes in adropin levels.

Cystamine reduces vascular stiffness in Western diet-fed female mice.

Consumption of diets high in fat, sugar, and salt (Western diet, WD) is associated with accelerated arterial stiffening, a major independent risk factor for cardiovascular disease (CVD). Women with obesity are more prone to develop arterial stiffening leading to more frequent and severe CVD compared with men. As tissue transglutaminase (TG2) has been implicated in vascular stiffening, our goal herein was to determine the efficacy of cystamine, a nonspecific TG2 inhibitor, at reducing vascular stiffness in female mice chronically fed a WD. Three experimental groups of female mice were created. One was fed regular chow diet (CD) for 43 wk starting at 4 wk of age. The second was fed a WD for the same 43 wk, whereas a third cohort was fed WD, but also received cystamine (216 mg/kg/day) in the drinking water during the last 8 wk on the diet (WD + C). All vascular stiffness parameters assessed, including aortic pulse wave velocity and the incremental modulus of elasticity of isolated femoral and mesenteric arteries, were significantly increased in WD- versus CD-fed mice, and reduced in WD + C versus WD-fed mice. These changes coincided with respectively augmented and diminished vascular wall collagen and F-actin content, with no associated effect in blood pressure. In cultured human vascular smooth muscle cells, cystamine reduced TG2 activity, F-actin:G-actin ratio, collagen compaction capacity, and cellular stiffness. We conclude that cystamine treatment represents an effective approach to reduce vascular stiffness in female mice in the setting of WD consumption, likely because of its TG2 inhibitory capacity.NEW & NOTEWORTHY This study evaluates the novel role of transglutaminase 2 (TG2) inhibition to directly treat vascular stiffness. Our data demonstrate that cystamine, a nonspecific TG2 inhibitor, improves vascular stiffness induced by a diet rich in fat, fructose, and salt. This research suggests that TG2 inhibition might bear therapeutic potential to reduce the disproportionate burden of cardiovascular disease in females in conditions of chronic overnutrition.

Voluntary Wheel Running Partially Compensates for the Effects of Global Estrogen Receptor-α Knockout on Cortical Bone in Young Male Mice.

Estrogen receptor-α knockout (ERKO) in female, but not male, mice results in an impaired osteogenic response to exercise, but the mechanisms behind this ability in males are unknown. We explored the main and interactive effects of ERKO and exercise on cortical geometry, trabecular microarchitecture, biomechanical strength, and sclerostin expression in male mice. At 12 weeks of age, male C57BL/6J ERKO and WT animals were randomized into two groups: exercise treatment (EX) and sedentary (SED) controls, until 22 weeks of age. Cortical geometry and trabecular microarchitecture were measured via µCT; biomechanical strength was assessed via three-point bending; sclerostin expression was measured via immunohistochemistry. Two-way ANOVA was used to assess sclerostin expression and trabecular microarchitecture; two-way ANCOVA with body weight was used to assess cortical geometry and biomechanical strength. ERKO positively impacted trabecular microarchitecture, and exercise had little effect on these outcomes. ERKO significantly impaired cortical geometry, but exercise was able to partially reverse these negative alterations. EX increased cortical thickness regardless of genotype. There were no effects of genotype or exercise on sclerostin expression. In conclusion, male ERKO mice retain the ability to build bone in response to exercise, but altering sclerostin expression is not one of the mechanisms involved.

Daily watermelon consumption decreases plasma sVCAM-1 levels in overweight and obese postmenopausal women.

Postmenopausal status is associated with an increase in total and abdominal body fat as well as increased incidence of insulin resistance and cardiovascular disease. The purpose of this study was to determine if watermelon supplementation affects select systemic markers of atherosclerosis and measures of insulin resistance in overweight and obese postmenopausal women. We hypothesized that overweight and obese postmenopausal women consuming 100% watermelon puree daily for 6 weeks would have improved levels of select systemic markers connected with cardiovascular disease without changing markers of insulin resistance. To test this hypothesis, overweight and obese postmenopausal women were recruited to participate in this study. Participants were randomly assigned to either the control group (no intervention) or the watermelon puree group (WM) for 6 weeks. Plasma concentration of markers connected with atherosclerosis and glycemic control were measured pre- and poststudy. A significant 6% decrease in soluble vascular cell adhesion molecule-1 occurred pre- to poststudy in WM, P = .003. The pattern of change in fasting blood glucose (P = .633), insulin (P = .158), and homeostatic model assessment-estimated insulin resistance (P = .174) did not differ between groups. Pre- to poststudy increases were measured in the fasting plasma concentration of l-arginine (8%, P = .005), cis-lycopene (32%, P = .003), and trans-lycopene (42%, P = .003) in WM. We conclude that 6 weeks of watermelon supplementation improved soluble vascular cell adhesion molecule-1 levels, a marker connected to atherogenesis, independent of changes in body composition or glycemic control.

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.

Western Diet-Fed, Aortic-Banded Ossabaw Swine: A Preclinical Model of Cardio-Metabolic Heart Failure.

The development of new treatments for heart failure lack animal models that encompass the increasingly heterogeneous disease profile of this patient population. This report provides evidence supporting the hypothesis that Western Diet-fed, aortic-banded Ossabaw swine display an integrated physiological, morphological, and genetic phenotype evocative of cardio-metabolic heart failure. This new preclinical animal model displays a distinctive constellation of findings that are conceivably useful to extending the understanding of how pre-existing cardio-metabolic syndrome can contribute to developing HF.

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.

Estrogen receptor-α signaling maintains immunometabolic function in males and is obligatory for exercise-induced amelioration of nonalcoholic fatty liver.

The role of estrogen receptor-α (ERα) signaling in immunometabolic function is established in females. However, its necessity in males, while appreciated, requires further study. Accordingly, we first determined whether lower metabolic function in male mice compared with females is related to reduced ERα expression. ERα protein expression in metabolically active tissues was lower in males than in females, and this lower expression was associated with worse glucose tolerance. Second, we determined whether ERα is required for optimal immunometabolic function in male mice consuming a chow diet. Despite lower expression of ERα in males, its genetic ablation (KO) caused an insulin-resistant phenotype characterized by enhanced adiposity, glucose intolerance, hepatic steatosis, and metaflammation in adipose tissue and liver. Last, we determined whether ERα is essential for exercise-induced metabolic adaptations. Twelve-week-old wild-type (WT) and ERα KO mice either remained sedentary (SED) or were given access to running wheels (WR) for 10 wk while fed an obesogenic diet. Body weight and fat mass were lower in WR mice regardless of genotype. Daily exercise obliterated immune cell infiltration and inflammatory gene transcripts in adipose tissue in both genotypes. In the liver, however, wheel running suppressed hepatic steatosis and inflammatory gene transcripts in WT but not in KO mice. In conclusion, the present findings indicate that ERα is required for optimal immunometabolic function in male mice despite their reduced ERα protein expression in metabolically active tissues. Furthermore, for the first time, we show that ERα signaling appears to be obligatory for exercise-induced prevention of hepatic steatosis.

Aerobic exercise training improves insulin-induced vasorelaxation in a vessel-specific manner in rats with insulin-treated experimental diabetes.

Vascular insulin resistance often precedes endothelial dysfunction in type 1 diabetes mellitus. Strategies to limit vascular dysfunction include intensive insulin therapy (4-9 mM) and aerobic training. To avoid the risk of hypoglycaemia, individuals often prescribed conventional insulin therapy (9-15 mM) and participate in resistance training. In a model of type 1 diabetes mellitus, this study examined insulin-induced vasomotor function in the aorta and femoral artery to determine (1) whether resistance training with conventional insulin therapy provides the same benefits as aerobic training with conventional insulin therapy, (2) whether aerobic training or resistance training, when paired with conventional insulin therapy, results in superior vasomotor function compared to intensive insulin therapy alone and (3) whether vessel-specific adaptations exist. Groups consisted of conventional insulin therapy, intensive insulin therapy, aerobic training with conventional insulin therapy and resistance training with conventional insulin therapy. Following multiple low doses of streptozotocin, male Sprague-Dawley rats were supplemented with insulin to maintain blood glucose concentrations (9-15 mM: conventional insulin therapy, aerobic training and resistance training; 4-9 mM: intensive insulin therapy) for 12 weeks. Aerobic training performed treadmill exercise and resistance training consisted of weighted climbing. Coinciding with increased Akt signalling, aerobic training resulted in enhanced insulin-induced vasorelaxation in the femoral artery. Intensive insulin therapy displayed increased mitogen-activated protein kinase signalling and no improvement in insulin-stimulated vasorelaxation compared to all other groups. These data suggest that aerobic training may be more beneficial for limiting the pathogenesis of vascular disease in type 1 diabetes mellitus than merely intensive insulin therapy.

Microvascular insulin resistance in skeletal muscle and brain occurs early in the development of juvenile obesity in pigs.

Impaired microvascular insulin signaling may develop before overt indices of microvascular endothelial dysfunction and represent an early pathological feature of adolescent obesity. Using a translational porcine model of juvenile obesity, we tested the hypotheses that in the early stages of obesity development, impaired insulin signaling manifests in skeletal muscle (triceps), brain (prefrontal cortex), and corresponding vasculatures, and that depressed insulin-induced vasodilation is reversible with acute inhibition of protein kinase Cß (PKCß). Juvenile Ossabaw miniature swine (3.5 mo of age) were divided into two groups: lean control ( n = 6) and obese ( n = 6). Obesity was induced by feeding the animals a high-fat/high-fructose corn syrup/high-cholesterol diet for 10 wk. Juvenile obesity was characterized by excess body mass, hyperglycemia, physical inactivity (accelerometer), and marked lipid accumulation in the skeletal muscle, with no evidence of overt atherosclerotic lesions in athero-prone regions, such as the abdominal aorta. Endothelium-dependent (bradykinin) and -independent (sodium nitroprusside) vasomotor responses in the brachial and carotid arteries (wire myography), as well as in the skeletal muscle resistance and 2A pial arterioles (pressure myography) were unaltered, but insulin-induced microvascular vasodilation was impaired in the obese group. Blunted insulin-stimulated vasodilation, which was reversed with acute PKCß inhibition (LY333-531), occurred alongside decreased tissue perfusion, as well as reduced insulin-stimulated Akt signaling in the prefrontal cortex, but not the triceps. In the early stages of juvenile obesity development, the microvasculature and prefrontal cortex exhibit impaired insulin signaling. Such adaptations may underscore vascular and neurological derangements associated with juvenile obesity.

Endothelial dysfunction occurs independently of adipose tissue inflammation and insulin resistance in ovariectomized Yucatan miniature-swine.

In rodents, experimentally-induced ovarian hormone deficiency increases adiposity and adipose tissue (AT) inflammation, which is thought to contribute to insulin resistance and increased cardiovascular disease risk. However, whether this occurs in a translationally-relevant large animal model remains unknown. Herein, we tested the hypothesis that ovariectomy would promote visceral and perivascular AT (PVAT) inflammation, as well as subsequent insulin resistance and peripheral vascular dysfunction in female swine. At sexual maturity (7 months of age), female Yucatan mini-swine either remained intact (control, n = 9) or were ovariectomized (OVX, n = 7). All pigs were fed standard chow (15-20 g/kg), and were euthanized 6 months post-surgery. Uterine mass and plasma estradiol levels were decreased by ∼10-fold and 2-fold, respectively, in OVX compared to control pigs. Body mass, glucose homeostasis, and markers of insulin resistance were not different between control and OVX pigs; however, OVX animals exhibited greater plasma triglycerides and triglyceride:HDL ratio. Ovariectomy enhanced visceral adipocyte expansion, although this was not accompanied by brachial artery PVAT adipocyte expansion, AT inflammation in either depot, or increased systemic inflammation assessed by plasma C-reactive protein concentrations. Despite the lack of AT inflammation and insulin resistance, OVX pigs exhibited depressed brachial artery endothelial-dependent vasorelaxation, which was rescued with blockade of endothelin receptor A. Together, these findings indicate that in female Yucatan mini-swine, increased AT inflammation and insulin resistance are not required for loss of ovarian hormones to induce endothelial dysfunction.

Ablation of eNOS does not promote adipose tissue inflammation.

Adipose tissue (AT) inflammation is a hallmark characteristic of obesity and an important determinant of insulin resistance and cardiovascular disease; therefore, a better understanding of factors regulating AT inflammation is critical. It is well established that reduced vascular endothelial nitric oxide (NO) bioavailability promotes arterial inflammation; however, the role of NO in modulating inflammation in AT remains disputed. In the present study, 10-wk-old C57BL6 wild-type and endothelial nitric oxide synthase (eNOS) knockout male mice were randomized to either a control diet (10% kcal from fat) or a Western diet (44.9% kcal from fat, 17% sucrose, and 1% cholesterol) for 18 wk (n= 7 or 8/group). In wild-type mice, Western diet-induced obesity led to increased visceral white AT expression of inflammatory genes (e.g., MCP1, TNF-α, and CCL5 mRNAs) and markers of macrophage infiltration (e.g., CD68, ITGAM, EMR1, CD11C mRNAs, and Mac-2 protein), as well as reduced markers of mitochondrial content (e.g., OXPHOS complex I and IV protein). Unexpectedly, these effects of Western diet on visceral white AT were not accompanied by decreases in eNOS phosphorylation at Ser-1177 or increases in eNOS phosphorylation at Thr-495. Also counter to expectations, eNOS knockout mice, independent of the diet, were leaner and did not exhibit greater white or brown AT inflammation compared with wild-type mice. Collectively, these findings do not support the hypothesis that reduced NO production from eNOS contributes to obesity-related AT inflammation.