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.

Aerobic capacity mediates susceptibility for the transition from steatosis to steatohepatitis.

KEY POINTS: Low intrinsic aerobic capacity is associated with increased all-cause and liver-related mortality in humans. Low intrinsic aerobic capacity in the low capacity runner (LCR) rat increases susceptibility to acute and chronic high-fat/high-sucrose diet-induced steatosis, without observed increases in liver inflammation. Addition of excess cholesterol to a high-fat/high-sucrose diet produced greater steatosis in LCR and high capacity runner (HCR) rats. However, the LCR rat demonstrated greater susceptibility to increased liver inflammatory and apoptotic markers compared to the HCR rat. The progressive non-alcoholic fatty liver disease observed in the LCR rats following western diet feeding was associated with further declines in liver fatty acid oxidation and mitochondrial respiratory capacity compared to HCR rats. ABSTRACT: Low aerobic capacity increases risk for non-alcoholic fatty liver disease and liver-related disease mortality, but mechanisms mediating these effects remain unknown. We recently reported that rats bred for low aerobic capacity (low capacity runner; LCR) displayed susceptibility to high fat diet-induced steatosis in association with reduced hepatic mitochondrial fatty acid oxidation (FAO) and respiratory capacity compared to high aerobic capacity (high capacity runner; HCR) rats. Here we tested the impact of aerobic capacity on susceptibility for progressive liver disease following a 16-week 'western diet' (WD) high in fat (45% kcal), cholesterol (1% w/w) and sucrose (15% kcal). Unlike previously with a diet high in fat and sucrose alone, the inclusion of cholesterol in the WD induced hepatomegaly and steatosis in both HCR and LCR rats, while producing greater cholesterol ester accumulation in LCR compared to HCR rats. Importantly, WD-fed low-fitness LCR rats displayed greater inflammatory cell infiltration, serum alanine transaminase, expression of hepatic inflammatory markers (F4/80, MCP-1, TLR4, TLR2 and IL-1ß) and effector caspase (caspase 3 and 7) activation compared to HCR rats. Further, LCR rats had greater WD-induced decreases in complete FAO and mitochondrial respiratory capacity. Intrinsic aerobic capacity had no impact on WD-induced hepatic steatosis; however, rats bred for low aerobic capacity developed greater hepatic inflammation, which was associated with reduced hepatic mitochondrial FAO and respiratory capacity and increased accumulation of cholesterol esters. These results confirm epidemiological reports that aerobic capacity impacts progression of liver disease and suggest that these effects are mediated through alterations in hepatic mitochondrial function.

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 UCP1 exacerbates Western diet-induced glycemic dysregulation independent of changes in body weight in female mice.

We tested the hypothesis that female mice null for uncoupling protein 1 (UCP1) would have increased susceptibility to Western diet-induced "whitening" of brown adipose tissue (AT) and glucose intolerance. Six-week-old C57BL/6J wild-type (WT) and UCP1 knockout (UCP1-/-) mice, housed at 25°C, were randomized to either a control diet (10% kcal from fat) or Western diet (45% kcal from fat and 1% cholesterol) for 28 wk. Loss of UCP1 had no effect on energy intake, energy expenditure, spontaneous physical activity, weight gain, or visceral white AT mass. Despite similar susceptibility to weight gain compared with WT, UCP1-/- exhibited whitening of brown AT evidenced by a striking ~500% increase in mass and appearance of large unilocular adipocytes, increased expression of genes related to inflammation, immune cell infiltration, and endoplasmic reticulum/oxidative stress (P < 0.05), and decreased mitochondrial subunit protein (COX I, II, III, and IV, P < 0.05), all of which were exacerbated by Western diet (P < 0.05). UCP1-/- mice also developed liver steatosis and glucose intolerance, which was worsened by Western diet. Collectively, these findings demonstrate that loss of UCP1 exacerbates Western diet-induced whitening of brown AT, glucose intolerance, and induces liver steatosis. Notably, the adverse metabolic manifestations of UCP1-/- were independent of changes in body weight, visceral adiposity, and energy expenditure. These novel findings uncover a previously unrecognized metabolic protective role of UCP1 that is independent of its already established role in energy homeostasis.

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.

Increased susceptibility to OVX-associated metabolic dysfunction in UCP1-null mice.

Premenopausal females are protected against adipose tissue inflammation and insulin resistance, until loss of ovarian hormone production (e.g., menopause). There is some evidence that females have greater brown adipose tissue (BAT) thermogenic capacity. Because BAT mass correlates inversely with insulin resistance, we hypothesized that increased uncoupling protein 1 (UCP1) expression contributes to the superior metabolic health of females. Given that UCP1 transiently increases in BAT following ovariectomy (OVX), we hypothesized that UCP1 may 'buffer' OVX-mediated metabolic dysfunction. Accordingly, female UCP1 knock-out (KO) and wild-type (Digby, et al.) mice received OVX or sham (SHM) surgeries at 12 weeks of age creating four groups (n=10/group), which were followed for 14 weeks and compared for: body weight and adiposity, food intake, energy expenditure and spontaneous physical activity (metabolic chambers), insulin resistance (HOMA-IR, ADIPO-IR, and glucose tolerance testing), and adipose tissue phenotype (histology, gene, and protein expression). Two-way ANOVA was used to assess main effects of genotype (G), OVX treatment (O), and genotype by treatment (GxO) interactions, which were considered significant when P<0.05. UCP1KO mice experienced a more adverse metabolic response to OVX than WT. Whereas OVX-induced weight gain was not synergistically greater for KO compared to WT (GxO, NS), OVX-induced insulin resistance was significantly exacerbated in KO compared to WT (GxO for HOMA-IR, P<0.05). These results suggest UCP1 is protective against metabolic dysfunction associated with loss of ovarian hormones and support the need for more research into therapeutics to selectively target UCP1 for prevention and treatment of metabolic dysfunction following ovarian hormone loss.

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.

Loss of Nlrp3 Does Not Protect Mice from Western Diet-Induced Adipose Tissue Inflammation and Glucose Intolerance.

We tested the hypothesis that loss of Nlrp3 would protect mice from Western diet-induced adipose tissue (AT) inflammation and associated glucose intolerance and cardiovascular complications. Five-week old C57BL6J wild-type (WT) and Nlrp3 knockout (Nlrp3-/-) mice were randomized to either a control diet (10% kcal from fat) or Western diet (45% kcal from fat and 1% cholesterol) for 24 weeks (n = 8/group). Contrary to our hypothesis that obesity-mediated white AT inflammation is Nlrp3-dependent, we found that Western diet-induced expression of AT inflammatory markers (i.e., Cd68, Cd11c, Emr1, Itgam, Lgals, Il18, Mcp1, Tnf, Ccr2, Ccl5 mRNAs, and Mac-2 protein) were not accompanied by increased caspase-1 cleavage, a hallmark feature of NLRP3 inflammasome activation. Furthermore, Nlrp3 null mice were not protected from Western diet-induced white or brown AT inflammation. Although Western diet promoted glucose intolerance in both WT and Nlrp3-/- mice, Nlrp3-/- mice were protected from Western diet-induced aortic stiffening. Additionally, Nlrp3-/- mice exhibited smaller cardiomyocytes and reduced cardiac fibrosis, independent of diet. Collectively, these findings suggest that presence of the Nlrp3 gene is not required for Western diet-induced AT inflammation and/or glucose intolerance; yet Nlrp3 appears to play a role in potentiating arterial stiffening, cardiac hypertrophy and fibrosis.