The Effect of Hops (Humulus lupulus L.) Extract Supplementation on Weight Gain, Adiposity and Intestinal Function in Ovariectomized Mice.

Estrogen decline during menopause is associated with altered metabolism, weight gain and increased risk of cardiometabolic diseases. The gut microbiota also plays a role in the development of cardiometabolic dysfunction and is also subject to changes associated with age-related hormone changes. Phytoestrogens are plant-based estrogen mimics that have gained popularity as dietary supplements for the treatment or prevention of menopause-related symptoms. These compounds have the potential to both modulate and be metabolized by the gut microbiota. Hops (Humulus lupulus L.) contain potent phytoestrogen precursors, which rely on microbial biotransformation in the gut to estrogenic forms. We supplemented ovariectomized (OVX) or sham-operated (SHAM) C57BL/6 mice, with oral estradiol (E2), a flavonoid-rich extract from hops, or a placebo carrier oil, to observe effects on adiposity, inflammation, and gut bacteria composition. Hops extract (HE) and E2 protected against increased visceral adiposity and liver triglyceride accumulation in OVX animals. Surprisingly, we found no evidence of OVX having a significant impact on the overall gut bacterial community structure. We did find differences in the abundance of Akkermansia muciniphila, which was lower with HE treatment in the SHAM group relative to OVX E2 treatment and to placebo in the SHAM group.

Monounsaturated fatty acids protect against palmitate-induced lipoapoptosis in human umbilical vein endothelial cells.

Diets high in saturated fatty acids are linked to increased cardiovascular disease risk, whereas monounsaturated fatty acids have been associated with improved cardiovascular outcomes. Accordingly, cell culture studies have demonstrated that saturated fatty acids, particularly long chain saturated fatty acids such as palmitate, induce dysfunction and cell death in a variety of cell types, and monounsaturated fatty acids may confer protection against palmitate-mediated damage. The aim of the present study was to examine whether monounsaturated fatty acids could protect against palmitate-mediated cell death in endothelial cells, to determine if AMPK inactivation and activation (via compound C and AICAR, respectively) underlies both palmitate-induced damage and monounsaturated fatty acid-mediated protection, and to explore the role of ER stress in this context. Human umbilical vein endothelial cells were examined for cell viability and apoptosis following treatment for 24 hours with palmitate (0.25 and 0.5mM) alone or in combination with the monounsaturated fatty acids oleate or palmitoleate (0.25 and 0.5mM), AICAR, compound C, 4µ8C, or TUDCA. Compared to control cells, palmitate significantly decreased cell viability and increased apoptosis in a dose-dependent manner. The monounsaturated fatty acids oleate and palmitoleate completely prevented the cytotoxic effects of palmitate. Although palmitate induced markers of ER stress, chemical inhibition of ER stress did not prevent palmitate-induced lipoapoptosis. Conversely, the AMPK activator AICAR (0.1 and 0.5mM) conferred protection from palmitate mediated-alterations in viability, apoptosis and ER stress, whereas the AMPK inhibitor compound C (20 and 40µM) significantly exacerbated palmitate-mediated damage. Lastly, co-incubation with palmitate, monounsaturated fatty acids, and compound C significantly mitigated the protective effects of both oleate and palmitoleate. In conclusion, monounsaturated fatty acids confer protection against the cytotoxic effects of palmitate in vascular endothelial cells; and palmitate-mediated damage, as well as monounsaturated-mediated protection, are due in part to inactivation and activation, respectively, of the metabolic regulator AMPK. These results may have implications for understanding the deleterious effects of high saturated fat diets on cardiovascular dysfunction and disease risk.

Estradiol-mediated improvements in adipose tissue insulin sensitivity are related to the balance of adipose tissue estrogen receptor α and ß in postmenopausal women.

We recently demonstrated that short-term estradiol (E2) treatment improved insulin-mediated suppression of lipolysis in postmenopausal women, but to a greater extent in those who were late compared to early postmenopausal. In this follow-up study we tested whether subcutaneous adipose tissue (SAT) expression of estrogen receptors (ER) α and ß differs between early and late postmenopausal women. We further tested whether the balance of ERα to ERß in SAT determined the effect of E2 on SAT insulin sensitivity. The present study included 35 women who were ≤6 years past menopause (EPM; n = 16) or ≥10 years past menopause (LPM; n = 19). Fasted SAT samples were taken following 1-week transdermal E2 treatment or placebo (PL) in a random cross-over design. Samples were analyzed for nuclear/cytosolic protein content and mRNA expression using Western blot and qPCR, respectively. While ESR1 increased slightly (~1.4-fold) following E2 treatment in both groups, ERα and ERß protein expression did not differ between groups at baseline or in response to E2. However, the balance of ERα/ERß protein in the SAT nuclear fraction increased 10% in EPM compared to a 25% decrease in LPM women (group x treatment interaction, p<0.05). A greater proportion of ERα/ERß protein in the nuclear fraction of SAT at baseline (placebo day) was associated with greater reduction in SAT insulin resistance (i.e., better suppression of lipolysis, EC50) in response to E2 (r = -0.431, p<0.05). In conclusion, there do not appear to be differences in the proportion of adipose tissue ERα/ERß protein in late, compared to early, postmenopausal women. However, the balance of ERα/ERß may be important for E2-mediated improvement in adipose tissue insulin sensitivity. TRIAL REGISTRATION: Clinical Trials#: NCT01605071.

Age- and menopause-related differences in subcutaneous adipose tissue estrogen receptor mRNA expression.

OBJECTIVES: Changes in estrogen receptor (ER) expression likely underlie differential metabolic effects of estrogen in pre- and postmenopausal women. The aim of the current study was to determine whether ER gene expression in abdominal and femoral subcutaneous adipose tissue (SAT) was associated with age, menopause, or regional adiposity. METHODS: We studied pre- and post-menopausal (n=23 and 22, respectively; age 35-65y) normal weight (mean±SD; BMI 23.7±2.5kg/m2) women with similar total fat mass. Abdominal and femoral SAT ERα (ESR1) and ERß (ESR2) mRNA expression was determined by qPCR. RESULTS: Total fat mass did not differ between pre- and postmenopausal women (22.7±5.3vs. 21.7±5.3kg). Compared to premenopausal women, ESR1 and the ratio of ESR1 to ESR2 were lower (p≤0.05) in postmenopausal abdominal and femoral SAT. ESR1 and ESR1:ESR2 were inversely associated with age in abdominal SAT (r=-0.380 and r=-0.463, respectively; p<0.05) and femoral SAT (r=-0.353 and r=-0.472, respectively; p<0.05). ESR2 was not related to age or menopause. The inverse association between ESR1 and age persisted after adjusting for trunk fat mass, estradiol, or leptin. CONCLUSION: Among healthy pre- and postmenopausal women, increased age was associated with a decreased balance of ERα to ERß in abdominal and femoral subcutaneous adipose tissue.

Fuzhuan tea reverses arterial stiffening after modest weight gain in mice.

OBJECTIVES: The aim of this study was to examine the effects of a Western diet (WD) and supplementation with Fuzhuan tea on large artery stiffness, as determined by aortic pulse wave velocity (aPWV). METHODS: Mice were subjected to a standard diet (SD; n = 12) or WD (n = 10) for 7 mo, and were then separated to receive nonsupplemented drinking water (SD-W and WD-W) or water supplemented with Fuzhuan tea (SD-T and WD-T) (200 mg/kg daily); mice were then maintained on their respective diets for an additional 2 mo. RESULTS: After the initial 7-mo feeding period, WD elicited a modest and significantly greater increase in body weight than did SD (39.6 ± 0.71 versus 34.5 ± 1.16 g; P < 0.01). PWV was significantly elevated in WD but not in SD (459.3 ± 4.8 versus 422.4 ± 6.4 cm/s; P < 0.001). Following an additional 2 mo, PWV continued to increase in WD-W, but returned to control levels in WD-T (WD-W: 519.8 ± 12.8; WD-T: 426.5 ± 18.6; SD-W: 429.7 ± 8.6; SD-T: 429.1 ± 6.1 cm/s; P < 0.001, WD-W versus all groups). The increase in PWV in WD-W was accompanied by an increase in aortic collagen (WD-W: 38.8 ± 4.6 versus SD-W: 17.5 ± 5.1 percent cross-sectional area; P < 0.05). CONCLUSION: The results of the present study suggest that the increase in arterial stiffness after modest, diet-induced weight gain can be reversed by supplementation with Fuzhuan tea.

Region-specific effects of oestradiol on adipose-derived stem cell differentiation in post-menopausal women.

The goal of this study was to determine the effect of acute transdermal 17ß-oestradiol (E2 ) on the adipogenic potential of subcutaneous adipose-derived stem cells (ASC) in post-menopausal women. Post-menopausal women (n = 11; mean age 57 ± 4.5 years) were treated for 2 weeks, in a randomized, cross-over design, with transdermal E2 (0.15 mg) or placebo patches. Biopsies of abdominal (AB) and femoral (FEM) subcutaneous adipose tissue (SAT) were obtained after each treatment and mature adipocytes were analysed for cell size and ASC for their capacity for proliferation (growth rate), differentiation (triglyceride accumulation) and susceptibility to tumour necrosis factor alpha-induced apoptosis. Gene expression of oestrogen receptors α and ß (ESR1 and ESR2), perilipin 1 and hormone-sensitive lipase (HSL), was also assessed. In FEM SAT, but not AB SAT, 2 weeks of E2 significantly (P = 0.03) increased ASC differentiation and whole SAT HSL mRNA expression (P = 0.03) compared to placebo. These changes were not associated with mRNA expression of oestrogen receptors α and ß, but HSL expression was significantly increased in FEM SAT with transdermal E2 treatment. Adipose-derived stem cells proliferation and apoptosis did not change in either SAT depot after E2 compared with placebo. Short-term E2 appeared to increase the adipogenic potential of FEM, but not AB, SAT in post-menopausal women with possible implications for metabolic disease. Future studies are needed to determine longer term impact of E2 on regional SAT accumulation in the context of positive energy imbalance.

Fuzhuan tea consumption imparts hepatoprotective effects and alters intestinal microbiota in high saturated fat diet-fed rats.

SCOPE: Nonalcoholic fatty liver disease is an obesity-related disorder characterized by lipid infiltration of the liver. Management is limited to lifestyle modifications, highlighting the need for alternative therapeutic options. The objective of this study was to examine if fermented Fuzhuan tea prevents metabolic impairments associated with development of hepatic steatosis. METHODS AND RESULTS: Rats consumed control (CON) or high saturated fat (SAT) diets with or without Fuzhuan tea for 8 weeks. Outcomes included enzymatic and gene expression measures of metabolic dysregulation in liver and adipose tissue. Pyrosequencing was used to assess intestinal microbiota adaptations. Fuzhuan tea prevented diet-induced inflammation in the liver. Liver triglycerides of ∼18 mg/g were observed in SAT-fed animals, but remained similar to CON diet levels (∼12 mg/g) when supplemented with Fuzhuan tea. In adipose tissue, tea treatment prevented SAT-induced inflammation and reduced plasma leptin approximately twofold. Fuzhuan tea also altered intestinal function and was associated with a threefold increase in two Lactobacillus spp. CONCLUSIONS: These data suggest that Fuzhuan tea protects against liver and adipose tissue stress induced by a high SAT diet and positively influences intestinal function. Further investigation of the molecular targets of Fuzhuan tea is warranted.

Increasing energy flux to decrease the biological drive toward weight regain after weight loss - A proof-of-concept pilot study.

OBJECTIVE: Weight loss induces compensatory biological adjustments that increase hunger and decrease resting metabolic rate (RMR), which increase propensity for weight regain. In non-obese adults high levels of physical activity coupled with high energy intake (high energy flux) are associated with higher RMR and reduced hunger. We tested the possibility that a high flux state attenuates the increase in hunger and the decrease in RMR characteristic of diet-induced weight loss. METHODS: Six obese adults [age (mean ± SE) = 42 ± 12 y; body mass index (BMI) = 35.7 ± 3.7 kg/m2] underwent measures of RMR, the thermic effect of a meal (TEM), and fasting and postprandial measures of hunger and fullness as well as plasma glucose and insulin. Following weight loss, subjects completed two 5-day conditions of energy balance in random order-Low Flux (LF): sedentary with energy intake (EI) = RMR (kcal/d) × 1.35; and High Flux (HF): net exercise energy cost of ∼500 kcal/d and EI = RMR (kcal/d) × 1.7. RMR was measured daily for each flux condition. The morning following each of the respective experimentally controlled HF and LF conditions (flux day 5), they underwent the same pre-weight loss tests and also reported their perceptions of hunger and fullness during the previous four days of HF and LF, respectively. RESULTS: Average daily RMR was higher during HF (1926 ± 138 kcal/day) compared to LF (1847 ± 126 kcal/day; P < 0.05). Perceived hunger at the end of day was lower (p < 0.03) and fullness throughout the day was higher (p < 0.02) in HF compared to LF conditions. On day 5 of each flux condition, the thermic effect of a meal and circulating glucose and insulin after the meal did not differ between HF and LF. CONCLUSION: Following weight loss, compared to a sedentary LF state of energy balance, a short-term HF energy balance state is associated with higher RMR, lower perceived hunger, and greater perceived fullness, all of which could help attenuate the biologic drive to regain weight. Given the pilot nature of this study and the relatively short period of time spent in the high and low flux states, future research is needed to address this research question in a larger sample over a longer time period.

Ovariectomy results in differential shifts in gut microbiota in low versus high aerobic capacity rats.

The increased risk for cardiometabolic disease with the onset of menopause is widely studied and likely precipitated by the decline in endogenous estradiol (E2), yet the precise mechanisms are unknown. The gut microbiome is involved in estrogen metabolism and has been linked to metabolic disease, suggesting its potential involvement in the postmenopausal phenotype. Furthermore, menopause-associated risk factors, as well as gut ecology, are altered with exercise. Therefore, we studied microbial changes in an ovariectomized (OVX vs. Sham) rat model of high (HCR) and low (LCR) intrinsic aerobic capacity (n = 8-10/group) in relation to changes in body weight/composition, glucose tolerance, and liver triglycerides (TG). Nine weeks after OVX, HCR rats were moderately protected against regional adipose tissue gain and liver TG accumulation (P < 0.05 for both). Microbial diversity and number of the Bacteroidetes phylum were significantly increased in LCR with OVX, but unchanged in HCR OVX relative to Sham. Plasma short-chain fatty acids (SCFA), produced by bacteria in the gut and recognized as metabolic signaling molecules, were significantly greater in HCR Sham relative to LCR Sham rats (P = 0.05) and were decreased with OVX in both groups. These results suggest that increased aerobic capacity may be protective against menopause-associated cardiometabolic risk and that gut ecology, and production of signaling molecules such as SCFA, may contribute to the mediation.

Femoral lipectomy increases postprandial lipemia in women.

Femoral subcutaneous adipose tissue (SAT) appears to be cardioprotective compared with abdominal SAT, possibly through better triglyceride (TG) sequestration. We hypothesized that removal of femoral SAT would increase postprandial TG through a reduction in dietary fatty acid (FA) storage. Normal-weight (means ± SD; BMI 23.9 ± 2.6 kg/m(2)) women (n = 29; age 45 ± 6 yr) were randomized to femoral lipectomy (LIPO) or control (CON) and followed for 1 yr. Regional adiposity was measured by DEXA and CT. A liquid meal labeled with [(14)C]oleic acid was used to trace the appearance of dietary FA in plasma (6-h postprandial TG), breath (24-h oxidation), and SAT (24-h [(14)C]TG storage). Fasting LPL activity was measured in abdominal and femoral SAT. DEXA leg fat mass was reduced after LIPO vs. CON (Δ-1.4 ± 0.7 vs. 0.1 ± 0.5 kg, P < 0.001) and remained reduced at 1 yr (-1.1 ± 1.4 vs. -0.2 ± 0.5 kg, P < 0.05), as did CT thigh subcutaneous fat area (-39.6 ± 36.6 vs. 4.7 ± 14.6 cm(2), P < 0.05); DEXA trunk fat mass and CT visceral fat area were unchanged. Postprandial TG increased (5.9 ± 7.7 vs. -0.6 ± 5.3 × 10(3) mg/dl, P < 0.05) and femoral SAT LPL activity decreased (-21.9 ± 22.3 vs. 10.5 ± 26.5 nmol·min(-1)·g(-1), P < 0.05) 1 yr following LIPO vs. CON. There were no group differences in (14)C-labeled TG appearing in abdominal and femoral SAT or elsewhere. In conclusion, femoral fat remained reduced 1 yr following lipectomy and was accompanied by increased postprandial TG and reduced femoral SAT LPL activity. There were no changes in storage of meal-derived FA or visceral fat. Our data support a protective role for femoral adiposity on circulating TG independent of dietary FA storage and visceral adiposity.

Oral nitrite therapy improves vascular function in diabetic mice.

AIM: We tested the hypothesis that short-term oral sodium nitrite supplementation would improve vascular dysfunction in obese, diabetic mice. METHODS AND RESULTS: Vascular function was determined in control mice and in db/db mice receiving drinking water with or without sodium nitrite (50 mg/L) for 5 weeks. Nitrite supplementation increased plasma nitrite concentrations in db/db mice (0.19±0.02 µM vs 0.80±0.26 µM; p < 0.05). Db/db mice had lower endothelium-dependent dilation (EDD) in response to increasing doses of acetylcholine versus heterozygous control mice (71.2% ± 14.3% vs 93% ± 7.0%; p < 0.05), and sodium nitrite supplementation restored endothelium-dependent dilation to control levels (92.9% ± 2.3% vs 93% ± 7.0%; p < 0.05). The improvement in endothelial function was accompanied by a reduction in intrinsic stiffness, but not by alterations in plasma or vascular markers of inflammation. CONCLUSION: These data suggest that sodium nitrite may be a novel therapy for treating diabetes-related vascular dysfunction; however, the mechanisms of improvement are unknown.

Postprandial triglycerides and adipose tissue storage of dietary fatty acids: impact of menopause and estradiol.

OBJECTIVES: Postprandial lipemia worsens after menopause, but the mechanism remains unknown. The hypothesized menopause-related postprandial lipemia would be (1) associated with reduced storage of dietary fatty acids (FA) as triglyceride (TG) in subcutaneous adipose tissue (SAT) and (2) improved by short-term estradiol (E2 ). METHODS: Twenty-three pre- (mean ± SD: 42 ± 4 years) and 22 postmenopausal (55 ± 4 years) women with similar total adiposity were studied. A subset of postmenopausal women (n = 12) were studied following 2 weeks of E2 (0.15 mg) and matching placebo in a random, cross-over design. A liquid meal containing (14) C-oleic acid traced appearance of dietary FA in: serum (postprandial TG), breath (oxidation), and abdominal and femoral SAT (TG storage). RESULTS: Compared to premenopausal women, healthy, lean, postmenopausal women had increased postprandial glucose and insulin and trend for higher TG but had similar dietary FA oxidation and storage. Adipocytes were larger in post- compared to premenopausal women, particularly in femoral SAT. Short-term E2 reduced postprandial TG and insulin but had no effect on oxidation or storage of dietary FA. E2 increased the proportion of small adipocytes in femoral (but not abdominal) SAT. CONCLUSIONS: Short-term E2 attenuated menopause-related increases in postprandial TG and increased femoral adipocyte hyperplasia but not through increased net storage of dietary FA.

Activation of the unfolded protein response in vascular endothelial cells of nondiabetic obese adults.

CONTEXT: Activation of the unfolded protein response (UPR) is emerging as an important molecular signature of cardiometabolic diseases associated with obesity. However, despite the well-established role of the vascular endothelium in obesity-related cardiometabolic dysfunction, it is unclear whether the UPR is activated in endothelial cells of obese adults. OBJECTIVE: The objective of the study was to determine whether markers of UPR activation are increased in endothelial cells (ECs) of nondiabetic obese adults with impaired endothelial function. DESIGN, SETTING, AND PARTICIPANTS: Endothelial cells were obtained from antecubital veins of the nondiabetic obese adults [body mass index (BMI) ≥ 30 kg/m(2), n = 12] with impaired endothelial function and from their nonobese peers (BMI < 30 kg/m(2), n = 14). MAIN OUTCOME VARIABLES: UPR activation via expression (quantitative immunofluorescence) of the proximal UPR sensors, inositol-requiring endoplasmic reticulum (ER)-to-nucleus signaling protein 1 (IRE1), RNA-dependent protein kinase-like ER eukaryotic initiation factor-2α kinase (PERK), and activating transcription factor 6 (ATF6), were the main outcome variables. RESULTS: IRE1 expression was greater in obese vs nonobese individuals (0.84 ± 0.09 vs 0.47 ± 0.02 IRE1 intensity/human umbilical vein EC (HUVEC) intensity (n = 10/8, P < .01). Obese individuals also had greater EC activation of UPR stress sensors PERK and ATF6, indicated by increased expression of phosphorylated PERK [p-PERK; 0.49 ± 0.05 vs 0.36 ± 0.03, p-PERK (threonine 981) intensity/HUVEC intensity, n = 10 men, 13 women, P < .05] and nuclear localization of ATF6 (0.38 ± 0.05 vs 0.23 ± 0.02, nuclear ATF6 intensity/HUVEC intensity, n = 5 men, 9 women, P < .01), respectively. Stepwise linear regression analysis revealed that indices of body fat (BMI and waist circumference) were the strongest independent predictors of all 3 UPR mediators, explaining between 18% and 59% of the variance in endothelial cell expression of IRE1, p-PERK, and nuclear ATF6 localization. CONCLUSION: These results provide novel evidence for UPR activation in the endothelial cells of nondiabetic obese adults with vascular endothelial dysfunction.

The effects of sex, metabolic syndrome and exercise on postprandial lipemia.

OBJECTIVE: Exercise has been suggested to have cardioprotective benefits due to a lowering of postprandial triglycerides (PPTG). We hypothesized that a morning exercise bout would significantly lower PPTG measured over a full day, in response to moderate fat meals (35% energy) in men more so than women, and in metabolic syndrome (MetS) relative to normal weight (NW) individuals. MATERIALS/METHODS: Participants completed two randomized study days; one control and one exercise day (60 min of morning exercise, 60% VO(2peak)). Meals were consumed at breakfast, lunch and dinner with the energy expended during exercise replaced on the active day. The areas (AUC) and incremental areas (IAUC) under the curve were calculated for total triglycerides, total cholesterol and other metabolites. RESULTS: Exercise did not significantly change the PPTG AUC & IAUC overall, or within, or between, each sex or group (NW and MetS). Exercise induced a 30% decrease in total cholesterol IAUC (p=0.003) in NW subjects. Overall, women had a lower IAUC for PPTG compared to men (p=0.037), with the greatest difference between MetS women and MetS men, due to a sustained drop in TG after lunch in the women. This suggests that PP, rather than fasting, lipid analyses may be particularly important when evaluating sex differences in metabolic risk. CONCLUSIONS: With energy replacement, moderate morning exercise did not result in a significant decrease in PPTG excursions. Exercise did elicit a significant decrease in PP cholesterol levels in NW subjects, suggesting a potential mechanism for the cardioprotective effects of exercise.

The antiobesity effects of centrally administered neuromedin U and neuromedin S are mediated predominantly by the neuromedin U receptor 2 (NMUR2).

Neuromedin U (NMU) and neuromedin S (NMS) are structurally related neuropeptides that have been reported to modulate energy homeostasis. Pharmacological data have shown that NMU and NMS inhibit food intake when administered centrally and that NMU increases energy expenditure. Additionally, NMU-deficient mice develop obesity, whereas transgenic mice overexpressing NMU are lean and hypophagic. Two high-affinity NMU/NMS receptors, NMUR1 and NMUR2, have been identified. NMUR1 is predominantly expressed in the periphery, whereas NMUR2 is predominantly expressed in the brain, suggesting that the effects of centrally administered NMU and NMS are mediated by NMUR2. To evaluate the role of NMUR2 in the regulation of energy homeostasis, we characterized NMUR2-deficient (Nmur2(-/-)) mice. Nmur2(-/-) mice exhibited a modest resistance to diet-induced obesity that was at least in part due to reduced food intake. Acute central administration of NMU and NMS reduced food intake in wild-type but not in Nmur2(-/-) mice. The effects on activity and core temperature induced by centrally administered NMU were also absent in Nmur2(-/-) mice. Moreover, chronic central administration of NMU and NMS evoked significant reductions in body weight and sustained reductions in food intake in mice. In contrast, Nmur2(-/-) mice were largely resistant to these effects. Collectively, these data demonstrate that the anorectic and weight-reducing actions of centrally administered NMU and NMS are mediated predominantly by NMUR2, suggesting that NMUR2-selective agonists may be useful for the treatment of obesity.