Pioglitazone Enhances β-Arrestin2 Signaling and Ameliorates Insulin Resistance in Classical Insulin Target Tissues

<b><i>Introduction:</i></b> Pioglitazone is a thiazolidinedione oral antidiabetic agent. This study aimed to investigate the effects of pioglitazone as insulin sensitizer on β-arrestin2 signaling in classical insulin target tissues. <b><i>Methods:</i></b> Experiments involved three groups of mice; the first one involved mice fed standard chow diet for 16 weeks; the second one involved mice fed high-fructose, high-fat diet (HFrHFD) for 16 weeks; and the third one involved mice fed HFrHFD for 16 weeks and received pioglitazone (30 mg/kg/day, orally) in the last four weeks of feeding HFrHFD. <b><i>Results:</i></b> The results showed significant improvement in the insulin sensitivity of pioglitazone-treated mice as manifested by significant reduction in the insulin resistance index. This improvement in insulin sensitivity was associated with significant increases in the β-arrestin2 levels in the adipose tissue, liver, and skeletal muscle. Moreover, pioglitazone significantly increased β-arrestin2 signaling in all the examined tissues as estimated from significant increases in phosphatidylinositol 4,5 bisphosphate and phosphorylation of Akt at serine 473 and significant decrease in diacylglycerol level. <b><i>Conclusion:</i></b> To the best of our knowledge, our work reports a new mechanism of action for pioglitazone through which it can enhance the insulin sensitivity. Pioglitazone increases β-arrestin2 signaling in the adipose tissue, liver, and skeletal muscle of HFrHFD-fed mice.

Impact of High-Fat Diet in Early-Life on Mammary Metabolic and Inflammatory Status in Later-Life in Mice

Objectives Emerging evidence indicates a potentially important role for early-life events and exposures in cancer development later in life. Moreover, accumulating evidence suggests that the incidence of cancers has reached a plateau in elders, whereas it continuously rises in young to middle adult. The present study aimed to investigate the potential impacts of high-fat diet in early-life, mimicking childhood/adolescent in humans, on mammary health in later-life of mice, equivalent to the young to middle age in human. Methods Female C57BL/8 mice (4 weeks of age) were fed a low-fat diet (LF: 10% kcal from fat) or a high-fat diet (HF: 60% kcal from fat) for 8 weeks, which is equivalent to child/adolescent age in humans. Mice in early-life groups were sacrificed after 8 weeks feeding, whereas mice in later-life groups were switched to standard chow diet (Lab Diet#5P76) and fed for additional 12 weeks before sacrifice. A panel of metabolic parameters, inflammatory cytokines, as well as gene expression related to tumorigenic Wnt-signaling were assessed by qPCR and immunoblotting analysis. Results Compared with LF group, the body weight in HF group was significantly elevated after 8-wk HF diet feeding (P &lt; 0.05). After switching to the standard chow diet for 12 weeks, the significance remained until 24 weeks of age although with a reduced degree of magnitude (P &lt; 0.05). For the metabolic factors, HFD reduced the expression levels of both Pparγ (P = 0.08) and adiponectin (P &lt; 0.05) at 12 weeks and the reductions remains at 24 weeks (P &lt; 0.01). Meanwhile, expressions of aromatase, estrogen receptor α and Tnf-α, Il-6, Il-10 as well as Cox2 among examined inflammatory mediators (Tnf-α, Il-6, Il-10, Il-2, Il-1β, Ifn-γ, Cox2) were significantly higher in HF than in LF group at 24 weeks (P &lt; 0.05). For Wnt-signaling target genes (Cyclin D1, C-Myc, and Axin 2), a significant increase for C-Myc was observed in HF group at 12 weeks (P &lt; 0.01). Conclusions Our results suggested that HF diet in early-life enhances adiposity and alters mammary metabolic and inflammatory status, creating a microenvironment in favor of breast tumorigenesis in later-life. Funding Sources This project was supported by USDA/Hatch (#1013548).

The Aging of Adipocytes Increases Expression of Pro-Inflammatory Cytokines Chronologically

Adipose tissue is a significant producer of pro-inflammatory cytokines in obese and old individuals. However, there is no direct evidence of whether and how aged adipocytes enhance the production of pro-inflammatory markers. We aimed to investigate whether the aging adipocytes increase pro-inflammatory markers. Swiss mouse embryonic-tissue-derived 3T3-L1 cells were differentiated into adipocytes and maintained for 60 days in the conditioned medium or 35 days in the unconditioned medium. Additionally, 20-month-old male C57BL/6 mice were fed a standard chow diet for 37 weeks until they were extremely aged, when ~75% of mice died because of aging. Accumulated lipids, pro-inflammatory markers, and nuclear factor kappa B (NF-κB) pathway markers from differentiated adipocytes were analyzed. Pro-inflammatory markers and NF-κB pathway markers of epididymal white adipose tissues (EWATs) and adipocytes from EWATs were also analyzed. We found that the aging adipocytes chronologically accumulated lipids and increased pro-inflammatory markers interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), and tumor necrosis factor-alpha (TNF-α); at the same time, NF-κB p50 markers were also increased while IκBα protein was decreased significantly in conditioned medium. Similar results were observed when differentiated adipocytes were maintained in the unconditioned medium and the adipocytes from EWATs of aged mice. We demonstrated that aging augmented chronic inflammation through the NF-κB signaling pathway in adipocytes and adipose tissue.

Dietary Epicatechin, A Novel Anti-aging Bioactive Small Molecule

: Epicatechin (EC), a flavonoid present in various foods including cocoa, dark chocolate, berries, and tea, has recently been reported to promote general health and survival of old mice fed a standard chow diet. This is considered a novel discovery in the field of identifying natural compounds to extend lifespan, given that presumably popular anti-aging natural agents including resveratrol, green tea extract, and curcumin had failed in extending the lifespan of standard chow-diet-fed mice. However, the anti-aging mechanism of EC is not fully understood, thus impeding the potential application of this natural compound in improving a healthy lifespan in humans. In this review, we first summarized the main dietary sources that contain a significant amount of EC and recent research regarding the absorption, metabolism and distribution of EC in humans and rodents. The review is then focused on the anti-aging effects of EC in cultured cells, animals and humans with the possible physiological, cellular and molecular mechanisms underlying its lifespan-extending effects.

A High Fat/High Sugar Diet Alters the Gastrointestinal Metabolome in a Sex Dependent Manner

The gut metabolome offers insight for identifying the source of diet related pathology. As such, the purpose of this study was to characterize alterations of the gut metabolome in female and male C57BL/6J mice randomly assigned to a standard “chow” diet (CHOW) or a high fat/high sugar diet (HFHS; 45% fat and 20% fructose drinking solution) for nine weeks. Cecal metabolites were extracted and an untargeted analysis via LC-MS/MS was performed. Partial Least Sums Discriminate Analysis (PLS-DA) presented significant differences between the two diet groups in a sex-dependent manner. Mann–Whitney U-tests revealed 2443 and 1669 features to be significantly different between diet groups in the females and males, respectively. The majority of altered metabolites were depleted within the cecum of the HFHS fed mice. Metabolic pathways associated with galactose metabolism, leukotriene metabolism, and androgen and estrogen biosynthesis and metabolism were differentially altered with an HFHS diet between sexes. We concluded the immense metabolite depletion and elevation of adverse metabolites associated with the HFHS diet is suggestive of poor gut health. Further, the differential alterations between female and male mice suggests that sex plays an important role in determining the effect of diet on the metabolome and host health.

Knockout of sulfatase 2 is associated with decreased steatohepatitis and fibrosis in a mouse model of nonalcoholic fatty liver disease

We report for the first time that in wild-type (WT) mice, fast-food diet (FFD) induced a threefold increase in hepatic Sulf2 mRNA and a 2.2-fold increase in sulfatase 2 (SULF2) protein expression compared with WT mice on standard chow diet (SC). We showed that knockout of SULF2 ameliorates FFD-induced obesity, hyperlipidemia, steatohepatitis, and fibrosis. These data, along with work from other laboratories, suggest that SULF2 may be critical to the ability of the liver to progress to nonalcoholic steatohepatitis and fibrosis in conditions of overnutrition.

Abstract P240: Activation Of Toll-like Receptor 4 (tlr4) Signaling In Mesenteric Arteries During Obesity Is Not Reversed By Weight Loss

Obesity in the United States is associated with overconsumption of the high fat and high carbohydrate western diet. It is a major risk factor for hypertension. The mechanisms by which obesity contributes to the development of hypertension remain unresolved. While obese women are three-fold more likely to develop hypertension than lean women, the majority of obesity-induced hypertension research has been conducted using male cohorts. Thus, there is an urgent need to investigate the pathophysiology of obesity-induced hypertension in women. A previous study from our lab demonstrated that endothelial dysfunction found in obese female rats is associated with TLR4 signaling activation in the aorta revealing an inflammatory state in a conduit artery during obesity. The goal of this present study is to investigate whether weight loss by reversal of western diet-induced obesity normalizes TLR4 signaling in conduit and resistance arteries. To address this question, eight-week-old female Wistar rats were randomized into three experimental groups: the Obese group (n=10) was fed a western diet (21% fat, 50% carbohydrate [34% sucrose], 20% protein) and the Lean group (n=10) was fed a standard chow diet (5% fat, 48.7% carbohydrate [3.2% sucrose], 24.1% protein) for 20 weeks. The weight loss group (n=10) was fed a western diet for 20 weeks and a standard chow diet for 8 weeks. While weight loss reduced BMI (0.65 ±0.03 vs.0.82 ±0.02 obese, p<0.01), systolic blood pressure consistently measured at 7pm by tail-cuff plethysmography remained elevated (139.83 ± 15.3 vs.150.06 ± 4.5, p=0.09). At the experimental endpoint, thoracic aortas and mesenteric arteries were obtained for molecular analysis of TLR4 signaling. As previously shown, aortic TLR4 signaling of the obese group was activated. Weight loss normalized TLR4 and MyD88 expression in thoracic aortas; however, expression of MyD88 remained increased in mesenteric arteries (2.8 fold increase, p<0.01, n=6). This suggests that persistent hypertension despite weight loss is associated with a failure of TLR4-MyD88 signaling to normalize in the resistance vessels.

Effects of High-Fat Diet and Exercise Intervention on the Metabolism Regulation of Infant Mice

Maternal exercise is crucial for promoting the health of the offspring. Previous studies showed that long-term maternal exercise improves energy metabolism during pregnancy. Whether swimming exercise can reverse the metabolic disorders caused by high-fat exposure in the early life of the offspring is yet to be elucidated. Three-week-old C57BL/6 female mice were randomly assigned to the standard chow diet group (SC), standard chow diet and exercise group (SC-Ex), high-fat diet group (HFD), and high-fat diet and exercise group (HFD-Ex). After swimming intervention for 13 weeks, male and female mice were caged, and the exercise intervention lasted until delivery. Then, the mothers were fed standard chow diet. A total of 8 offsprings/group were randomly selected after 4 weeks of lactation for GTT and ITT. After body composition analysis, the mice were sacrificed to obtain specimens. The levels of metabolism factors and IL-6 were measured by suspension microarray. Subsequently, 15 min after starting the GTT and ITT, the curve detected significant difference between the HFD and other groups. The body fat percentage of the HFD-Ex offspring was significantly lower than that of HFD offspring (p<0.05) irrespective of the gender. The levels of IL-6 and TG in the male offspring in the HFD-Ex group were improved significantly (p<0.05). Compared to the HFD offspring, serum glucose and GIP in the female offspring in the HFD-Ex group was significantly reduced (p<0.05). Long-term exercise of the mother effectively improved the metabolic disorder caused by high-fat exposure in the infant offspring. Thus, the metabolic inheritance of the offspring is gender-dependent; the maternal metabolism can make male offspring genetically susceptible.

Imaging Fibrogenesis in a Diet-Induced Model of Nonalcoholic Steatohepatitis (NASH)

Purpose. Liver fibrosis is the hallmark of chronic nonalcoholic steatohepatitis (NASH) and is characterised by the excessive deposition of extracellular matrix proteins. Early detection and accurate staging of liver fibrosis is critically important for patient management. One of the earliest pathological markers in NASH is the activation of hepatic stellate cells (HSCs) which may be exploited as a marker of fibrogenesis. Activated HSCs secreting factors such as integrin αvβ3 propagate fibrosis. The purpose of the current study was to assess the utility of the integrin αvβ3 imaging agent [18F]FtRGD for the early detection of fibrosis in a diet-induced model of NASH longitudinally using PET imaging. Procedures. Mice were fed with either standard chow diet (SD), high-fat diet (HFD), or a choline-deficient, L-amino acid-defined high-fat fibrogenic diet (CDAHFD) to mimic the clinical pathology of liver disease and followed longitudinally for 10 weeks to assess the development of liver fibrosis using [18F]FtRGD positron emission tomography (PET) imaging. Standard blood biochemistry, histological measures, and qPCR were used to quantify integrin αvβ3, smooth muscle actin, and collagen types 1 and 6 to assess the extent of NASH pathology and accurately stage liver fibrosis. Results. The CDAHFD fibrogenic diet predictably developed hepatic inflammation and steatosis over the 10 weeks studied with little NASH pathology detected in high fat diet-treated animals. Stage 1 fibrosis was detected early by histology at day 21 and progressed to stage 2 by day 35 and stage 3 by day 56 in mice fed with CDAHFD diet only. Noninvasive imaging with [18F]FtRGD correlated well with integrin αvβ3 and was able to distinguish early mild stage 2 fibrosis in CDAHFD animals compared with standard chow diet-fed animals at day 35. When compared with high fat diet-fed animals, [18F]FtRGD was only able to distinguish later moderate stage 2 fibrosis in CDAHFD animals at day 49. Conclusions. The diet-induced progression of liver fibrosis was confirmed using histology and correlated well with the mRNA of integrin αvβ3 and extracellular matrix protein expression. [18F]FtRGD showed very good correlation between liver uptake and integrin αvβ3 expression and similar detection sensitivity to the current clinical gold standard modalities for staging of liver fibrosis.

Effects of different diets used in diet-induced obesity models on insulin resistance and vascular dysfunction in C57BL/6 mice

The aim of the present study was to compare different diets used to induce obesity in a head-to-head manner with a focus on insulin resistance and vascular dysfunction. Male C57BL/6J mice were put on standard chow diet (SCD), normal-fat diet (NFD), cafeteria diet (CAF) or high-fat diet (HFD) for 12 weeks starting at the age of 6 weeks. Both CAF and HFD led to obesity (weight gain of 179% and 194%, respectively), glucose intolerance and insulin resistance to a comparable extent. In aortas containing perivascular adipose tissue (PVAT), acetylcholine-induced vasodilation was best in the NFD group and worst in the CAF group. Reduced phosphorylation of endothelial nitric oxide synthase at serine 1177 was observed in both CAF and HFD groups. Plasma coagulation activity was highest in the HFD group and lowest in the SCD group. Even the NFD group had significantly higher coagulation activity than the SCD group. In conclusions, CAF and HFD are both reliable mouse diets in inducing visceral obesity, glucose intolerance and insulin resistance. CAF is more effective than HFD in causing PVAT dysfunction and vascular dysfunction, whereas hypercoagulability was mostly evident in the HFD group. Coagulation activity was higher in NFD than NCD group.