Comparative hepatic proteome analysis between lean and obese rats fed a high-fat diet reveals the existence of gender differences

Gender differences in obesity stem from metabolic and hormonal differences between sexes and contribute to differences between women and men in health risks attributable to obesity. We hypothesized that liver may be an ideal target for the evaluation of gender differences in obesity development in response to a high-fat diet (HFD). Therefore, to test this hypothesis, we performed a global proteome analysis in the liver of lean and obese rats of both genders who were fed an HFD through 2-DE combined with MALDI-TOF-MS. When rats were exposed to HFD, male rats gained more body weight with increased values of plasma biochemical parameters than female rats. Image analysis and further statistical analysis of a 2-DE protein map allowed for the detection and identification of 34 proteins that were significantly modulated in a gender-dependent manner. We found 19 proteins showing identical gender-different regulation in both normal diet (ND) and HFD. Five proteins also showed clear gender differences in both ND and HFD; however, their regulation modes in HFD were opposite to those in ND. Of particular interest, 10 proteins showed gender differences only in either ND or HFD rats. Present proteomic insight into gender-dimorphic protein modulation in liver would aid in the improvement of gender awareness in the health-care system and in implementation of evidence-based gender-specific clinical recommendations.     

Differential expression of skeletal muscle proteins in high‐fat diet‐fed rats in response to capsaicin feeding

In this study, the effects of capsaicin on expression of skeletal muscle proteins in Sprague–Dawley rats fed with a high‐fat diet (HFD) were investigated. Rats were fed a HFD with or without capsaicin treatment for 8 wk. After HFD feeding, capsaicin‐treated rats weighed an average of 8% less than those of the HFD control group. Gastrocnemius muscle tissue from lean and obese rats with or without capsaicin treatment was arrayed using 2‐DE for detection of HFD‐associated markers. Proteomic analysis using 2‐DE demonstrated that 36 spots from a total of approximately 600 matched spots showed significantly different expression; 27 spots were identified as gastrocnemius muscle proteins that had been altered in response to capsaicin feeding, and 6 spots could not be identified by mass fingerprinting. Expression of various muscle proteins was determined by immunoblot analysis for the determination of molecular mechanisms, whereby capsaicin caused inhibition of adipogenesis. Immunoblot analysis revealed increased uncoupling protein 3 (UCP3) protein expression in HFD‐fed rats, whereas contents were reduced with capsaicin treatment. Compared with the HFD control group, capsaicin treatment increased phosphorylation of AMP‐activated protein kinase (AMPIC) CP3 and acetyl‐CoA carboxylase (ACC). To support this result, we also analyzed in vitro differential protein expression in L6 skeletal muscle cells. These data suggest that the AMPK‐ACC‐malonyl‐CoA metabolic signaling pathway is one of the targets of capsaicin action. To the best of our knowledge, this is the first proteomic study to report on analysis of diet‐induced alterations of protein expression that are essential for energy expenditure in rat muscle.     

Pharmacological and nutritional agents promoting browning of white adipose tissue

The role of brown adipose tissue in the regulation of energy balance and maintenance of body weight is well known in rodents. Recently, interest in this tissue has re-emerged due to the realization of active brown-like adipose tissue in adult humans and inducible brown-like adipocytes in white adipose tissue depots in response to appropriate stimuli (“browning process”). Brown-like adipocytes that appear in white fat depots have been called “brite” (from brown-in-white) or “beige” adipocytes and have characteristics similar to brown adipocytes, in particular the capacity for uncoupled respiration. There is controversy as to the origin of these brite/beige adipocytes, but regardless of this, induction of the browning of white fat represents an attractive potential strategy for the management and treatment of obesity and related complications. Here, the different physiological, pharmacological and dietary determinants that have been linked to white-to-brown fat remodeling and the molecular mechanisms involved are reviewed in detail. In the light of available data, interesting therapeutic perspectives can be expected from the use of specific drugs or food compounds able to induce a program of brown fat differentiation including uncoupling protein 1 expression and enhancing oxidative metabolism in white adipose cells. However, additional research is needed, mainly focused on the physiological relevance of browning and its dietary control, where the use of ferrets and other non-rodent animal models with a more similar adipose tissue organization and metabolism to humans could be of much help. This article is part of a Special Issue entitled Brown and White Fat: From Signaling to Disease.     

Adiponutrin mRNA expression in white adipose tissue is rapidly induced by meal-feeding a high-sucrose diet

Adiponutrin is a recently identified gene of unknown function that is expressed exclusively in adipose tissues. To provide information about its physiological regulation and possible function, the effect of meal-feeding rats on the expression of adiponutrin mRNA in white adipose tissue was studied. A high-sucrose meal increased adiponutrin mRNA levels by at least 5-fold within 3 h. Post-meal levels returned to pre-meal levels with a half-life of about 5 h. The induction was prevented by injection of actinomycin-D prior to the meal. This pattern of expression was very similar to that seen for leptin mRNA. There were only minimal, or no, effects on acrp30/adiponectin, resistin, adipsin, or stearoyl-CoA desaturase 1. Adiponutrin appears more like leptin with respect to its acute regulation by meal-feeding than to any of the other adipokines or to enzymes directly involved in lipogenesis. This suggests that adiponutrin could be involved in overall energy homeostasis, as is leptin.     

Effect of high-fat diet feeding on leptin receptor expression in white adipose tissue in rats: depot- and sex-related differential response

Previous studies have illustrated the importance of leptin receptor (OB-Rb) mediated action on adipocytes in the regulation of body weight. The aim of the present study was to investigate in male and female rats the effects of high-fat (HF) diet feeding on the expression levels of OB-Rb in different depots of white adipose tissue (WAT), and its relation to fatty acid oxidation capacity. Male and female Wistar rats were fed until the age of 6 months with a normal-fat (NF) or non-isocaloric HF-diet (10 and 45% calories from fat, respectively). At this age, the weight of three different fat depots (retroperitoneal, mesenteric and inguinal) and the expression levels of OB-Rb, PPARα and CPT1 in these depots were measured. HF-diet feeding resulted in an increase in the weight of the different fat depots, the retroperitoneal depot being the one with the greatest increase in both sexes. In this depot, HF-diet feeding resulted in a significant decrease in OB-Rb mRNA levels, more marked in male than in female rats. In the mesenteric depot, the effects of HF-diet feeding on OB-Rb mRNA levels were sex-dependent: they decreased in males rats (associated with a decrease in PPARα and CPT1 mRNA levels), but increased in female rats. In the inguinal depot, OB-Rb expression was not affected by HF-diet feeding. These results show that a chronic intake of an HF-diet altered the expression of OB-Rb in WAT in a depot and sex-dependent manner. The decreased expression of OB-Rb in the internal depots of male rats under HF-diet feeding, with the resulting decrease in leptin sensitivity, can help to explain the higher tendency of males to suffer from obesity-linked disorders under HF-diet conditions.     

Chronic high-fat feeding impairs adaptive induction of mitochondrial fatty acid combustion-associated proteins in brown adipose tissue of mice

Since brown adipose tissue (BAT) is involved in thermogenesis using fatty acids as a fuel, BAT activation is a potential strategy for treating obesity and diabetes. However, whether BAT fatty acid combusting capacity is preserved in these conditions has remained unclear. We therefore evaluated expression levels of fatty acid oxidation-associated enzymes and uncoupling protein 1 (Ucp1) in BAT by western blot using a diet-induced obesity C57BL/6J mouse model. In C57BL/6J mice fed a high-fat diet (HFD) over 2–4 weeks, carnitine palmitoyltransferase 2 (Cpt2), acyl-CoA thioesterase (Acot) 2, Acot11 and Ucp1 levels were significantly increased compared with baseline and control low-fat diet (LFD)-fed mice. Similar results were obtained in other mouse strains, including ddY, ICR and KK-Ay, but the magnitudes of the increase in Ucp1 level were much smaller than in C57BL/6J mice, with decreased Acot11 levels after HFD-feeding. In C57BL/6J mice, increased levels of these mitochondrial proteins declined to near baseline levels after a longer-term HFD-feeding (20 weeks), concurrent with the accumulation of unilocular, large lipid droplets in brown adipocytes. Extramitochondrial Acot11 and acyl-CoA oxidase remained elevated. Treatment of mice with Wy-14,643 also increased these proteins, but was less effective than 4 week-HFD, suggesting that mechanisms other than peroxisome proliferator-activated receptor α were also involved in the upregulation. These results suggest that BAT enhances its fatty acid combusting capacity in response to fat overload, however profound obesity deprives BAT of the responsiveness to fat, possibly via mitochondrial alteration.     

Brown and white adipose tissue lipid composition in three successive progenies of rats: Effects of ethanol exposure

The effect of ethanol exposure on the fatty acid composition of brown and white adipose tissue in three successive rat progenies at the end of an experimental period (24 weeks) was studied. Ethanol‐treated rats received a standard rat chow diet and 5, 10 and 15% ethanol in the ad libitum drinking fluid over 3 successive weeks. Then a concentration of 20% ethanol was maintained for 5 additional weeks up to the end of the experimental period. The males and females in the ethanol treated group were mated to obtain the 1st generation of offspring. Then female and male rats from the 1st generation were mated to obtain the 2nd generation. Finally, males and females from the 2nd generation were mated to obtain the 3rd generation of ethanol treated rats. Another group served as control and received only water and a standard rat chow diet. The control group was handled in the same way as the other experimental groups. In the 1st and 2nd generations the percentage of stearic acid (18:0) decreased and palmitoleic (16:ln7) and oleic acid (18:ln9) increased in both adipose tissues of ethanol‐treated rats with respect to control. Additionally, n‐3 and n‐6 series were reduced both in brown and white adipose tissues. In the 3rd generation the fatty acid composition of the white adipose tissue was similar to that of control rats. Thus, no significant difference in essential fatty acids and oleic acid (18:ln9) were found. However, the fatty acid composition of the brown adipose tissue, in the 3rd generation, was similar to that observed in the 1st and 2nd generation. Thus, a decrease in essential fatty acids and an increase in oleic acid (18:ln9) was found. This suggests adaptation to ethanol consumption during successive progenies in white adipose tissue. However, in brown adipose tissue the values indicate a triglyceride storing during the thermogenesis, which is more important to newborns.     

Postnatal expression of myostatin propeptide cDNA maintained high muscle growth and normal adipose tissue mass in transgenic mice fed a high-fat diet

Myostatin plays a robust, negative role in controlling muscle mass. A disruption of myostatin function by transgenic expression of its propeptide (the 5'region, 866 nucleotides) results in significant muscle growth (Yang et al., 2001. Mol Rep Dev 60:351-361). Studies from myostatin and the propeptide transgene mRNA indicated that myostatin mRNA was detected at day 10.5 postcoitum in fetal mice. Its level remained low, but increased by 180% during the postnatal fast-growth period (day 0-10). An early, high-level postnatal expression of the transgene was identified as being responsible for a highly muscled phenotype. High-fat diet induces adiposity in rodents. To study the effects of dietary fat on muscle growth and adipose tissue fat deposition in the transgenic mice, we challenged the mice with a high-fat diet (45% kcal fat) for 21 weeks. Transgenic mice showed 24%-50% further enhancement of growth on the high-fat diet compared to the normal-fat diet (P = 0.004) from 17 to 25 weeks of age. The total mass of the main muscles of transgenic mice showed a 27% increase on the high-fat diet compared to the normal-fat diet (P = 0.004), while the white adipose tissue mass of the transgenic mice was not significantly different from that of wild-type mice fed a normal-fat diet (P = 0.434). The high-fat diet induced wild-type mice developed 190% greater mass of white adipose tissues compared to the normal-fat diet (P = 0.008), which primarily resulted from enlarged adipocytes. These results demonstrate that disruption of myostatin function by its propeptide shifted dietary fat utilization toward muscle tissues with minimal effects on adiposity. These results suggest that enhancing muscle growth by myostatin propeptide or other means during the early developmental stage may serve as an effective means for obesity prevention.     

Effects of diet and exercise on metabolic disturbances in high-fat diet-fed mice

Consumption of a high-fat diet (HFD) is associated with white adipose tissue (WAT) inflammation, which contributes to key components of the metabolic syndrome, including insulin resistance (IR) and hepatic steatosis (HS). To determine the differential effects of exercise training (EX), low-fat diet (LFD), and their combination on WAT inflammation, Balb/cByJ male mice (n = 34) were fed an HFD for 12 wks before they were randomized into one of four intervention groups: HFD-EX, LFD-EX, HFD-sedentary (SED), or LFD-SED. EX mice performed 12 wks of exercise training on a motorized treadmill (1 h/d, 5 d/wk, 12 m/min, 5% grade, 65% VO₂ max), while SED mice remained sedentary in their home cages. WAT gene expression of adipokines was assessed using rt-PCR. IR was measured using HOMA-IR, and HS via hepatic triglyceride content. EX significantly reduced (53%) WAT gene expression of MCP-1, and LFD significantly reduced (50%) WAT gene expression of the macrophage specific marker, F4/80 as well as the adipocytokine IL-1ra (25%). EX independently improved IR, while both EX and LFD improved HS. These findings suggest that both diet and exercise have unique beneficial effects on WAT inflammatory markers and the mechanism by which each treatment improves metabolic complications associated with chronic consumption of an HFD may be different.     

Brown adipose tissue: Updates in cellular and molecular biology

Brown adipose tissue (BAT) is mainly composed of adipocytes, it is highly vascularized and innervated, and can be activated in adult humans. Brown adipocytes are responsible for performing non-shivering thermogenesis, which is exclusively mediated by uncoupling protein (UCP) -1 (a protein found in the inner mitochondrial membrane), the hallmark of BAT, responsible for the uncoupling of the proton leakage from the ATP production, therefore, generating heat (i.e. thermogenesis). Besides UCP1, other compounds are essential not only to thermogenesis, but also to the proliferation and differentiation of BAT, including peroxisome proliferator-activated receptor (PPAR) family, PPARgamma coactivator 1 (PGC1)-alpha, and PRD1-BF-1-RIZ1 homologous domain protein containing protein (PRDM) -16. The sympathetic nervous system centrally regulates thermogenesis through norepinephrine, which acts on the adrenergic receptors of BAT. This bound leads to the initialization of the many pathways that may activate thermogenesis in acute and/or chronic ways. In summary, this mini-review aims to demonstrate the latest advances in the knowledge of BAT.     

GDF1 is a novel mediator of macrophage infiltration in brown adipose tissue of obese mice

We previously demonstrated a marked upregulation in the bone morphogenic protein (BMP)/growth differentiation factor (GDF) family member, GDF5, which is capable of promoting brown adipogenesis, in brown adipose tissue (BAT) of obese mice. In this study, we identified other GDF family members, besides GDF5 that are responsive to different obesogenic signals in BAT using inborn and acquired obesity animal models. In BAT from leptin-deficient ob/ob mice, GDF1 expression was preferentially downregulated, whereas the expression of several other genes in the BMP/GDF family, including GDF5, was upregulated. Moreover, in cultured brown adipocytes exposed to tunicamycin and hydrogen peroxide, at concentrations not affecting cellular viability, GDF1 expression was significantly downregulated. Recombinant GDF1 failed to significantly alter brown adipogenesis, despite the promoted phosphorylation of Smad1/5/8 in cultured brown adipocytes, but accelerated Smad1/5/8 phosphorylation with a concomitant increase in the number of migrating cells during exposure in a manner sensitive to activin-like kinase inhibitors in macrophagic RAW264.7 cells. Similarly, accelerated migration was observed in murine peritoneal macrophages exposed to GDF1. These results indicate that obesity could lead to predominant downregulation of GDF1 expression in BAT, which can modulate cellular migration through a mechanism relevant to activation of the downstream Smad signaling pathway in adjacent macrophages.     

Modulation of adipose tissue thermogenesis as a method for increasing energy expenditure

There is a renewed interest in the role of adipose tissue in energy utilization and thermogenesis and its potential application in the treatment of metabolic disorders such as obesity and diabetes. The last few years have seen the identification of brown adipose tissue capable of metabolic activation in adult humans, the possibility of recruiting ‘beige’ adipocytes to increase energy expenditure, and the implication of molecules such as FGF21 and irisin in inducing increases in energy expenditure in adipose tissue. The translation of these findings into human trials to deliver safe, efficacious medicines remains a challenge.     

Plasma proteome analysis in diet-induced obesity-prone and obesity-resistant rats

One of the major issues in the field of obesity is why some humans become obese and others resist development of obesity when exposed to high-calorie diets. Despite the same genetic background, namely obesity-prone (OP) and -resistant (OR) rats, differing responses have been demonstrated in a high fat diet-induced rodent model. The aim of the present study was to discover novel obesity-related biomarkers for susceptibility and/or resistance to obesity by proteomic analysis of OP and OR rat plasma. After feeding of high fat diet, OP rats gained approximately 25% more body weight than OR rats and were used for proteomic analysis using 2-DE combined with MALDI-TOF-MS. We categorized identified proteins into three groups by analysis of both average spot density in each group and individual spot density of six rats as a function of body weight. Consequently, category (1) included inter-α-inhibitor H4 heavy chain and fetuin B precursor, which can be used as novel plasma biomarkers for risk of obesity. Nine proteins of category (2) and (3) can also be plausible plasma markers in the study of obesity. This proteomic study is an important advancement over the previous steps needed for identification of OP and OR rats.     

Interaction between heat acclimation and exogenous insulin in brown adipose tissue of rats

Seventy-one male Wistar strain rats (7 weeks old) were kept at 5, 25, or 34° C, respectively, for 2 weeks with or without insulin administration. Insulin (Novo Lente MC) was given subcutaneously in a dose of 3.62 nmol/125 µl saline per 100 g body weight. An apparent effect of insulin treatment was noted only in heat-exposed rats, resulting in a remarkable gain in inter-scapular brown adipose tissue (BAT) mass of heat-acclimated, insulin-treated rats in terms of weight or weight per unit body weight. The BAT from heat-acclimated, insulin-treated rats had significantly higher levels of protein, DNA, RNA, and triglyceride than BAT from heat-acclimated, saline-treated rats. Therefore, it seems likely that the growth of BAT in heat-acclimated, insulin-treated rats was mostly due to the anabolic effects of insulin. The uncoupling protein mRNA was, however, present in BAT of heat-acclimated, insulin-treated rats at rather a depressed level, explaining a corresponding decrease in cold tolerance. On the other hand, the expression of insulin receptor mRNA was attenuated in BAT of rats from all the insulin-treated groups, possibly due to the down-regulation of insulin. Thus, there appeared to be some linkage among BAT, heat acclimation, and insulin.     

棕色脂肪组织与心血管疾病的关系

棕色脂肪组织 (BAT)是由棕色脂肪细胞和血管基质成分 (SVF)组成,其中SVF中包括内皮细胞、淋巴细胞、成纤维细胞以及具有多种分化功能的干细胞,在调节心血管健康与疾病上扮演着重要角色,参与心血管疾病的发生与发展。一方面,BAT通过能量代谢以及自分泌或旁分泌方式分泌的细胞因子对机体产生重要影响。BAT可以作用于心血管系统,发挥抗炎、抗心室重构等作用,从而对心血管起到保护作用。另一方面,脂肪组织来源干细胞的发现与应用,也为心血管疾病的治疗提供了有效途径。本文回顾了BAT自发现以来在人体及动物模型上的相关研究进展,论述了其与心血管损伤的相关性。     

Altered estrogen receptor expression in skeletal muscle and adipose tissue of female rats fed a high-fat diet

Estrogen receptors (ERs) are expressed in adipose tissue and skeletal muscle, with potential implications for glucose metabolism and insulin signaling. Previous studies examining the role of ERs in glucose metabolism have primarily used knockout mouse models of ERα and ERβ, and it is unknown whether ER expression is altered in response to an obesity-inducing high-fat diet (HFD). The purpose of the current study was to determine whether modulation of glucose metabolism in response to a HFD in intact and ovariectomized (OVX) female rats is associated with alterations in ER expression. Our results demonstrate that a 6-wk HFD (60% calories from fat) in female rats induces whole body glucose intolerance with tissue-specific effects isolated to the adipose tissue, and no observed differences in insulin-stimulated glucose uptake, GLUT4, or ERα protein expression levels in skeletal muscle. In chow-fed rats, OVX resulted in decreased ERα with a trend toward decreased GLUT4 expression in adipose tissue. Sham-treated and OVX rats fed a HFD demonstrated a decrease in ERα and GLUT4 in adipose tissue. The HFD also increased activation of stress kinases (c-jun NH?-terminal kinase and inhibitor of κB kinase β) in the sham-treated rats and decreased expression of the protective heat shock protein 72 (HSP72) in both sham-treated and OVX rats. Our findings suggest that decreased glucose metabolism and increased inflammation in adipose tissue with a HFD in female rats could stem from a significant decrease in ERα expression.     

Plasma marker proteins associated with the progression of lung cancer in obese mice fed a high-fat diet

Recent studies have indicated that obesity increases the risk of developing several types of cancers including lung cancer, which is the leading cause of cancer death worldwide. In the present study, we attempted to discover marker proteins associated with lung cancer progression mediated by treatment of a high-fat diet (HFD) using 2DE combined with MALDI-TOF-MS. Image analysis and further statistical analysis allowed for the detection and identification of 14 proteins, which consequently were classified into two groups based on their regulation patterns in response to diet and tumor. Interestingly, the protein abundances of ten proteins exhibited a synergistic effect when treated with HFD in tumor-bearing mice (Group I). Proteins that had a higher abundance in the plasma of tumor-bearing mice included FGB, Tf, Hpx, Cp, and Hp and the proteins that had a lower abundance included A1AT precursor, PON1, TTRt, and α2-M. These proteins can be used as molecular markers that contribute simultaneously to both obesity and cancer. Four other proteins showed an increase (complement C3 and FGA) or decrease (Apo H and AT III precursor) in the only tumor-bearing mice independently of diet (Group II). The marker proteins identified here may lead to the development of new therapeutics for obesity-causative treatment of lung cancer.