Magdalena Gianotti

Gender dimorphism in high-fat-diet-induced insulin resistance in skeletal muscle of aged rats.

Muscle resistance to insulin plays a key role in the metabolic dysregulation associated to obesity. A pro-inflammatory and pro-oxidant status has been proposed to be the link between dietary obesity and insulin resistance. Given the gender differences previously found in mitochondrial function and oxidative stress, the aim of the present study was to investigate whether this gender dimorphism leads to differences in the development of high-fat-diet-induced insulin resistance in rat skeletal muscle. Male and female rats of 15 months of age were fed with a high-fat-diet (32% fat) for 14 weeks. Control male rats showed a more marked insulin resistance status compared to females, as indicated by the glucose tolerance curve profile and the serum insulin, resistin and adiponectin levels. High-fat-diet feeding induced an excess of body weight of 16.2% in males and 38.4% in females, an increase in both muscle mitochondrial hydrogen peroxide production and in oxidative damage, together with a decrease in the Mn-superoxide dismutase activity in both genders. However, high-fat-diet fed female rats showed a less marked insulin resistance profile than males, higher mitochondrial oxygen consumption and cytochrome c oxidase activity, and a better capacity to counteract the oxidative-stress-dependent insulin resistance through an overexpression of both muscle UCP3 and GLUT4 proteins. These results point to a gender dimorphism in the insulin resistance status and in the response of skeletal muscle to high-fat-diet feeding which could be related to a more detrimental effect of age in male rats.

Sex Steroid Receptor Expression Profile in Brown Adipose Tissue. Effects of Hormonal Status

Recent investigations suggest that sex hormones play an important role in the brown adipose tissue (BAT) thermogenic program by acting on several steps of the lipolytic signal cascade and on the UCP1 transcription control. However, the number of studies focusing on steroid receptor status in brown adipose tissue is negligible. In the present study, we analyze steroid receptor mRNA levels in brown adipose tissue in male and female rats and in pregnant and lactating females, all of them models with a different hormonal background. The direct effect of sex hormones on the expression of their receptors was studied in vitro in primary culture of brown adipocytes. Oestrogen receptor (ERα) and androgen receptor (AR) densities were higher in male than in female BAT. PR A+B mRNA expression was downregulated in lactation, suggesting a role of progesterone signalling in thermogenesis impairment at this stage. In vitro studies showed that progesterone decreased PR A+B mRNA and that testosterone downregulated ERα mRNA. The results highlighted in this study demonstrate the presence of steroid receptor mRNA in BAT and in brown cell cultured adipocytes, supporting the idea that changes in steroid receptor expression would be important for the understanding of sex hormone effects on BAT physiology.

Involvement of the retinoblastoma protein in brown and white adipocyte cell differentiation: Functional and physical association with the adipogenic transcription factor C/EBPα

We investigated the expression of the retinoblastoma protein (pRB) in adipocytes and its possible interaction with the adipogenic transcription factor CCAAT/enhancer-binding protein alpha (C/EBPalpha) in controlling the acquisition of the terminally differentiated adipocyte phenotype. The pRB was expressed (as measured by immunoblotting and/or immunofluorescence) in mice brown and white adipose tissue and in cultured adipocytes that showed lipid accumulation and expressed specific differentiation markers such as aP2 (measured using a specific cDNA probe) and in the case of brown adipocytes UCP-1 (measured using specific antibodies), but was undetectable in proliferative undifferentiated preadipocytes. Transient transfection experiments revealed a functional interaction between pRB and C/EBPalpha affecting transcription from the ucp-1 gene promoter. Thus, in immortalized brown adipocytes, co-transfection of both a C/EBPalpha and a pRB expression vectors maximally enhanced the expression of reporter chloramphenicol acetyltransferase driven by the ucp-1 promoter. Interestingly, C/EBPalpha inhibited reporter gene expression in CHO cells in an effect that was also potentiated in the presence of pRB. A positive effect of pRB on transcription from the ucp-1 promoter could be detected in C/EBPalpha-/-fibroblasts only after forced to overexpress C/EBPalpha, suggesting that the effect of pRB is dependent on its interaction with C/EBPalpha. We also found evidence that pRB and C/EBPalpha can directly bind to each other in vitro. Our results show that the expression of pRB is restricted to differentiated adipocytes, and provide evidence of a physical and functional interaction between pRB and C/EBPalpha that affects the transcriptional activity of the later on a brown adipocyte-specific gene.

Long-term High-fat-diet Feeding Impairs Mitochondrial Biogenesis in Liver of Male and Female Rats

Background/Aims: Mitochondrial biogenesis includes both mitochondrial proliferation and differentiation and its regulation under different physiological conditions is not clear. Given the sexual dimorphism previously found in mitochondrial function, the aim of this study was to investigate the gender-dependent effect of chronic high-fat-diet (HFD) feeding on rat liver mitochondrial function and biogenesis. Methods: Ten-week old male and female rats were fed a HFD (26% fat) or a control diet (2.9% fat) for 26 weeks. Mitochondrial morphology was studied. Mitochondrial DNA and protein content, hydrogen peroxide production, oxidative capacity, antioxidant defenses, as well as markers of oxidative damage and mitochondrial biogenesis were analyzed. Results: Female rats showed higher levels of mitochondrial protein and an enhanced oxidative capacity per mitochondrion than males. In both genders, HFD feeding increased mtDNA content and decreased mitochondrial differentiation markers. Conclusion: In comparison to male rats, females show higher oxidative capacity as a consequence of their greater mitochondrial differentiation under both control and obese status. In response to HFD feeding, the oxidative capacity of the whole mitochondrial population is maintained in both genders. This is obtained by means of an enhancement of mitochondrial proliferation, which counteracts the diet-induced impairment of the function of each mitochondrion.

Sex differences in the effect of high-fat diet feeding on rat white adipose tissue mitochondrial function and insulin sensitivity.

Obesity-induced mitochondrial dysfunction in white adipose tissue (WAT) leads to a dysregulation of adipokine secretion, which is involved in insulin resistance development. Taking into account the sex differences previously found both in mitochondrial function and for the insulin sensitivity profile in different tissues, the aim of this study was to investigate whether a sex-dependent effect of a long-term high-fat diet (HFD) feeding exists on WAT mitochondrial function. Indeed, HFD effects on the levels of the key components of the insulin and adiponectin signaling pathways, and the consequences of these effects on the systemic profile of insulin sensitivity were also studied. Wistar rats of both sexes were fed a standard diet or an HFD. Serum markers of insulin sensitivity, protein, and mRNA levels of the main elements of the insulin and adiponectin signaling pathways, and the markers of mitochondrial function and biogenesis, were measured. Our results indicate that different physiological strategies are adopted by male and female rats in response to HFD. In this regard, HFD induced mitochondrial proliferation in males and mitochondrial differentiation in females, as well as a greater retroperitoneal WAT expandability capacity, which allows them to preserve a better insulin sensitivity profile than male rats for both control and HFD groups. Moreover, female WAT showed a decrease in adiponectin and insulin signaling pathway element levels. This sexual dimorphism suggests that there are different strategies for retroperitoneal WAT to maintain the energetic and metabolic homeostasis in response to HFD feeding.

Skeletal muscle and liver oxidative metabolism in response to a voluntary isocaloric intake of a high fat diet in male and female rats.

High fat diets (HFD) usually lead to hyperphagia and body weight gain. However, macronutrient proportions in the diet can modulate energy intake and body fat deposition. The aim of the study was to investigate muscle and liver oxidative metabolism in response to an isocaloric intake of a HFD and to elucidate the possible gender-dependent response. Eight week-old male and female rats were fed either standard chow or HFD for 14 weeks. Energy intake, body weight and whole animal oxygen consumption were determined periodically. Mitochondrial oxygen consumption, hydrogen peroxide production, TBARS levels, Cytochrome-c-oxidase, Citrate synthase and antioxidant enzyme activities were measured in muscle and liver. UCP1 and UCP3 protein levels were analyzed in brown adipose tissue and muscle, respectively. Male rats showed higher energy efficiency, enhanced adiposity, greater hydrogen peroxide production and less effective antioxidant machinery compared to females. HFD feeding increased energy expenditure but did not modify either tissue oxidative metabolism or oxidative damage in either gender. HFD animals over-expressed uncoupling proteins in order to maintain energy balance (brown adipose tissue UCP1) and to avoid oxidative stress (skeletal muscle UCP3), thus counteracting the alterations induced by the modification of the proportion of macronutrients in the diet.

Estradiol stimulates mitochondrial biogenesis and adiponectin expression in skeletal muscle.

Sexual dimorphism has been found in mitochondrial features of skeletal muscle, with female rats showing greater mitochondrial mass and function compared with males. Adiponectin is an insulin-sensitizing adipokine whose expression has been related to mitochondrial function and that is also expressed in skeletal muscle, where it exerts local metabolic effects. The aim of this research was to elucidate the role of sex hormones in modulation of mitochondrial function, as well as its relationship with adiponectin production in rat skeletal muscle. An in vivo study with ovariectomized Wistar rats receiving or not receiving 17β-estradiol (E2) (10 μg/kg per 48 h for 4 weeks) was carried out, in parallel with an assay of cultured myotubes (L6E9) treated with E2 (10 nM), progesterone (Pg; 1 μM), or testosterone (1 μM). E2 upregulated the markers of mitochondrial biogenesis and dynamics, and also of mitochondrial function in skeletal muscle and L6E9. Although in vivo E2 supplementation only partially restored the decreased adiponectin expression levels induced by ovariectomy, these were enhanced by E2 and Pg treatment in cultured myotubes, whereas testosterone showed no effects. Adiponectin receptor 1 expression was increased by E2 treatment, both in vivo and in vitro, but testosterone decreased it. In conclusion, our results are in agreement with the sexual dimorphism previously reported in skeletal muscle mitochondrial function and indicate E2 to be its main effector, as it enhances mitochondrial function and diminishes oxidative stress. Moreover, our data support the idea of the existence of a link between mitochondrial function and adiponectin expression in skeletal muscle, which could be modulated by sex hormones.

Gender related differences in paraoxonase 1 response to high-fat diet-induced oxidative stress.

Objective: To evaluate the influence of the pro‐oxidant and proinflammatory state related to dietary obesity on serum paraoxonase 1 (PON1) activity in male and female rats.

Opposite effects of 17-β estradiol and testosterone on mitochondrial biogenesis and adiponectin synthesis in white adipocytes

Sexual dimorphism has been found in both mitochondrial functionality and adiponectin expression in white adipose tissue, with female rats presenting more functional mitochondria than males and greater adiponectin expression. However, little is known about the role of sex hormones in this dimorphism. The aim was to elucidate the role of sex hormones in mitochondrial biogenesis and dynamics and in adiponectin synthesis in white adipocytes, and also to provide new evidence of the link between these processes. 3T3-L1 preadipocytes were differentiated and treated either with 17-β estradiol (E₂; 10  nM), progesterone (Pg), testosterone (1  μM both), or a combination of Pg or testosterone with flutamide (FLT; 10  μM) or E₂ (1  μM). The markers of mitochondrial biogenesis and dynamics and adiponectin expression were analyzed. E₂ induced mitochondrial proliferation and differentiation in 3T3-L1, although testosterone showed opposite effects. Pg treatment stimulated proliferation but impaired differentiation. In concerns mitochondrial dynamics, these hormones promoted fusion over fission. FLT treatment indicated that Pg elicits its effects on mitochondrial dynamics through the androgen receptor. E₂ coadministration with testosterone or Pg reversed its effects. In conclusion, our results show that E₂ induces stimulation of mitochondrial biogenesis in white adipocytes in vitro, especially in situations that imply an impairment of mitochondrial function, whereas testosterone would have opposite effects. Moreover, testosterone and Pg alter mitochondrial dynamics by promoting fusion over fission, while E₂ stimulates both processes. All these alterations run in parallel with changes in adiponectin expression, thus suggesting the existence of a link between mitochondrial biogenesis and dynamics and adiponectin synthesis in white adipocytes.

Isocaloric intake of a high-fat diet modifies adiposity and lipid handling in a sex dependent manner in rats

BackgroundHigh-fat (HF) diet feeding usually leads to hyperphagia and body weight gain, but macronutrient proportions in the diet can modulate energy intake and fat deposition. The mechanisms of fat accumulation and mobilization may differ significantly between depots, and gender can also influence these differences.AimTo investigate, in rats of both sexes, the effect of an isocaloric intake of a diet with an unbalanced proportion of macronutrients on fatty acid composition of visceral and subcutaneous adipose tissues and how this is influenced by both dietary fatty acids and levels of proteins involved in tissue lipid handling.MethodsEight-week-old Wistar rats of both sexes were fed a control diet (3% w/w fat) or high-fat diet (30% w/w fat) for 14 weeks. Fatty acid composition was analyzed by gas-chromatography and levels of LPL, HSL, α2-AR, β3-AR, PKA and CPT1 were determined by Western blot.ResultsThe HF diet did not induce hyperphagia or body weight gain, but promoted an increase of adiposity index only in male rats. HF diet produced an increase of the proportion of MUFA and a decrease in that of PUFA in both adipose depots and in both sexes. The levels of proteins involved in the adrenergic control of the lipolytic pathway increased in the gonadal fat of HF females, whereas LPL levels increased in the inguinal fat of HF males and decreased in that of females.ConclusionSexual dimorphism in adiposity index reflects a differential sex response to dietary fatty acid content and could be related to the levels of the proteins involved in tissue lipid management.