Francisca Serra

Expression of Adipose MicroRNAs Is Sensitive to Dietary Conjugated Linoleic Acid Treatment in Mice

Background Investigation of microRNAs (miRNAs) in obesity, their genetic targets and influence by dietary modulators is of great interest because it may potentially identify novel pathways involved in this complex metabolic disorder and influence future therapeutic approaches. This study aimed to determine whether miRNAs expression may be influenced by conjugated linoleic acid (CLA), currently used to induce fat loss. Methodology/Principal Findings We determined retroperitoneal adipose tissue (rWAT) expression of five miRNAs related to adipocyte differentiation (miRNA-143) and lipid metabolism (miRNA-103 and -107) and altered in obesity (miRNA-221 and -222), using the TaqMan®MicroRNA Assay (Applied-Biosystems). In the first experiment, mice were fed with a standard fat diet and orally treated with sunflower oil (control group) and 3 or 10 mg CLA/day for 37 days. In the second experiment, mice were fed with a high fat diet for 65 days. For the first 30 days, mice received the same doses of CLA described above and, from that time onwards, animals received a double dose. Results showed that expression of selected miRNAs was modified in response to CLA treatment and metabolic status. Interestingly, a strong correlation was observed between miR-103 and -107 expression, as well as miR-221 and -222 in both experiments. Moreover, changes in miRNAs expression correlated with several adipocyte gene expressions: miR-103 and -107 correlated with genes involved in fatty acid metabolism whereas miR-221 and miR-222 correlated with the expression of adipocytokines. Regarding the minor changes observed in miR-143 expression, no differences in expression of adipogenic markers were observed. Conclusions/Significance Although elucidating the functional implications of miRNAs is beyond the scope of this study, these findings provide the first evidence that miRNAs expression may be influenced by dietary manipulation, reflecting or even contributing to the new metabolic state originated by CLA treatment.

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.

Moderate doses of conjugated linoleic acid isomers mix contribute to lowering body fat content maintaining insulin sensitivity and a noninflammatory pattern in adipose tissue in mice.

Conjugated linoleic acid (CLA) modulates body composition, especially by reducing adipose tissue. However, despite the increasing knowledge about CLAs beneficial effects on obesity management, the mechanism of action is not yet fully understood. Furthermore, in some human studies fat loss is accompanied by impairment in insulin sensitivity, especially when using the trans-10,cis-12 isomer. The aim of this work was to study the effects of moderate doses of CLA on body fat deposition, cytokine profile and inflammatory markers in mice. Mice were orally treated with a mixture of CLA isomers, cis-9,trans-11 and trans-10,cis-12 (50:50), for 35 days with doses of CLA1 (0.15 g CLA/kg body weight) and CLA2 (0.5 g CLA/kg body weight). CLA had discrete effects on body weight but caused a clear reduction in fat mass (retroperitoneal and mesenteric as the most sensitive depots), although no other tissue weights were affected. Glucose and insulin were not altered by CLA treatment, and maintenance of glucose homeostasis was observed even under insulin overload. The study of gene expression (Emr1, MCP-1, IL-6, TNFalpha, PPARgamma2 and iNOS) either in adipocytes and/or in the stromal vascular fraction indicated that CLA does not lead to the infiltration of macrophages in adipose tissue or to the induction of expression of pro-inflammatory cytokines. The use of a mixture of both isomers, as well as moderate doses of CLA, is able to induce a reduction of fat gain without an impairment of adipose tissue function while preserving insulin sensitivity.

Retinoic acid modulates retinoid X receptor α and retinoic acid receptor α levels of cultured brown adipocytes

A novel potential regulatory pathway of brown adipose tissue (BAT) thermogenesis was recently recognized after identifying retinoic acid (RA) as a transcriptional activator of the uncoupling protein (UCP) gene. Here we provide evidence that the UCP responsiveness to RA in primary cultures of brown adipocytes involves RA receptor α (RARα), and show, in the same system and also in CHO cells, that RA down‐regulates the steady‐state levels of RARα and especially of retinoid X receptor α, suggesting autoregulation of the retinoid pathway and therefore supporting the idea of a physiological role for it in controlling the thermogenic capacity of BAT.

Resistin expression in different adipose tissue depots during rat development.

Resistin is a hormonal factor synthesised by adipocytes that was first thought to be related with the resistance to insulin in obesity, but whose function is not yet completely established. Here we have studied the ontogenic pattern of resistin mRNA expression in different white adipose tissue depots (WAT) – epididymal, inguinal, mesenteric and retroperitoneal – and in brown adipose tissue (BAT), as well as the circulating resistin levels, in rats of different ages (from the suckling period to one year of age). Resistin mRNA was determined by Northern blotting, and serum levels by enzyme immunoassay. In WAT, resistin expression remains almost constant with age, except in early development, where there is a peak of expression in the epididymal and retroperitoneal depots, and a decrease in the inguinal one, while the expression remains constant for the mesenteric depot. Moreover, there is a site-specific difference regarding resistin expression: all the depots express characteristic levels of mRNA, especially at the age of 2 months, the moment when resistin mRNA levels are significantly higher in the epididymal and the retroperitoneal than in the inguinal and mesenteric WAT and than in the BAT. The transient increased resistin expression in the epididymal and the retroperitoneal WAT at a period of time in which there is a change in diet (from milk to chow) suggests a common nutritional regulation of the resistin gene. Circulating resistin levels increase with age probably reflecting the increase in the body fat content.

Moderate doses of conjugated linoleic acid reduce fat gain, maintain insulin sensitivity without impairing inflammatory adipose tissue status in mice fed a high-fat diet

BackgroundThe enrichment of diet with nutrients with potential benefits on body composition is a strategy to combat obesity. Conjugated linoleic acid (CLA) due its beneficial effects on body composition and inflammatory processes becomes an interesting candidate, since the promotion and impairment of obesity is closely linked to a low-grade inflammation state of adipose tissue. Previously we reported the favourable effects of moderate doses of CLA mixture on body composition and inflammatory status of adipose tissue in mice fed a standard-fat diet. In the present study we assessed the potential beneficial effects of CLA mixture (cis-9, trans-11 and trans-10, cis-12, 50:50) in mice fed a high-fat diet.MethodsTwo doses were assayed: 0.15 g (CLA1) and 0.5 g CLA/kg body weight (CLA2) for the first 30 days of the study and then animals received a double amount for another 35 days.ResultsThe lowest dose (CLA1) had minor effects on body composition, plasma parameters and gene expression. However, a clear reduction in fat accumulation was achieved by CLA2, accompanied by a reduction in leptin, adiponectin and non-esterified fatty acids (NEFA) plasma concentrations. Insulin sensitivity was maintained despite a slight increase in fasting glucose and insulin plasma concentrations. The study of gene expression both in adipocytes and in the stromal vascular fraction (SVF) suggested that CLA may reduce either the infiltration of macrophages in adipose tissue or the induction of expression of pro-inflammatory cytokines.ConclusionIn conclusion, the use of moderate doses of an equimolar mix of the two main CLA isomers reduces body fat content, improves plasma lipid profile, maintains insulin sensitivity (despite a moderate degree of hyperinsulinaemia) without the promotion of inflammatory markers in adipose tissue of mice fed a high-fat diet.

Dietary l-leucine supplementation of lactating rats results in a tendency to increase lean/fat ratio associated to lower orexigenic neuropeptide expression in hypothalamus

The aim of this study was to assess the effects of dietary leucine supplementation in lactating dams, particularly on energy homeostasis through signaling mechanisms in the central nervous system. Dams were fed ad libitum with standard diet during pregnancy (control dams) or supplemented with 2% leucine (leucine-supplemented dams) from delivery onwards. Food intake, body weight and composition were periodically recorded. Hypothalamus was collected at the end of lactation, and the expression of neuropeptide Y (NPY), agouti-related protein (AgRP) pro-opiomelanocortin (POMC), cocaine and amphetamine regulated transcript (CART), insulin receptor (InsR), ghrelin receptor (GSHR), melanocortin receptor (MCR4), leptin receptor (Ob-Rb) and suppressor of cytokine signaling 3 (SOCS3) were analyzed. Dietary leucine supplementation to lactating rats increased plasma leucine by 56%, modulated body composition and contributed to a tendency of higher ratio of lean/fat mass content of dams during lactation, without affecting food intake, thermogenesis capacity or body or tissue/organs weights. No differences in body weight of offspring from control and leucine-supplemented dams were found. The expression of orexigenic peptides (NPY and AgRP) decreased in leucine-dams, whereas the expression of anorexigenic peptides (POMC and CART), the hypothalamic receptors of insulin, ghrelin, melanocortin and leptin and SOCS3 did not change by leucine supplementation. In conclusion, increased leucine intake during lactation may contribute to a healthier profile of body composition in dams, without compromising the growth and development of the progeny by a mechanism associated with lower expression of orexigenic neuropeptides in hypothalamus.

Conjugated Linoleic Acid Supplementation under a High-Fat Diet Modulates Stomach Protein Expression and Intestinal Microbiota in Adult Mice.

The gastrointestinal tract constitutes a physiological interface integrating nutrient and microbiota-host metabolism. Conjugated linoleic acids (CLA) have been reported to contribute to decreased body weight and fat accretion. The modulation by dietary CLA of stomach proteins related to energy homeostasis or microbiota may be involved, although this has not been previously analysed. This is examined in the present study, which aims to underline the potential mechanisms of CLA which contribute to body weight regulation. Adult mice were fed either a normal fat (NF, 12% kJ content as fat) or a high-fat (HF, 43% kJ content as fat) diet. In the latter case, half of the animals received daily oral supplementation of CLA. Expression and content of stomach proteins and specific bacterial populations from caecum were analysed. CLA supplementation was associated with an increase in stomach protein expression, and exerted a prebiotic action on both Bacteroidetes/Prevotella and Akkermansia muciniphila. However, CLA supplementation was not able to override the negative effects of HF diet on Bifidobacterium spp., which was decreased in both HF and HF+CLA groups. Our data show that CLA are able to modulate stomach protein expression and exert a prebiotic effect on specific gut bacterial species.

Calcium supplementation modulates gut microbiota in a prebiotic manner in dietary obese mice.

SCOPE Dietary calcium has been inversely associated with body fat and energy balance. The main scope of this study has been to assess the potential contribution of gut microbiota on energy regulation mediated by calcium. METHODS AND RESULTS Gut microbiota in C57BL/6J mice receiving calcium supplementation under a high-fat (HF) diet were analysed by PCR and their relationships with host metabolic parameters were determined. Calcium conferred a prebiotic-like effect on gut microbiota, and animals presented lower plasmatic endotoxin levels, increased expression of angiopoietin-like 4 in intestine and lower hepatic lipid content, although increased expression of stress markers in adipose tissue and of inflammation in liver was also found. To determine whether slimming effects could be transferred to obese mice, a faecal microbial transplant (FMT) was carried out, showing that host bacteria grown under a HF diet could not be superseded by those from calcium-fed animals. Therefore, FMT was not able to transfer the beneficial effects of calcium. CONCLUSION In conclusion, calcium modulated gut microbiota in a prebiotic manner, establishing a host cross-talk and promoting a healthier metabolic profile. However, lack of effectiveness of FMT suggests the need of further appropriate dietary factors in addition to the bacteria per se.

Effect of calcium-enriched high-fat diet on calcium, magnesium and zinc retention in mice.

The aim of this work was to assess the effects of a high-fat diet enriched in Ca, which accompanies lower body fat deposition, on mineral depots, as well as to assess the potential role of adaptive thermogenesis in mice. Male mice were fed ad libitum a high-fat (43 %) diet with a Ca content of 4 g/kg from calcium carbonate (control group) or 12 g/kg (42 % from milk powder and the rest from calcium carbonate) (Ca group) for 56 d. Body weight, food intake and urine were periodically collected. Tissue samples were collected when the mice were killed and the composition was determined. Expression of uncoupling proteins was determined by Western blotting. Mineral content was measured by flame atomic absorption spectrometry. Lower body weight gain and fat accretion was found in the Ca group. This could not be attributable to lower gross energy intake or to activation of adaptive thermogenesis. Although significant urine mineral loss was found in the Ca group, preservation of mineral depots in bone was observed. Our data support the fact that adding more Ca to the diet, using a combination of calcium carbonate plus milk powder containing among other things higher Zn and Mg, contributes to counteracting obesity and improving lipid metabolism.