Ana M. Rodríguez

Brown adipose tissue response to cafeteria diet-feeding involves induction of the UCP2 gene and is impaired in female rats as compared to males

Abstract. Noradrenaline-dependent brown adipose tissue (BAT) thermogenesis is activated by the cold and excess energy intake, largely depends on the activity of the uncoupling protein 1 (UCP1), and is mediated mainly through the β3-adrenoceptor (β3-AR). We investigated the expression of ucp2, a gene that encodes a putative UCP1-like uncoupling protein, along with that of ucp1 and β3-ar, in the interscapular BAT (IBAT) of male and female rats chronically fed a cafeteria diet. After 3 months on this diet, male rats attained a 34% excess body mass and showed IBAT hypertrophy and increased IBAT thermogenic potential, in terms of both UCP1 and UCP2 mRNA expression (both by 1.6-fold), UCP1 protein expression (by 1.75-fold) and GDP binding to IBAT mitochondria (by 2.2-fold); female rats attained a larger excess body weight (50%) and their IBAT, although hypertrophied, showed no signs of increased thermogenic potential per gram of tissue. Interestingly, the IBAT of female rats was already activated compared to males. Treatment of mouse brown adipocytes in primary culture with noradrenaline also triggered a dose-dependent increase of the levels of UCP1 mRNA and UCP2 mRNA. Retroregulatory down-regulation of the β3-AR mRNA levels was found in the two models used. The results support a physiological role for UCP2, along with UCP1, in rodent BAT thermogenesis.

Adiponectin and Resistin Response in the Onset of Obesity in Male and Female Rats

Objective: Studying the sex‐dependent response of adiponectin and resistin adipose tissue expression and circulating levels in the onset of dietary obesity.

Regional differences in the expression of genes involved in lipid metabolism in adipose tissue in response to short- and medium-term fasting and refeeding

The aim of this study was to analyze regional differences in the time-course response to fasting and refeeding in the expression of genes involved in lipid metabolism in retroperitoneal, mesenteric and inguinal adipose tissue. Rats were studied under different feeding conditions: feeding state; after 4, 8 or 24 h of fasting; and after 3 h of refeeding following 8 h of fasting. The expression of lipogenesis-related genes decreased by fasting in adipose tissue, and the retroperitoneal depot showed the fastest response: mRNA levels of peroxisome proliferator-activated receptor gamma 2 (PPARgamma2) decreased after 4 h of fasting and those of sterol regulatory element binding protein 1c (SREBP1c), fatty acid synthase (FAS), GPAT and glucose transporter 4 (GLUT4) decreased after 8 h. In the inguinal depot, mRNA levels of SREBP1c, acetyl-coenzyme A carboxylase alpha, FAS and lipoprotein lipase decreased after 8 h of fasting, while in the mesenteric depot, only GLUT4 and FAS mRNA levels decreased after 8 and 24 h, respectively. Concerning lipolytic and fatty acid oxidation genes, only adipose triglyceride lipase and carnitine palmitoyltransferase 1a expression increased after 24 h of fasting in the retroperitoneal depot. Three hours of refeeding restored the expression of the lipogenic transcription factors PPARgamma2 and SREBP1c in the retroperitoneal depot and of PPARgamma2 in the inguinal depot. This period of refeeding was ineffective in changing the expression of genes related with lipid mobilization and fatty acid oxidation, except hormone-sensitive lipase, whose expression decreased in the mesenteric depot. It is suggested that different regulations of the expression of genes related with lipid metabolism between internal and subcutaneous depots to feeding and fasting conditions are site-specific metabolic features of white adipose tissue.

Gene expression patterns in visceral and subcutaneous adipose depots in rats are linked to their morphologic features.

The aim was to characterize the expression pattern of genes involved in lipid metabolism in internal (retroperitoneal, mesenteric) and subcutaneous (inguinal) adipose tissue depots in rats and their relation with site-specific morphological- and metabolic-features. Gene expression by RT-qPCR, western blot and morphometric analyses were performed. Lipogenesis-related genes (PPARγ2, SREBP1c, ACC1, GPAT, LPL, CD36, GLUT4) showed higher mRNA levels in the retroperitoneal depot versus the mesenteric and the inguinal depots; the expression of PPARgamma;2, ACC1, CD36, and GLUT4 in the mesenteric depot was also higher than in the inguinal depot. HKII was similarly expressed in the retroperitoneal and mesenteric depots and higher than in the inguinal one. The expression of lipolysis-related genes (HSL, ATGL) was higher in the retroperitoneal than in the mesenteric and inguinal depots, while the expression of fatty-acid oxidation-related genes (PPARα, CPT1) was lower in the retroperitoneal depot compared with the mesenteric and the inguinal depots. Thus, a higher expression of lipogenesis- and lipolysis-related genes and lower expression of fatty-acid oxidation-related genes in internal depots (particularly in the retroperitoneal, which also presents the largest adipocyte size) can explain its higher triacylglyceride turnover rates and hence account for the differential behavior of fat depots in physiological situations and its involvement in obesity-linked metabolic disorders.

Induction of NPY/AgRP orexigenic peptide expression in rat hypothalamus is an early event in fasting: relationship with circulating leptin, insulin and glucose.

Hypothalamus is crucial in the control of energy intake and expenditure in mammals, presenting two interconnected populations of neurons producing orexigenic NPY/AgRP (neuropeptide Y; agouti related peptide) and anorexigenic POMC/CART (pro-opiomelanocortin; cocaine and amphetamine regulated transcript) neuropeptides. We aimed to shed more light on the response and sensitivity in the production of these neuropeptides to face nutritional changes, particularly food deprivation, and on the signals that regulate them. Male Wistar rats were fasted for 0, 4, 8 and 24h and refed for 3h after 8h fasting. mRNA levels of gastric and adipose tissue (retroperitoneal, mesenteric and inguinal) leptin, and of hypothalamic NPY, AgRP, POMC, CART, leptin receptor, SOCS3 (suppressor of cytokine signaling 3) and insulin receptor were analyzed. Gastric and circulating leptin, and circulating insulin, glucose and ghrelin were also determined. The only neuropeptide mRNAs that responded (increasing) to the short-term periods of fasting used were those of NPY (transiently) and AgRP, and these changes were accompanied by an increase in leptin receptor mRNA levels and by a decrease in adipose and gastric leptin expression and in the circulating levels of leptin, insulin and glucose, but without changes in circulating ghrelin. The elevation in AgRP and leptin receptor mRNA levels and the drop in circulating leptin were not reverted with refeeding. It is suggested that the induction of expression of the orexigenic molecules in NPY/AgRP neurons is an early event upon fasting, related with changes in leptin, insulin and glucose levels, but with the role of leptin signaling in particular.

Resistin as a putative modulator of insulin action in the daily feeding/fasting rhythm

Resistin and adiponectin are adipokines with postulated opposite functions. Resistin has been related with insulin resistance in obesity, while adiponectin could be associated to higher insulin sensitivity. We have determined whether the production of these two adipokines during the day is related to the feeding rhythm in rats. Resistin mRNA levels in adipose tissue correlated positively with the gastric contents and serum insulin concentration, showing higher levels during the dark phase (period of the highest food intake), especially in the mesenteric depot, while levels decreased during the light phase. The diurnal pattern of resistin expression was not directly reflected in the circulating levels, but it showed a 6-h delay and correlated negatively with the gastric contents and serum insulin. Adiponectin expression followed an opposite pattern, not apparently related to feeding or insulin release, and not translated into changes in circulating levels. Moreover, considering that insulin stimulates resistin expression and that circulating resistin follows a contrary circadian pattern in comparison to insulin, resistin, apart from its role in the increased insulin resistance associated to obesity, could also act as a putative modulator of insulin in the daily feeding/fasting rhythm through a negative feedback regulation of its action.

Free fatty acid effects on myokine production in combination with exercise mimetics.

SCOPE We aimed to study the effects of free fatty acids (FFAs) alone and combined with the exercise mimetics adrenaline and 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) in the production of IL6, IL15 and Irisin in muscle cells, using a time-sequential model. METHODS AND RESULTS Differentiated C2C12 myotubes were treated with FFA, adrenaline or AICAR alone for 0, 1, 3, 8, 12 and 24 h and with double or triple combinations for 0, 3 and 24 h. Levels of mRNA in cells and protein in the medium were measured. Adrenaline, AICAR and FFA showed no significant effects on Irisin expression, while the presence in the culture of adrenaline and/or AICAR decreased IL15 mRNA expression. On contrary, the three signals showed a deep, rapid impact on the IL6 induction, especially when both AICAR and FFA were present. CONCLUSION The different response in IL6 versus IL15 regulation may be explained by their different energy-activating versus muscle-cell-hypertrophy suggested roles, considering that adrenaline and AMPK are involved in the activation of energy-generating pathways. Moreover, the results suggest FFAs are components that may regulate IL6 production and may have a role in muscle-adipose tissue crosstalk.

Time-course effects of increased fatty acid supply on the expression of genes involved in lipid/glucose metabolism in muscle cells.

Fatty acid (FA) oversupply in skeletal muscle is related with metabolic disorders associated to obesity, and also with normal physiological responses. We studied, in vivo and in vitro, the chronological response to physiological increases of FA, analyzing the expression of selected genes important for glucose/lipid metabolism. An in vivo sequential model of fasting (known to increase circulating FA) and refeeding was used in male Wistar rats to study soleus (more oxidative) and gastrocnemius (more glycolytic) muscles, and a chronological study was made in C2C12 muscle cells under treatment of oleic/linoleic FA mixture, at physiological concentration. Body weight, muscle glycogen and blood parameters (glucose, insulin, free fatty acids -FFA-, triglycerides) were monitored. mRNA levels of muscle carnitine palmitoyl transferase 1 (mCPT1), GLUT 4, insulin receptor (InsR), MyoD1, peroxisome proliferator activated receptor (PPAR) γ coactivator 1α (PGC1α) and β (PGC1β), PPARα, PPARδ, pyruvate dehydrogenase kinase 4 (PDK4) and uncoupling proteins (UCPs) 2 and 3 were analyzed by quantitative RT-PCR. The main results were the quick induction of PGC1α, UCP3 and PDK4 in vivo (more marked in gastrocnemius) and of PGC1α, PGC1β, InsR, PDK4, UCP2 and UCP3 in vitro. It is concluded that FA are able to rapidly induce the expression in muscle cells of key genes involved in their catabolism and that the oleic/linoleic acid mixture has a positive role increasing the expression of master metabolic regulators and their downstream target genes, facilitating the transition from a more glycolytic to a more lipid-oxidative metabolism.

Synergic effect of overweight and cold on uncoupling proteins expression, a role of α2/β3 adrenergic receptor balance?

Abstract. The effect of cold exposure, being overweight and their interaction was investigated on the response of uncoupling proteins UCP1, UCP2 and UCP3 and the α2/β3 adrenergic receptor (AR) balance in brown adipose tissue (BAT), as well as the involvement of leptin gene expression in white adipose tissues, in control and overweight male rats of the dietary obesity model known as the post-cafeteria model. UCP1, UCP2 and UCP3 mRNAs were up-regulated by cold, with a synergic effect of cold exposure and being overweight on UCP1 mRNA levels (with the related UCP1 protein response), and with UCP2 mRNA showing a parallel response. Furthermore, the BAT α2/β3 AR ratio was diminished in overweight rats. The results suggest that the UCP1-dependent thermogenic capacity in BAT of post-cafeteria overweight rats has a more sensitive response to cold exposure and that UCP2 and UCP3 could be somehow involved in the thermogenic response but differentially regulated. Moreover, the diminished α2/β3 AR ratio in BAT could be one of the factors involved in the more sensitive response of overweight rats to cold in terms of BAT thermogenesis-related parameters.

The steroid RU486 induces UCP1 expression in brown adipocytes.

RU486 (mifepristone), a potent antagonist at progesterone and glucocorticoid receptors (PR and GR), is well known for its use in the termination of unwanted pregnancies, the potential development of oral contraceptives, treatment of certain cancers and other activities. Potentially, it could also play a role in obesity control, although the few studies that have addressed this aspect have focused mainly on its central and anti-glucocorticoid effects. We have shown previously that it could have a direct effect on brown adipocytes in culture when administered together with progesterone. The aim of the present work was to analyse the effects of RU486 on the expression of uncoupling proteins (UCPs) in brown adipocytes. In culture-grown, differentiated brown adipocytes, placed in a serum-free medium to exclude the presence of progesterone or glucocorticoids, RU486 stimulated UCP1 expression at both the mRNA and protein levels. These effects could be mediated by PR, GR or other unknown mechanisms but do not seem to be due to its anti-progestin or anti-glucocorticoid actions. The results suggest that the steroid RU486 has a direct action on adipocytes which could be useful for stimulating non-shivering BAT thermogenesis and therefore is of interest in obesity studies.