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.     

Gender-dependent differences in serum profiles of insulin and leptin in caloric restricted rats

In the present study, we have investigated whether differences between male and female rats described in response to 40% caloric restriction (CR) were influenced by circulating level variations of sex hormones and/or insulin and leptin. Body weights (BW), organ weights, and adipose depot weights (ADW) were also measured. The most affected tissues by CR were the fat depots. Metabolically active organs were the least affected, especially more in females than in males (male weight lost: 24.3% vs. female: 17.3%). Testosterone and estradiol circulating levels did not show changes by CR. Insulin levels were decreased by CR in both genders, but was more evident in female rats than males. Leptin serum levels were higher in male rats than in females, and CR caused a circulating leptin level reduction only in males. In conclusion, our results indicate that leptin and insulin could be one of the keys of the different hormonal control of energy homeostasis in response to CR between female and male rats. In this sense, leptin serum levels correlated statistically with BW and with individual ADW only in male rats, whereas insulin serum levels correlated statistically with BW and with any of the ADW studied only in females.     

Adipose triglyceride lipase expression and fasting regulation are differently affected by cold exposure in adipose tissues of lean and obese Zucker rats

Adipose triglyceride lipase (ATGL) hydrolyzes triacylglycerols to diacylglycerols in the first step of lipolysis, providing substrates for hormone-sensitive lipase (HSL). Here we studied whether ATGL messenger RNA (mRNA) and protein levels were affected by 24-h cold exposure in different white adipose tissue depots and in interscapular brown adipose tissue of lean and obese Zucker rats submitted to feeding and 14-h fasting conditions. HSL mRNA expression was also studied in selected depots. In both lean and obese rats, as a general trend, cold exposure increased ATGL mRNA and protein levels in the different adipose depots, except in the brown adipose tissue of lean animals, where a decrease was observed. In lean rats, cold exposure strongly improved fasting up-regulation of ATGL expression in all the adipose depots. Moreover, in response to fasting, in cold-exposed lean rats, there was a stronger positive correlation between circulating nonesterified fatty acids (NEFA) and ATGL mRNA levels in the adipose depots and a higher percentage increase of circulating NEFA in comparison with control animals not exposed to cold. In obese rats, fasting-induced up-regulation of ATGL was impaired and was not improved by cold. The effects of obesity and cold exposure on HSL mRNA expression were similar to those observed for ATGL, suggesting common regulatory mechanisms for both proteins. Thus, cold exposure increases ATGL expression and improves its fasting-up-regulation in adipose tissue of lean rats. In obese rats, cold exposure also increases ATGL expression but fails to improve its regulation by fasting, which could contribute to the increased difficulty for mobilizing lipids in these animals.     

Response of leptin mRNA to 24-h food deprivation and refeeding is influenced by age in rats

To obtain an insight into the influence of aging on leptin gene expression, the responses of leptin mRNA in retroperitoneal and epididymal adipose tissues and plasma leptin concentrations to 24-h food deprivation and refeeding were examined in 2-, 10- and 24-month-old normal rats. The basal level of leptin gene expression in retroperitoneal adipose tissue was significantly higher in 10- and 24-month-old rats than that in 2-month-old rats, while the level in epididymal adipose tissue was highest in 10-month-old rats for all three age groups. The basal concentrations of plasma leptin was significantly higher in 10- and 24-month-old rats than those in 2-month-old rats. The 24-h food deprivation was followed by a significant reduction in leptin mRNA expression in both retorperitoneal and epididymal adipose tissues for all three age groups. The leptin gene expression was restored to control levels 24 h following refeeding in the 2- and 10-month-old rats, but failed to be restored in the 24-month-old rats. In addition, the time course of recovery for leptin mRNA expression by refeeding to the control levels differed between the retroperitoneal and the epididymal adipose tissue in 2- and 10-month-old rats. The concentrations of plasma leptin 24 h following refeeding were compatible with the leptin mRNA levels in adipose tissues in three age groups. These results suggest that the expression of the leptin gene in response to food-deprivation and refeeding is influenced by an animal's age and that this expression is different for different regions of white adipose tissue.     

Differential effect of long-term food restriction on fatty acid synthase and leptin gene expression in rat white adipose tissue.

Long-term food restriction (85%, 70% and 50% of ad libitum energy intake for one month) induced a substantial fall in serum leptin concentration and leptin mRNA levels in epididymal white adipose tissue in rats. Surprisingly, this suppression was not reversed by refeeding ad libitum for 48 h. The reduction in serum leptin concentration and leptin mRNA level did not strictly correlate with reduction in fat or body mass. Unlike serum leptin concentration and epididymal adipose tissue leptin mRNA levels, fatty acid synthase activity, fatty acid synthase protein abundance and fatty acid synthase mRNA levels increased significantly in white adipose tissue after refeeding rats subjected to food restriction. The increase in serum insulin concentration was observed in all groups on different degrees of food restriction and refed ad libitum for 48 h compared to controls. A decrease in serum insulin concentration was found in the rats not refed before sacrifice. Long-term food restriction did not significantly affect serum glucose concentrations in either refed or non-refed rats. The data reported in this paper indicate that there is no rapid rebound in serum leptin concentration or leptin gene expression in contrast to the increase in serum insulin concentration and fatty acid gene expression in white adipose tissue of rats refed ad libitum after one month's food restriction.     

Evidence that triiodothyronine decreases rat serum leptin concentration by down-regulation of leptin gene expression in white adipose tissue

Conflicting results have been reported regarding the effect of triiodothyronine (T(3)) on serum leptin and adipose tissue leptin gene expression in human and animals. The aim of the present study was to evaluate the effect of administration of increasing doses of T(3) on serum leptin concentration and on leptin mRNA abundance in white adipose tissue of rats. The results presented in this paper indicate that administration of single different doses of T(3) to euthyroid rats resulted dose dependent increases of serum total T(3) concentrations which are associated with a decrease in white adipose tissue leptin mRNA level. The leptin mRNA level in white adipose tissue was negatively correlated with serum total T(3) concentration (r=-0.8, p<0.001). Like white adipose tissue leptin mRNA level, serum leptin concentration decreased after T(3) administration, and was also negatively correlated with the serum T(3) concentration (r=-0.8, p<0.001). In contrast, administration of T(3) to the same rats led to a significant increase in white adipose tissue expression of the malic enzyme gene (malic enzyme activity and malic enzyme mRNA level), a known target gene for T(3). The results indicate that T(3) exerts a selective inhibitory effect on white adipose tissue leptin gene expression in vivo. A conclusion is that T(3) decreases rat serum leptin concentration by down-regulation of leptin gene expression in white adipose tissue.     

Effect of exercise training on adipocyte-size-dependent expression of leptin and adiponectin

AimsOur aim was to evaluate the effect of exercise training (TR) on adipocyte-size-dependent expression of leptin and adiponectin.Main methodsMale Wistar rats were divided into 2 groups, sedentary control (CR) and TR group, and both monitored for 9 weeks. Adipocytes isolated from epididymal, retroperitoneal, and inguinal fat depots were independently separated into 3 fractions of different cell size, and the relationships between adipocyte size and either leptin or adiponectin mRNA were determined by real-time RT-PCR analysis.Key findingsIn epididymal and inguinal adipose tissue, positive relationships between adipocyte size and both leptin and adiponectin mRNA expression were found. Comparison of TR and CR rats showed no significant effect of TR on the slopes of the linear regression lines of correlation between leptin mRNA and adipocyte size in either adipose tissue, whereas the slopes of the regression line of correlation between adipocyte size and adiponectin mRNA were greater in TR group. Leptin levels per milliliter of plasma were significantly lower in TR than CR rats, whereas leptin levels adjusted to the 3 fat depots did not differ. TR did not affect adiponectin levels in plasma, whereas adiponectin levels adjusted to the 3 fat depots were significantly greater in TR than CR group.SignificanceTR-induced reduction in leptin mRNA expression was closely associated with smaller adipocyte size. However, TR amplified the adipocyte-size-dependent expression of adiponectin mRNA, suggesting that TR-induced alterations in adiponectin mRNA may also be mediated by factor(s) other than adipocyte size.     

The role of the sympathetic nervous system in the regulation of leptin synthesis in C57BL/6 mice

The objectives of this study were to determine whether leptin synthesis is regulated by the sympathetic nervous system and if so whether beta-adrenergic receptors mediate this effect. We show that sympathetic blockade by reserpine increases leptin mRNA levels in brown but not white adipose tissue, while acute cold-exposure decreases leptin expression 10-fold in brown adipose tissue and 2-fold in white adipose tissue. The cold-induced reduction in leptin mRNA can be prevented by a combination of propranolol and SR 59230A but not by either antagonist alone, indicating that beta3-adrenergic receptors and classical beta1/beta2-adrenergic receptors both mediate responses to sympathetic stimulation. Circulating leptin levels reflect synthesis in white adipose tissue but not in brown adipose tissue.     

Dehydroepiandrosterone prevents age-associated alterations, increasing insulin sensitivity

The age decline in DHEA levels has been associated with the appearance of age-related disorders such as obesity and insulin resistance. The aim of this study was to analyse the effect of chronic administration (13 weeks) of DHEA (5 g/kg diet) to old female rats fed on a high-fat diet on body weight and adiposity, and concretely on the expression of the adipokines related to obesity and insulin resistance, such as leptin, adiponectin and resistin. DHEA treatment induced a decrease in body weight, adipose tissue mass and serum insulin, adiponectin and leptin levels. Adiponectin mRNA expression in visceral fat depots decreased with aging, but this reduction was attenuated by DHEA treatment. DHEA treatment also stimulated resistin gene expression in the ovaric and renal adipose depots, which is associated with an increase in its circulating levels. In conclusion, DHEA treatment decreases body weight and adiposity in old female rats fed a high-fat diet, leading to an improvement of the HOMA index for insulin sensitivity, with decreasing circulating insulin levels, and preventing the age-associated decline of visceral-adipose adiponectin expression.     

Effects of retinoic acid administration and dietary vitamin A supplementation on leptin expression in mice: lack of correlation with changes of adipose tissue mass and food intake

Retinoic acid (RA) administration and chronic vitamin A supplementation were reported to inhibit adipose tissue leptin expression in rodents, but the impact of this effect on food intake and its relationship with changes of body adiposity was not analyzed. Here, we have studied the effects of RA administration at three different doses on body weight, adipose tissue mass, food intake, adipose tissue leptin expression and circulating leptin levels in NMRI mice; the effects of chronic vitamin A supplementation with a 40-fold excess retinyl palmitate on the same parameters in NMRI and C57BL/6J mice; and the effects of RA and retinoid receptors agonists on leptin expression in brown and white adipocyte cell model systems. The results show that vitamin A down-regulates leptin expression in white and brown adipose tissue and circulating leptin levels independently of changes of adipose tissue mass and, for the first time to our knowledge, that this effect does not correlate with increased food intake. They also demonstrate a direct inhibitory effect of RA on leptin expression in both white and brown adipocyte cell cultures, and constitute first proof of the involvement of both RA receptors (RARs) and rexinoid receptors (RXRs) in this effect. Reduction of leptin levels by specific nutrients is of potential interest from a clinical point of view.     

Nicotine infusion alters leptin and uncoupling protein 1 mRNA expression in adipose tissues of rats

We attempted to clarify whether leptin and uncoupling protein 1 (UCP1) are involved in the action of nicotine on the energy balance. Male Wistar rats were infused subcutaneously with nicotine (12 mg x kg(-1) x day(-1)) for 4 or 14 days. At the end of the 4-day period, the plasma concentrations of leptin of the nicotine-treated and pair-fed rats were lower than those of the freely fed rats, although the levels of leptin mRNA expression in various white adipose tissues did not differ among the three groups. At the end of the 14-day nicotine infusion period, plasma concentrations of leptin were higher, and leptin mRNA expression in the omentum and epididymal and retroperitoneal adipose tissues was stronger in the nicotine-treated rats than in the pair-fed and freely fed rats. UCP1 mRNA expression in the brown adipose tissue of nicotine-treated was stronger than that of the pair-fed rats. These results suggest that continuous nicotine infusion differentially affects the synthesis and secretion of leptin according to the duration of infusion and stimulates UCP1 mRNA expression, probably in a manner independent of leptin.     

Effects of central or peripheral leptin administration on norepinephrine turnover in defined fat depots

Leptin preserves lean tissue but decreases adipose tissue by increasing lipolysis and/or inhibiting lipogenesis. The sympathetic nervous system (SNS) is a primary regulator of lipolysis, but it is not known if leptin increases norepinephrine turnover (NETO) in white adipose tissue. In this study, we examined the effect of leptin administered either as a chronic physiological dose (40 microg/day for 4 days from ip miniosmotic pumps) or as an acute injection in the third ventricle (1.5 microg injected two times daily for 2 days) on NETO and the size of brown and white fat depots in male Sprague Dawley rats. NETO was determined from the decline in tissue norepinephrine (NE) during 4 h following administration of the NE synthesis inhibitor alpha-methyl-para-tryrosine. The centrally injected leptin-treated animals demonstrated more dramatic reductions in food intake, body weight, and fat pad size and an increase in NETO compared with the peripherally infused animals. Neither route of leptin administration caused a uniform increase in NETO across all fat pads tested, and in both treatment conditions leptin decreased the size of certain fat pads independent of an increase in NETO. Similar discrepancies in white fat NETO were found for rats pair fed to leptin-treated animals. These results demonstrate that leptin acting either centrally or peripherally selectively increases sympathetic outflow to white fat depots and that a leptin-induced change in fat pad weight does not require an increase in NETO.     

Adipose Tissue Promotes a Serum Cytokine Profile Related to Lower Insulin Sensitivity after Chronic Central Leptin Infusion

Obesity is an inflammatory state characterized by an augment in circulating inflammatory factors. Leptin may modulate the synthesis of these factors by white adipose tissue decreasing insulin sensitivity. We have examined the effect of chronic central administration of leptin on circulating levels of cytokines and the possible relationship with cytokine expression and protein content as well as with leptin and insulin signaling in subcutaneous and visceral adipose tissues. In addition, we analyzed the possible correlation between circulating levels of cytokines and peripheral insulin resistance. We studied 18 male Wistar rats divided into controls (C), those treated icv for 14 days with a daily dose of 12 μg of leptin (L) and a pair-fed group (PF) that received the same food amount consumed by the leptin group. Serum leptin and insulin were measured by ELISA, mRNA levels of interferon-γ (IFN-γ), interleukin-2 (IL-2), IL-4, IL-6, IL-10 and tumor necrosis factor-α (TNF-α) by real time PCR and serum and adipose tissue levels of these cytokines by multiplexed bead immunoassay. Serum leptin, IL-2, IL-4, IFN-γ and HOMA-IR were increased in L and TNF-α was decreased in PF and L. Serum leptin and IL-2 levels correlate positively with HOMA-IR index and negatively with serum glucose levels during an ip insulin tolerance test. In L, an increase in mRNA levels of IL-2 was found in both adipose depots and IFN-γ only in visceral tissue. Activation of leptin signaling was increased and insulin signaling decreased in subcutaneous fat of L. In conclusion, leptin mediates the production of inflammatory cytokines by adipose tissue independent of its effects on food intake, decreasing insulin sensitivity.     

Regulation of leptin by steroid hormones in rat adipose tissue.

We investigated if steroid hormones regulate the secretion and the expression of leptin in female and male rat adipose tissue fragments in vitro. Dexamethasone time and dose-dependently increased the secretion and mRNA expression of leptin with a half-maximal stimulation of approximately 1 nM. A time-course revealed a maximal stimulatory effect of 17 beta-estradiol after 24 hours. In male adipose tissue 17 beta-estradiol increased leptin secretion (32% by 50 nM 17 beta-estradiol, P = 0.07 and 34% by 500 nM 17 beta-estradiol, P < 1780.05) after 24 hours. An additional effect of estrogen was seen in the dexamethasone (50 nM) stimulated cells (38% with 50 nM 17 beta-estradiol, P < 0.05 and 48% by 500 nM 17 beta-estradiol, P < 0.05). Basal secretion of leptin was equal in female and male adipose tissue, whereas the effects of 17 beta-estradiol (50 nM) and dexamethasone were significantly increased in female as compared with male adipose tissue. Progesterone, testosterone, dihydrotestosterone and dehydroepiandrostendione-sulfate neither affected leptin secretion in male nor female adipose tissue in vitro. Furthermore, to investigate the effect of estrogen female rats were ovariectomized (OVX) and the adipose tissue was incubated in vitro and compared with adipose tissue leptin secretion from sham operated rats (SHAM), and with ovariectomized rats treated with 17 beta-estradiol (EST). A decreased basal and dexamethasone-stimulated leptin secretion from OVX rats compared with SHAM rats was found (P < 0.005) whereas 17 beta-estradiol treatment of ovariectomized rats maintained a normal leptin secretion. However, the dexamethasone stimulation was equally increased above basal levels in SHAM, OVX and EST rats (3.7 +/- 1.2, 2.9 +/- 0.8, 4.2 +/- 1.4, NS, ANOVA) respectively.     

Circulating leptin response to feeding and exogenous infusion of insulin in sheep exposed to thermoneutral and cold environments

Leptin has been shown to regulate feed intake and energy expenditure. Insulin stimulates leptin secretion in rodents, but its action on leptin secretion is still obscure in ruminants. If insulin stimulates leptin secretion in ruminants, circulating leptin concentrations may change during exposure to cold, because of fluctuating insulin secretion and action in the cold environment. The present experiment was designed to determine whether feeding or exogenous administration of insulin affects circulating leptin levels in sheep exposed to thermoneutral and cold environments. Suffolk rams that were shorn and fed a diet once daily were subjected to a thermoneutral (20 degrees C) or cold (0 degrees C) environment for at least 1 week. Overall mean concentrations of plasma leptin in the feeding experiment were lower (P<0.05) in the cold environment than in the thermoneutral environment. Plasma leptin levels remained relatively unchanged after feeding in both environments, though plasma insulin response to feeding in both environments increased (P<0.01). The euglycemic clamps (insulin infusion rate: 4 mUkgBW(-1)min(-1) for 2 h) increased (P<0.01) circulating leptin concentrations in the thermoneutral, but not in the cold environment. These results suggest that lower circulating leptin levels in ruminants exposed to the cold environment could be partly due to the depressed insulin action on leptin secretion.     

The gender- and fat depot-specific regulation of leptin, resistin and adiponectin genes expression by progesterone in rat

Progesterone affects lipid metabolism in adipose tissue and influences fat distribution in human. The aim of the study was to analyze the effect of progesterone on rat body and fat mass and on expression of genes encoding adipokines involved in the regulation of energy homeostasis. The results presented here indicate that progesterone administration to females caused increase in body and inguinal white adipose tissue mass. The increase of inguinal white adipose tissue mass is associated with the hypertrophy of adipocyte. The same dose of progesterone caused increase of its circulating concentration in males, however it barely reached the value observed in non-treated control females and did not have any effect on body and fat mass. The elevated circulating progesterone concentration was associated with an approximately 6- and 2-fold increase of leptin and resistin mRNA level respectively, and 2-fold decrease of adiponectin mRNA level only in inguinal white adipose tissue of females. RU 486, specific antagonist of progesterone receptor, abolished the effect of progesterone on the adipokine mRNA level in inguinal adipose tissue. In males, the elevated circulating progesterone concentration showed no effects on leptin, resistin or adiponectin mRNA level in inguinal, retroperitoneal or epididymal adipose tissue. Moreover, the results presented in this paper demonstrate a relatively high level of progesterone receptor mRNA in inguinal white adipose tissue of females, which was down-regulated in response to progesterone administration. In retroperitoneal adipose tissue of control females progesterone receptor mRNA level was approximately 3-fold lower as compared to inguinal adipose tissue. In inguinal, epididymal and retroperitoneal white adipose tissue of males progesterone receptor mRNA was hardly detected. Our results suggest that depot- and sex-dependent responsiveness of adipose tissue to the pharmacological dose of progesterone is controlled by both circulating concentration of progesterone and the white adipose tissue progesterone receptor level.     

Tissue leptin and plasma insulin are associated with lipoprotein lipase activity in severely obese patients

The development of metabolic complications of obesity has been associated with the existence of depot-specific differences in the biochemical properties of adipocytes. The aim of this study was to investigate, in severely obese men and women, both gender- and depot-related differences in lipoprotein lipase (LPL) expression and activity, as well as the involvement of endocrine and biometric factors and their dependence on gender and/or fat depot. Morbidly obese, nondiabetic, subjects (9 men and 22 women) aged 41.1+/-1.9 years, with a body mass index (BMI) of 54.7+/-1.7 kg/m(2) who had undergone abdominal surgery were studied. Both expression and activity of LPL and leptin expression were determined in adipose samples from subcutaneous and visceral fat depots. In both men and women, visceral fat showed higher LPL mRNA levels as well as lower ob mRNA levels and tissue leptin content than the subcutaneous one. In both subcutaneous and visceral adipose depots, women exhibited higher protein content, decreased fat cell size and lower LPL activity than men. The gender-related differences found in abdominal fat LPL activity could contribute to the increased risk for developing obesity-associated diseases shown by men, even in morbid obesity, in which the massive fat accumulation could mask these differences. Furthermore, the leptin content of fat depots as well as plasma insulin concentrations appear in our population as the main determinants of adipose tissue LPL activity, adjusted by gender, depot and BMI.     

Zinc deficiency reduces leptin gene expression and leptin secretion in rat adipocytes

The present study was conducted to measure ob mRNA abundance in the zinc-deficient (ZD) rats and the secretion of leptin from adipose tissue obtained from ZD, zinc-adequate (ZA), and pair-fed (PF) rats. It was found that ob mRNA abundance was greatest (P < 0.05) in adipose tissue obtained from ZA and PF rats. Ob mRNA abundance was similar in PF and ZD rats. To study leptin secretion from adipose tissue in a cell culture model, a method was developed to use excised epididymal adipose tissue from ZD, ZA, and PF rats. Tissue was incubated in Opti-modified Eagle's medium (MEM) cell culture medium in which concentrations of zinc and insulin were manipulated. It was observed that leptin secretion was higher (P < 0.05) in adipose tissue obtained from ZA than ZD and PF rats. Secretion of leptin was higher in adipose tissue of PF than ZD rats (P < 0.05). Surprisingly, media zinc content in this ex vivo model tended to suppress secretion of leptin. This suppression seems to be zinc specific and might be caused by the sequestration of insulin in the culture medium. Our results indicate that the reduction in serum leptin observed in ZD rats is likely caused by not only a reduction in body fat, but also by a decrease in leptin synthesis and secretion per gram of adipose tissue. Taking these results into account along with a prior study (1), it is possible that even a marginal zinc deficiency could affect leptin secretion and serum leptin concentrations. Impaired leptin secretion caused by zinc deficiency might be one factor contributing to hypogonadism observed in zinc deficiency.