Effect of clofibrate on malic enzyme and leptin mRNAs level in rat brown and white adipose tissue.

Two previous studies have reported contradictory results regarding the effect of fibrates treatment on obese (ob) gene expression in rodents. The purpose of the present study was to reinvestigate this issue. We examined the effect of clofibrate (fibrate derivative) administration for 14 days to rats on malic enzyme (as an adequate control of fibrates action) and leptin mRNAs level in the white and brown adipose tissues (WAT and BAT, respectively). The malic enzyme activity and malic enzyme mRNA level in white adipose tissue increased significantly after clofibrate feeding. In brown adipose tissue, the drug treatment resulted in depression of malic enzyme activity and malic enzyme mRNA level. Under the same conditions, leptin mRNA level did not change in these tissues. The results presented in this paper provide further evidence that the clofibrate (activator of peroxisome proliferator activated receptor alpha), feeding is without effect on ob gene expression in rat white and brown adipose tissue. Furthermore, the present study demonstrates that clofibrate causes opposite effects on malic enzyme gene expression in WAT (up-regulation) and BAT (down-regulation).     

Pharmacological doses of triiodothyronine upregulate lipogenic enzyme gene expression in rat white adipose tissue.

Triiodothyronine (T (3)) is known to increase liver lipogenic enzyme gene expression both in vivo and in tissue culture. Conflicting results have been reported on the effect of T (3) on lipogenic enzyme gene expression in white adipose tissue. The results presented in this paper indicate that administration of pharmacological doses of T (3) in rats leads to increased fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC), ATP-citrate lyase (ACL) and malic enzyme (ME) activity in white adipose tissue. The increase in lipogenic enzyme activity was associated with increased FAS, ACC, ACL and ME mRNA levels. The response was dose-dependent. Activity of lipogenic enzyme and the lipogenic enzyme mRNA levels were positively correlated to serum T (3) concentration. The in vivo effect of T (3) on lipogenic enzyme gene expression could be reproduced in primary white rat adipocyte culture. In conclusion, the results presented in this paper indicate that T (3) exerts a stimulatory effect on lipogenic enzyme gene expression in white adipose tissue both in vivo and in tissue culture. Significant effects of T (3) on lipogenic enzyme gene expression were only observed in the presence of relatively high (pharmacological) concentrations of the hormone.     

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.     

The age-related differences in obese and fatty acid synthase gene expression in white adipose tissue of rat

To determine if the age-dependent increase of adiposity is directly related to altered obese (ob) and fatty acid synthase (FAS) gene expression, we assessed an adiposity index, leptin and FAS mRNA levels, FAS activity in perirenal adipose tissue and serum leptin concentration in rats aged 1, 2, 3, 6 and 20 months. The results indicate that there are two distinct phases of changes in perirenal white adipose tissue leptin mRNA level and serum leptin concentration. The first phase, between 1 and 3 months of the animals' lives, was characterized by a strong positive correlation between adiposity index and leptin mRNA level as well as serum leptin concentration. In the second phase (over 3 months) no significant changes of leptin mRNA and serum concentration occurred. A close correlation between the age-induced increase of leptin mRNA abundance and serum leptin concentration and the age-induced suppression of FAS gene expression in the same tissue was observed. This suggests that the changes of FAS gene expression occur in response to serum leptin concentration and that in mature rats the high level of ob gene expression and consequently the high leptin concentration protect the white adipose tissue cells against fat overload by two independent mechanisms: (a) preventing an increase of food intake through the leptin action on the hypothalamus; (b) inhibiting FAS gene expression and consequently decreasing the rate of lipogenesis.     

Blood leptin homeostasis: sex-associated differences in circulating leptin levels in rats are independent of tissue leptin expression

Circulating leptin levels are higher in women than in men. The aim of the study has been to determine in rats the putative existence of sex-associated differences in leptin expression in different adipose tissue depots (gonadal, retroperitoneal, mesenteric and inguinal white adipose tissue and interscapular brown adipose tissue) and the relationship with circulating leptin levels. Adult male and female Wistar rats acclimated to 22 degrees C or to 28 degrees C were used. Leptin mRNA expression was assessed by northern blot and serum leptin levels by enzyme-linked immunosorbent assay (ELISA). Contrary to what happens in humans, we report here that male rats acclimated to standard animal house conditions (22 degrees C) have a higher leptin concentration in blood than female rats. This situation cannot be explained by a greater size of the fat depots in males, because the adiposity index is similar in both genders, but are rather associated to higher leptin specific mRNA expression by the white adipose tissue. Around thermoneutral conditions (28 degrees C), sex related differences in leptin mRNA expression disappear, but the gender difference in circulating leptin levels remains. In addition, leptin mRNA expression is higher in both genders in thermoneutral conditions but this is not reflected in changes in the circulating leptin levels. In conclusion, this study shows that rat circulating leptin levels are finely regulated, and not exclusively dependent on leptin mRNA expression, but other mechanisms are also involved, possibly regarding leptin rate of degradation.     

Effect of acute cold exposure on the expression of the adiponectin, resistin and leptin genes in rat white and brown adipose tissues.

Leptin, adiponectin, and resistin are key hormones produced by adipose tissue. In the present study, we have examined the effects of acute cold exposure (18 h at 6 degrees C) on the expression of the genes encoding these hormones in both brown and white fat of rats. Acute cold exposure resulted in a significant (p < 0.001) increase in the level of UCP1 and metallothionein-1 mRNAs in brown adipose tissue, indicative of an activation of thermogenesis. Leptin mRNA was decreased (p < 0.001) in brown fat in the cold, and there was also a small but statistically significant (p < 0.05) decrease in adiponectin mRNA; resistin mRNA did not change significantly (p > 0.05). In white fat, the level of leptin mRNA also fell in the cold (p < 0.05), but there was no significant change (p > 0.05) in either adiponectin or resistin mRNA. The serum concentration of adiponectin was unchanged following acute cold exposure. We conclude that while leptin gene expression is inhibited by exposure to cold, there is no major effect on the expression of either the adiponectin or resistin genes in white or brown fat despite the cold-induced stimulation of sympathetic activity and fatty acid flux. Thus, adiponectin and resistin are unlikely to play a key role in the extensive metabolic adaptations to cold.     

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.     

Dietary vitamin A supplementation in rats: suppression of leptin and induction of UCP1 mRNA

All-trans-retinoic acid (RA), an active metabolite of vitamin A, induces the gene expression of uncoupling protein 1 (UCP1) in brown adipose tissue (BAT) and suppresses leptin gene expression in white adipose tissue (WAT) when given as an acute dose. These contrasting effects of RA leave in doubt the overall effect of chronic RA or vitamin A supplementation on energy homeostasis. To investigate the effects of dietary vitamin A supplementation on leptin and UCP1 gene expression, rats were fed either a normal diet (2.6 retinol/kg diet) or a vitamin A-supplemented diet (129 mg retinol/kg diet) for 8 weeks, and adiposity, serum leptin levels, leptin mRNA levels in perirenal WAT, UCP1 and UCP2 mRNA levels in BAT, and beta3-adrenergic receptor mRNA levels in BAT and WAT were examined. Rats on both diets gained a similar amount of weight, but there was a small 9% decrease in the adiposity index in the vitamin A-supplemented rats. Dietary vitamin A supplementation increased UCP1 gene expression in BAT by 31%, but suppressed leptin gene expression by 44% and serum leptin levels by 65%. UCP2 and beta3-adrenergic receptor gene expression in BAT and perirenal WAT were unchanged by the vitamin A diet. These data suggest that dietary vitamin A has a role in regulating energy homeostasis by enhancing UCP1 gene expression and decreasing serum leptin levels.     

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.     

Daily rhythms of plasma melatonin, but not plasma leptin or leptin mRNA, vary between lean, obese and type 2 diabetic men

Melatonin and leptin exhibit daily rhythms that may contribute towards changes in metabolic physiology. It remains unclear, however, whether this rhythmicity is altered in obesity or type 2 diabetes (T2DM). We tested the hypothesis that 24-hour profiles of melatonin, leptin and leptin mRNA are altered by metabolic status in laboratory conditions. Men between 45-65 years old were recruited into lean, obese-non-diabetic or obese-T2DM groups. Volunteers followed strict sleep-wake and dietary regimes for 1 week before the laboratory study. They were then maintained in controlled light-dark conditions, semi-recumbent posture and fed hourly iso-energetic drinks during wake periods. Hourly blood samples were collected for hormone analysis. Subcutaneous adipose biopsies were collected 6-hourly for gene expression analysis. Although there was no effect of subject group on the timing of dim light melatonin onset (DLMO), nocturnal plasma melatonin concentration was significantly higher in obese-non-diabetic subjects compared to weight-matched T2DM subjects (p<0.01) and lean controls (p<0.05). Two T2DM subjects failed to produce any detectable melatonin, although did exhibit plasma cortisol rhythms comparable to others in the group. Consistent with the literature, there was a significant (p<0.001) effect of subject group on absolute plasma leptin concentration and, when expressed relative to an individual's 24-hour mean, plasma leptin showed significant (p<0.001) diurnal variation. However, there was no difference in amplitude or timing of leptin rhythms between experimental groups. There was also no significant effect of time on leptin mRNA expression. Despite an overall effect (p<0.05) of experimental group, post-hoc analysis revealed no significant pair-wise effects of group on leptin mRNA expression. Altered plasma melatonin rhythms in weight-matched T2DM and non-diabetic individuals supports a possible role of melatonin in T2DM aetiology. However, neither obesity nor T2DM changed 24-hour rhythms of plasma leptin relative to cycle mean, or expression of subcutaneous adipose leptin gene expression, compared with lean subjects.     

The obesity in bilateral ovariectomized rats is related to a decrease in the expression of leptin receptors in the brain.

We investigated the expression levels of leptin receptors in the brain of ovariectomized (OVX) rats. The mean expression level of ob mRNA in adipose tissues of OVX rats was significantly (P < 0.01) lower than that in the SHAM operation group rats, and the mean body weight of OVX rats was significantly (P < 0.01) greater than that in the SHAM group rats. However, there were no differences between serum leptin concentrations in these two groups. The mean level of leptin receptor (OB-R) mRNA expression in the brain tissue and the mean level of long form type OB-R (OB-RL) mRNA expression in the hypothalamus of the OVX rats were significantly (P < 0.05) lower than those in the SHAM group rats. These changes were cancelled by supplementation with 17 beta-estradiol in OVX rats. These results suggested that not only changes in the expression level of ob mRNA in adipose tissue and the serum leptin concentration but also changes in the OB-R mRNA in the brain are involved in the body weight increase in OVX rats and that a decrease in OB-R makes transmission of signals to suppress the amount of food intake difficult, thus leading to an increase in body weight.     

Altered mRNA expression of hepatic lipogenic enzyme and PPARalpha in rats fed dietary levan from Zymomonas mobilis

Levan or high molecular beta-2,6-linked fructose polymer is produced extracellularly from sucrose-based substrates by bacterial levansucrase. In the present study, to investigate the effect of levan feeding on serum leptin, hepatic lipogenic enzyme and peroxisome proliferation-activated receptor (PPAR) alpha expression in high-fat diet-induced obese rats, 4-week-old Sprague-Dawley male rats were fed high-fat diet (beef tallow, 40% of calories as fat), and, 6 weeks later, the rats were fed 0%, 1%, 5% or 10% levan-supplemented diets for 4 weeks. Serum leptin and insulin level were dose dependently reduced in levan-supplemented diet-fed rats. The mRNA expressions of hepatic fatty acid synthase and acetyl CoA carboxylase, which are the key enzymes in fatty acid synthesis, were down-regulated by dietary levan. However, dietary levan did not affect the gene expression of hepatic malic enzyme, phosphatidate phosphohydrolase and HMG CoA reductase. Also, the lipogenic enzyme gene expression in the white adipose tissue (WAT) was not affected by the diet treatments. However, hepatic PPARalpha mRNA expression was dose dependently up-regulated by dietary levan, whereas PPARgamma in the WAT was not changed. The results suggest that the in vivo hypolipidemic effect of dietary levan, including anti-obesity and lipid-lowering, may result from the inhibition of lipogenesis and stimulation of lipolysis, accompanied with regulation of hepatic lipogenic enzyme and PPARalpha gene expression.     

Central stimulatory effect of leptin on T3 production is mediated by brown adipose tissue type II deiodinase

To assess whether intracerebroventricular leptin administration affects monodeiodinase type II (D2) activity in the tissues where it is expressed [cerebral cortex, hypothalamus, pituitary, and brown adipose tissue (BAT)], hepatic monodeiodinase type I (D1) activity was inhibited with propylthiouracil (PTU), and small doses of thyroxine (T4; 0.6 nmol. 100 g body wt(-1). day(-1)) were supplemented to compensate for the PTU-induced hypothyroidism. Two groups of rats were infused with leptin for 6 days, one of them being additionally treated with reverse triiodothyronine (rT3), an inhibitor of D2. Control rats were infused with vehicle and pair-fed the amount of food consumed by leptin-infused animals. Central leptin administration produced marked increases in D2 mRNA expression and activity in BAT, changes that were likely responsible for increased plasma T3 and decreased plasma T4 levels. Indeed, plasma T3 and T4 concentrations were unaltered by central leptin administration in the presence of rT3. The additional observation of a leptin-induced increased mRNA expression of BAT uncoupling protein-1 suggested that the effect on BAT D2 may be mediated by the sympathetic nervous system.     

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.     

Developmental patterns of serum leptin levels, leptin gene expression in adipose tissue and Ob-Rb gene expression in hypothalamus of Erhualian and Large White pigs

~~Developmental patterns of serum leptin levels, leptin gene expression in adipose tissue and Ob-Rb gene expression in hypothalamus of Erhualian and Large White pigs~~     

Central leptin regulates the UCP1 and ob genes in brown and white adipose tissue via different beta-adrenoceptor subtypes

The three known subtypes of beta-adrenoreceptors (beta(1)-AR, beta(2)-AR, and beta(3)-AR) are differentially expressed in brown and white adipose tissue and mediate peripheral responses to central modulation of sympathetic outflow by leptin. To assess the relative roles of the beta-AR subtypes in mediating leptin's effects on adipocyte gene expression, mice with a targeted disruption of the beta(3)-adrenoreceptor gene (beta(3)-AR KO) were treated with vehicle or the beta(1)/beta(2)-AR selective antagonist, propranolol (20 microgram/g body weight/day) prior to intracerebroventricular (ICV) injections of leptin (0.1 microgram/g body weight/day). Leptin produced a 3-fold increase in UCP1 mRNA in brown adipose tissue of wild type (FVB/NJ) and beta(3)-AR KO mice. The response was unaltered by propranolol in wild type mice, but was completely blocked by this antagonist in beta(3)-AR KO mice. In contrast, ICV leptin had no effect on leptin mRNA in either epididymal or retroperitoneal white adipose tissue (WAT) from beta(3)-AR KOs. Moreover, propranolol did not block the ability of exogenous leptin to reduce leptin mRNA in either WAT depot site of wild type mice. These results demonstrate that the beta(3)-AR is required for leptin-mediated regulation of ob mRNA expression in WAT, but is interchangeable with the beta(1)/beta(2)-ARs in mediating leptin's effect on UCP1 mRNA expression in brown adipose tissue.     

Regulation of leptin gene expression and secretion by steroid hormones.

Previous work has shown that 17 beta-estradiol is the primary ovarian signal regulating body weight and adiposity, although its mechanisms of action remain unclear. We hypothesized that 17 beta-estradiol could enhance leptin levels as a mechanism of its anorectic effects. Administration of 5 microg 17 beta-estradiol subcutaneously (s.c.) for 2 days significantly elevated leptin mRNA levels in adipose tissue as compared to vehicle controls (P < 0.003). A time-course administration of estrogen showed increased mRNA levels in adipose tissue between 6 and 12 h after estrogen injection as compared to vehicle controls (P < 0.03). Corresponding to the increased leptin mRNA levels at 6 and 12 h, elevated plasma leptin levels were observed at 12 h after estrogen administration as compared to controls (P < 0.05). Administration of progesterone (1 mg/rat) after estradiol injection did not enhance the elevated leptin mRNA levels in adipose tissue. Serum leptin levels from cycling rats did not differ significantly between metestrous and proestrous animals. In conclusion, the present studies demonstrate that 17 beta-estradiol can regulate leptin gene expression and secretion in the female rat, thus providing a better understanding of the possible anorectic effect of estrogens.