Atrophic change of rat salivary gland during adenovirus-induced hyperleptinemia

Sustained hyperleptinemia in normal rats induced by infusing a recombinant adenovirus containing the rat leptin cDNA (AdCMV-leptin) exhibited a remarkable reduction in food intake (AdCMV-leptin, 9.3 +/- 2.6 vs untreated, 20.6 +/- 1.0 g/day) and ablated body fat without any significant changes in wet weight of liver and left ventricle. In those hyperleptinemic rats, we found a 52% reduction in wet weight of salivary gland compared with that in the pair-fed AdCMV-beta-gal-treated rats, which received a recombinant virus containing the beta-galactosidase gene (AdCMV-beta-gal) and were fed on the same amount of food as had been consumed by the AdCMV-leptin-treated group on the previous day. Microscopic examination with hematoxylin-eosin staining revealed that atrophic change was induced in both serous and mucous gland only in the AdCMV-leptin-treated group, but not in the pair-fed controls. Thus, the atrophic changes in hyperleptinemic rats were due to neither a decrease of food intake nor disuse of the salivary gland related with anorexia. Our data suggested that size of the salivary gland was controlled, at lease in part, by "non-anorexic" effect of leptin.     

Slow recovery of body fat lost during adenovirus-induced hyperleptinemia

In normal rats, adenovirus-induced hyperleptinemia causes disappearance of visible body fat, downregulation of lipogenic enzymes, and upregulation of oxidative enzymes and thermogenic proteins. In addition, preadipocyte markers replace mature adipocyte markers, suggesting dedifferentiation. In weight loss induced by caloric restriction, by contrast, the lipogenic machinery is essentially intact. To determine if the radical changes induced by leptin would slow the reappearance of body fat, we compared normal lean rats made hyperleptinemic by infusing an adenovirus-leptin construct with diet-matched littermates. Initially, in plasma leptin the hyperleptinemic rats averaged approximately 50x the controls and, although it declined progressively, it was still slightly elevated at 150 days (P < 0.05). In the hyperleptinemics, body fat mass, quantified by magnetic resonance spectroscopy, remained below the pretreatment value for 60 days, while in diet-matched controls it exceeded the pretreatment value. Epididymal fat pad weight in hyperleptinemics was still 28% below paired controls at 150 days posttreatment. Histologic examination revealed adipocytes of hyperleptinemic animals to be smaller 60 days after treatment. At 60 days, adipose tissue UCP-2 gene expression in hyperleptinemics was still above controls, but expression of other lipogenic and oxidative enzymes had returned to baseline expression levels. We conclude that in normal rats recovery of body fat following adenovirus-induced hyperleptinemia is much slower than after caloric restriction, possibly because of persistent upregulation of adipocyte UCP-2.     

Hyperleptinemia depletes fat from denervated fat tissue.

Adenovirus-mediated transfer of the leptin gene causes severe hyperleptinemia with rapid disappearance of visible body fat. To determine if this dramatic lipopenic action is mediated by neurotransmitted signals from the central nervous system, we transplanted the right epididymal fat pad of normal rats to the anterior abdominal wall. Four weeks later, rats were infused with either adenovirus-leptin cDNA (AdCMV-leptin) or adenovirus-beta-galactosidase (AdCMV-beta-gal). Eight days later, plasma leptin averaged 23 +/- 12 ng/ml in the former and 1.2 +/- 0.4 ng/ml in the latter. The fat transplant was intact in all 4 AdCMV-beta-gal-infused rats but had disappeared in all 4 hyperleptinemic rats. Tyrosine hydroxylase staining of the fat pad remnant was negative, excluding regrowth of sympathetic nerves. Thus, the lipopenic action of severe hyperleptinemia on adipocytes is not mediated by neurotransmitters, but must have resulted either from direct action of leptin and/or from leptin-mediated neurohormones.     

Effects of leptin,troglitazone, and dietary fat on stearoyl CoA desaturase

Leptin, troglitazone, and high fat feeding profoundly influence the lipid content of various tissues. To determine if they affect the expression of stearoyl CoA desaturase (SCD)-1 and -2, their mRNA was measured in livers of normal, hyperleptinemic, troglitazone-treated, and fat-fed rats. Hyperleptinemia, which reduces tissue TG by downregulating lipogenic enzymes and upregulating fatty acid oxidation, lowered SCD-1 96% below controls and reduced SCD-2 slightly. By contrast, hepatic SCD-1 mRNA of leptin-resistant fa/fa rats was five times wild-type controls, but SCD-2 mRNA was 66% lower. High fat feeding lowered SCD-1 by 80%, possibly by inducing hyperleptinemia. Troglitazone treatment, which reduces nonadipose tissue TG of fa/fa rats without downregulating lipogenic enzymes, raised SCD-2 13-fold but lowered SCD-1 by 25%. The findings suggest that leptin controls SCD-1 expression and that troglitazone's antilipotoxic action may involve SCD-2 upregulation.     

Leptin resistance of adipocytes in obesity: role of suppressors of cytokine signaling

Liver-derived hyperleptinemia induced in normal rats by adenovirus-induced gene transfer causes rapid disappearance of body fat, whereas the endogenous adipocyte-derived hyperleptinemia of obesity does not. Here we induce liver-derived hyperleptinemia in rats with adipocyte-derived hyperleptinemia of acquired obesity caused by ventromedial hypothalamus lesioning (VMH rats) or by feeding 60% fat (DIO rats). Liver-derived hyperleptinemia in obese rats caused only a 5-7% loss of body weight, compared to a 13% loss in normoleptinemic lean animals; but in actual grams of weight lost there was no significant difference between obese and lean groups, suggesting that a subset of cells remain leptin-sensitive in obesity. mRNA and protein of a putative leptin-resistance factor, suppressor of cytokine signaling (SOCS)-1 or -3, were both increased in white adipose tissues (WAT) of VMH and DIO rats. Since transgenic overexpression of SOCS-3 in islets reduced the lipopenic effect of leptin by 75%, we conclude that the increased expression of SOCS-1 and -3 in WAT of rats with acquired obesity could have blocked leptin's lipopenic action in the leptin-resistant WAT population.     

Adipose tissue cellularity in hypo-and hyperthyroid rats

To determine adipose tissue cellularity in hypo- and hyperthyroidism, male rats were thyroidectomized after weaning (T) and injected daily with either 0, 0.1, 1.8 or 25 microgram of L-thyroxine/100 g body weight for 40 days. They were compared with intact controls (C). Both epididymal fat-pad weight and adipocyte diameter were reduced in T+0, T+0.1 and T+25 animals. When corrected per unit of body weight, the diameters of adipocytes from T+0 and T+0.1 animals were larger than in the other groups. Those same animals have reduced absolute adipocyte number but not when corrected per unit of body weight. The fat-pad protein concentration varied conversely with the fat cell diameter. These findings indicate that thyroid hormone deficiency reduces the proliferation of fat cells in parallel with body growth while hyperthyroidism causes reduction in the size, but not the number, of fat cells which corresponds to its depletion of fat storage.     

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.     

Combined leptin actions on adipose tissue and hypothalamus are required to deplete adipocyte fat in lean rats: implications for obesity treatment

Intense hyperleptinemia completely depletes adipocyte fat of normal rats within 14 days. To determine the mechanism, epididymal fat pads from normal wild-type (+/+) and obese (fa/fa) Zucker Diabetic Fatty (ZDF) donor rats were transplanted into normal +/+ and fa/fa ZDF recipients. Hyperleptinemia induced by adenovirus-leptin administration depleted all fat from native fat pads and from fat transplants from +/+ donors but not from transplants from ZDF(fa/fa) donors with defective leptin receptors. In both native and transplanted +/+ fat pads, large numbers of mitochondria were apparent, and genes involved in fatty acid oxidation were up-regulated. However, +/+ fat pads transplanted into fa/fa recipients did not respond to hyperleptinemia, suggesting lack of an essential leptin-stimulated cohormone(s). In +/+ but not in fa/fa rats, plasma catecholamine levels rose, and both P-STAT3 and P-CREB increased in adipose tissue, suggesting that both direct and indirect (hypothalamic) leptin receptor-mediated actions of hyperleptinemia are involved in depletion of adipocyte fat.     

Retinol-binding protein 4 and nicotinamide phosphoribosyltransferase/visfatin in rat obesity models.

Retinol-binding protein 4 (RBP4) and nicotinamide phosphoribosyltransferase/visfatin (Nampt/visfatin) are adipocyte-secreted proteins (adipokines) whose relevance to the metabolic syndrome and regulation in obesity remain controversial. Here, we tested the hypothesis that adipose tissue expression and circulating levels of these two adipokines are elevated in obesity by analyzing their changes in both a genetic and a diet-induced model of obesity in the rat (obese FA/ FA Zucker rats and Wistar rats fed a cafeteria diet, respectively). Compared with lean controls, obese FA/ FA rats were hyperleptinemic, hyperinsulinemic, and insulin resistant and had reduced RBP4 serum levels and mRNA levels in adipose depots, unchanged Nampt/visfatin serum levels, and reduced Nampt/visfatin mRNA levels selectively in the inguinal adipose depot. Cafeteria diet-induced obesity resulted in increased fed blood glucose levels, a variable degree of insulin resistance, unchanged serum Nampt/visfatin and RBP4 levels, and reduced mRNA levels of both adipokines in several adipose depots. Hence, increases in RBP4 or Nampt/visfatin do not accompany obesity and insulin resistance in the models examined.     

Hyperleptinemia in obese adolescents deregulates neuropeptides during weight loss

Leptin has emerged over the past decade as a key hormone not only in energy balance regulation but also in neuroendocrine and inflammatory processes. The aim of the present study was to evaluate whether hyperleptinemia deregulates neuropeptides during weight loss. A total of 86 post-pubertal obese adolescents (with or without hyperleptinemia) participated in one year of interdisciplinary weight loss therapy (clinical, nutritional, psychological and exercise-related). Adipokine and neuropeptide concentrations were measured by ELISA, visceral fat was measured by ultrasound and body composition was measured by pletismography. The hyperleptinemic patients presented a lower alpha-MSH concentration and higher NPY/AgRP ratio while the adiponectin/leptin (A/L) ratio was lower compared with the non-hyperleptinemic group. After therapy, significant improvements in BM, BMI, body fat mass, visceral and subcutaneous fat, HOMA-IR, QUICKI, total cholesterol and triglycerides were observed in both groups. Indeed, we observed significant increases in adiponectin and A/L as well as reductions in leptin and NPY/AgRP ratio in the hyperleptinemic group. In the stepwise multiple linear regression analysis with leptin concentration as the dependent variable, α-MSH and body fat mass (%) were the independent predictors to explain leptin concentration. For the entire group, we found positive correlations between leptinemia and BMI and body fat mass (%) as well as a negative correlation with free fat mass (%) and alpha-MSH. Finally, we verified negative correlations between adiponectin/leptin ratio with total cholesterol and LDL-c, only in hyperleptinemic patients. In conclusion, the hyperleptinemia in obese adolescents deregulates neuropeptides during weight loss.     

Specific expression of lactase in the jejunum and colon during postnatal development and hormone treatments in the rat.

The effects of hyperinsulinaemia imposed on normal rats on the subsequent insulin-responsiveness in vivo of 2-deoxy-D-glucose uptake of white adipose tissue and of various muscle types were investigated. This was done by treating normal rats with insulin via osmotic minipumps, and by comparing them with saline-infused controls. Hyperinsulinaemia produced by prior insulin treatment resulted in a well-tolerated hypoglycaemia. At the end of the treatment, the glucose utilization index of individual tissues was determined by euglycaemic/hyperinsulinaemic clamps associated with the labelled 2-deoxy-D-glucose method. Prior insulin treatment resulted in increased insulin-responsiveness of the glucose utilization index of white adipose tissue, and in increased total lipogenesis in white adipose tissue and fat-pad weight. In contrast, prior insulin treatment resulted in a decreased glucose utilization index of several muscles. These opposite effects of hyperinsulinaemia on glucose utilization in white adipose tissue and muscles persisted when the hypoglycaemia-induced catecholamine output was prevented (adrenomedullectomy, propranolol treatment), as well as when hypoglycaemia was normalized by concomitant insulin treatment and glucose infusion. Insulin suppressed hepatic glucose production during the clamps in insulin-treated rats as in the respective controls, whereas total hepatic lipid synthesis and liver fat content were greater in rats treated with insulin than in controls. It is concluded that hyperinsulinaemia itself could be one of the driving forces responsible for producing increased glucose utilization by white adipose tissue, increased total lipid synthesis with fat accumulation in adipose tissue and the liver, together with an insulin-resistant state at the muscular level.     

The chemical chaperones tauroursodeoxycholic and 4-phenylbutyric acid accelerate thyroid hormone activation and energy expenditure

Exposure of cell lines endogenously expressing the thyroid hormone activating enzyme type 2 deiodinase (D2) to the chemical chaperones tauroursodeoxycholic acid (TUDCA) or 4-phenylbutiric acid (4-PBA) increases D2 expression, activity and T3 production. In brown adipocytes, TUDCA or 4-PBA induced T3-dependent genes and oxygen consumption (∼2-fold), an effect partially lost in D2 knockout cells. In wild type, but not in D2 knockout mice, administration of TUDCA lowered the respiratory quotient, doubled brown adipose tissue D2 activity and normalized the glucose intolerance associated with high fat feeding. Thus, D2 plays a critical role in the metabolic effects of chemical chaperones.     

Central leptin gene therapy suppresses body weight gain, adiposity and serum insulin without affecting food consumption in normal rats: a long-term study

The weight-reducing effects of leptin are predominantly mediated through the hypothalamus in the brain. Gene therapy strategies designed for weight control have so far tested the short-term effect of peripherally delivered viral vectors encoding the leptin gene. In order to circumvent the multiple peripheral effects of hyperleptinemia and to overcome the age-related development of leptin resistance due to multiple factors, including defective leptin transport across the blood brain barrier, we determined whether delivery of viral vectors directly into the brain is a viable therapeutic strategy for long-term weight control in normal wild-type rats. A recombinant adeno-associated virus (rAAV) vector encoding rat leptin (Ob) cDNA was generated (rAAV-betaOb). When administered once intracerebroventricularly (i.c.v.), rAAV-betaOb suppressed the normal time-related weight gain for extended periods of time in adult Sprague-Dawley rats. The vector expression was confirmed by immunocytochemical localization of GFP and RT-PCR analysis of leptin in the hypothalamus. This sustained restraint on weight gain was not due to shifts in caloric consumption because food-intake was similar in rAAV-betaOb-treated and rAAV-GFP-treated control rats throughout the experiment. Weight gain suppression, first apparent after 2 weeks, was a result of reduced white fat depots and was accompanied by drastically reduced serum leptin and insulin concentrations in conjunction with normoglycemia. Additionally, there was a marked increase in uncoupling protein-1 (UCP1) mRNA expression in brown adipose tissue, thereby indicating increased energy expenditure through thermogenesis. Seemingly, a selective enhancement in energy expenditure following central delivery of the leptin gene is a viable therapeutic strategy to control the age-related weight gain and provide protection from the accompanying multiple peripheral effects of hyperleptinemia and hyperinsulinemia.     

Induction of uncoupling protein-2 (UCP2) gene expression on the differentiation of rat preadipocytes to adipocytes in primary culture

We have examined uncoupling protein-2 (UCP2) gene expression in the adipose tissue of obese and normal rats and mice, and also in differentiated rat adipocytes in primary culture. Expression of the UCP2 gene was examined in rat and mouse adipose tissues using both RT-PCR and Northern blotting. Although the RT-PCR was not quantitative, the band corresponding to the UCP2 mRNA was stronger in white adipose tissue than in brown fat, regardless of the body weight of the rats. In agreement with the RT-PCR data, there was a higher level of UCP2 mRNA in the white adipocytes than in brown adipocytes, the level being greater in obese mice. Fibroblastic preadipocytes were obtained from the inguinal fat pad of suckling rats. Lipid droplets developed inside the cells upon differentiation and adipsin and UCP2 mRNAs were detected by Northern blotting. Both mRNAs were evident in the adipocytes at 4, 6, and 10 d after the induction of differentiation. There was no indication that the expression of UCP2 was markedly affected by the addition of leptin, dexamethasone or isoprenaline.     

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.     

Short-term treatment with estrone oleate in liposomes (Merlin-2) does not affect the expression of the ob gene in Zucker obese rats

Young female Zucker fa/fa rats of 370-430 g were implanted with osmotic minipumps releasing 3.5 mol/dayúkg of estrone oleate in liposomes (Merlin-2) into the bloodstream for up to 14 days. Merlin-2 induced a sustained loss of appetite, and a decrease in body weight of 3.5%, which contrasts with the 8.2% increase in controls during the period studied. Plasma insulin, glucose and urea decreased, and liver glycogen increased with Merlin-2 treatment. Plasma ACTH and corticosterone increased to a maximum at the end of the experiment. The expression of the ob gene in adipose tissue was unchanged, and plasma leptin levels were also unchanged by treatment. Estrone levels increased more than 1500-fold, and estrone oleate rose 100-fold during treatment. The fact that estrone oleate had no effect on the leptin levels or expression in obese rats, in contrast with the marked inhibition observed in the lean suggests that the functionality of the leptin receptor is essential for estrone oleate inhibition of the ob gene. This also suggests that leptin may control ob gene expression in white adipose tissue and that estrone oleate may activate this process. The slimming effect of estrone oleate is, thus, not directly dependent on leptin, since both normoleptinemic and hyperleptinemic animals lose fat following treatment nor are the effects on appetite and energy expenditure mediated by leptin. However, leptin levels and the expression of the ob gene are directly linked with estrone oleate function. A possible involvement of leptin in estrone oleate action is postulated. The results support the participation of estrone oleate in the control of body weight and hint at the complexity of its regulation by leptin and glucocorticoids.     

Effects of hyper- and hypothyroidism on thyroid hormone concentrations in regions of the rat brain

The purpose of this study was to investigate the effects of hyper- and hypothyroidism on thyroid hormone concentrations and deiodinase activities in nine regions of the rat brain. Four weeks of treatment with 75 microg thyroxine (T4)/kg body wt induced a two- to threefold increase in T4 levels in all of these brain regions, whereas the 3,5,3'-triiodothyronine (T3) concentrations were reduced in five brain regions and remained unchanged in four. Even after 8 wk treatment with 300 microg T4/kg, the T3 concentrations remained normal in cortical areas, the hippocampus and amygdala, and were elevated only in areas in which inner-ring deiodinase activity was low or absent, and in the hypothalamus. At the subcellular level, nuclear concentrations of T3 were diminished in hypothyroidism but remained unaltered in hyperthyroidism in all areas except the hypothalamus, where they were enhanced. Cortical mitochondrial succinate dehydrogenase activity was reduced in both hypo- and hyperthyroidism in spite of normal T3 concentrations in hyperthyroid animals. The results show that nuclear T3 concentrations fall in hypothyroidism but do not change during severe hyperthyroidism in any brain region except the hypothalamus. Further research is thus needed to clarify the mechanisms mediating the numerous biochemical and psychological effects of hyperthyroidism.     

Effects of diet and phenotype on adipose cellularity and 5'-deiodinase activity of liver and brown adipose tissue of diabetic SHR/N-cp rats.

1. Groups of lean and obese male SHR/N-cp rats were fed isoenergetic diets containing 54% carbohydrate as cornstarch (CS) or sucrose (SU) plus other nutrients from 5 weeks of age, and measures of adiposity, thyroxine 5' deiodinase (T4-5'DI) activity, and tissue and plasma triiodothyronine (T3) content determined at 9.5 months of age. 2. Body weights (BW) of obese greater than lean, and were greater when fed the SU than CS diet in both phenotypes. Phenotype effects (obese greater than lean) were present for fat pad weights and adipose cellularity in most primary adipose tissue depots, and diet effects (SU greater than CS) were present for epididymal and retroperitoneal depots in both phenotypes. 3. Interscapular brown adipose tissue (IBAT) and IBAT:BW ratios of obese greater than lean, and diet effects (SU greater than CS) were present for lean but not obese rats. Liver T4-5'DI activity and plasma and tissue T3 of lean greater than obese, while IBAT 5'DI activity of obese greater than lean in the CS diet. 4. These results indicate that obesity occurs in the SHR/N-cp rat as the result of hypertrophy and hyperplasia of adipose tissue, and that isoenergetic substitution of simple for complex carbohydrate exaggerates fat accretion in lean but not obese rats. Moreover, the obesity occurs in spite of greater mass, cellularity, and T4-5'DI activity of IBAT, consistent with a thermogenic defect in the obese phenotype of this strain.     

PPAR-gamma,TNF-alpha messenger RNA levels and lipase activity in the pregnant and lactating rat

Dramatic alternations in maternal metabolism occur during gestation and lactation, especially glucose and fat metabolism. For example, in rats, the amount of body fat mass increases during gestation, then decreases just prior to delivery, and remains low after parturition. To investigate the factors involved in such changes in maternal fat mass, messenger ribonucleic acid (mRNA) levels of adipocytokines, peroxisome proliferator-activated receptor-gamma (PPAR-gamma) and tumor necrosis factor-alpha (TNF-alpha), were examined in the intraabdominal adipose tissue of non-pregnant rats, pregnant rats and postpartum rats. We also examined the issue of whether apoptosis, which could be promoted by PPAR-gamma and TNF-alpha, is involved in any of the changes in maternal fat mass The activity of lipoprotein lipase (LPL) and hormone sensitive lipase (HSL) in adipose tissue was also measured. PPAR-gamma and TNF-alpha mRNA levels remained constant during the gestational and postpartum periods. Apoptosis was not detected at any time as evidenced by DNA laddering and in situ staining. LPL activity was significantly increased at day 5 and remained elevated until day 14 of gestation. HSL activity was significantly increased at day 10 of gestation and then decreased after delivery, at day 10 of lactation. In conclusion, during the gestational and postpartum period of rats, changes in maternal fat mass did not directly correlate with the levels of expression of PPAR-gamma and TNF-alpha mRNA. Apoptosis also does not appear to influence on fat mass change. The changes in LPL and HSL activities during gestation suggest that these enzymes might be regulators of maternal adipose tissue level.