Kinetic parameters for plasma beta-endorphin in lean and obese Zucker rats

To determine plasma clearance kinetics for beta-endorphin (BE) by empirical compartmental analysis, a bolus of radioactive labeled 125I-BE was rapidly injected into a carotid artery catheter of unanesthetized lean (L) and obese (O) Zucker rats. The plasma disappearance of 125I was followed over a 3-h period. A 3-component exponential equation provided the best fit for plasma data. Plasma transit times were very short (10 s); however, plasma fractional catabolic rate was much slower. Plasma mean residence time was similar for both groups (50 min) as was recycle time (1.3 min). These data suggest that BE plasma disappearance kinetics are similar in L and O rats.     

Serum hormone levels and tissue metabolism in pair-fed lean and obese Zucker rats.

Obese Zucker rats were either pair-fed to their lean litter-mates or fed ad lib, to determine the effect of hyperphagia on serum hormone levels and tissue metabolism as indicated by enzyme activities and in vitro metabolite flux. Hyperphagia was shown to be non-essential for the elevation in serum insulin and suppression in serum growth hormone and prolactin in the genetically obese rat. It was also shown that the increased liver cell lipogenic rate was not dependent on hyperphagia in the obese rat and that adipose cell lipogenesis was not significantly altered in the pair-fed obese rat. The utilization of alanine for glucose synthesis in vitro was similar for both lean and obese rats, but its utilization for fatty acid synthesis was higher in the obese rat. Data is presented which suggest that the inhibitory effect of glucagon on liver lipogenesis is blunted in the obese rat.     

CCK-resistance in Zucker obese versus lean rats

Obese Zucker rats are less sensitive to the satiety effect of CCK than lean litter mates. The present studies further characterised this CCK resistance. Subcutaneous injection of the CCK agonist caerulein dose-dependently decreased food intake in Zucker obese and lean rats whereas the CCK-B agonist gastrin-17 did not. Caerulein at 4 μg/kg, which resulted in CCK plasma bioactivity slightly above postprandial levels, decreased food intake in lean rats but not in obese rats. The decrease in food intake was also more marked at higher caerulein doses (20–100 μg/kg) in lean versus obese rats. In lean animals the satiety effects of the “near physiological” 4 μg/kg caerulein dose was abolished after blockade of vagal afferents with capsaicin, whereas the effects of higher caerulein doses were not. CCK-stimulated amylase secretion from pancreatic acini and binding capacity of ¹²⁵I- labelled CCK-8 were decreased in obese versus lean rats. The CCK-A antagonist loxiglumide at 20 mg/kg, a dose which abolished the action of all caerulein doses on food intake, failed to alter the food intake either in obese or in lean rats when given without an agonist. The results suggest that the satiety effects of “near physiological” doses of caerulein in lean rats are mediated by vagal afferents whereas pharmacological doses act via non-vagal mechanisms. The differences in CCK's satiety effect between lean and obese rats may be due to differences in CCK-receptor binding and action at peripheral vagal sites. However, the failure of the CCK-A antagonist to increase food intake questions whether any of the effects of exogenous CCK are of physiological relevance.     

Decreased noradrenergic and serotonergic reactivity of vas deferens of newborn rats from mothers treated with the serotonin reuptake inhibitor fluoxetine during pregnancy and breast-feeding

Female Wistar rats were treated with the serotonin reuptake inhibitor fluoxetine (10 mg/kg/i.p/day), during pregnancy and breast-feeding, for the study of the corresponding newborn rats. At the end of the preweaning period, the 30-day old litters had their vas deferens removed for testing peripheral sympathetic reactivity, through the following experiments in vitro: (a) concentration-contraction curves for serotonin and for the adrenergic agonists noradrenaline, phenylephrine, clonidine and dopamine or for the indirect agonist tyramine (b) contractions induced by electric field stimulation, as an indicator of sympathetic neurotransmission (c) release of endogenous noradrenaline, measured by real-time determinations on HPLC (d) Ca(+2) time-contraction curves, to check for changes on Ca(+2) translocation. Our results showed that the affinity (pD(2)) for serotonin was strikingly decreased by about 1.5 log units. The pD(2) for adrenergic agonists was decreased by about 0.5 log units, except for dopamine and clonidine. The maximum effects and intrinsic activity were decreased only for dopamine. On the other hand, the response to Ca(+2) and the release of noradrenaline from nerve terminals were not modified. In additional experiments, the mother's body weights were measured, showing a decrease during gestation and a recovery during lactation while the offspring's weights were lower than controls. It is concluded that, besides the alterations on body weights, changes on noradrenergic and serotonergic mechanisms were observed and persisted in the newborn, at least one month after parturition.     

Effects of intracerebroventricular leptin administration on feeding and sexual behaviors in lean and obese female Zucker rats

Obese Zucker rats (fa/fa) are characterized by inadequate leptin signaling caused by a mutation in the leptin receptor gene. Obese Zucker females are infertile and hyporesponsive to the inductive effects of ovarian hormones on sexual behaviors. Leptin treatment reverses aspects of reproductive dysfunction due to perturbations in energy balance in other animal models. Our first experiment tested the hypothesis that intracerebroventricular (icv) leptin administration would enhance the display of sexual behaviors in obese Zucker females. A second experiment compared lean and obese Zucker females' responses to leptin, during fed and fasted conditions. Ovariectomized (OVX) Zucker rats were implanted with lateral ventricular cannulae. In Experiment 1, fasted, obese females received estradiol benzoate, progesterone, and icv injections of 3, 18, or 36 microg murine leptin or vehicle. Leptin administration reduced food intake, but did not enhance sexual behaviors. In Experiment 2, steroid-replaced, OVX lean and obese females (from a different source than those in Experiment 1) received icv injections of vehicle or 3 or 36 microg leptin under fed and fasted conditions. Leptin treatment reduced food intake and weight gain in the fed, but not the fasted, condition in both genotypes. Sexual receptivity and locomotion were not affected, but icv leptin injections reduced proceptive behaviors in ad libitum-fed rats. These data confirm previous reports that centrally administered leptin decreases food intake and weight gain in obese Zucker rats; results from Experiment 2 suggest that lean and obese females are similarly responsive to these actions of leptin. Contrary to our hypothesis, leptin treatment did not stimulate sexual behaviors; rather, the hormone appears to inhibit the display of sexual proceptivity in ad libitum-fed lean and obese Zucker female rats.     

Hormonal studies of young lean and obese Zucker rats

Plasma concentrations of insulin, corticosterone, T3, T4 and glucose were measured at 6 hour intervals throughout 24 hours in undisturbed, 34-day-old lean (Fa/?) and genetically obese (fa/fa) Zucker rats. fa/fa rats had higher plasma concentrations of insulin at all sampling times and higher plasma concentrations of corticosterone at 0300 and 0900 hours. Neither T3 nor T4 levels differed between phenotypes at any sampling time. Fasting for 24 hours at 34 days abolished the hyperinsulinaemia of fa/fa rats and raised the plasma corticosterone concentrations of both phenotypes. Before weaning there were no phenotypic differences in the plasma insulin or corticosterone concentrations measured at two sampling times in undisturbed rats. Following an intra-gastric glucose load however, fa/fa rats became hyper-insulinaemic compared with similarly treated Fa/? animals. Pancreatic insulin contents were higher in fa/fa rats at 34 days of age, but not before weaning. Somatostatin contents of the pancreas, hypothalamus and cerebral cortex did not differ between phenotypes at either 18 or 34 days of age. In conclusion, the elevated plasma concentrations of insulin and corticosterone in young fa/fa rats may contribute to their greater lipid deposition and lower protein deposition.     

In Zucker diabetic fatty rats plasma leptin levels are correlated with plasma insulin levels rather than with body weight.

The obese (ob) gene product leptin, secreted from adipose tissue, acts in the hypothalamus to regulate body energy stores. In vitro experiments showed that insulin increases both leptin mRNA expression and leptin secretion by adipocytes. Here, we report on the relationship between plasma insulin and plasma leptin in a longitudinal in vivo study. In Zucker diabetic fatty (ZDF) rats, an animal model for non-insulin-dependent diabetes mellitus (NIDDM), and in ZDF control rats, blood glucose, body weight, plasma insulin and plasma leptin levels were measured from 10 to 25 weeks of age. In ZDF control rats, body weight, plasma leptin and plasma insulin levels increased gradually during the study period. In ZDF rats, the time course of plasma leptin was similar to that of plasma insulin, but did not parallel that of body weight. Calculation of partial correlation coefficients revealed that in ZDF control rats plasma leptin correlated with body weight rather than with plasma insulin. However, in ZDF rats, plasma leptin correlated with plasma insulin rather than with body weight, suggesting an important role for insulin in the modulation of leptin secretion in this animal model for NIDDM.     

Higher levels of aggression are observed in socially dominant toadfish treated with the selective serotonin reuptake inhibitor, fluoxetine

The following study set out to test the hypothesis that acute treatment with the selective serotonin reuptake inhibitor, fluoxetine, would result in a rise in circulating 5-HT levels and consequently a decrease in territorial aggression in the Gulf toadfish, Opsanus beta. Size-matched pairs of toadfish were implanted intraperitoneally with the same dose of fluoxetine (0, 10 or 25 μg g^− 1). After a social interaction between a pair of fish, circulating levels of serotonin (5-HT; 5-hydroxytryptamine) and cortisol were measured and relative mRNA expression of the 5-HT_1A receptor in the toadfish brain was determined using quantitative (real-time) PCR (qPCR). Behavioral endpoints such as the number of aggressive acts and swimming activity were also quantified so that dominant and subordinate fish could be identified. Fluoxetine treatment resulted in an increase in circulating levels of 5-HT, regardless of social status. Circulating cortisol concentrations were unaffected by fluoxetine, but were significantly higher in subordinate individuals when compared to dominant fish. Toadfish brain 5-HT_1A receptor mRNA expression was not affected by treatment or social status. Lastly and contrary to our predictions, fluoxetine treatment resulted in an increase in the number of aggressive acts made by dominant individuals, with no differences in the level of aggression or swimming activity of subordinate fish. This study is the first to describe elevated aggression in a teleost fish with elevated circulating levels of 5-HT.     

Tissue and serum somatostatin-like immunoreactivity in lean and obese Zucker rats

Tissue and serum somatostatin levels were measured in genetically lean and obese Zucker rats. Immunoreactive somatostatin content was decreased in three central nervous system regions (hypothalamus, septum and preoptic area and thalamus) of obese rats but was increased in cerebral cortex. No differences were observed in antral or colonic somatostatin content but obese animals had significantly elevated pancreatic levels. Portal vein somatostatin-like immunoreactivity in contrast was significantly lower in obese rats. The widespread alterations in tissue and serum somatostatin-like immunoreactivity suggest either a diffuse abnormality of somatostatin physiology or a response to a generalised feature of the obese hyperinsulinaemic state.     

Insulin binding in the hypothalamus of lean and genetically obese Zucker rats

Recent reports have suggested that the obesity and hyperphagia of the genetically obese Zucker rat may be related to defective insulin action or binding in the hypothalamus. We used quantitative autoradiography to determine if insulin binding is altered in specific hypothalamic nuclei associated with food intake. Insulin binding was measured in the arcuate (ARC), dorsomedial (DMN), and ventromedial (VMN) hypothalamic nuclei of 3–4-month-old lean (Fa/Fa) and genetically obese (fa/fa) Zucker rats. A consistently reproducible 15% increase in the total specific binding of 0.1 nM [¹²⁵I]-insulin was found in the ARC of the obese genotype. A slight increase in insulin binding in the DMN was also found. No difference in specific insulin binding was found between genotypes in the VMN. Nonlinear least squares analysis of competitive binding studies showed that the K_d of the ARC insulin binding site was 33% higher in the lean rats than in the obese rats, indicating an increased affinity for insulin. No difference in site number (B_max) was found in the ARC, DMN or VMN, and no evidence was found for reduced insulin binding in the hypothalamus of the obese (fa/fa) genotype. The results suggest that hyperphagia and obesity of the obese (fa/fa) Zucker rat genotype may be associated with increased insulin binding in the arcuate nucleus.     

Lipoprotein lipase and cholesterol transfer activities of lean and obese Zucker rats.

Adult female lean and obese Zucker rats maintained under standard conditions were used for the estimation of plasma, liver and white adipose tissue (WAT) activity of lipoprotein lipase, plasma and liver hepatic lipase and plasma lecithin-cholesterol acyltransferase. No differences in plasma or tissue levels of lipoprotein lipase between lean and obese rats were detected, but the larger WAT size of the obese rats resulted in higher lipase activity per unit of rat weight. Hepatic lipase levels in plasma were higher in the obese, but in liver, the higher activity was found in lean rats. No significant differences were found for lecithin-cholesterol acyltransferase activity, except when the levels in the HDL fraction were expressed per unit of protein weight, showing lower activity in the obese rats. In conclusion, the essentially maintained enzyme activities in obese rat tissues suggest that they cannot explain the deficient lipoproteins processing of obese rats, and, consequently their dislipidaemia.     

Behavioral thermoregulation in obese and lean Zucker rats in a thermal gradient

Genetically obese Zucker (Z) rats have been reported to display a body core temperature (Tb) that is consistently below that of their lean littermates. We asked the question whether the lower Tb was a result of deficits in thermoregulation or a downward resetting of the set point for Tb. For a period of 45 consecutive hours, lean and obese Z rats were free to move within a thermal gradient with an ambient temperature (T(a)) range of 15-35 degrees C, while subjected to a 12:12-h light-dark cycle. Tb was measured using a miniature radio transmitter implanted within the peritoneal cavity. Oxygen consumption (VO2) was measured using an open flow technique. Movements and most frequently occupied position in the gradient (preferred T(a)) were recorded using a series of infrared phototransmitters. Obese Z rats were compared with lean Z rats matched for either age (A) or body mass (M). Our results show that obese Z rats have a lower Tb [37.1 +/- 0.1 degrees C (SD) vs. 37.3 +/- 0.1 degrees C, P < 0.001] and a lower VO2 (25.3 +/- 1.9 ml x kg(-1) x h(-1)) than lean controls [33.1 +/- 3.7 (A) and 33.9 +/- 3.9 (M) ml x kg(-1) x h(-1), P < 0.001]. Also, the obese Z rats consistently chose to occupy a cooler T(a) [20.9 +/- 0.6 degrees C vs. 22.7 +/- 0.6 degrees C (A) and 22.5 +/- 0.7 degrees C (M), P < 0.001] in the thermal gradient. This suggests a lower set point for Tb in the obese Z rat, as they refused the option to select a warmer T(a) that might allow them to counteract any thermoregulatory deficiency that could lead to a low Tb. Although all rats followed a definite circadian rhythm for both Tb and VO2, there was no discernible circadian pattern for preferred T(a) in either obese or lean rats. Obese Z rats tended to show a far less definite light-dark activity cycle compared with lean rats.     

Plasma amino acids of lean and obese Zucker rats subjected to a cafeteria diet after weaning.

Plasma amino acids of Zucker obese (fa/fa) and lean (Fa/?) rats fed either a reference nonpurified pellet or a cafeteria diet have been studied from 30 to 60 days after birth. Obese rats showed higher plasma branched chain amino acid levels but similar total amino acids, urea and glucose concentrations. The ingestion of a cafeteria diet induced higher levels in many amino acids, as well as in the composite figure in lean rats, but failed to alter total 2-amino nitrogen concentrations in obese rats, despite high levels in several non-essential amino acids and lower values in essential amino acids; urea levels were much lower in rats fed the cafeteria diet. The results are consistent with an impairment of amino acid nitrogen elimination via urea cycle in cafeteria diet-fed rats. This is independent of the hyperinsulinemia-driven plasma accumulation of several essential amino acids induced by genetic obesity. The effects were, then additive.     

Hepatic conversion of thyroxine to triiodothyronine in obese and lean Zucker rats

The in vitro conversion of thyroxine (T4) to triiodothyronine (T3) was studied in liver homogenates from fed and fasted lean and obese Zucker rats. T3 generation was decreased in fed young (2 month) obese rats as compared to values in fed lean controls. This was not corrected by the addition of dithiothreitol (DTT), suggesting a deficiency in 5'-deiodinase activity in young obese rats. Both lean and obese 2 month old rats responded to a 2 day fast by decreasing hepatic T3 generation as is always observed in other strains of rats. The hepatic conversion rate was not decreased in older (5 month) fed obese rats when compared to age-matched lean controls. Hepatic conversion of T4 to T3 was markedly decreased in 5 month old lean Zucker rats fasted for 4 days. In contrast, a 4 day fast had no effect on the hepatic conversion rate in the 5 month old obese rats. The hepatic conversion rate was assessed in 5 month old obese rats fasted for up to 28 days and hepatic conversion still did not decrease. This paradoxical response of the 5 month old obese rat may provide a new model to further evaluate the control of hepatic T3 generation from T4.     

Effects of CCK on gastrointestinal function in lean and obese Zucker rats

Cholecystokinin (CCK), a hormone affecting several gastrointestinal functions, has also been shown to elicit satiety and affect daily meal patterns. Since Zucker obese rats are less sensitive to the satiety effects of CCK, two experiments were designed to determine if they are also less sensitive to the gastric emptying and intestinal transit rate effects of CCK. In the first experiment phenol red was administered to 5.5 hr fasted rats 15 minutes after intraperitoneal injection of CCK-8 or saline. Rats were sacrificed after 30 minutes, the stomach and small intestine were removed, and phenol red content was measured. More phenol red was in the stomach of obese but not lean rats treated with CCK-8. The rate of transit of the contents of the small intestine was increased by CCK-8 and the percent of phenol red in the fourth quarter of the small intestine was greater in obese than lean rats (91 vs 37%, p<0.05). In the second experiment gastrointestinal transit of ferric oxide was measured during the light and dark phases of the diurnal cycle, and when obese rats were ad lib or yoke-fed to lean pair-mates. Total gastrointestinal transit time of the ferric oxide was decreased 15% when CCK-8 was administered to yoke-fed obese rats in either the light or dark portions of the diurnal cycle but was not affected in ad lib-fed obese rats or lean rats. Thus, while Zucker obese rats are less sensitive to satiety effects of CCK, they appear to be more sensitive to the gastrointestinal effects of CCK, and therefore it is not clear what role these gastrointestinal responses have on the feeding behavior responses.     

Mono- and diacylglycerol lipases in spinal cord of lean and obese Zucker rats

1. Genetically obese Zucker rats (fa/fa) contain 2-3 times higher activities mono- and diacylglycerol lipases in their spinal cords than their lean littermates. 2. When rats were exercised (1 hr daily, 5 days/week) on a treadmill for 6 months, there was a decrease of about 30% (P less than 0.05) in the activities of mono- and diacylglycerol lipases in lean rats but not in obese animals. 3. High activities of lipases in Zucker obese rats may be related to the elevated levels of beta-endorphin present in these animals. 4. The activities of arylsulfatase, beta-N-acetylhexosaminidase and alkaline phosphatase, tested to check the stability of spinal cord extracts, were similar in lean and obese rat spinal cords.     

Effects of 2 G on adiposity, leptin, lipoprotein lipase, and uncoupling protein-1 in lean and obese Zucker rats.

Male Zucker rats were exposed to 2 G for 8 wk to test the hypothesis that the leptin regulatory pathway contributes to recovery from effects of 2 G on feeding, growth, and nutrient partitioning. After initial hypophagia, body mass-independent food intake of the lean rats exposed to 2 G surpassed that of the lean rats maintained at 1 G, but food intake of the obese rats exposed to 2 G remained low. After 8 wk at 2 G, body mass and carcass fat were less in both genotypes. Leptin and percent fat were lower in lean rats exposed to 2 G vs. 1 G but did not differ in obese rats exposed to 2 G vs. 1 G. Although exposure to 2 G did not alter uncoupling protein-1 levels, it did elicit white fat pad-specific changes in lipoprotein lipase activity in obese but not lean rats. We conclude that 2 G affects both genotypes but that the lean Zucker rats recover their food intake and growth rate and retain "normal" lipoprotein lipase activity to a greater degree than do the obese rats, emphasizing the importance of a functional leptin regulatory pathway in this acclimation.     

Ammonium uptake and urea production in hepatocytes from lean and obese Zucker rats

The metabolic differences in vitro between genetic and dietary obese rats in the uptake of ammonium and amino acids by the liver and their use for ureogenesis have been assayed using hepatocytes isolated from Lean, Obese Zucker (Genetic obese) rats and Dietary obese rats. The hepatocytes of genetic obese animals took up more ammonium and produced higher amounts of urea from ammonium and alanine than those of lean and dietary obese groups (2 and 5 times more respectively). In the lean and dietary obese groups urea synthesis accounted for almost all the nitrogen taken up as ammonium. Thus, dietary and genetic obesity show a widely different handling of nitrogen, and the genetic obese rats need to break down protein to maintain their hepatocyte function.     

Feeding response to ghrelin agonist and antagonist in lean and obese Zucker rats

Ghrelin is a new orexigenic and adipogenic peptide primarily produced by the stomach and the hypothalamus. In the present experiment, we determined the circulating ghrelin levels in 60-week old fa/fa Zucker rats with a well-established obesity (n = 12) and in their lean (FA/FA) counterparts (n = 12). We also tested the feeding response of both groups to intra-peritoneal (I.P.) injection of ghrelin agonist and antagonist. Obese rats ate significantly more than the lean rats (21.7 +/- 1.1 vs. 18.3 +/- 0.3 g/day; p < 0.01). Their plasma ghrelin concentration was 35% higher than that in the lean homozygous rats (p < 0.025). GHRP-6 (1 mg/kg I.P, a GHS-R agonist) stimulated food intake in lean but not in obese rats (p < 0.01), whereas [D-Lys)]-GHRP-6 (12 mg/kg I.P., a GHS-R antagonist) decreased food intake in both groups (p < 0.0001). These results indicate that the obese Zucker rat is characterized by an increase in plasma ghrelin concentrations and by an attenuated response to a GHS-R agonist. They support a role for ghrelin in the development of obesity in the absence of leptin signaling.