Intraventricular neuropeptide Y injections stimulate food intake in lean, but not obese Zucker rats.

We examined the effect of acute third intraventricular (IVT) injections of either saline or NPY (0.95, 3.0, 9.5, or 30.0 micrograms in 1 microliter) on the 1-, 4-, and 22-hour postinjection food and water intake of female obese (fa/fa), heterozygous lean (Fa/fa), and homozygous lean (Fa/Fa) Zucker rats. None of the doses of NPY had an effect on either food or water intake of fa/fa rats. A significant increase of food intake was seen in Fa/Fa rats at 1 and 4 hours after the 3.0 micrograms injection of NPY and at 1, 4, and 22 hours after the 9.5 micrograms injection of NPY. Both 3.0 and 9.5 micrograms of NPY also stimulated 1- and 4-hour postinjection food intake of Fa/fa rats, although this effect was significant only at 4 hours after the 3.0 micrograms dose. NPY had a less reliable effect on water intake; 3.0 micrograms of NPY stimulated 1-hour postinjection water intake of Fa/fa rats and 4-hour postinjection water intake of Fa/Fa rats. These results indicate that lean, but not obese Zucker rats, respond by eating more to centrally administered NPY. This deficit is similar to the effects seen with IVT insulin injections and may be a result of a common receptor-mediated mechanism.     

Daily rhythms of feeding in the genetically obese and lean Zucker rats

Feeding patterns were examined in obese (fa/fa) and lean (Fa/-) adult Zucker rats over the light-dark cycle during 14 days. Obese rats eat more than lean rats especially during the dark phase. Light and dark feeding expressed as percentage of 24 hr intake showed no significant differences between the lean and obese groups. The higher food intake in obese rats is mainly caused by larger meals since obese rats ate fewer meals than lean rats. Only for the obese group differences were observed between mean meal size in light and dark phase. There is some indication that the circadian controlled temporal distribution of meals is different in obese rats compared to lean rats since obese rats eat fewer but larger meals during the first half of the dark phase. During this phase meal size increases gradually in the obese rats, suggesting that the circadian influence on feeding motivation is increased.     

The effect of leptin receptor deficiency and fasting on cannabinoid receptor 1 mRNA expression in the rat hypothalamus, brainstem and nodose ganglion

Despite ample evidence for the involvement of the endocannabinoid system in the control of appetite, food intake and energy balance, relatively little is known about the regulation of cannabinoid receptor 1 (CB₁R) expression in respect to leptin signalling and fasting. In the present study, we examined CB₁R mRNA levels in lean (Fa/?) and obese (fa/fa) male Zucker rats under basal and food-restricted conditions. Using stereological sampling principles coupled with semi-quantitative radioactive in situ hybridization we provide semi-quantitative estimates of CB₁R mRNA expression in key appetite regulatory hypothalamic and brainstem areas, as well as in the nodose ganglia. Whereas no effect of fasting were determined on CB₁R mRNA levels in the paraventricular (PVN) and ventromedial hypothalamic (VMH) nucleus, in the brainstem dorsal vagal complex or nodose ganglion of lean Zucker rats, CB₁R mRNA levels were consistently elevated in obese Zucker rats pointing to a direct influence of disrupted leptin signalling on CB₁R mRNA regulation.     

Acute and chronic administration of immunomodulators induces anorexia in Zucker rats

To investigate the possible involvement of leptin signaling in lipopolysaccharide (LPS) anorexia, we compared the anorectic effect of LPS in genetically obese (fa/fa) Zucker rats and in their lean (Fa/?) counterparts. The effects of interleukin-1beta (IL-1beta) and muramyl dipeptide (MDP) were also tested. LPS [100 microg/kg body weight (BW)], IL-1beta (2 microg/kg BW) and MDP (2.2 mg/kg BW) injected intraperitoneally (i.p.) at lights out reduced food intake similarly in obese and lean rats. LPS injection at 500 or 1000 microg/kg BW (i.p.) also reduced food intake and BW similarly in obese and lean rats, but obese regained BW faster than lean rats. LPS (2.45 microg or 9.8 microg/h/rat) administered chronically with i.p. implanted osmotic pumps reduced food intake similarly on experimental day 1, regardless of the genotype. After day 3, the lean rats' anorectic response and recovery were dose-dependent, whereas the anorectic response in obese rats was minimally affected by dose (significant dose effect only on day 3). Again, obese rats regained lost BW faster than lean rats. These results do not support a role for leptin as the sole mediator of anorexia induced by bacterial products (LPS and MDP) and IL-1beta.     

Differential expression of nicotinamide adenine dinucleotide phosphate-diaphorase in hypothalamic areas of obese Zucker rats

Several studies have suggested that the activity of nitric oxide synthase (NOS) may be involved in the regulation of food intake in the genetically obese Zucker rats. In the present study, we investigated the expression of NOS in various hypothalamic regions of obese and lean Zucker rats using nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry. Obese Zucker rats showed significantly lower staining intensities of NADPH-diaphorase-positive neurons in the paraventricular nucleus (PVN), lateral hypothalamic area (LHA) and ventromedial hypothalamic nucleus (VMH) than lean Zucker rats did. The differences in staining intensities between obese and lean Zucker rats were large in both the PVN and LHA, but such differences were relatively small in the VMH.     

Chronic infusion of the amylin antagonist AC 187 increases feeding in Zucker fa/fa rats but not in lean controls

Numerous studies have established the pancreatic B-cell hormone amylin as an important anorectic peptide affecting meal-ending satiety. In the present study, we investigated the effect of a chronic infusion of the amylin antagonist AC 187 on food intake. The studies were performed using obese Zucker fa/fa rats, which are hyperamylinemic but have a defective leptin and insulin signaling system. A chronic intraperitoneal infusion of the amylin antagonist AC 187 (10 microg/kg/h) significantly increased dark phase and total food intake in Zucker but not in lean control rats. During the 8-day infusion experiment, AC 187 had no clear effect on body weight gain in either group. After acute administration, amylin and its agonist salmon calcitonin (sCT) equally reduced food intake in Zucker and lean control rats while cholecystokinin's (CCK) anorectic effect was weaker in the Zucker rats. We provide evidence for amylin being a potential long-term regulator of food intake because AC 187 increased food intake in obese fa/fa rats but not in lean control animals, which have low baseline amylin levels. Amylin may play some role as lipostatic feedback signal similar to leptin and insulin at least when the leptin and insulin feedback signaling systems are deficient. Despite basal hyperamylinemia in the Zucker rats, they do not seem to be less sensitive to the anorectic effects of amylin or its agonist sCT than respective controls. This contrasts with CCK whose anorectic action is reduced in Zucker rats when compared with lean controls.     

Insulin stimulates inositol incorporation in hippocampus of lean but not obese Zucker rats

We have previously reported that intraventricular insulin is ineffective in decreasing the body weight of obese (fa/fa) Zucker rats. In the present study, we report that insulin at concentrations of 1 and 5 nM significantly stimulates incorporation of 3H-myoinositol into inositol phosphates (29 +/- 5% and 20 +/- 6% stimulation over control levels) and membrane inositol lipids (29 +/- 4% and 20 +/- 6% stimulation over control levels) within hippocampal slices from lean (Fa/Fa) Zucker rats (n = 3 preparations). Smaller but significant stimulations were observed in the Fa/fa Zucker rat (n = 5) hippocampus [10 +/- 3 and 13 +/- 4% stimulation over control levels (inositol phosphates) and 10 +/- 4 and 14 +/- 3% stimulation over control levels (inositol lipids) with 1 and 5 nM insulin]. Insulin had no effect on 3H-inositol incorporation into hippocampal slices from obese (fa/fa) Zucker rats (n = 5). No difference in insulin binding to hippocampal membranes was observed among the three genotypes. These findings confirm the behavioral observation of insulin insensitivity within the fa/fa Zucker CNS and suggest that this insensitivity may be gene-dose-related.     

Food motivated behavior in genetically obese and hypothalamic-hyperphagic rats and mice

The food-associated behavior of genetically obese Zucker rats, fa/fa and yellow obese mice, aAy, was studied using classic operant procedures. When obese Zucker rats and yellow mice are compared to their lean littermates and lean littermates made obese by electrolytic lesions and chemical lesions, respectively, the naturally obese animals do not display the behavioral patterns associated with rodents made obese by hypothalamic damage. These experiments point out the necessity for careful selection of animal models in studying behaviors associated with regulatory disturbances of normal food intake.     

Feeding response of weanling zucker obese rats to cholecystokinin and bombesin

Zucker weanling obese rat meal size is greater than in lean litter-mates by 4 weeks of age, indicating a possible decreased sensitivity to satiety signals. Adult Zucker obese rats are less sensitive to the putative satiety signal octapeptide of cholecystokinin (OP-CCK) when injected after a normal intermeal interval. In these experiments were compared responses of Zucker lean and obese rats from 3–11 weeks of age to OP-CCK and bombesin (BBS), another recently reported putative satiety agent. Injection of 2.0 and 4.0 μg/kg OP-CCK in 4–5 week olds had no effect on food intake of obese rats while decreasing 60-min food intake in lean rats 29 and 28 percent, respectively. However, 8.0 μg/kg OP-CCK decreased food intake of obese and lean rats similarly, indicating decreased, rather than lack of, sensitivity in the obese. The doses of 2.0 and 4.0 μg/kg BBS decreased food intake similarly in the obese and lean rats, but 1.0 μg/kg, although having no effect in lean rats, increased food intake in obese rats approximately 17 percent. Thus, while Zucker obese weanling rats appear to be less sensitive to OP-CCK, shown to decrease food intake in lean rats, they appear to be equally sensitive to the satiety effect of similar doses of BBS, but at low doses BBS stimulated food intake in obese but not lean rats.     

Sympathoadrenal function in genetically obese Zucker rats.

The effects of genetic obesity on the actions and alterations of the sympathetic nervous system were studied in 10-12-month-old obese (fa/fa) and lean (Fa/-) Zucker rats. Blood glucose, plasma insulin, epinephrine (E), norepinephrine (NE), and free fatty acids (FFA) concentrations were measured in blood samples taken through a permanent heart catheter before, during, and after exercise or intravenous infusion of E and NE. Baseline plasma FFA and insulin levels were markedly increased in the obese animals. Exercise, i.e., strenuous swimming against a counter current for 15 min, led to reduction of plasma insulin concentrations and an increase of all other blood components in lean Zucker rats. In obese animals, an exaggerated increase of blood glucose and a large suppression of plasma insulin occurred. Plasma FFA levels tended to decline during exercise. Plasma catecholamine patterns in the exercising fatty Zuckers were not different to those of the lean animals. Infusion of E caused an increase of blood glucose and a decrease of plasma insulin concentrations in both groups of animals. The increase in blood glucose in the obese animals was significantly larger compared to the changes in the lean animals. Infusion of NE significantly reduced plasma insulin concentration in obese but not in lean animals. The results revealed that activation of the sympathetic system, expressed as exercise-induced alterations in plasma E and NE levels, is normal in obese Zucker rats. However, postsynaptic receptor effects of catecholamines on glycogenolysis and lipolysis are different in obese and lean animals, which points to permanent changes in adrenoceptor mechanisms on adipocytes, hepatocytes, and muscle cells in obesity.     

Decreased hypothalamic concentration of neuropeptide Y correlates with onset of hyperphagia in fa/fa rats on postnatal day 12

An increased action of hypothalamic neuropeptide Y (NPY) has been proposed as a major factor in the pathophysiology of the obesity syndrome in Zucker (fa/fa) rats. Using a developmental strategy to test this hypothesis, we showed previously that significantly more arcuate NPY was expressed in fa/fa pups than in lean littermates on postnatal day (P) 2 and throughout the preweaning period [Physiol. Behav. 67 (1999) 521], and that hyperphagia first appeared on P12 [Am. J. Physiol. 275 (1998) R1106]. To test the hypothesis further, we used a specific radioimmunoassay to measure the concentration of hypothalamic NPY peptide in lean (+/+ and +/fa) and obese fa/fa Zucker rat pups on P9, P10, and P12. The concentration of NPY in fa/fa pups was not significantly different from that of the other genotypes. There was, however, a significant decrease in NPY concentration from P9 to P12 in fa/fa pups, but not in lean pups. The combination of increased NPY message and decreasing concentration of NPY peptide in fa/fa pups with age is consistent with, but does not prove, increased release of hypothalamic NPY in fa/fa pups just before and on P12 when hyperphagia emerges. These results provide further support for the importance of hypothalamic NPY in the phenotypic expression of hyperphagia in the fa/fa pups during the second postnatal week.     

Response of brown adipose tissue to electrical stimulation of hypothalamic centres in intact and adrenalectomized Zucker rats

Electrical stimulation of the ventromedial nucleus of the hypothalamus (VMN) increased brown adipose tissue (BAT) temperature in lean and obese Zucker rats, whereas stimulation of the paraventricular nucleus (PVN) was without effect in either genotype, before or after adrenalectomy. The prolonged duration of the increase in BAT temperature in the obese (fa/fa) rat observed after VMN stimulation was normalized after adrenalectomy.     

Serotonergic dorsal raphe neurons from obese zucker rats are hyperexcitable

Release of serotonin (5-HT) from dorsal raphe nucleus (DRN) neurons projecting to the ventromedial hypothalamus (VMH) has a modulatory effect on the neural pathway involved in feeding, hunger, and satiety. The obese Zucker rat, an animal model of genetic obesity, exhibits differences in serotonin signaling as well as a mutated leptin receptor. To evaluate possible mechanisms underlying this difference in serotonin signaling, we have compared electrophysiological responses of DRN neurons from 14- to 25-day-old male lean (Fa/Fa) and obese (fa/fa) Zucker rats using the whole-cell patch clamp technique on cells in brain slices from these animals. We found that the resting properties of these neurons are not different, but the DRN neurons from obese rats are hyperexcitable in response to current injection. This hyperexcitability is not accompanied by an increase in the depolarization caused by current injection or by changes in the threshold for spiking. However, the hyperexcitability is accompanied by reduction in the size and time course of the afterhyperpolarization (AHP) following an action potential. DRN neurons of obese rats recover from the AHP faster due to a smaller amplitude AHP and a faster time constant (tau) of decay of the AHP. These deficits are not due to changes in the spike waveform, as the spike amplitude and duration do not differ between lean and obese animals. In summary, we provide evidence that serotonergic DRN neurons from obese Zucker rats are intrinsically hyperexcitable compared with those from lean rats. These results suggest a potential mechanism for the reported increase in 5-HT release at the VMH of obese rats during feeding, and provide the first direct evidence of changes in the intrinsic activity of serotonergic neurons, which are crucial regulators of feeding behavior, in a genetic model of obesity.     

Impairment of Hormone-Stimulated cardiac adenylate cyclase activity in the genetically obese (fa/fa) Zucker rat

The age-related development of the capacity of the cardiac adenylate cyclase system to be stimulated with secretin, vasoactive intestinal peptide (VIP), glucagon, the β-adrenergic agonist isoproterenol, Gpp(NH)p, and NaF was compared in obese (fa/fa) Zucker rats and their lean (FA/?) littermates. The obese (fa/fa) Zucker rats tested developed postweaning obesity associated with marked hypertriglyceridemia, mild hyperglycemia, and hyperinsulinism. At 4 weeks, there was already a 57% reduction in secretin-VIP-stimulated adenylate cyclase activity in fa/fa rats. At 12 weeks, the secretin-VIP-stimulation was reduced by 77%, and glucagon-and isoproterenol-stimulations by 16–21%. At 45 weeks, secretin-VIP-stimulation was reduced by 91%, glucagon- and isoproterenol stimulations by 34–42%, and Gpp(NH)p- and NaF-stimulations by 16–23%. The reductions of isoproterenol-, Gpp(NH)p-, and NaF-stimulations were totally or partially reversed in 30-week old fa/fa animals submitted for 5 weeks to severe food restriction that almost normalized the altered blood parameters. In sharp contrast, food restriction imposed a further decrease in secretin-VIP- and glucagon-stimulated adenylate cyclase activities. This pattern of impaired secretin-VIP-stimulated adenylate cyclase activity appeared limited to cardiac membranes in obese animals as the responses of liver, brain and anterior pituitary adenylate cyclase activities to secretin and/or VIP were unaltered. These results suggest that secretin-VIP receptors coupled to adenylate cyclase were rapidly and specifically altered in the heart of fa/fa Zucker rats.     

Ontogeny of feeding behavior in the Zucker obese rat

Increased food intake in Zucker obese rats has been reported as early as 16 days of age. To determine if the feeding behavior of Zucker obese rats differs from that of lean rats by weaning, or if the differences develop with age, feeding patterns of Zucker obese and lean rats were compared from 3–10 weeks of age. Increased food intakes of obese rats at 3 weeks of age were due to a trend toward increased frequency while at 4 weeks of age were due to increased meal size. Meal size subsequently increased at a faster rate in obese than lean rats and meal frequencies did not differ. While always greater in obese than lean rats meal durations decreased and rates of eating increased with age and were not different for obese and lean rats. The adult patterns of diurnal variation in obese and lean rats were apparent by 3 weeks of age. Thus, in the Zucker obese rat the characteristic of increased meal size did not occur until 4 weeks of age after increased food intake and body weight were evident, and the characteristic of decreased meal frequency did not occur by 10 weeks of age. While increased meal size is associated with early differences in feeding behaviors, decreased meal frequency may be a consequence of obesity.     

Interrelationships among activity,food intake and weight gain in genetically obese and lean Zucker rats

In Experiment 1, food deprivation resulting in a 30% reduction in body weight produced significant increases in wheel running in both obese and lean female Zucker rats. In Experiment 2, a new technique, food contingent activity (FR, VI), dramatically increased wheel running in both obese and lean female Zucker rats. This increase in activity was achieved primarily during the dark period. Regardless of changes in activity levels, food intake and body weight gain remained similar to controls. When food was again available ad lib, activity levels rapidly decreased for obese but not lean rats. These results indicate that behavioral interventions alone are not sufficient to correct the obesity of the genetically obese rat.     

Weight gain and food intake in corticotropin releasing factor immunized Zucker rats

In Zucker obese rats (fa/fa) there are disturbances in the regulation of ACTH and corticosterone. In addition, beta-endorphin concentrations are higher in the pituitary and hypothalamus in obese than in lean rats. Since ACTH and beta-endorphin are thought to be controlled by corticotropin releasing factor (CRF), these effects may be due to abnormalities in CRF regulation. This possibility was investigated by immunizing rats against CRF. Obese rats immunized against CRF developed higher titer antibodies than lean rats. Hypothalamic CRF concentrations were higher in CRF-immunized obese but not lean rats compared with those of control rats, suggesting that compensation for sequestration of peripheral CRF developed in obese rats. In obese, but not lean rats, immunization against CRF decreased weight gains during weeks 1-4 and increased gains during weeks 9-12 and food intakes were decreased during weeks 5-8 compared with those for obese rats immunized against bovine serum albumin (BSA). Adrenal glands weighed 30% less in both obese and lean rats immunized against CRF compared with those immunized against BSA. These responses to immunization against CRF occurred even though plasma, hypothalamic and pituitary concentrations of ACTH and beta-endorphin were unaffected at the end of the study.     

Feeding and drinking behavior responses of adult Zucker obese rats to cholecystokinin

Increased meal size in obese animals may occur because of decreased sensitivity to satiety factors. Feeding and drinking behavior responses of Zucker obese and lean rats to two forms of cholecystokinin (CCK), a putative satiety factor, were compared in these experiments. Octapeptide of CCK (CCK-8) in both obese and lean rats decreased meal size (47 and 65%) and rate of eating but not meal duration, and increased satiety ratio but not postmeal interval. Impure CCK decreased first meal size after a 6-hr fast similarly in obese and lean rats (33 and 40%, respectively); however, meal duration was decreased only in lean rats, and rate of eating was decreased only in obese rats. Postmeal interval was decreased in lean rats, while satiety ratio was increased only in obese rats. In spite of decreased first meal size daily food intakes were increased by both impure CCK and CCK-8 in lean rats. In 6-hr water-deprived rats injected with CCK-8, decreased water intake was associated with decreased food intake. However, while impure CCK in lean rats elicited similar responses as CCK-8, impure CCK in obese rats decreased food but not water intake. Feeding behavior response to the putative satiety agent, cholecystokinin, depended on form of the peptide administered and phenotype.     

Repeated hypothalamic stimulation with neuropeptide Y increases daily carbohydrate and fat intake and body weight gain in female rats

Neuropeptide Y (NPY), repeatedly injected in the hypothalamic paraventricular nucleus (PVN), produces dramatic obesity and overeating in female rats maintained on a single nutritionally complete diet. In the present study, we investigated whether these effects could also be obtained in animals with a choice of three pure macronutrients: protein, carbohydrate, and fat. Female rats with indwelling PVN cannulas were injected with NPY (235 pmol) or its saline vehicle every 8 hr for 6 days. A third group was left undisturbed. Consumption of each macronutrient and body weight were measured every 24 hr for 6 days preinjection, 6 days during injections, and 21 days after the injections were terminated. Relative to vehicle or preinjection rates of body weight gain (approximately 1.5 g/day), NPY dramatically enhanced weight gain to a rate of 9.3 g/day and more than doubled total daily food intake. This augmentation was accounted for by increases in carbohydrate intake (+26.4 kcal/day) and fat intake (+48.5 kcal/day), with no significant potentiation of protein consumption. When the NPY injections were terminated, body weight and macronutrient intake returned to control levels within 1 or 2 weeks. These findings are consistent with a role for NPY in hypothalamic mechanisms of macronutrient intake and body weight regulation and suggest that disturbances in brain NPY may contribute to the development of eating and weight disorders.     

Hyperinsulinemia of preobese and obese fa/fa rats is partly vagus nerve mediated

In vivo glucose-induced insulin secretion was greater in preweaned preobese 17-day-old Zucker rats than in the corresponding controls. This hypersecretion of insulin was reversed to normal by acute pretreatment with atropine. A short-lived (30 s) electrical stimulation of the vagus nerve preceding a glucose load potentiated the in vivo glucose-induced insulin release in adult animals (6-9 wk) and more so in obese Zucker (fa/fa) than in lean rats. This suggested the existence of enhanced sensitivity and/or responsiveness of the B cells of obese animals to the parasympathetic system. That the parasympathetic tone was increased in adult obese Zucker (fa/fa) rats was corroborated by the observation that acute vagotomy of these animals resulted in a significant decrease in glucose-induced insulin secretion, whereas no such effect was seen in lean rats. Also, perfused pancreases from adult obese (fa/fa) rats oversecreted insulin during a stimulation by arginine when compared with controls, an oversecretion that was restored toward normal by superimposed infusion of atropine. It is concluded that a) the increased insulin secretion of preobese Zucker fa/fa rats is an early abnormality that is mediated by the vagus nerve, and b) increased secretion of insulin in adult obese fa/fa rats continues to be partly vagus-nerve mediated, although a decreased sympathetic tone and other unknown defects could conceivably play a role as well.