Develop Diet-induced Obesity Rats Research Articles

IL-6 ameliorates defective leptin sensitivity in DIO ventromedial hypothalamic nucleus neurons.

Rats selectively bred to develop diet-induced obesity (DIO) have an early onset reduction in the sensitivity of their ventromedial hypothalamic nucleus (VMN) neurons to leptin compared with diet-resistant (DR) rats. This reduced sensitivity includes decreased leptin receptor (Lepr-b) mRNA expression, leptin receptor binding, leptin-induced phosphorylation of STAT3 (pSTAT3), and impaired leptin excitation (LepE) of VMN neurons. When administered exogenously, the pancreatic peptide, amylin, acts synergistically to reduce food intake and body weight in obese, leptin-resistant DIO rats by increasing VMN leptin signaling, likely by stimulation of microglia IL-6, which acts on its receptor to increase leptin-induced pSTAT3. Here, we demonstrate that incubation of cultured VMN neurons of outbred rats with IL-6 increases their leptin sensitivity. Control, dissociated DIO VMN neurons express 66% less Lepr-b and 75% less Bardet Biedl Syndrome-6 (BBS6) mRNA and have reduced leptin-induced activation of LepE neurons compared with DR neurons. Incubation for 4 days with IL-6 increased DIO neuron Lepr-b expression by 77% and BBS6 by 290% and corrected their defective leptin activation of LepE neurons to DR levels. Since BBS6 enhances trafficking of Lepr-b to the cell membrane, the increases in Lepr-b and BBS6 expression appear to account for correction of the reduced leptin excitation of DIO LepE neurons to that of control DR rats. These data support prior findings suggesting that IL-6 mediates the leptin-sensitizing effects of amylin on VMN neurons and that the inherent leptin resistance of DIO rats can be effectively reversed at a cellular level by IL-6.

Large Litter Rearing Enhances Leptin Sensitivity and Protects Selectively Bred Diet-induced Obese Rats from Becoming Obese

Abstract Because rearing rats in large litters (LLs) protects them from becoming obese, we postulated that LL rearing would protect rats selectively bred to develop diet-induced obesity (DIO) from becoming obese by overcoming their inborn central leptin resistance. Male and female DIO rats were raised in normal litters (10 pups/dam) or LLs (16 pups/dam) and assessed for anatomical, biochemical, and functional aspects of leptin sensitivity at various ages when fed low-fat chow or a 31% fat high-energy (HE) diet. LL rearing reduced plasma leptin levels by postnatal d 2 (P) 2 and body weight gain by P8. At P16, LL DIO neonates had increased arcuate nucleus (ARC) binding of leptin to its extracellular receptors and at P28 an associated increase of their agouti-related peptide and α-MSH axonal projections to the paraventricular nucleus. Reduced body weight persisted and was associated with increased ARC leptin receptor binding and sensitivity to the anorectic effects of leptin, reduced adiposity, and enhanced insulin sensitivity in LL DIO rats fed chow until 10 wk of age. The enhanced ARC leptin receptor binding and reduced adiposity of LL DIO rats persisted after an additional 5 wk on the HE diet. Female LL DIO rats had similar reductions in weight gain on both chow and HE diet vs. normal litter DIO rats. We postulate that LL rearing enhances DIO leptin sensitivity by lowering plasma leptin levels and thereby increasing leptin receptor availability and that this both enhances the ARC-paraventricular nucleus pathway development and protects them from becoming obese.

Large litter rearing enhances leptin sensitivity and protects selectively bred diet-induced obese rats from becoming obese.

Because rearing rats in large litters (LLs) protects them from becoming obese, we postulated that LL rearing would protect rats selectively bred to develop diet-induced obesity (DIO) from becoming obese by overcoming their inborn central leptin resistance. Male and female DIO rats were raised in normal litters (NLs; 10 pups/dam) or LLs (16 pups/dam) and assessed for anatomical, biochemical, and functional aspects of leptin sensitivity at various ages when fed low-fat chow or a 31% fat high-energy (HE) diet. LL rearing reduced plasma leptin levels by postnatal day 2 (P2) and body weight gain by P8. At P16, LL DIO neonates had increased arcuate nucleus (ARC) binding of leptin to its extracellular receptors and at P28 an associated increase of their agouti-related peptide and alpha-MSH axonal projections to the paraventricular nucleus. Reduced body weight persisted and was associated with increased ARC leptin receptor binding and sensitivity to the anorectic effects of leptin, reduced adiposity, and enhanced insulin sensitivity in LL DIO rats fed chow until 10 wk of age. The enhanced ARC leptin receptor binding and reduced adiposity of LL DIO rats persisted after an additional 5 wk on the HE diet. Female LL DIO rats had similar reductions in weight gain on both chow and HE diet vs. normal litter DIO rats. We postulate that LL rearing enhances DIO leptin sensitivity by lowering plasma leptin levels and thereby increasing leptin receptor availability and that this both enhances the ARC-paraventricular nucleus pathway development and protects them from becoming obese.

Three weeks of postweaning exercise in DIO rats produces prolonged increases in central leptin sensitivity and signaling

In rats selectively bred to develop diet-induced obesity (DIO) 3 wk of postweaning exercise reduces weight and adipose regain for 10 wk after exercise cessation, despite intake of 31% fat high-energy (HE) diet. To test the hypothesis that this effect is due to increased central leptin sensitivity, 4-wk-old DIO rats were fed the HE diet and left sedentary (Sed), exercised for 3 wk, and then remained sedentary for 10 additional weeks (Ex/Sed) or continued exercise for a full 13 wk (Ex). After 3 wk, leptin (5 mg/kg ip) induced a 36% decrease in 24-h food intake in Ex rats, while Sed rats had no change in 24-h intake. Ex rats also had 23% more leptin-induced phospho-STAT3 (pSTAT3)-expressing neurons in the arcuate nucleus (ARC) and 95% and 68% higher (125)I-labeled leptin receptor binding in the ventromedial and dorsomedial nuclei than did Sed rats, respectively. At 7 wk after onset, leptin decreased 24-h intake by 20% in Ex and 24% in Ex/Sed rats without altering Sed intake. After a total of 13 wk, compared with Sed rats, Ex and Ex/Sed rats had 58% and 38% less fat, respectively, but leptin failed to decrease food intake in any group. Nevertheless, Ex, but not Ex/Sed rats, still had 32% more ARC leptin-induced pSTAT3-expressing neurons than Sed rats. These data suggest that brief postweaning exercise in DIO rats that are inherently leptin resistant causes a sustained resistance to obesity on HE diet, which is, in part, due to increased central leptin sensitivity.

Ventromedial nucleus neurons are less sensitive to leptin excitation in rats bred to develop diet-induced obesity

Maternal obesity accentuates offspring obesity in dams bred to develop diet-induced obesity (DIO) on a 31% fat, high energy (HE) diet but has no effect on offspring of diet-resistant (DR) dams. Only DIO dams became obese on HE diet when they and DR dams were fed 5% fat chow or HE diets throughout gestation and lactation. Leptin sensitivity of dissociated arcuate (ARC) and ventromedial (VMN) hypothalamic nucleus neurons from the 3- to 4-wk-old offspring was assessed using fura-2 calcium imaging to monitor leptin-induced changes in intracellular calcium ([Ca(2+)](i)) as an index of neuronal activity. At 0.1, 1, 10 fmol/l leptin, approximately 4 times more VMN and ARC neurons were excited than inhibited by leptin. In the VMN, leptin excited up to 41% fewer neurons, and these excited neurons were less sensitive to increasing doses of leptin in DIO compared with DR offspring. Also, maternal HE diet intake decreased the percentage of leptin-excited VMN neurons in both DIO and DR offspring and decreased the percentage of leptin-inhibited VMN neurons by 36% only in DIO offspring. In the ARC, there were no genotype or maternal diet effects on the percentage of ARC neurons excited by leptin. However, those DR neurons that were leptin excited were more sensitive to leptin than were those from DIO offspring. These data suggest that reduced responsiveness of DIO VMN neurons to leptin's excitatory effects may be an important contributing factor to the reduced anorectic and thermogenic leptin responsiveness of DIO rats in vivo.

Three weeks of early-onset exercise prolongs obesity resistance in DIO rats after exercise cessation

We assessed the effect of early-onset exercise as a means of preventing childhood obesity using juvenile male rats selectively bred to develop diet-induced obesity (DIO) or to be diet resistant (DR) when fed a 31% fat high-energy diet. Voluntary wheel running begun at 36 days of age selectively reduced adiposity in DIO vs. DR rats. Other 4-wk-old DIO rats fed a high-energy diet and exercised (Ex) for 13 wk increased their core temperature, gained 22% less body weight, and had 39% lighter fat pads compared with sedentary (Sed) rats. When wheels were removed after 6 wk (6 wk Ex/7 wk Sed), rats gained less body weight over the next 7 wk than Sed rats and still had comparable adipose pad weights to 13-wk-exercised rats. In fact, only 3 wk of exercise sufficed to prevent obesity for 10 wk after wheel removal. Terminally, the 6-wk-Ex/7-wk-Sed rats had a 55% increase in arcuate nucleus proopiomelanocortin mRNA expression vs. Sed rats, suggesting that this contributed to their sustained obesity resistance. Finally, when Sed rats were calorically restricted for 6 wk to weight match them to Ex rats (6 wk Rstr/7 wk Al), they increased their intake and body weight when fed ad libitum and, after 7 wk more, had higher leptin levels and adiposity than Sed rats. Thus, early-onset exercise may favorably alter, while early caloric restriction may unfavorably influence, the development of the hypothalamic pathways controlling energy homeostasis during brain development.

Short-term post-weaning exercise increases central leptin sensitivity in diet-induced obese rats fed a high-energy diet even after exercise termination.

We previously showed that if 4-week old rats bred to develop diet-induced obesity (DIO) exercised in a running wheel for only 3 weeks (Ex), they did not become obese on a 31% fat diet for another 10 weeks of remaining sedentary (Sed). We postulated that increased leptin sensitivity caused this resistance to weight regain. In fact, while Sed rats had no response to leptin, (5 mg/kg, i.p.), DIO rats exercised for 3 week ate 36% less in 24 h (11.9+1.1 g) than their baseline intake (18.6+0.8g; P =0.001) and had a 23% increase in the number of arcuate nucleus neurons expressing pSTAT3 following leptin treatment. Furthermore, their increased leptin sensitivity persisted 4 weeks after exercise termination (3 weeksEx/ 4 weeksSed); rats left Sed for 7 weeks still had no response to leptin, while 3 weekEx/4 weekSed rats at 24% less (20.1+1.4g) after leptin than their 24 h baselines (26.6+1.0 g; P =0.001). However, increased leptin sensitivity was not responsible for the long-term reduction in body fat, since neither 13 weeks Sed nor rats exercised for 3 weeks and held Sed for 10 weeks responded to the anorectic effects of leptin, despite the fact that the latter maintained lower carcass adiposity. These data suggest that, 3 weeks of post-weaning exercise lowers the defended body weight in leptin-resistant DIO rats by increasing their leptin sensitivity for several weeks after exercise cessation. However, this increased sensitivity alone cannot fully explain their long-term resistance to obesity. We postulate that early onset exercise increases in leptin sensitivity may promote this long-term effect by favorably altering the development of neural pathways involved in energy homeostasis.

Altered Hypothalamic Leptin, Insulin, and Melanocortin Binding Associated with Moderate-Fat Diet and Predisposition to Obesity

Rats with a genetic predisposition to develop diet-induced obesity (DIO) have a preexisting reduction in central leptin and insulin sensitivity. High-fat diets also reduce sensitivity to leptin, insulin, and melanocortin agonists. We postulated that such reduced sensitivities would be associated with decreased binding to the hypothalamic leptin, insulin, and melanocortin receptors in selectively bred DIO rats and in rats fed a high-energy (HE; 31% fat) diet for 7 wk. On HE diet, DIO rats gained 15% more weight and had 121% heavier fat pads and 70% higher leptin levels than low fat chow-fed DIO rats. Diet-resistant (DR) rats gained no more weight on HE diet but had 48% heavier fat pads and 70% higher leptin levels than chow-fed DR rats. Compared with DR rats, DIO (125)I-leptin binding was 41, 36, and 40% lower in the hypothalamic dorsomedial, arcuate, and dorsomedial portion of the ventromedial nuclei, respectively, and arcuate (125)I-insulin binding was 31% lower independent of diet. In contrast, hypothalamic melanocortin binding did not differ between DIO and DR rats. However, HE diet intake lowered lateral hypothalamic melanocortin-3 and melanocortin-4 receptor and hippocampal insulin binding of both DIO and DR rats and hypothalamic paraventricular nucleus melanocortin-4 receptor binding only in DR rats. Neither genotype nor diet affected substantia nigra or ventral tegmental area binding. These results corroborate our previous findings demonstrating a preexisting decrease in DIO hypothalamic leptin and insulin signaling and demonstrate that HE diet intake reduces hypothalamic melanocortin and hippocampal insulin binding.

Reduced anorexic effects of insulin in obesity-prone rats fed a moderate-fat diet

Rats prone to develop diet-induced obesity (DIO) have reduced central sensitivity to many metabolic and hormonal signals involved in energy homeostasis. High-fat diets produce similar defects in diet-resistant (DR) rats. To test the hypothesis that genotype and diet exposure would similarly affect central insulin signaling, we assessed the anorectic effects of 8 mU third ventricular (iv3t) insulin before and after 4 wk intake of a 31% fat, high-energy (HE) diet intake in outbred (OutB) rats. Rats were retrospectively designated as DR or DIO by their low or high weight gains on HE diet. Before the HE diet, iv3t insulin reduced 4-h and 24-h chow intake by 53% and 69% in DR rats but by only 17% and 27% in DIO rats, respectively. Also, the anorectic response to iv3t insulin in OutB rats was inversely correlated (r = 0.72, P = 0.002) with subsequent 4-wk weight gain on the HE diet. Similarly, in selectively bred (SB) chow-fed DR rats, 8 mU iv3t insulin reduced 4-h and 24-h intake by 21% and 22%, respectively, but had no significant effect in SB DIO rats. Four-week HE diet intake reduced 4-h and 24-h insulin-induced anorexia by 45% in OutB DR rats and completely abolished it in SB DR rats. Reduced insulin responsiveness was unassociated with differences in arcuate nucleus insulin receptor mRNA expression between DIO and DR rats or between rats fed chow or HE diet. These data suggest that DIO rats have a preexisting reduction in central insulin signaling, which might contribute to their becoming obese on the HE diet. However, since the HE diet reduced central insulin sensitivity in DR rats but did not make them obese, it is likely that other brain areas are involved in insulin's anorectic action or that other pathways contribute to the development and maintenance of obesity.

Obesity-prone rats have preexisting defects in their counterregulatory response to insulin-induced hypoglycemia.

Rats that develop diet-induced obesity (DIO) on a 31% fat [high-energy (HE)] diet have defective sensing and responding to altered glucose levels compared with diet-resistant (DR) rats. Thus we postulated that they would also have defective counterregulatory responses (CRR) to insulin-induced hypoglycemia (IIH). Chow-fed selectively bred DIO and DR rats underwent three sequential 60-min bouts of IIH separated by 48 h. Glucose levels fell comparably, but DIO rats had 22-29% lower plasma epinephrine (Epi) levels during the first two bouts than DR rats. By the third trial, despite comparable Epi levels, DIO rats had lower 30-min glucose levels and rebounded less than DR rats 85 min after intravenous glucose. Although DIO rats gained more carcass and fat weight after 4 wk on an HE diet than DR rats, they were unaffected by prior IIH. Compared with controls, DR rats with prior IIH and HE diet had higher arcuate nucleus neuropeptide Y (50%) and proopiomelanocortin (POMC; 37%) mRNA and an inverse correlation (r = 0.85; P = 0.004) between POMC expression and body weight gain on the HE diet. These data suggest that DIO rats have a preexisting defect in their CRR to IIH but that IIH does not affect the expression of their hypothalamic neuropeptides or weight gain as it does in DR rats.

Obesity-prone rats have normal blood-brain barrier transport but defective central leptin signaling before obesity onset.

Rats selectively bred to develop diet-induced obesity (DIO) were compared with those bred to be diet resistant (DR) on a 31% fat high-energy diet with regard to their central leptin signaling and blood-brain barrier (BBB) transport. Peripheral leptin injection (15 mg/kg ip) into lean 4- to 5-wk-old rats produced 54% less anorexia in DIO than DR rats. DIO rats also had 21, 63, and 64% less leptin-induced immunoreactive phosphorylated signal transducer and activator of transcription 3 (pSTAT3) expression in the hypothalamic arcuate, ventromedial, and dorsomedial nuclei, respectively. However, hindbrain leptin-induced nucleus tractus solitarius pSTAT3 and generalized sympathetic (24-h urine norepinephrine) activation were comparable. Reduced central leptin signaling was not due to defective BBB transport since transport did not differ between lean 4- to 5-wk-old DIO and DR rats. Conversely, DIO leptin BBB transport was reduced when they became obese at 23 wk of age on low-fat chow or after 6 wk on high-energy diet. In addition, leptin receptor mRNA expression was 23% lower in the arcuate nuclei of 4- to 5-wk-old DIO compared with DR rats. Thus a preexisting reduction in hypothalamic but not brain stem leptin signaling might contribute to the development of DIO when dietary fat and caloric density are increased. Defects in leptin transport appear to be an acquired defect associated with the development of obesity and possibly age.

Abnormalities of leptin and ghrelin regulation in obesity-prone juvenile rats.

Rats selectively bred to develop diet-induced obesity (DIO) spontaneously gain more body weight between 5 and 7 wk of age than do those bred to be diet resistant (DR). Here, chow-fed DIO rats ate 9% more and gained 19% more body weight from 5 to 6 wk of age than did DR rats but had comparable leptin and insulin levels. However, 6-wk-old DIO rats had 29% lower plasma ghrelin levels at dark onset but equivalent levels 6 h later compared with DR rats. When subsequently fed a high-energy (HE; 31% fat) diet for 10 days, DIO rats ate 70% more, gained more body and adipose depot weight, had higher leptin and insulin levels, and had 22% lower feed efficiency than DR rats fed HE diet. In DIO rats on HE diet, leptin levels increased significantly at 3 days followed by increased insulin levels at 7 days. These altered DIO leptin and ghrelin responses were associated with 10% lower leptin receptor mRNA expression in the arcuate (ARC), dorsomedial (DMN), and ventromedial hypothalamic nuclei and 13 and 15% lower ghrelin receptor (GHS-R) mRNA expression in the ARC and DMN than in the DR rats. These data suggest that increased ghrelin signaling is not a proximate cause of DIO, whereas reduced leptin sensitivity might play a causal role.

Ontogeny of diet-induced obesity in selectively bred Sprague-Dawley rats.

Outbred Sprague-Dawley rats selectively bred for their propensity to develop diet-induced obesity (DIO) become heavier on low-fat diet than those bred to be diet resistant (DR) beginning at approximately 5 wk of age. Here we assessed the development of metabolic and neural functions for insights into the origins of their greater weight gain. From week 5 to week 10, chow-fed DIO rats gained 15% more body weight and ate approximately 14% more calories but had only slightly greater adiposity and plasma leptin than DR rats. From day 3 through week 10, DIO and DR rats had similar mRNA expression of arcuate nucleus neuropeptide Y, proopiomelanocortin, agouti-related peptide, and all splice variants of the leptin receptor (OB-R). When fed a high-energy (HE; 31% fat) diet, 7-wk-old DIO rats had a 240% increase in plasma leptin levels after only 3 days. Despite this early leptin rise, they maintained a persistent hyperphagia and became more obese than chow-fed DIO rats and DR rats fed chow or HE diet. Their failure to reduce caloric intake, despite high levels of leptin, suggests that selectively bred DIO rats might have reduced leptin sensitivity similar to that seen in the outbred DIO parent strain.

Dysregulation of hypothalamic serotonin turnover in diet-induced obese rats

When outbred Sprague–Dawley rats are placed on a diet relatively high in fat and calories (HE diet), half develop diet-induced obesity (DIO), while the rest are diet-resistant (DR). When fed a low fat chow diet from weaning, DIO- and DR-prone rats weigh the same, but DIO-prone rats have a number of abnormalities of neural function, many of which are normalized when they become obese after chronic exposure to a HE diet. Because of its important role in the regulation of energy homeostasis in the hypothalamus, we examined the ratio of serotonin (5-HT) to its metabolite, 5-hydroxyindolacetic acid (5HIAA), as an index of transmitter turnover in micropunches from various brain areas in these rats. While still on chow, both DIO- and DR-prone rats showed lower 5-HT turnover in most brain areas sampled during the last hour of the light phase, when animals become active and begin foraging for food, as compared to the first hour of the light phase, when animals are generally quiescent and not eating. However, unlike DR-prone rats, DIO-prone rats did not show a significant time-dependent difference in 5-HT turnover in either the arcuate or paraventricular hypothalamic nuclei. When fasted for 48 h, both DIO- and DR-prone rats showed a generalized 16–46% decrease in 5-HT turnover in the dorsomedial nucleus, perifornical lateral hypothalamus, dentate gyrus and motor cortex as compared to their free-fed counterparts. However, fasted DIO-prone rats showed a 53% greater reduction in the ventromedial nucleus turnover than fasted DR-prone rats. Finally, when DIO rats became obese after 14 weeks on the HE diet, their abnormalities in hypothalamic 5-HT turnover at the end of the light phase were normalized. Thus, DIO-prone rats show abnormalities of diurnal and fasting-induced alterations in brain 5-HT turnover which may predispose them to become obese when dietary fat and caloric density are increased. Once obesity develops, these abnormalities, like those of several other hypothalamic transmitters and peptides, are normalized. This may contribute to the persistence of obesity once it develops.

Arcuate NPY neurons and energy homeostasis in diet-induced obese and resistant rats.

The neuropeptide Y (NPY) neurons in the hypothalamic arcuate nucleus regulate and are regulated by short-term changes in energy homeostasis. Both outbred and inbred strains of rats that develop diet-induced obesity (DIO) or are diet resistant (DR) when fed a diet relatively high in energy, fat, and sucrose content (HE diet) were used to study arcuate NPY mRNA expression during long-term changes in energy balance. Outbred, chow-fed obesity-prone rats had 59% higher NPY levels than obesity-resistant rats. After 14 wk on HE diet, DIO rats had 17% lower NPY levels than DR rats made comparably obese on a highly palatable diet. When switched to chow, obese DR rats spontaneously reduced their intake and their body weights fell to control levels in association with a 10% decrease in NPY levels. DIO rats lost weight only with energy restriction associated with a 21% increase in their NPY levels. When again fed ad libitum, the weight and NPY levels in the rats returned to those of unrestricted DIO rats. Chow-fed, inbred DIO rats weigh more and are fatter than age-matched inbred DR rats. As with outbred DIO rats fed the HE diet, inbred DIO rats had 20% lower NPY levels than DR rats. Thus preobese, outbred DIO rats have high levels of NPY message that are not susceptible to metabolic regulation. When obesity develops in both inbred and outbred rats, the levels of NPY mRNA fall but become responsive to alterations in energy availability.

Reduced glucose-induced neuronal activation in the hypothalamus of diet-induced obese rats

Rats predisposed to develop diet-induced obesity (DIO) preferentially activate their sympathetic nervous system during intracarotid glucose infusion [B.E. Levin, Intracarotid glucose-induced norepinephrine response and the development of diet-induced obesity, Int. J. Obesity 16 (1992) 451–457.] but their brains are generally less responsive to glucose than diet-resistant rats (DR) [B.E. Levin, K.L. Brown, A.A. Dunn-Meynell, Differential effects of diet and obesity on high and low affinity sulfonylurea binding sites in the rat brain, Brain Res. 739 (1996) 293–300.; B.E. Levin, B. Planas, Defective glucoregulation of brain α 2-adrenoceptors in obesity-prone rats, Am. J. Physiol. 264 (1993) R305–R311.; B.E. Levin, A.C. Sullivan, Glucose-induced norepinephrine levels and obesity resistance, Am. J. Physiol. 253 (1987) R475–R481.; B.E. Levin, A.C. Sullivan, Glucose-induced sympathetic activation in obesity-prone and resistant rats, Int. J. Obesity 13 (1989) 235–246.]. Here, 1 h intracarotid glucose infusions (4 mg/kg/min) selectively increased Fos-like immunoreactivity (FLIR) in the hypothalamic paraventricular, ventromedial, dorsomedial and arcuate nuclei of inbred DR but not DIO rats. This suggests that enhanced glucose-induced sympathetic activation in DIO rats is related to a failure of glucose to produce neuronal activation in these areas.

Defense of differing body weight set points in diet-induced obese and resistant rats.

Among outbred Sprague-Dawley rats, approximately one-half develop diet-induced obesity (DIO) and one-half are diet resistant (DR) on a diet relatively high in fat and energy content (HE diet). Here we examined the defense of body weight in these two phenotypes. After HE diet for 13 wk, followed by chow for 6 wk, DR rats gained weight comparably but their plasma leptin levels fell to 54% of chow-fed controls. When a palatable liquid diet (Ensure) was added for 13 wk, other DR rats became obese. But when switched to chow, their intakes fell by 60%, and body and retroperitoneal (RP) fat pad weights and plasma leptin and insulin levels all declined for 2 wk and then stabilized at control levels after 6 wk. In contrast, comparably obese DIO rats decreased their intake by only 20%, and their weights plateaued when they were switched to chow after 13 wk on HE diet. When a subgroup of these DIO rats was restricted to 60% of prior intake, their weights fell to chow-fed control levels over 2 wk. But their leptin and insulin levels both fell disproportionately to 30% of controls. When no longer restricted, their intake and feed efficiency rose immediately, and their body and RP pad weights and leptin and insulin levels rose to those of unrestricted DIO rats within 2 wk. Thus diet and genetic background interact to establish high (DIO) or low (DR) body weight set points, which are then defended against subsequent changes in diet composition and/or energy availability. If leptin affects energy homeostasis, it does so differentially in DIO vs. DR rats since comparably low and high levels were associated with differing patterns of weight change between the two phenotypes.

Selective breeding for diet-induced obesity and resistance in Sprague-Dawley rats.

In outbred Sprague-Dawley rats, about one-half develop diet-induced obesity (DIO) on a diet relatively high in fat and energy (HE diet). The rest are diet resistant (DR), gaining weight and fat at the same rate as chow-fed controls. Here we selectively bred for high (DIO) and low (DR) weight gainers after 2 wk on HE diet. By the F5 generation, both male and female inbred DIO rats gained > 90% more weight than inbred DR rats on HE diets. Even on low-fat chow diet, DIO males were 31% and females were 22% heavier than their respective DR rats. Full metabolic characterization in male rats showed that weight-matched, chow-fed DIO-prone rats had similar energy intakes and feed efficiency [body weight (kg0.75)/energy intake (kcal)] but 44% more carcass fat than comparable DR-prone rats. Their basal plasma insulin and glucose levels in the fed state were 70 and 14% higher, respectively. But, when fasted, DIO-prone oral glucose tolerance results were comparable to DR-prone rats. Chow-fed DIO-prone males also had 42% greater 24-h urine norepinephrine levels than DR-prone males. During 2 wk on HE diet, DIO rats ate 25% more, gained 115% more weight, had 36% more carcass fat, and were 42% more feed efficient than comparable DR rats. Fasted HE diet-fed DIO rats developed frank glucose intolerance during a glucose tolerance test with 55 and 158% greater insulin and glucose areas under the curve, respectively. Thus the DIO and DR traits in the outbred Sprague-Dawley population appear to be due to a polygenic pattern of inheritance.

Location and effect of obesity on putative anorectic binding sites in the rat brain.

Anorectic drugs such as mazindol bind to a class of low-affinity, sodium-sensitive sites in the brain which are affected by ambient glucose concentrations and a predisposition to develop diet-induced obesity (DIO). This study used quantitative autoradiography of 10 nM 3H-mazindol binding to identify the cellular location of these putative anorectic binding sites in the brain and to assess the way in which the development of DIO affected their binding. We previously showed that chow-fed, obesity-prone rats have widespread increases in brain 3H-mazindol binding to these low-affinity sites as compared with diet-resistant (DR) rats. Here, low-affinity 3H-mazindol binding was assessed in the brains of eight rats which developed DIO vs. eight which were DR after three months on a high-energy diet. DIO rats gained 89% more weight and had 117% higher plasma insulin levels but no difference in plasma glucose levels compared with DR rats. Along with these differences, low-affinity 3H-mazindol binding in DIO rats was identical to that in DR rats in all of the 23 brain areas assessed. This suggested that this binding was downregulated by the development of obesity in DIO rats. In other chow-fed rats, stereotaxic injections of 5,7-dihydroxytryptamine and 6-hydroxydopamine (6OHDA) to ablate serotonin and catecholamine nerve terminals in the ventromedial nucleus of the hypothalamus (VMN) had no effect on 3H-mazindol binding. However, ibotenic acid injected into the VMN, substantia nigra, pars reticulata, and pars compacta destroyed intrinsic neurons and/or their local processes and decreased low-affinity 3H-mazindol binding by 13%-22%. Destruction of dopamine neurons in the substantia nigra, pars compacta, and noradrenergic neurons in the locus ceruleus with 6OHDA also reduced 3H-mazindol binding in those areas by 9% and 12%, respectively. This suggested that up to 22% of putative anorectic binding sites may be located on the cell bodies of dopamine, norepinephrine, and other neurons, but not on serotonin or catecholamine nerve terminals in the brain. Binding to these sites may be downregulated by the development of DIO, possibly as a result of the concomitant hyperinsulinemia.

Dysregulation of arcuate nucleus preproneuropeptide Y mRNA in diet-induced obese rats.

Neuropeptide Y (NPY) neurons in the hypothalamic arcuate nucleus (ARC) produce metabolic and physiological effects that promote the development and maintenance of obesity. In turn, NPY metabolism in these neurons is inhibited by dopamine release. In this study, ARC prepro-NPY mRNA and ARC/median eminence (ME) dopamine turnover were assessed in chow-fed male Sprague-Dawley rats prone to develop diet-induced obesity (DIO) or to be diet resistant (DR) when fed a high-energy (HE) diet. By in situ hybridization, DIO-prone rats had 39% more ARC NPY mRNA expression than DR-prone rats under chow-fed conditions. DIO-prone rat ARC/ME dopamine levels were 14% higher, but dopamine half-life was 176% longer and turnover was 59% less than DR-prone rats. Neither a 48-h fast nor 50% energy intake restriction for 5 days affected the already increased ARC NPY mRNA levels in DIO-prone rats. Both manipulations increased NPY expression to the level of DIO-prone rats in DR-prone rats by 23 and 35%, respectively. Finally, when fed HE diet for 2 wk, neither DIO- nor DR-prone rats altered their ARC NPY expression despite the development of obesity and hyperinsulinemia in DIO rats. Thus DIO-prone rats overexpress and fail to regulate ARC NPY mRNA to energy restriction or hyperinsulinemia. This dysregulation is possibly secondary to reduced inhibition because of defective ARC/ME dopamine turnover. Both may be important predisposing factors in the development of DIO.