Search

Selectively bred diet-induced obese (DIO) rats become obese on a high-fat diet and are leptin resistant before becoming obese. Compared with diet-resistant (DR) neonates, DIO neonates have impaired leptin-dependent arcuate (ARC) neuropeptide Y/agouti-related peptide (NPY/AgRP) and α-melanocyte-stimulating hormone (α-MSH; from proopiomelanocortin (POMC) neurons) axon outgrowth to the paraventricular nucleus (PVN). Using phosphorylation of STAT3 (pSTAT3) as a surrogate, we show that reduced DIO ARC leptin signaling develops by postnatal day 7 (P7) and is reduced within POMC but not NPY/AgRP neurons. Since amylin increases leptin signaling in adult rats, we treated DIO neonates with amylin during postnatal hypothalamic development and assessed leptin signaling, leptin-dependent ARC-PVN pathway development, and metabolic changes. DIO neonates treated with amylin from P0–6 and from P0–16 increased ARC leptin signaling and both AgRP and α-MSH ARC-PVN pathway development, but increased only POMC neuron number. Despite ARC-PVN pathway correction, P0–16 amylin-induced reductions in body weight did not persist beyond treatment cessation. Since amylin enhances adult DIO ARC signaling via an IL-6-dependent mechanism, we assessed ARC-PVN pathway competency in IL-6 knockout mice and found that the AgRP, but not the α-MSH, ARC-PVN pathway was reduced. These results suggest that both leptin and amylin are important neurotrophic factors for the postnatal development of the ARC-PVN pathway. Amylin might act as a direct neurotrophic factor in DIO rats to enhance both the number of POMC neurons and their α-MSH ARC-PVN pathway development. This suggests important and selective roles for amylin during ARC hypothalamic development.

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.

The objective of this study was to determine the potential role of astrocyte-derived ketone bodies in regulating the early changes in caloric intake of diet induced-obese (DIO) versus diet-resistant (DR) rats fed a 31.5% fat high-energy (HE) diet. After 3 days on chow or HE diet, DR and DIO rats were assessed for their ventromedial hypothalamic (VMH) ketone bodies levels and neuronal ventromedial hypothalamic nucleus (VMN) sensing using microdialysis coupled to continuous food intake monitoring and calcium imaging in dissociated neurons, respectively. DIO rats ate more than DR rats over 3 days of HE diet intake. On day 3 of HE diet intake, DR rats reduced their caloric intake while DIO rats remained hyperphagic. Local VMH astrocyte ketone bodies production was similar between DR and DIO rats during the first 6 h after dark onset feeding but inhibiting VMH ketone body production in DR rats on day 3 transiently returned their intake of HE diet to the level of DIO rats consuming HE diet. In addition, dissociated VMN neurons from DIO and DR rats were equally sensitive to the largely excitatory effects of β-hydroxybutyrate. Thus while DR rats respond to increased VMH ketone levels by decreasing their intake after 3 days of HE diet, this is not the case of DIO rats. These data suggest that DIO inherent leptin resistance prevents ketone bodies inhibitory action on food intake.

Amylin phosphorylates ERK (p-ERK) in the area postrema to reduce eating and synergizes with leptin to phosphorylate STAT3 in the arcuate (ARC) and ventromedial (VMN) hypothalamic nuclei to reduce food intake and body weight. The current studies assessed potential amylin and amylin-leptin ARC/VMN interactions on ERK signaling and their roles in postnatal hypothalamic pathway development. In amylin knockout mice, the density of agouti-related protein (AgRP)-immunoreactive (IR) fibers in the hypothalamic paraventricular nucleus (PVN) was increased, while the density of α-melanocyte–stimulating hormone (αMSH) fibers was decreased. In mice deficient of the amylin receptor components RAMP1/3, both AgRP and αMSH-IR fiber densities were decreased, while only αMSH-IR fiber density was decreased in rats injected neonatally in the ARC/VMN with an adeno-associated virus short hairpin RNA against the amylin core receptor. Amylin induced p-ERK in ARC neurons, 60% of which was present in POMC-expressing neurons, with none in NPY neurons. An amylin-leptin interaction was shown by an additive effect on ARC ERK signaling in neonatal rats and a 44% decrease in amylin-induced p-ERK in the ARC of leptin receptor–deficient and of ob/ob mice. Together, these results suggest that amylin directly acts, through a p-ERK–mediated process, on POMC neurons to enhance ARC-PVN αMSH pathway development.

Metabolic sensing neurons in the ventromedial hypothalamus (VMH) alter their activity when ambient levels of metabolic substrates, such as glucose and fatty acids (FA), change. To assess the relationship between a high-fat diet (HFD; 60%) intake on feeding and serum and VMH FA levels, rats were trained to eat a low-fat diet (LFD; 13.5%) or an HFD in 3 h/day and were monitored with VMH FA microdialysis. Despite having higher serum levels, HFD rats had lower VMH FA levels but ate less from 3 to 6 h of refeeding than did LFD rats. However, VMH β-hydroxybutyrate (β-OHB) and VMH-to-serum β-OHB ratio levels were higher in HFD rats during the first 1 h of refeeding, suggesting that VMH astrocyte ketone production mediated their reduced intake. In fact, using calcium imaging in dissociated VMH neurons showed that ketone bodies overrode normal FA sensing, primarily by exciting neurons that were activated or inhibited by oleic acid. Importantly, bilateral inhibition of VMH ketone production with a 3-hydroxy-3-methylglutaryl-CoA synthase inhibitor reversed the 3- to 6-h HFD-induced inhibition of intake but had no effect in LFD-fed rats. These data suggest that a restricted HFD intake regimen inhibits caloric intake as a consequence of FA-induced VMH ketone body production by astrocytes.

Hypothalamic “metabolic-sensing” neurons sense glucose and fatty acids (FAs) and play an integral role in the regulation of glucose, energy homeostasis, and the development of obesity and diabetes. Using pharmacologic agents, we previously found that ∼50% of these neurons responded to oleic acid (OA) by using the FA translocator/receptor FAT/CD36 (CD36). For further elucidation of the role of CD36 in neuronal FA sensing, ventromedial hypothalamus (VMH) CD36 was depleted using adeno-associated viral (AAV) vector expressing CD36 short hairpin RNA (shRNA) in rats. Whereas their neuronal glucosensing was unaffected by CD36 depletion, the percent of neurons that responded to OA was decreased specifically in glucosensing neurons. A similar effect was seen in total-body CD36-knockout mice. Next, weanling rats were injected in the VMH with CD36 AAV shRNA. Despite significant VMH CD36 depletion, there was no effect on food intake, body weight gain, or total carcass adiposity on chow or 45% fat diets. However, VMH CD36–depleted rats did have increased plasma leptin and subcutaneous fat deposition and markedly abnormal glucose tolerance. These results demonstrate that CD36 is a critical factor in both VMH neuronal FA sensing and the regulation of energy and glucose homeostasis.

International audience; The delivery of blood-borne molecules conveying metabolic information to neural networks that regulate energy homeostasis is restricted by brain barriers. The fenestrated endothelium of median eminence microvessels and tight junctions between tanycytes together compose one of these. Here, we show that the decrease in blood glucose levels during fasting alters the structural organization of this blood-hypothalamus barrier, resulting in the improved access of metabolic substrates to the arcuate nucleus. These changes are mimicked by 2-deoxyglucose-induced glucoprivation and reversed by raising blood glucose levels after fasting. Furthermore, we show that VEGF-A expression in tanycytes modulates these barrier properties. The neutralization of VEGF signaling blocks fasting-induced barrier remodeling and significantly impairs the physiological response to refeeding. These results implicate glucose in the control of blood-hypothalamus exchanges through a VEGF-dependent mechanism and demonstrate a hitherto unappreciated role for tanycytes and the permeable microvessels associated with them in the adaptive metabolic response to fasting.

A major side effect of insulin treatment of diabetes is weight gain, which limits patient compliance and may pose additional health risks. Although the mechanisms responsible for this weight gain are poorly understood, it has been suggested that there may be a link to the incidence of recurrent episodes of hypoglycemia. Here we present a rodent model of marked weight gain associated with weekly insulin-induced hypoglycemic episodes in the absence of diabetes. Insulin treatment caused a significant increase in both body weight and fat mass, accompanied by reduced motor activity, lowered thermogenesis in response to a cold challenge, and reduced brown fat uncoupling protein mRNA. However, there was no effect of insulin treatment on total food intake nor on hypothalamic neuropeptide Y or proopiomelanocortin mRNA expression, and insulin-treated animals did not become insulin-resistant. Our results suggest that repeated iatrogenic hypoglycemia leads to weight gain, and that such weight gain is associated with a multifaceted deficit in metabolic regulation rather than to a chronic increase in caloric intake.

Unique subpopulations of specialized metabolic sensing neurons reside in a distributed network throughout the brain and respond to alterations in ambient levels of various metabolic substrates by altering their activity. Variations in local brain substrate levels reflect their transport across the blood- and cerebrospinal-brain barriers as well as local production by astrocytes. There are a number of mechanisms by which such metabolic sensing neurons alter their activity in response to changes in substrate levels, but it is clear that these neurons cannot be considered in isolation. They are heavily dependent on astrocyte and probably tanycyte metabolism and function but also respond to hormones (e.g. leptin and insulin) and cytokines that cross the blood-brain barrier from the periphery as well as hard-wired neural inputs from metabolic sensors in peripheral sites such as the hepatic portal vein, gastrointestinal tract, and carotid body. Thus, these specialized neurons are capable of monitoring and integrating multiple signals from the periphery as a means of regulating peripheral energy homeostasis.

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.

Maternal obesity accentuates offspring obesity in dams bred to develop diet-induced obesity (DIO) on a 31% fat, high-sucrose, high-energy (HE) diet but has no effect on offspring of diet-resistant (DR) dams. Also, only DIO dams become obese when they and DR dams are fed HE diet throughout gestation and lactation. We assessed glucose and oleic acid (OA) sensitivity of dissociated ventromedial hypothalamic nucleus (VMN) neurons from 3- to 4-wk old offspring of DIO and DR dams fed chow or HE diet using fura-2 calcium imaging to monitor intracellular calcium fluctuations as an index of neuronal activity. Offspring of DIO dams fed chow had ∼2-fold more glucose-inhibited (GI) neurons than did DR offspring. This difference was eliminated in offspring of DIO dams fed HE diet. At 2.5 mM glucose, offspring of chow-fed DIO dams had more GI neurons that were either excited or inhibited by OA than did DR offspring. Maternal HE diet intake generally increased the percentage of neurons that were excited and decreased the percentage that were inhibited by OA in both DIO and DR offspring. However, this effect was more pronounced in DIO offspring. These data, as well as concentration-dependent differences in OA sensitivity, suggest that genotype, maternal obesity, and dietary content can all affect the sensitivity of offspring VMN neurons to glucose and long-chain fatty acids. Such altered sensitivities may underlie the propensity of DIO offspring to become obese when fed high-fat, high-sucrose diets.

Although several studies implicate small declines in blood glucose levels as stimulus for spontaneous meal initiation, no mechanism is known for how these dips might initiate feeding. To assess the role of ventromedial hypothalamus (VMH) (arcuate plus ventromedial nucleus) glucosensing neurons as potential mediators of spontaneous and glucoprivic feeding, meal patterns were observed, and blood and VMH microdialysis fluid were sampled in 15 rats every 10 min for 3.5 h after dark onset and 2 h after insulin (5 U/kg, i.v.) infusion. Blood glucose levels declined by 11% beginning ∼5 min before 65% of all spontaneous meals, with no fall in VMH levels. After insulin, blood and VMH glucose reached nadirs by 30–40 min, and the same rats ate 60% faster and spent 84% more time eating during the ensuing hypoglycemia. Although 83% of first hypoglycemic meals were preceded by 5 min dips in VMH (but not blood) glucose levels, neither blood nor VMH levels declined before second meals, suggesting that low glucose, rather than changing levels, was the stimulus for glucoprivic meals. Furthermore, altering VMH glucosensing by raising or lowering glucokinase (GK) activity failed to affect spontaneous feeding, body or adipose weights, or glucose tolerance. However, chronic depletion by 26–70% of VMH GK mRNA reduced glucoprivic feeding. Thus, although VMH glucosensing does not appear to be involved in either spontaneous feeding or long-term body-weight regulation, it does participate in glucoprivic feeding, similar to its role in the counter-regulatory neurohumoral responses to glucoprivation.

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 125I-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.

OBJECTIVE—The counterregulatory response to insulin-induced hypoglycemia is mediated by the ventromedial hypothalamus (VMH), which contains specialized glucosensing neurons, many of which use glucokinase (GK) as the rate-limiting step in glucose's regulation of neuronal activity. Since conditions associated with increased VMH GK expression are associated with a blunted counterregulatory response, we tested the hypothesis that increasing VMH GK activity would similarly attenuate, while decreasing GK activity would enhance the counterregulatory response to insulin-induced hypoglycemia. RESEARCH DESIGN AND METHODS—The counterregulatory response to insulin-induced hypoglycemia was evaluated in Sprague-Dawley rats after bilateral VMH injections of 1) a GK activator drug (compound A) to increase VMH GK activity, 2) low-dose alloxan (4 μg) to acutely inhibit GK activity, 3) high-dose alloxan (24 μg), or 4) an adenovirus expressing GK short hairpin RNA (shRNA) to chronically reduce GK expression and activity. RESULTS—Compound A increased VMH GK activity sixfold in vitro and reduced the epinephrine, norepinephrine, and glucagon responses to insulin-induced hypoglycemia by 40–62% when injected into the VMH in vivo. On the other hand, acute and chronic reductions of VMH GK mRNA or activity had a lesser and more selective effect on increasing primarily the epinephrine response to insulin-induced hypoglycemia by 23–50%. CONCLUSIONS—These studies suggest that VMH GK activity is an important regulator of the counterregulatory response to insulin-induced hypoglycemia and that a drug that specifically inhibited the rise in hypothalamic GK activity after insulin-induced hypoglycemia might improve the dampened counterregulatory response seen in tightly controlled diabetic subjects.

In rats selectively bred to develop diet-induced obesity (DIO) or to be diet-resistant (DR), DIO maternal obesity selectively enhances the development of obesity and insulin resistance in their adult offspring. We postulated that the interaction between genetic predisposition and factors in the maternal environment alter the development of hypothalamic peptide systems involved in energy homeostasis regulation. Maternal obesity in the current studies led to increased body and fat pad weights and higher leptin and insulin levels in postnatal day 16 offspring of both DIO and DR dams. However, by 6 wk of age, most of these intergroup differences disappeared and offspring of obese DIO dams had unexpected increases in arcuate nucleus leptin receptor mRNA, peripheral insulin sensitivity, diet- and leptin-induced brown adipose temperature increase and 24-h anorectic response compared with offspring of lean DIO, but not lean DR dams. On the other hand, while offspring of obese DIO dams did have the highest ventromedial nucleus melanocortin-4 receptor expression, their anorectic and brown adipose thermogenic responses to the melanocortin agonist, Melanotan II (MTII), did not differ from those of offspring of lean DR or DIO dams. Thus, during their rapid growth phase, juvenile offspring of obese DIO dams have alterations in their hypothalamic systems regulating energy homeostasis, which ameliorates their genetic and perinatally determined predisposition toward leptin resistance. Because they later go onto become more obese, it is possible that interventions during this time period might prevent the subsequent development of obesity.

Amylin enhances arcuate (ARC) and ventromedial (VMN) hypothalamic nuclei leptin signaling and synergistically reduces food intake and body weight in selectively bred diet-induced obese (DIO) rats. Since DIO 125I-amylin dorsomedial nucleus-dorsomedial VMN binding was reduced, we postulated that this contributed to DIO ventromedial hypothalamus (VMH) leptin resistance, and that impairing VMH (ARC + VMN) calcitonin receptor (CTR)-mediated signaling by injecting adeno-associated virus (AAV) expressing a short hairpin portion of the CTR mRNA would predispose diet-resistant (DR) rats to obesity on high-fat (45%) diet (HFD). Depleting VMH CTR by 80–90% in 4-wk-old male DR rats reduced their ARC and VMN 125I-labeled leptin binding by 57 and 51%, respectively, and VMN leptin-induced phospho-signal transducer and activator of transcription 3-positive neurons by 59% vs. AAV control rats. After 6 wk on chow, VMH CTR-depleted DR rats ate and gained the equivalent amount of food and weight but had 18% heavier fat pads (relative to carcass weight), 144% higher leptin levels, and were insulin resistant compared with control AAV DR rats. After 6 wk more on HFD, VMH CTR-depleted DR rats ate the same amount but gained 28% more weight, had 60% more carcass fat, 254% higher leptin levels, and 132% higher insulin areas under the curve during an oral glucose tolerance test than control DR rats. Therefore, impairing endogenous VMH CTR-mediated signaling reduced leptin signaling and caused DR rats to become more obese and insulin resistant, both on chow and HFD. These results suggest that endogenous VMH amylin signaling is required for full leptin signaling and protection from HFD-induced obesity.

While the neural control of glucoregulatory responses to insulin-induced hypoglycemia is beginning to be elucidated, brain sites responsible for behavioral responses to hypoglycemia are relatively poorly understood. To help elucidate central control mechanisms associated with hypoglycemia unawareness, we first evaluated the effect of recurrent hypoglycemia on a simple behavioral measure, the robust feeding response to hypoglycemia, in rats. First, food intake was significantly, and similarly, increased above baseline saline-induced intake (1.1 ± 0.2 g; n = 8) in rats experiencing a first (4.4 ± 0.3; n = 8) or third daily episode of recurrent insulin-induced hypoglycemia (IIH, 3.7 ± 0.3 g; n = 9; P < 0.05). Because food intake was not impaired as a result of prior IIH, we next developed an alternative animal model of hypoglycemia-induced behavioral arousal using a conditioned place preference (CPP) model. We found that hypoglycemia severely blunted previously acquired CPP in rats and that recurrent hypoglycemia prevented this blunting. Pretreatment with a brain penetrant, selective orexin receptor-1 antagonist, SB-334867A, blocked hypoglycemia-induced blunting of CPP. Recurrently hypoglycemic rats also showed decreased preproorexin expression in the perifornical hypothalamus (50%) but not in the adjacent lateral hypothalamus. Pretreatment with sertraline, previously shown to prevent hypoglycemia-associated glucoregulatory failure, did not prevent blunting of hypoglycemia-induced CPP prevention by recurrent hypoglycemia. This work describes the first behavioral model of hypoglycemia unawareness and suggests a role for orexin neurons in mediating behavioral responses to hypoglycemia.

Glucosensing is a property of specialized neurons in the brain that regulate their membrane potential and firing rate as a function of ambient glucose levels. These neurons have several similarities to β- and α-cells in the pancreas, which are also responsive to ambient glucose levels. Many use glucokinase as a rate-limiting step in the production of ATP and its effects on membrane potential and ion channel function to sense glucose. Glucosensing neurons are organized in an interconnected distributed network throughout the brain that also receives afferent neural input from glucosensors in the liver, carotid body, and small intestines. In addition to glucose, glucosensing neurons can use other metabolic substrates, hormones, and peptides to regulate their firing rate. Consequently, the output of these “metabolic sensing” neurons represents their integrated response to all of these simultaneous inputs. The efferents of these neurons regulate feeding, neuroendocrine and autonomic function, and thereby energy expenditure and storage. Thus, glucosensing neurons play a critical role in the regulation of energy homeostasis. Defects in the ability to sense glucose and regulatory hormones like leptin and insulin may underlie the predisposition of some individuals to develop diet-induced obesity.

There is growing evidence that the postnatal environment can have a major impact on the development of obesity and insulin resistance in offspring. We postulated that cross-fostering obesity-prone offspring to lean, obesity-resistant dams would ameliorate their development of obesity and insulin resistance, while fostering lean offspring to genetically obese dams would lead them to develop obesity and insulin resistance as adults. We found that obesity-prone pups cross-fostered to obesity-resistant dams remained obese but did improve their insulin sensitivity as adults. In contrast, obesity-resistant pups cross-fostered to genetically obese dams showed a diet-induced increase in adiposity, reduced insulin sensitivity, and associated changes in hypothalamic neuropeptide, insulin, and leptin receptors, which might have contributed to their metabolic defects. There was a selective increase in insulin levels and differences in fatty acid composition of obese dam milk which might have contributed to the increased adiposity, insulin resistance, and hypothalamic changes in obesity-resistant cross-fostered offspring. These results demonstrate that postnatal factors can overcome both genetic predisposition and prenatal factors in determining the development of adiposity, insulin sensitivity, and the brain pathways that mediate these functions.

To test the hypothesis that glucokinase is a critical regulator of neuronal glucosensing, glucokinase activity was increased, using a glucokinase activator drug, or decreased, using RNA interference combined with calcium imaging in freshly dissociated ventromedial hypothalamic nucleus (VMN) neurons or primary ventromedial hypothalamus (VMH; VMN plus arcuate nucleus) cultures. To assess the validity of our approach, we first showed that glucose-induced (0.5-2.5 mmol/l) changes in intracellular Ca(2+) concentration ([Ca(2+)](i)) oscillations, using fura-2 and changes in membrane potential (using a membrane potential-sensitive dye), were highly correlated in both glucose-excited and -inhibited neurons. Also, glucose-excited neurons increased (half-maximal effective concentration [EC(50)] = 0.54 mmol/l) and glucose-inhibited neurons decreased (half-maximal inhibitory concentration [IC(50)] = 1.12 mmol/l) [Ca(2+)](i) oscillations to incremental changes in glucose from 0.3 to 5 mmol/l. In untreated primary VMH neuronal cultures, the expression of glucokinase mRNA and the number of demonstrable glucosensing neurons fell spontaneously by half over 12-96 h without loss of viable neurons. Transfection of neurons with small interfering glucokinase RNA did not affect survival but did reduce glucokinase mRNA by 90% in association with loss of all demonstrable glucose-excited neurons and a 99% reduction in glucose-inhibited neurons. A pharmacological glucokinase activator produced a dose-related increase in [Ca(2+)](i) oscillations in glucose-excited neurons (EC(50) = 0.98 mmol/l) and a decrease in glucose-inhibited neurons (IC(50) = 0.025 micromol/l) held at 0.5 mmol/l glucose. Together, these data support a critical role for glucokinase in neuronal glucosensing.

To determine the effect of exercise on weight gain and adiposity in obesity-prone and -resistant rats.Body weight gain, fat pad weights, food intake, plasma leptin and insulin levels were assessed in outbred male Sprague-Dawley rats, which remained sedentary or were given unrestricted access to running wheels either before or after they developed diet-induced obesity (DIO) or diet-resistance (DR) on a high energy (HE; 31% fat) diet.When fed a low fat (4.5%) chow diet, rats which would later develop DIO (n=6) after 3 weeks on HE diet ran the same amount as DR rats (n=6). Other rats were first made DIO (n=12) or DR (n=12) after 10 weeks on HE diet and then either kept sedentary or given running wheels for 4 weeks on HE diet. DIO and DR rats ran comparable amounts but only the DIO rats reduced their body weight gain, fat pad relative to body weights and plasma leptin levels significantly, compared to their sedentary controls. Exercise had no effect on food intake in either DIO or DR rats but reduced feed efficiency (weight gain/caloric intake) in both.Although DIO and DR rats ran similar amounts, the greater reduction in body weight gain and adiposity of exercising DIO rats suggests that they are more sensitive to some metabolic or physiologic system that prevents them from increasing their intake sufficiently to compensate for their net reduction in energy stores.

Hypothalamic fatty acid (FA) sensing neurons alter their activity utilizing the FA translocator/receptor, FAT/CD36. Depletion of ventromedial hypothalamus (VMH) CD36 with adeno-associated viral vector expressing CD36 shRNA (AAV CD36 shRNA) leads to redistribution of adipose stores and insulin resistance in outbred rats. This study assessed the requirement of VMH CD36-mediated FA sensing for the regulation of energy and glucose homeostasis in postnatal day 5 (P5) and P21 selectively bred diet-induced obese (DIO) and diet-resistant (DR) rats using VMH AAV CD36 shRNA injections. P5 CD36 depletion altered VMH neuronal FA sensing predominantly in DIO rats. After 10 wk on a 45% fat diet, DIO rats injected with VMH AAV CD36 shRNA at P21 ate more and gained more weight than DIO AAV controls, while DR AAV CD36 shRNA-injected rats gained less weight than DR AAV controls. VMH CD36 depletion increased inguinal fat pad weights and leptin levels in DIO and DR rats. Although DR AAV CD36 shRNA-injected rats became as obese as DIO AAV controls, only DIO control and CD36 depleted rats became insulin-resistant on a 45% fat diet. VMH CD36 depletion stunted linear growth in DIO and DR rats. DIO rats injected with AAV CD36 shRNA at P5 had increased fat mass, mostly due to a 45% increase in subcutaneous fat. They were also insulin-resistant with an associated 71% increase of liver triglycerides. These results demonstrate that VMH CD36-mediated FA sensing is a critical factor in the regulation of energy and glucose homeostasis and fat deposition in DIO and DR rats.

We previously created a novel F-DIO rat strain derived by crossing rats selectively bred for the diet-induced obesity (DIO) phenotype with obesity-resistant Fischer F344 rats. The offspring retained the DIO phenotype through 3 backcrosses with F344 rats but also had exaggerated insulin responses to oral glucose before they became obese on a 31% fat high-energy (HE) diet. Here, we demonstrate that chow-fed rats from the subsequent randomly bred progeny required 57% lower glucose infusions to maintain euglycemia during a hyperinsulinemic clamp in association with 45% less insulin-induced hepatic glucose output inhibition and 80% lower insulin-induced glucose uptake than F344 rats. The DIO phenotype and exaggerated insulin response to oral glucose in the nonobese, chow-fed state persisted in the F6 generation. Also, compared with F344 rats, chow-fed F-DIO rats had 68% higher arcuate nucleus proopiomelanocortin mRNA expression which, unlike the increase in F344 rats, was decreased by 26% on HE diet. Further, F-DIO lateral hypothalamic orexin expression was 18% lower than in F344 rats and was increased rather than decreased by HE diet intake. Finally, both maternal obesity and 30% caloric restriction during the third week of gestation produced F-DIO offspring which were heavier and had higher leptin and insulin levels than lean F-DIO dam offspring. Third-gestational week dexamethasone also produced offspring with higher leptin and insulin levels but with lower body weight. Thus F-DIO rats represent a novel and potentially useful model for the study of DIO, insulin resistance, and perinatal factors that influence the development and persistence of obesity.

Glucosensing neurons are specialized cells that use glucose as a signaling molecule to alter their action potential frequency in response to variations in ambient glucose levels. Glucokinase (GK) appears to be the primary regulator of most neuronal glucosensing, but other regulators almost certainly exist. Glucose-excited neurons increase their activity when glucose levels rise, and most use GK and an ATP-sensitive K+ channel as the ultimate effector of glucose-induced signaling. Glucose-inhibited (GI) neurons increase their activity at low glucose levels. Although many use GK, it is unclear what the final pathway of GI neuronal glucosensing is. Glucosensing neurons are located in brain sites and respond to and integrate a variety of hormonal, metabolic, transmitter, and peptide signals involved in the regulation of energy homeostasis and other biological functions. Although it is still uncertain whether daily fluctuations in blood glucose play a specific regulatory role in these physiological functions, it is clear that large decreases in glucose availability stimulate food intake and counterregulatory responses that restore glucose levels to sustain cerebral function. Finally, glucosensing is altered in obesity and after recurrent bouts of hypoglycemia, and this altered sensing may contribute to the adverse outcomes of these conditions. Thus, although much is known, much remains to be learned about the physiological function of brain glucosensing neurons.

Glucokinase (GK) is hypothesized to be the critical glucosensor of pancreatic β-cells and hypothalamic glucosensing neurons. To understand the role of GK in glucoprivic counterregulatory responses, we injected alloxan, a GK inhibitor and toxin, into the third ventricle (3v) to target nearby GK-expressing neurons. Four and 6 days after 3v, but not 4v, alloxan injection, alloxan-treated rats ate only 30% and their blood glucose area under the curve was only 28% of saline controls’ after systemic 2-deoxy-d-glucose. In addition, their hyperglycemic response to hindbrain glucoprivation induced with 5-thio-glucose was impaired, whereas fasting blood glucose levels and food intake after an overnight fast were elevated. These impaired responses were associated with the destruction of 3v tanycytes, reduced glial fibrillary acidic protein-immunoreactivity surrounding the 3v, neuronal swelling, and decreased arcuate nucleus neuropeptide Y (NPY) mRNA. Nevertheless, hypothalamic GK mRNA was significantly elevated. Two weeks after alloxan injection, 3v tanycyte destruction was reversed along with restoration of feeding and hyperglycemic responses to both systemic and hindbrain glucoprivation. At this time there were significant decreases in GK, NPY, and proopiomelanocortin mRNA. Thus, neural substrates near and around the 3v affected by alloxan may be critically involved in the expression of these glucoprivic responses.

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.

Previous breeding for the diet-induced obese (DIO) trait from outbred Sprague-Dawley rats produced a substrain with selection characteristics suggesting a polygenic mode of inheritance. To assess this issue further, selectively bred DIO male rats were crossed with obesity-resistant inbred Fischer F344 dams. Male offspring were crossed twice more against female F344 dams. The resultant N3 (F.DIO) rats were then inbred three more times. On low-fat chow, 10-wk-old male and female DIO rats weighed 86 and 59% more than respective F344 rats. By the N3 (F.DIO) generation, they were only 12 and 10% heavier, respectively. After three additional inbreeding cycles, chow-fed F.DIO males had an exaggerated insulin response to oral glucose compared with F344 rats. After 3 wk on a 31% fat (high-energy) diet, male N3 F.DIO rats gained 16-20% more carcass and adipose weight with 98% higher plasma leptin levels, whereas F.DIO females gained 36-54% more carcass and adipose weight with 130% higher leptin levels than comparable F344 rats. After three inbreeding cycles, F.DIO males still gained more weight on high-energy diet and developed a threefold greater insulin response to oral glucose than F344 males. Preservation of the DIO and glucose intolerance traits through successive backcrosses and inbreeding cycles to produce the F.DIO strain lends further support to the idea that they inherited in a polygenic fashion.

On low-fat chow diet, rats prone to diet-induced obesity (DIO) have increased arcuate nucleus neuropeptide Y (NPY) expression but similar leptin levels compared with diet-resistant (DR) rats (19). Here, body weight and leptin levels rose in DIO rats, and they defended their higher body weight after only 1 wk on a 31% fat high-energy (HE) diet. However, DIO NPY expression did not fall to DR levels until 4 wk when plasma leptin was 168% of DR levels. When switched to chow, DIO rats lost carcass fat (18). By 10 wk, leptin levels fell to 148% and NPY expression again rose to 150% of DR levels. During 4 wk of food restriction, DIO leptin fell by approximately 50% while NPY increased by 30%. While both returned to control levels by 8 wk, DIO rats still regained all lost weight when fed ad libitum. Finally, the anorexic effect of intracerebroventricular leptin (10 microg) was inversely correlated with subsequent 3-wk weight gain on HE diet. Thus NPY expression and food intake are less sensitive to the leptin's suppressive effects in DIO rats. While this may predispose them to develop DIO, it does not fully explain their defense of a higher body weight on HE diet.

Amylin acts acutely via the area postrema to reduce food intake and body weight, but it also interacts with leptin over longer periods of time, possibly via the ventromedial hypothalamus (VMH), to increase leptin signaling and phosphorylation of STAT3. We postulated that amylin enhances VMH leptin signaling by inducing interleukin (IL)-6, which then interacts with its gp130 receptor to activate STAT3 signaling and gene transcription downstream of the leptin receptor. We found that components of the amylin receptor (RAMPs1–3, CTR1a,b) are expressed in cultured VMH astrocytes, neurons, and microglia, as well as in micropunches of arcuate and ventromedial hypothalamic nuclei (VMN). Amylin exposure for 5 days increased IL-6 mRNA expression in VMH explants and microglia by two- to threefold, respectively, as well as protein abundance in culture supernatants by five- and twofold, respectively. Amylin had no similar effects on cultured astrocytes or neurons. In rats, 5 days of amylin treatment decreased body weight gain and/or food intake and increased IL-6 mRNA expression in the VMN. Similar 5-day amylin treatment increased VMN leptin-induced phosphorylation of STAT3 expression in wild-type mice and rats infused with lateral ventricular IgG but not in IL-6 knockout mice or rats infused with ventricular IL-6 antibody. Lateral ventricular infusion of IL-6 antibody also prevented the amylin-induced decrease of body weight gain. These results show that amylin-induced VMH microglial IL-6 production is the likely mechanism by which amylin treatment interacts with VMH leptin signaling to increase its effect on weight loss.

The brain contains a subpopulation of glucosensing neurons that alter their firing rate in response to elevated glucose concentrations. In pancreatic beta-cells, glucokinase (GK), the rate-limiting enzyme in glycolysis, mediates glucose-induced insulin release by regulating intracellular ATP production. A similar role for GK is proposed to underlie neuronal glucosensing. Via in situ hybridization, GK mRNA was localized to hypothalamic areas that are thought to contain relatively large populations of glucosensing neurons (the arcuate, ventromedial, dorsomedial, and paraventricular nuclei and the lateral area). GK also was found in brain areas without known glucosensing neurons (the lateral habenula, the bed nucleus stria terminalis, the inferior olive, the retrochiasmatic and medial preoptic areas, and the thalamic posterior paraventricular, interpeduncular, oculomotor, and anterior olfactory nuclei). Conversely, GK message was not found in the nucleus tractus solitarius, which contains glucosensing neurons, or in ependymal cells lining the third ventricle, where others have described its presence. In the arcuate nucleus, >75% of neuropeptide Y-positive neurons also expressed GK, and most GK+ neurons also expressed KIR6.2 (the pore-forming subunit of the ATP-sensitive K+ channel). The anatomic distribution of GK mRNA was confirmed in micropunch samples of hypothalamus via reverse transcription-polymerase chain reaction (RT-PCR). Nucleotide sequencing of the recovered PCR product indicated identity with nucleotides 1092-1411 (within exon 9 and 10) of hepatic and beta-cell GK. The specific anatomic localization of GK mRNA in hypothalamic areas known to contain glucosensing neurons and the coexpression of KIR6.2 and NPY in GK+ neurons support a role for GK as a primary determinant of glucosensing in neuropeptide neurons that integrate multiple signals relating to peripheral energy metabolism.

Half of Sprague-Dawley rats develop and defend diet-induced obesity (DIO) or diet resistance (DR) when fed a high-energy (HE) diet. Here, adult male rats were made DIO or DR after 10 wk on HE diet. Then half of each group was food restricted for 8 wk on chow to maintain their body weights at 90% of their respective baselines. Rate and magnitude of weight loss were comparable, but maintenance energy intake and the degree of sympathetic activity (24-h urine norepinephrine) inhibition were 17 and 29% lower, respectively, in restricted DR than DIO rats. Restricted DIO rats reduced adipose depot weights, plasma leptin, and insulin levels by 35%. Restricted DR rats reduced none of these. When fed ad libitum, both DR and DIO rats returned to the body weights of their respective chow-fed phenotype controls within 2 wk. This was associated with increased adipose mass and leptin and insulin levels only in DIO rats. Thus DR rats appear to alter primarily their lean body mass, whereas DIO rats primarily alter their adipose mass during chronic caloric restriction and refeeding.

Both high and low affinity sulfonylurea receptors (SURs) reside on glucose responsive neurons where they influence cell firing and neurotransmitter release via the adenosinetriphosphate (ATP)-sensitive K + (katp) channel. Here, the effect of diabetes on [ 3 H] glyburide binding to SURs was assessed in male obesity-resistant Sprague-Dawley rats rendered diabetic with streptozotocin (65 mg/kg, i.p.). Additional streptozotocin-treated rats were supplemented with insulin (1.5 U/kg/ day). Streptozotocin reduced plasma insulin to 13% of control associated with hyperglycemia (25.3 ± 1.7 mmol/1), while insulin lowered plasma glucose (9.56 ± 1.78 mmol/l) to near control levels (7.65 ± 0.22 mmol/l). Over 7 days, all streptozotocin-treated rats lost 12% of their initial body wt. while controls gained 1%. Despite equivalent wt. loss, streptozotocin-induced diabetes selectively increased high affinity [ 3 H] glyburide binding in the hypothalamic dorsomedial nuclei (DMN) and ventromedial nuclei (VMN) and lateral area (LH). This was prevented by insulin injections. Low affinity binding was similarly increased in the DMN and VMN, as well as two amygdalar subnuclei but decreased in the substantia nigra, pars compacta. Insulin fully prevented these changes only in the DMN and one amygdalar nucleus and the substantia nigra. Therefore, binding to (SURs) appears to be generally upregulated in the face of hypoinsulinemia with hyperglycemia and this is prevented by insulin treatment. These and other data suggest that this combination of abnormalities in diabetes should have an adverse effect on the glucose sensing capacity of the brain.

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.

The model of lateral, rigid impact traumatic brain injury is widely used but remains relatively poorly characterized by comparison with fluid percussion injury models. Thus, whilst the gross morphological changes that occur over the short- and long-term post-injury have been described, more subtle measures of neuronal injury and activation, and markers of axonal and glial reactions have not been investigated, complicating interpretation of data from this model. To address this issue, a variety of neurohistological markers were examined in adult male rats which had been subjected to open brain, lateral rigid impact injury. A piston device was unilaterally driven 3.0 mm into the somatosensory cortex at a speed of 3.2 m/s. Neuronal activation evidenced by Fos-like immunoreactivity showed a complex pattern at 3 h after injury which appeared to be related both to proximity to the impact site and cortical efferent connectivity. At 24 h after injury, acid fuchsin staining demonstrated dying neurons in the margin of the injury and in ipsilateral hippocampus and dorsal thalamus. Injured cells identified by heat-shock protein immunoreactivity showed a similar distribution. Axonal injury demonstrated with 68 kDa neurofilament immunoreactivity was more widely distributed. Less axonal damage was found with increasing distance from the injury site. At 7 days post-injury, glial fibrillary acidic protein immunoreactive astrocytes were prolific in the ipsilateral thalamus, hippocampus and striatum and throughout the injured cortex. In general, controlled, lateral rigid impact injury provides a more focused injury than is seen with lateral fluid percussion which may have implications for the behavioral deficits seen in this injury model.

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.

Norepinephrine (NE) has been implicated in recovery of function following traumatic brain injury (TBI). While bilateral decreases in brain NE turnover occur at 6–24 h after TBI, it is unknown what effects unilateral TBI might have on brain NE turnover over the first few minutes after injury. Here male Sprague-Dawley rats had unilateral contusions of either the right or left somatosensory cortex produced by an air driven piston. At 30min after TBI, brain NE turnover was assessed by measuring the ratio of 3-methoxy-4-hydroxyphenylglycol (MHPG) to NE levels in various brain regions. Both right and left TBI produced 32–103% increases in NE turnover at the injury site and in the ipsilateral cerebral cortex surrounding, rostral and caudal to the injury as compared to the contralateral, uninjured site or to the homologous sites in uninjured controls. NE turnover was also altered selectively in some brain areas not affected by right TBI. Left TBI decreased NE turnover by 29% in the frontal cortex contralateral to the injury and by 24% bilaterally in the hypothalamus while increasing locus coeruleus NE turnover by 72% compared to uninjured controls. Thus, unilateral cortical TBI produced predominantly ipsilateral increases in cortical NE turnover but variable, bilateral changes in NE turnover in subcortical areas which were dependent upon the side of injury. These subcortical differences may explain some of the lateralized effects of cortical injury on post-injury behavior.

To discover hypothalamic genes that might play a role in regulating energy balance, we carried out a microarray screen for genes induced by a 48-h fast in male C57Bl/6J mouse hypothalamus. One such gene was Fkbp51 (FK506 binding protein 5; Locus NP_034350). The product of this gene is of interest because it blocks glucocorticoid action, suggesting that fasting-induced elevation of this gene in the hypothalamus may reduce glucocorticoid negative feedback, leading to elevated glucocorticoid levels, thus promoting obese phenotypes. Subsequent analysis demonstrated that a 48-h fast induces Fkbp51 in ventromedial, paraventricular, and arcuate hypothalamic nuclei of mice and rats. To assess if hypothalamic Fkbp51 promotes obesity, the gene was transferred to the hypothalamus via an adeno-associated virus vector. Within 2 wk following Fkbp51 overexpression, mice on a high-fat diet exhibited elevated body weight, without hyperphagia, relative to mice receiving the control mCherry vector. Body weight remained elevated for more than 8 wk and was associated with elevated corticosterone and impaired glucose tolerance. These studies suggest that elevated hypothalamic Fkbp51 promotes obese phenotypes.

The effect of right sensorimotor traumatic brain injury (TBI) in male Sprague-Dawley rats on brain norepinephrine (NE) turnover was assessed by measuring the decline of endogenous NE levels following tyrosine hydroxylase inhibition produced with α-methyl-p-tyrosine. Right sensorimotor cortex contusions were produced by a pneumatically driven piston which depressed the dural surface by 2 mm at 3.2 m/s. TBI rats were compared to uninjured, anesthetized controls at 6 h and 24 h after surgery. While NE turnover was not affected at the lesion site at 6 h after TBI, it was either abolished or decreased by 33–75% bilaterally in the hypothalamus and in the cerebral cortex surrounding and rostral to the lesion site. In the cortex caudal to the lesion site, NE turnover was completely abolished. NE turnover in cerebral cortex opposite the lesion site and in the contralateral cerebellum was decreased by 51 and 43%, respectively, at 6 h. At 24 h, NE turnover was either abolished or decreased bilaterally by 45–92% in all cortical areas, in the hypothalamus, cerebellum, locus coeruleus and medulla. Thus, right sensorimotor cortex contusion causes a marked, early and widespread depression of brain NE turnover. Since amphetamine increases NE turnover, this may explain the dramatic improvement in behavioral deficits which occurs following amphatamine administration at 24 h after such lesions.

Norepinephrine (NE) has been implicated in cerebral plasticity and recovery of function after brain injury. To examine the status of noradrenergic mechanisms in the brain following traumatic brain injury (TBI), male Sprague-Dawley rats underwent right sensorimotor cortex contusions and were observed for the next 30 days for recovery of motor function by measurement of the time taken to perform a modified beam walking task! At 30 days, their brains were assayed by receptor autoradiography for αr- and α2-adrenoceptor binding with 1 nM [3H]prazosin and 1 nM [3H]paraminoclonidine, respectively. One day after contusion, TBI rats took 60% longer to run the beam than sham-lesioned controls. Run times were directly proportional (r = 0.784; P = 0.012) to lesion volume determined at 30 days. The motor deficit persisted for 8 days, after which TBI and control rats had similar run times, largely due to increased run times in sham rats. At 30 days, TBI rats had a generalized, bilateral decrease in [3H]prazosin binding across all brain areas read (F[l,13] = 9.23; P = 0.009) with specific 12%-21% decreases in the cortex contralateral to the lesion and bilaterally in the dorsomedial hypothalamic and three thalamic nuclei. On the other hand, [3H]paraminoclonidine binding did not differ from sham lesion controls in any brain area of TBI rats. Thus, unilateral TBI is followed by widespread, bilateral changes in α1-adrenoceptor binding which would leave the animal vulnerable to any factors which reduced the access of NE to its postsynaptic adrenoceptors. This is compatible with the observation that α1-antagonists and α2-agonists can transiently reinstate the motor deficit after recovery has occurred.

Plastic change occurs in the adult rat barrel receptor complex following peripheral deafferentation by removal of facial vibrissae (vibrissectomy) and can be prevented by prior depletion of brain norepinephrine. Growth-associated protein (GAP-43, B50, F1, pp46), a marker for synaptic reorganization, increases in the barrel cortex of adult rats following both peripheral and central deafferentation. Here we followed changes in GAP-43 mRNA expression in the barrel receptor system following vibrissectomy. Adult rats had unilateral total vibrissectomy with sparing of the central (C3) vibrissa. By in situ hybridization, GAP-43 mRNA first increased at 24h (9%, P < 0.05) in the ipsilateral trigeminal complex. Levels remained elevated (up to 25% of the unlesioned side) over the next 6 days, decreased to 88% at 7 days and returned to control levels at 14 days. Contralateral barrel cortex levels of GAP-43 mRNA increased by 14% at 4-5 days remained elevated through 7 days and returned to control levels by 14 days. Increased GAP-43 mRNA levels 6 days after vibrissectomy were reproduced by complete transection of the infraorbital nerve and were blocked by depletion of brain norepinephrine. No change occurred in ventrobasal thalamus GAP-43 mRNA at any time. Dot blot and Northern blot hybridizations of GAP-43 mRNA after vibrissectomy showed a 43% increase in the ipsilateral trigeminal complex and a 16% increase in the contralateral barrel cortex at 3 days and an 84% increase in ipsilateral trigeminal and 50% increase in contralateral barrel cortex GAP-43 mRNA at 6 days, respectively. Thus, deafferentation-induced plasticity in the barrel pathway depends upon norepinephrine and is associated with increase in both GAP-43 mRNA and protein suggesting that this may involve a structural change.

The present studies aimed to identify mechanisms contributing to amylin/leptin synergy in reducing body weight and adiposity. We reasoned that if amylin/leptin harnessed complementary neuronal pathways, then in the leptin-sensitive state, amylin should augment leptin signaling/binding and that in the absence of endogenous amylin, leptin signaling should be diminished. Amylin (50 μg/kg, ip) amplified low-dose leptin-stimulated (15 μg/kg, ip) phosphorylated signal transducer and activator of transcription-3 signaling within the arcuate nucleus (ARC) in lean rats. Amylin (50 μg/kg · d) or leptin (125 μg/kg · d) infusion to lean rats decreased 28-d food intake (14 and 10%, respectively), body weight (amylin by 4.3%, leptin by 4.9%), and epididymal fat (amylin by 19%, leptin by 37%). Amylin/leptin co-infusion additively decreased food intake (by 26%) and reduced body weight (by 15%) and epididymal fat (by 78%; all P < 0.05 vs. all groups) in a greater than mathematically additive manner, consistent with synergy. Amylin increased leptin binding within the ventromedial hypothalamus (VMN) by 35% and dorsomedial hypothalamus by 47% (both P < 0.05 vs. vehicle). Amylin/leptin similarly increased leptin binding in the VMN by 40% and ARC by 70% (P < 0.05 vs. vehicle). In amylin-deficient mice, hypothalamic leptin receptor mRNA expression was reduced by 50%, leptin-stimulated phosphorylated signal transducer and activator of transcription-3 within ARC and VMN was reduced by 40%, and responsiveness to leptin’s (1 mg/kg · d for 28 d) weight-reducing effects was attenuated (all P < 0.05 vs. wild-type controls). We suggest that amylin/leptin’s marked weight- and fat-reducing effects are due to activation of intrinsic synergistic neuronal signaling pathways and further point to the integrated neurohormonal therapeutic potential of amylin/leptin agonism in obesity.

Both genetic and environmental factors contribute to individual differences in body weight regulation. The present study examined a possible role for the dendritic arbor of hypothalamic ventromedial nucleus (VMH) neurons in a model of diet-induced obesity (DIO) in male rats. Rats were screened and selectively bred for being either susceptible, i.e., exhibiting DIO, or diet resistant (DR) when exposed to a 31% fat diet. A 2 × 2 experimental design was used, based on these two strains of rats and exposure to rat chow versus the 31% fat diet for seven weeks. Golgi-impregnated neurons were measured for soma size and dendrite parameters, including number, length, and direction. As previously observed, each VMH neuron had a single long primary dendrite. Genetic background and diet did not affect soma size or the number of dendrites of VMH neurons. However, genetic background exerted a main effect on the length of the long primary dendrites. In particular, the long primary dendrites were approximately 12.5% shorter on the VMH neurons in the DIO rats compared with DR rats regardless of diet. This effect was isolated to the long primary dendrites extending in the dorsolateral direction, with these long primary dendrites 19% shorter for the DIO group compared with the DR group. This finding implicates the connectivity of the long primary dendrites on VMH neurons in the control of energy balance. The functional significance of these shortened dendrites and their afferents warrants further study.

We assessed the mechanisms by which specialized hypothalamic ventromedial nucleus (VMN) neurons utilize both glucose and long-chain fatty acids as signaling molecules to alter their activity as a potential means of regulating energy homeostasis. Fura-2 calcium (Ca2+) and membrane potential dye imaging, together with pharmacological agents, were used to assess the mechanisms by which oleic acid (OA) alters the activity of dissociated VMN neurons from 3- to 4-wk-old rats. OA excited up to 43% and inhibited up to 29% of all VMN neurons independently of glucose concentrations. In those neurons excited by both 2.5 mM glucose and OA, OA had a concentration-dependent effective excitatory concentration (EC50) of 13.1 nM. Neurons inhibited by both 2.5 mM glucose and OA had an effective inhibitory concentration (IC50) of 93 nM. At 0.5 mM glucose, OA had markedly different effects on these same neurons. Inhibition of carnitine palmitoyltransferase, reactive oxygen species formation, long-chain acetyl-CoA synthetase and ATP-sensitive K+channel activity or activation of uncoupling protein 2 (UCP2) accounted for only ∼20% of OA's excitatory effects and ∼40% of its inhibitory effects. Inhibition of CD36, a fatty acid transporter that can alter cell function independently of intracellular fatty acid metabolism, reduced the effects of OA by up to 45%. Thus OA affects VMN neuronal activity through multiple pathways. In glucosensing neurons, its effects are glucose dependent. This glucose-OA interaction provides a potential mechanism whereby such “metabolic sensing” neurons can respond to differences in the metabolic states associated with fasting and feeding.

The hypothesis that fetal tissue grafts may exert a trophic influence on damaged catecholaminergic fibers was examined. Ascending dopamine and norepinephrine axons normally innervate frontal cortex targets in the intact rat brain. These and other ascending catecholaminergic fibers were disrupted with stereotaxic injections of 6-hydroxydopamine into the medial forebrain bundle (mfb), followed after 1 or 14 days by grafts of fetal neocortical tissue placed into the injection site, or by sham grafts. Glyoxylic acid histofluorescence techniques were then used to examine catecholaminergic fiber distribution. When such lesions were made without subsequent grafting, virtually no growth of catecholaminergic fibers occurred beyond the injection site and frontal cortex norepinephrine levels were depleted to 15% of control levels. However, when grafts of fetal neocortical tissue were made into the lesion site and animals examined 3 months later, catecholaminergic fibers grew through the lesion site to ramify within the graft tissue. Catecholaminergic fibers were seen in all portions of most grafts, though they were most dense on the caudal and ventral edges of the graft, close to the path of the mfb. Similar densities of graft innervation were seen 3 months after animals received grafts placed into the same site without prior lesioning of catecholaminergic fibers. Fetal neocortical grafts thus induce collateral sprouting from intact host catecholaminergic axons and may also promote regenerative sprouting when such fibers are otherwise irreparably damaged.

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 ([Ca2+]i) as an index of neuronal activity. At 0.1, 1, 10 fmol/l leptin, ∼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.

Neurons in the ventromedial and arcuate hypothalamic nuclei (VMN and ARC, respectively) mediate many of leptin’s effects on energy homeostasis. Some are also glucosensing, whereby they use glucose as a signaling molecule to regulate their firing rate. We used fura-2 calcium (Ca 2 ) imaging to determine the interactions between these two important mediators of peripheral metabolism on individual VMN neurons and the mechanisms by which leptin regulates neuronal activity in vitro. Leptin excited 24%, inhibited 20%, and had a biphasic response in 10% of VMN neurons. Excitation occurred with a EC50 of 5.2 fmol/liter and inhibition with a IC50 of 4.2 fmol/liter. These effects were independent of the ambient glucose levels, and both glucosensing and non-glucosensing neurons were affected by leptin. In contrast, the ARC showed a very different distribution of leptin-responsive neurons, with 40% leptin excited, 10% leptin inhibited, and 2% having a biphasic response ( 2 60.2; P < 0.0001). Using pharmacological manipulations we found that leptin inhibits VMN neurons via activation of phosphoinositol-3 kinase and activation of the ATP-sensitive K channel. In addition, leptin inhibition was antagonized by 5-AMP-activated protein kinase activation in 39% of neurons but was unaffected by 5AMP-activated protein kinase inhibition. No mechanism was delineated for leptin-induced excitation. Thus, within the physiological range of brain glucose levels, leptin has a differential effect on VMN vs. ARC neurons, and acts on both glucosensing and non-glucosensing VMN neurons in a glucose-independent fashion with inhibition primarily dependent upon activation of the ATP-sensitive K channel. (Endocrinology 149: 5146–5154, 2008)

Horseradish peroxidase was injected unilaterally into the optic tectum of the channel catfish, Ictalurus punctatus. The sources of tectal afferents were thereby revealed by retrogradely labeled neurons in various brain centers. Retrogradely labeled cells were seen in both the ipsilateral and contralateral telencephalon. The superficial pretectal area was labeled on both sides of the brain. Ipsilateral projections were also observed coming from the entopeduncular nucleus. Both the anterior thalamic nucleus and the ventro-medial thalamic nucleus projected to the ipsilateral optic tectum. Cells in the ipsilateral nucleus of the posterior commissure were seen to project to the tectum. Labeled fibers were visualized in the lateral geniculate nucleus ipsilateral to the injected tectum, however, no labeled cell bodies were observed. Therefore, tectal cells project to the lateral geniculate nucleus, but this projection is not reciprocal. No labeled cells were found in the cerebellum. Labeled cells occurred in both the ipsilateral and contralateral medial reticular formation; they were also observed in the ipsilateral nucleus isthmi. A projection was seen coming from the dorsal funicular nucleus. Furthermore, labeled cells were shown in the inferior raphe nucleus.