Melanocortin activity in the amygdala controls appetite for dietary fat.

The amygdala is rich in melanocortin 4 receptors. Because the reduction in dietary fat intake after enterostatin is injected in the central nucleus of the amygdala (CeA) is blocked by a melanocortin 4 receptor antagonist, we investigated the role of melanocortin activity in the CeA in regulating food intake and macronutrient choice. Sprague-Dawley rats, fitted with CeA cannulas, were fed either chow, a high-fat (HF) diet, or adapted to a two-choice HF or low-fat (LF) diet. Injections of the MC4R agonist melanotan II (MTII) in the CeA had a dose-dependent inhibitory effect on food intake that lasted for at least 24 h. This response was greater in rats fed a HF diet. The inverse agonist agouti-related protein (AgRP) and antagonist SHU-9119 increased food intake in a dose-dependent manner, with the hyperphagia lasting for 60 h. In rats adapted to a two-choice HF/LF diet, MTII decreased HF consumption but had no effect on LF consumption, resulting in a long-lasting decrease in total calorie intake (-35.5% after 24 h, P < 0.05). Total calorie intake increased in both AgRP- and SHU-9119-treated rats (32 and 109% after 24 h, respectively) as the result of increased intake of HF diet. There was no modification of LF consumption with AgRP treatment and a transient nonsignificant decrease with SHU-9119 treatment. Amygdala brain-derived neurotrophic factor expression was increased by AgRP in fed rats. These results identify the amygdala as a site of action for the melanocortin system to control food intake and dietary preferences.

High-fat diets induce a rapid loss of the insulin anorectic response in the amygdala.

Intracerebroventricular insulin decreases food intake (FI). The central bed nucleus of the amygdala (CeA), as other regions of the brain regulating feeding behavior, expresses insulin receptors. Our objectives were to show an insulin anorectic response in the amygdala, study the effect of high-fat diets on this response, and map the neural network activated by CeA insulin using c-Fos immunohistochemistry. Sprague-Dawley (SD) rats fitted with unilateral CeA cannulas were adapted to a low-fat (LFD) diet before they were fed a high-fat diet (HFD). Their feeding response to CeA saline or insulin (8 mU) was tested after 24 h, 72 h, or 7 days of being on a HFD. In a second experiment, SD rats were fed the HFD for 3, 7, or 49 days and were then refed with the LFD. They were tested for their insulin response before and after an HFD and every 3 days for the following weeks. Insulin tolerance tests were performed in a parallel group of rats. The CeA insulin stimulation c-Fos expression was studied to identify the distribution of activated neuronal populations. Feeding an HFD for 72 h or more induced a CeA, but not peripheral, insulin resistance, which was slowly reversed by LFD refeeding. The duration of HFD feeding determined the time frame for reversal of the insulin resistance. CeA insulin increased c-Fos in multiple brain regions, including the arcuate nucleus/paraventricular nucleus region of the hypothalamus. We conclude that the amygdala may be an important site for insulin regulation of food intake and may have a significant role in determining susceptibility to HFD-induced obesity.

Enterostatin affects cyclic AMP and ERK signaling pathways to regulate Agouti-related protein (AgRP) expression.

Enterostatin, a gut-brain peptide, inhibits dietary fat intake in rats. The purpose of this study was to identify the intracellular signaling pathways that are responsive to enterostatin and that modulate the effects of enterostatin on the expression of Agouti-related protein (AgRP). We used the hypothalamic GT1-7 neuronal cell line to identify the effects of enterostatin on cyclic AMP and ERK signaling using conventional immunoassays or Western blots to assay the activity of these pathways. Enterostatin enhanced the level of cyclic AMP, PKA(RIIbeta) and phospho-CREB and increased pERK levels in GT 1-7 cells. The effects on pERK were rapid (7.5 min) and dose-dependent. These signaling responses were blocked by an antibody to the enterostatin receptor (beta subunit of F1-ATPase), by the pERK inhibitor U0126 and by the P2Y receptor antagonist Suramin. Enterostatin showed a biphasic effect on AgRP mRNA, initially increasing but subsequently decreasing the levels. The cyclic AMP activator Sp-cAMP increased AgRP mRNA expression. Transfection of a wild type ERK construct reduced AgRP mRNA levels. Enterostatin inhibited expression of Krüppel-like factor 4 (KLF4), a transcriptional regulator of AgRP. KLF4 gene expression was increased by Sp-cAMP but decreased by wild-type ERK expression. U0126 blocked the effect of enterostatin on KLF4 expression. We conclude that enterostatin binding to its receptor activates the pERK pathway to inhibit AgRP gene expression but may enhance AgRP expression through activation of the cyclic AMP pathway. These pathways probably mediate the enterostatin inhibition of dietary fat intake.

Leucine regulation of glucokinase and ATP synthase sensitizes glucose-induced insulin secretion in pancreatic beta-cells.

We have recently shown that leucine culture upregulates ATP synthase beta-subunit (ATPSbeta) and increases ATP level, cytosolic Ca(2+), and glucose-induced insulin secretion in rat islets. The aim is to test whether glucokinase expression is also affected in rat islets and its role in glucose sensitization during leucine culture. Leucine culture increased glucose-induced NAD(P)H level at 1 and 2 days but not at 1 week. The half-maximal effective concentration of the glucose response curve for NAD(P)H was left-shifted from 5-7 to 2-3 mmol/l. The effect was dose dependent and rapamycin insensitive. Leucine culture did not affect glyceraldehyde effects on NAD(P)H. Leucine pretreatment for 30 min had no effects on NAD(P)H levels. Leucine culture for 2 days also increased glucose-induced cytosolic Ca(2+) elevation, ATP level, and insulin secretion. Leucine increase of glucokinase mRNA levels occurred as early as day 1 and lasted through 1 week. That of ATPSbeta did not occur until day 2 and lasted through 1 week. Leucine effects on both mRNAs were dose dependent. The upregulation of both genes was confirmed by Western blotting. Leucine culture also increased glucose-induced insulin secretion, ATP level, glucokinase, and ATPSbeta levels of type 2 diabetic human islets. In conclusion, leucine culture upregulates glucokinase, which increases NAD(P)H level, and ATPSbeta, which increases oxidation of NADH and production of ATP. The combined upregulation of both genes increases glucose-induced cytosolic Ca(2+) and insulin secretion.

Enterostatin inhibition of dietary fat intake is modulated through the melanocortin system.

Enterostatin injected into the amygdala selectively reduces dietary fat intake by an action that involves a serotonergic component in the paraventricular nucleus. We have investigated the role of melanocortin signaling in the response to enterostatin by studies in melanocortin 4 receptor (MC4R) knock out mice and by the use of the MC4R and MC3R antagonist SHU9119, and by neurochemical phenotyping of enterostatin activated cells. We also determined the effect of enterostatin in vivo on the expression of AgRP in the hypothalamus and amygdala of rats and in culture on a GT1-7 neuronal cell line. Enterostatin had no effect on food intake in MC4R knock out mice. SHU9119 i.c.v. blocked the feeding response to amygdala enterostatin in rats. Amygdala enterostatin induced fos activation in alpha-melanocyte stimulating hormone (alpha-MSH) neurons in the arcuate nucleus. Enterostatin also reduced the expression of AgRP in the hypothalamus and amygdala and in GT1-7 cells. These data suggest enterostatin inhibits dietary fat intake through a melanocortin signaling pathway.

Neuropeptide Y administration into the amygdala alters high fat food intake.

The orexigenic effects of neuropeptide Y (NPY) are mediated through the hypothalamus, while the anxiolytic effects of NPY appear to be mediated through the amygdala. We hypothesized that intra-amygdalar administration of NPY might alter food preference without changing total food intake. Neuropeptide Y was administered into the central nucleus of the amygdala in both satiated and overnight-fasted rats, and intake and preference for a high fat diet (56%)/low carbohydrate (20%) diet or a low fat (10%)/high carbohydrate (66%) diet were measured. Intra-amygdalar NPY administration in satiated rats did not change total caloric intake, but it did produce a dose-dependent decrease in intake of and preference for high fat diet relative to low fat diet over 24 h. In overnight-fasted rats, intra-amygdalar NPY also decreased the intake and preference for a high fat diet relative to low fat diet over 24 h, without altering total caloric intake. Intra-amygdalar NPY administration did not produce conditioned taste aversions to a novel saccharin solution. These results suggest that amygdalar NPY may have a role in macronutrient selection, without altering total caloric intake.

Increased expression of mu opioid receptors in animals susceptible to diet-induced obesity.

Stimulation of mu opioid receptors preferentially increases the intake of a high fat diet. In this paper we investigated whether there was a difference in the expression of mu opioid receptors between animals susceptible (Osborne-Mendel) or resistant (S5B/Pl) to obesity induced by eating a high fat diet. Immunohistochemical studies demonstrated that Osborne-Mendel rats eating a chow diet had an increased number of mu opioid receptors in the arcuate nucleus when compared to S5B/Pl rats. These immunohistochemical findings were supported by Real Time-PCR which demonstrated that the mRNA level of mu opioid receptors was also increased in the hypothalamus of Osborne-Mendel rats compared to S5B/Pl rats. Low doses of the mu opioid receptor agonist DAMGO [d-Ala(2)-N-Me-Phe(4)-Glycol(5)]-enkephalin administered to Osborne-Mendel rats caused a significant increase in the preference for a diet high in fat. The same doses of DAMGO switched the diet preference of S5B/Pl rats to high fat but did not significantly increase food intake. The combination of these findings suggests that the increased levels of hypothalamic mu opioid receptors in Osborne-Mendel rats may contribute to their preference for a diet high in fat and increase their susceptibility to becoming obese.

Procolipase gene expression in the rat brain: source of endogenous enterostatin production in the brain.

Enterostatin is a pentapeptide released from its precursor protein procolipase, which is synthesized in the exocrine pancreas and gastric mucosa. As central injection of enterostatin has potent effects on feeding, we hypothesized that the procolipase may also be expressed in the brain. We confirmed the presence of preprocolipase gene expression in amygdala by reverse transcription-polymerase chain reaction and Northern blot analysis and of protein expression by Western blots. Immunohistochemical analysis using antibodies for procolipase and enterostatin identified their immunoreactivity (IR) in rat brain. Procolipase IR was present in the cytoplasm of paraventricular, amygdala, and the dorsal thalamus nucleus. Enterostatin IR was evident in the fibers of the dorsal thalamus and arcuate nucleus. In vivo injection of enterostatin antibody into rat amygdala increased food intake. These data suggest that procolipase and enterostatin are synthesized within specific regions of the brain that function in the regulation of food intake centrally.

Pharmacological characterization of alpha1- and beta-adrenergic synergism of 5'DII activity in rat brown adipocytes.

The role of adrenoceptor subtypes was studied in rat brown adipose tissue (BAT). The type II 5'-deiodinase (5'DII) was activated in response to simultaneous stimulation by beta3- and alpha1-adrenergic agonists, BRL 37344 or CGP 12177, and cirazoline, in brown adipocytes. Inhibition of the alpha1- and beta-adrenergic phenylephrine-stimulated 5'DII activity was obtained by the alpha1-adrenergic antagonists in the order of prazosin >/= wb 4101 > 5-methylurapidil. In comparison, the binding of [3H]prazosin to rat BAT plasma membranes was inhibited by alpha1-adrenergic antagonists in the order of prazosin > WB 4101 = benoxathian > 5-methylurapidil. Although the order of the alpha1-adrenergic competition seemed to be rather typical for the alpha1B-adrenergic receptors, a molecular analysis on adrenoceptor mRNAs should be made to confirm the exact alpha1-adrenergic subtypes at the level of brown adipocytes, since the possibility of a mixture of different receptor subtypes in brown fat cells and/or tissue may interact with the pharmacological characterization. Thus, specific alpha1- and beta-adrenoceptor subtypes participate in the regulation of 5'DII activity in the rat brown adipocytes, and therefore, an impaired alpha1- and beta-adrenergic co-work may be involved in a defective BAT function, e.g., in obese Zucker rats, too. An interesting possibility is that the decreased number of alpha1-adrenoceptors in the BAT of obese Zucker rats is due to the decrease in the alpha1B-adrenoceptor subtype which would further be involved especially in the regulation of BAT 5'DII activity.

Effects of altered amygdalar neuropeptide Y expression on anxiety-related behaviors.

Neuropeptide Y (NPY) decreases anxiety-related behaviors in various animal models of anxiety. The purpose of the present study was to examine the role of the amygdalar NPY system in anxiety-related responses in the elevated plus maze. The first experiment determined if herpes virus-mediated alterations in amygdalar NPY levels would alter anxiety-related behaviors in the elevated plus maze. Viral vectors encoding NPY, NPY antisense, or LacZ (control virus) were bilaterally injected into the amygdala, and 4 days postinjection, rats were tested in the elevated plus maze test. NPY-like immunoreactivity (NPY-ir) was measured in the amygdala of these rats. In rats injected with the viral vector encoding NPY, reduced anxiety-related behaviors in the elevated plus maze accompanied by moderate increases in NPY-ir were detected compared to NPY-antisense viral vector-treated subjects. Elevated plus maze behavior did not differ compared to LacZ-treated controls. NPY overexpression at this time point was also suggested by enhanced NPY mRNA expression seen in the amygdala 4 days postinjection using real-time polymerase chain reaction analysis. Experiment 2 was conducted to provide further evidence for a role of amygdalar NPY in regulating anxiety-related behaviors in the elevated plus maze test. The nonpeptide NPY Y1 receptor antagonist, BIBP 3226 (1.5 microg/microl), was bilaterally injected into the amygdala and rats were tested in the elevated plus maze test. Rats receiving BIBP 3226 exhibited increased anxiety-related behaviors in this test. The results of these experiments provide further support for the role of amygdalar NPY in anxiety-related behaviors.

5-HT1B receptors modulate the feeding inhibitory effects of enterostatin.

Serotonin (5-HT) is considered to play an important role in control of appetite. Enterostatin has been shown to alter 5-HT release in the brain, and non-specific 5-HT antagonists blocked the anorectic response to icv enterostatin. The aim of this study was to further identify which 5-HT receptor subtype mediates the enterostatin feeding behavior and whether this effect occurs due to action in the PVN. Wild-type and 5-HT2C receptor-/- (KO) mice and normal Sprague-Dawley rats were used in these experiments. All animals were fed a high fat diet. Enterostatin (120 nmol, i.p.) reduced the intake of high fat diet in 5-HT2C receptor mutant mice (saline 4.54 +/- 0.47 kcal vs. Ent 2.53 +/- 0.76 kcal) 1 h after injection. A selective 5-HT1B antagonist (GR55526, 40 mg/kg body weight, i.p.) blocked the enterostatin hypophagic effects in these KO mice. Rats were implanted with cannulas into the amygdala and the ipsilateral PVN. The 5-HT receptor antagonists metergoline (non-specific receptor subtypes 1 and 2), or ritanserin (selective 2C), or GR55562 (selective l B) was injected into the PVN prior to enterostatin (0.01 nmol) injection into the amygdala. Enterostatin reduced food intake (saline: 5.80 +/- 0.59 g vs. enterostatin 3.47 +/- 0.56 g, P < 0.05 at l h). Pretreatment with either metergoline (10 nmol) or GR55526 (10 nmol) but not ritanserin (10 nmol) into the PVN attenuated the anorectic response to amygdala enterostatin. The data imply that the enterostatin anorectic response may be modulated by 5-HT1B receptors and that a neuronal pathway from the amygdala to the PVN regulates the enterostatin response through activation of 5-HTlB receptors in PVN.

Regulation of ghrelin gene expression in stomach and feeding response to a ghrelin analogue in two strains of rats.

Ghrelin is a peptide produced by the stomach and released into the circulation. As a natural ligand of the growth hormone secretagogue (GHS) receptor, it stimulates growth hormone secretion but it also stimulates feeding in humans and rodents. The orexigenic effect of ghrelin has been related to AgRP/NPY and orexin pathways. We proposed that ghrelin might be involved in the susceptibility to diet induced obesity and in the regulation of macronutrient selection. We have investigated these hypotheses in two strains of rat, the Osborne-Mendel (OM) rat that prefers diets high in fat and is sensitive to dietary obesity and the S5B/P1 (S5B) rat that prefers a low fat diet and is resistant to high fat diet induced obesity. OM and S5B rats were adapted to a choice of high fat (HF) and low fat (LF) diet for 2 weeks. GHRP-2, an analogue of ghrelin, was injected intraperitoneally into satiated and 24 h fasted rats at doses of 10, 30 and 90 nmol. Food intake was measured over the next 4 h period. In satiated S5B rats, GHRP-2 stimulated intake of the LF diet in a dose dependent manner but did not affect the intake of the HF diet. In satiated OM rats, 90 nmol of GHRP-2 stimulated HF intake. In contrast, neither fasted OM nor S5B rats increased the intake of either HF or LF diet in response to GHRP-2. Fasting for 18 h induced a large rise in ghrelin mRNA in stomach of OM rats but not in S5B rats. There were no significant differences in plasma total ghrelin. An increase in ghrelin mRNA in stomach immediately before the onset of the dark cycle was observed in OM but not in S5B rats. Active ghrelin level was significantly affected by different feeding conditions in both OM and S5B rats adapted on HF diet with a trend to increase after 48 h of fasting and to decline to basal levels following 10 h of refeeding. These data suggest that ghrelin stimulates the intake of the preferred macronutrient. In addition, a differential regulation of ghrelin gene expression between OM and S5B rats may be important in their differential sensitivity to HF diet-induced obesity.

Comparative study of enterostatin sequence in five rat strains and enterostatin binding proteins in rat and chicken serum.

Enterostatin, a pentapeptide derived from the precursor protein procolipase has been shown to inhibit dietary fat intake and to reduce body fat after chronic administration in rats. We repeat that the enterostatin amino acid sequence from the genomic DNA of 5 different rat strains is APGPR. 125I-APGPR bound to three proteins (300, 205 and 60 kDa) in rat serum and one 60 kDa protein in chicken serum. These serum binding proteins were also eluted by APGPR affinity chromatography. Western blot analysis of serum protein identified enterostatin-like immunoreactivity associated with the same molecular weight bands. Our results demonstrate the enterostatin sequence in rat is APGPR and suggest the presence of enterostatin binding proteins in rat and chicken serum.

The F1-ATPase beta-subunit is the putative enterostatin receptor.

It has been suggested that the F1-ATPase beta-subunit is the enterostatin receptor. We investigated the binding activity of the purified protein with a labeled antagonist, beta-casomorphin1-7, in the absence and presence of cold enterostatin. 125I-beta-casomorphin1-7 weakly binds to the rat F1-ATPase beta-subunit. Binding was promoted by low concentrations of cold enterostatin but displaced by higher concentrations. To study the relationship between binding activity and feeding behavior, we examined the ability of a number of enterostatin analogs to affect beta-casomorphin1-7 binding to the F1-ATPase beta-subunit. Peptides that suppressed food intake promoted beta-casomorphin1-7 binding whereas peptides that stimulated food intake or did not affect the food intake displaced beta-casomorphin1-7 binding. Surface plasmon resonance measurements show that the beta-subunit of F1-ATPase binds immobilized enterostatin with a dissociation constant of 150 nM, where no binding could be detected for the assembled F1-ATPase complex. Western blot analysis showed the F1-ATPase beta-subunit was present on plasma and mitochondrial membranes of rat liver and amygdala. The data provides evidence that the F1-ATPase beta-subunit is the enterostatin receptor and suggests that enterostatin and beta-casomorphin1-7 bind to distinct sites on the protein.

Amygdala enterostatin induces c-Fos expression in regions of hypothalamus that innervate the PVN.

Enterostatin selectively inhibits the intake of the dietary fat after both central and peripheral administration. Our previous studies have shown that a central site of action is the central nucleus of amygdala. Serotonergic agonists administered into the paraventricular nucleus (PVN) inhibit fat intake and serotonergic antagonists block the feeding suppression induced by amygdala enterostatin, suggesting that there are functional connections between the PVN and amygdala that affect the feeding response to enterostatin. Our purpose was to identify the anatomic and functional projections from the amygdala to the PVN and hypothalamic area that are responsive to enterostatin, by using a retrograde tracer fluorogold (FG) and c-Fos expression. Rats were injected with fluorogold unilaterally into the PVN and a chronic amygdala cannula was implanted ipsilaterally. After 10 days recovery, rats were injected with either enterostatin (0.1 nmol) or saline vehicle (0.1 microl) into the amygdala and sacrificed 2 h later by cardiac perfusion under anesthesia. The brains were subjected to dual immunohistochemistry to visualize both FG and c-Fos-positive cells. FG/c-Fos double-labeled cells were found in forebrain regions including the PVN, amygdala, lateral hypothalamus (LH), ventral medial hypothalamus (VMH) and arcuate nucleus (ARC). The data provides the first anatomical evidence that enterostatin activates amygdala neurons that have functional and anatomic projections directly to the PVN and also activates neurons in the arcuate, LH and VMH, which innervate the PVN.

Regulation of feeding behavior, gastric emptying, and sympathetic nerve activity to interscapular brown adipose tissue by galanin and enterostatin: the involvement of vagal-central nervous system interactions.

Galanin and enterostatin, which are distributed in both the central nervous system and the gastrointestinal tract, regulate the feeding behavior. In the first set of experiments, we investigated the effects of galanin and enterostatin, injected into the third ventricle, on food intake, gastric emptying, and the sympathetic activity of nerves innervating interscapular brown adipose tissue in rats. Galanin dose-dependently increased the intake of a high-fat diet after overnight starvation, but it did not affect low-fat diet intake. In contrast, enterostatin suppressed the intake of the high-fat diet, while intake of the low-fat diet was not affected. Galanin significantly and dose-dependently suppressed gastric emptying rate. However, gastric emptying showed no response to enterostatin. Galanin produced a dose-dependent suppression of sympathetic firing rate. In rats fed a high-fat diet, the injection of enterostatin showed a dose-dependent increase in firing rate. In contrast, animals fed a chow diet showed almost no response. In the second set of experiments, we investigated the role of the hepatic vagus nerve in modulating the peripheral response to enterostatin in rats. Intraperitoneal (i.p.) enterostatin reduced the intake of a high-fat diet. Immunohistochemical identification indicated that the Fos protein was present in the nucleus tractus solitarius, and parabrachial, paraventricular, and supraoptic nuclei after IP enterostatin. These responses to i.p. enterostatin were blocked by hepatic vagotomy. These results suggest that galanin and enterostatin coordinate to regulate feeding behavior, gastric emptying, and sympathetic activity to interscapular brown adipose tissue via central and peripheral sites of action, one of which was the interaction which was found to exist through the vagal system.

Effect of a beta-3 agonist on food intake in two strains of rats that differ in susceptibility to obesity.

CONTEXT: Beta-3 agonists acutely reduce food intake, but the mechanism is not well understood. OBJECTIVE: To evaluate the effect of a beta3 agonist on food intake in two strains of rats that differ in their sensitivity to becoming obese while eating a high-fat (HF) diet. METHODS: Male Osborne-Mendel (OM) and S5B/Pl (S5B) rats were treated with a beta3-adrenergic agonist (CL 316,243) at 8 weeks of age, after an adaptation to either an HF (56% fat energy) or a low-fat (LF; 10% fat energy) diet that was equicaloric for protein (24% energy). Ad-lib-fed rats were injected intraperitoneally with CL 316,243, at doses of 0.03, 0.1, 0.3, 1.0 or 3.0 mg/kg, or with vehicle at the beginning of the dark cycle. Food intake was measured at 1, 3, 6 and 24 h after injections. RESULTS: The beta3 agonist CL 316,243 significantly decreased food intake at all timepoints in both strains of rats eating both diets. However, this inhibition of food intake was significantly greater in the S5B rat. CL 316,243 significantly decreased serum leptin and serum glucose in both the OM and the S5B rats, and again, the inhibition was greater in the S5B rat. Whereas CL 316,243 increased serum insulin levels in the OM rat, it decreased them in the S5B rat on an LF diet. In a second experiment, chow-fed rats were implanted with vascular ports into the jugular vein and allowed to recover. When CL 316,243 was injected into the animals that were fasted overnight, rats of both strains significantly increased their serum insulin at 30 min, but the increase was much more pronounced in the S5B rat. Serum glucose was decreased significantly at both the 30- and 60-min timepoints in the OM rat and at 30 min in the S5B rat. CONCLUSION: These experiments demonstrate that a beta3 agonist (CL 316,243) has a much greater effect in a strain of rats that resist fat-induced obesity.

PKCθ over expression in the central nucleus of the amygdala or hypothalamus has differential effects on energy balance and peripheral glucose homeostasis.

The increase in PKCθ expression in the amygdala of rats fed high fat diet (HFD) has been related to the loss of the anorectic response to insulin injections into the central nucleus of the amygdala (CeA) in these animals. PKCθ overexpression in the CeA increases food intake, body weight and body fat and inhibits insulin stimulation of Akt signaling. To study the effects of bilateral overexpression of PKCθ in the CeA of rats on peripheral metabolism, rats were injected into the CeA or 3rd ventricle with a lentiviral (LV)-PKCθ construct or LV-Green fluorescent protein (GFP) construct as a control and fed either LFD or HFD. Insulin and glucose tolerance tests were undertaken and hepatic AMPK activation, Pepck, Srebp1c gene expression and lipid levels assayed. CeA LV-PKCθ injected rats increased food intake, body weight and body fat and increased hepatic, but not serum, triglyceride levels compared to control rats that received a CeA-LV-GFP construct. Hepatic AMP-kinase activity was reduced but expression of Pepck increased while serum insulin decreased, glucose tolerance improved and the hypoglycemic response to insulin was enhanced in CeA LV-PKCθ injected rats. In contrast, rats that received LV-PKCθ injections into the 3rd Ventricle did not show any changes in food intake or body weight although serum, but not hepatic, triglyceride levels were increased and glucose tolerance was impaired. The data suggest that activation of PKCθ in the CeA and hypothalamus have different effects on energy balance and peripheral metabolism and that insulin signaling in the amygdala regulates peripheral metabolism.

The effect of high fat diet and saturated fatty acids on insulin signaling in the amygdala and hypothalamus of rats.

Insulin injections into the central nucleus of the amygdala (CeA) inhibit food intake but this response is lost quickly on feeding a high fat diet. The purpose of the studies described in this manuscript was to identify the potential mechanism for the development of this insulin resistance. High fat diets (HFD) induced PKCθ activation and blocked the stimulation of Akt but not mTOR phosphorylation in the amygdala in response to CeA insulin injections. Infusions of palmitic acid onto the CeA had identical effects to HFD on PKCθ expression and insulin signaling in the amygdala. CeA insulin also induced an increase in Akt phosphorylation in the hypothalamus but had no effect on hypothalamic mTOR phosphorylation. Feeding HFD but not CeA palmitate infusions reversed the hypothalamic Akt signaling response to CeA insulin. These data, which show the independence of Akt and mTOR signaling responses to insulin in the amygdala and the effect of insulin signaling in the CeA on hypothalamic Akt signaling, suggest that the amygdala might also have a significant role in regulating hypothalamic responses to dietary fat.