Neuropeptide Y and leptin sensitivity is dependent on diet composition.

Rats on different free-choice (fc) diets for 1 week of either chow, saturated fat and liquid sugar (fcHFHS), chow and saturated fat (fcHF), or chow and liquid sugar (fcHS) have differential levels of neuropeptide Y (NPY) mRNA in the arcuate nucleus. Because these differences were not explained by plasma leptin levels but did predict subsequent feeding behaviour, in the present study, we first examined whether leptin sensitivity could explain these differences. Second, we focused on the role of NPY on feeding behaviour, and measured NPY mRNA levels and sensitivity to NPY after 4 weeks on the different choice diets. To determine leptin sensitivity, we measured food intake after i.p. leptin or vehicle injections in male Wistar rats subjected to the fcHFHS, fcHS, fcHF or Chow diets for 7 days. Next, we measured levels of arcuate nucleus NPY mRNA with in situ hybridisation in rats subjected to the choice diets for 4 weeks. Finally, we studied NPY sensitivity in rats subjected to the fcHFHS, fcHS, fcHF or Chow diet for 4 weeks by measuring food intake after administration of NPY or vehicle in the lateral ventricle. Leptin decreased caloric intake in rats on Chow, fcHS and fcHF but not in rats on the fcHFHS diet. After 4 weeks, rats on the fcHFHS diet remained hyperphagic, whereas fcHS and fcHF rats decreased caloric intake to levels similar to rats on Chow. By contrast to 1 week, after 4 weeks, levels of NPY mRNA were not different between the diet groups. Lateral ventricle administration of NPY resulted in higher caloric intake in fcHFHS rats compared to rats on the other choice diets or rats on Chow. Our data show that consuming a combination of saturated fat and liquid sugar results in leptin resistance and increased NPY sensitivity that is associated with persistent hyperphagia.

GHS-R1a signaling in the DMH and VMH contributes to food anticipatory activity.

BACKGROUND: Rats that have restricted access to food at a fixed time point of the circadian phase display high levels of food anticipatory activity (FAA). The orexigenic hormone ghrelin has been implicated in the regulation of FAA. However, it is not known via which brain area ghrelin exerts this effect. Growth hormone secretagogue receptor 1a (GHS-R1a) is highly expressed in the hypothalamus, including the dorsomedial hypothalamus (DMH) and the ventromedial hypothalamus (VMH). These two hypothalamic areas have been reported to play a role in FAA. AIM OF THE STUDY: To examine the role of GHS-R1a signaling in the DMH and VMH in FAA. DESIGN: Adeno-associated virus expressing a shRNA directed against GHS-R1a was used to establish local knockdown of GHS-R1a in the DMH and VMH in rats. Rats were subsequently subjected to a restricted feeding schedule (RFS). RESULTS: Under ad libitum conditions, knockdown of GHS-R1a in the VMH increased food intake and body weight gain. In addition, GHS-R1a knockdown in VMH and DMH reduced body temperature and running wheel activity (RWA). When rats were subjected to a RFS, the main effect of GHS-R1a knockdown in both DMH and VMH was a decrease in RWA and an attenuation of body weight loss. Rats with knockdown of GHS-R1a in DMH and VMH showed a delay in onset of FAA. In addition, GHS-R1a knockdown in DMH resulted in a reduction of FAA amplitude. CONCLUSION: This is the first study to investigate the effect of local hypothalamic knockdown of GHS-R1a on FAA. Our results implicate hypothalamic GHS-R1a signaling in the regulation of FAA. Nevertheless, some FAA remained, suggesting that a distributed network of brain areas and signaling pathways is involved in the development of FAA.

A free-choice high-fat high-sugar diet induces glucose intolerance and insulin unresponsiveness to a glucose load not explained by obesity.

OBJECTIVES: In diet-induced obesity, it is not clear whether impaired glucose metabolism is caused directly by the diet, or indirectly via obesity. This study examined the effects of different free-choice, high-caloric, obesity-inducing diets on glucose metabolism. In these free-choice diets, saturated fat and/or a 30% sugar solution are provided in an addition to normal chow pellets. METHOD: In the first experiment, male rats received a free-choice high-fat high-sugar (HFHS), free-choice high-fat (HF) or a chow diet. In a second experiment, male rats received a free-choice high-sugar (HS) diet or chow diet. For both experiments, after weeks 1 and 4, an intravenous glucose tolerance test was performed. RESULTS: Both the HFHS and HF diets resulted in obesity with comparable plasma concentrations of free fatty acids. Interestingly, the HF diet did not affect glucose metabolism, whereas the HFHS diet resulted in hyperglycemia, hyperinsulinemia and in glucose intolerance because of a diminished insulin response. Moreover, adiposity in rats on the HF diet correlated positively with the insulin response to the glucose load, whereas adiposity in rats on the HFHS diet showed a negative correlation. In addition, total caloric intake did not explain differences in glucose tolerance. To test whether sugar itself was crucial, we next performed a similar experiment in rats on the HS diet. Rats consumed three times as much sugar when compared with rats on the HFHS diet, which resulted in obesity with basal hyperinsulinemia. Glucose tolerance, however, was not affected. CONCLUSION: Together, these results suggest that not only obesity or total caloric intake, but the diet content also is crucial for the glucose intolerance that we observed in rats on the HFHS diet.

Melanocortin receptor-mediated effects on obesity are distributed over specific hypothalamic regions.

OBJECTIVE: Reduction of melanocortin signaling in the brain results in obesity. However, where in the brain reduced melanocortin signaling mediates this effect is poorly understood. DESIGN: We determined the effects of long-term inhibition of melanocortin receptor activity in specific brain regions of the rat brain. Melanocortin signaling was inhibited by injection of a recombinant adeno-associated viral (rAAV) vector that overexpressed Agouti-related peptide (AgRP) into the paraventricular nucleus (PVN), the ventromedial hypothalamus (VMH), the lateral hypothalamus (LH) or the accumbens shell (Acc). RESULTS: Overexpression of AgRP in the rat PVN, VMH or LH increased bodyweight, the percentage of white adipose tissue, plasma leptin and insulin concentrations and food intake. Food intake was mainly increased because of an increase in meal size in the light and dark phases, after overexpression of AgRP in the PVN, LH or VMH. Overexpression of AgRP in the PVN or VMH reduced average body core temperature in the dark on day 40 post injection, whereas AgRP overexpression in the LH did not affect temperature. In addition, overexpression of AgRP in the PVN, LH or VMH did not significantly alter mRNA expression of AgRP, neuropeptide Y (NPY), pro-opiomelanocortin (POMC) or suppressor of cytokine signaling 3 (SOCS3) in the arcuate. Overexpression of AgRP in the Acc did not have any effect on the measured parameters. CONCLUSIONS: Reduction of melanocortin signaling in several hypothalamic regions increased meal size. However, there were brain area-specific effects on other parameters such as core temperature and plasma leptin concentrations. In a previous study, where NPY was overexpressed with an rAAV vector in the PVN and LH, meal frequency and meal size were increased respectively, whereas locomotor activity was reduced by NPY overexpression at both nuclei. Taken together, AgRP and NPY have complementary roles in energy balance.

Suppressor of cytokine signaling 3 knockdown in the mediobasal hypothalamus: counterintuitive effects on energy balance.

An increase in brain suppressor of cytokine signaling 3 (SOCS3) has been implicated in the development of both leptin and insulin resistance. Socs3 mRNA is localized throughout the brain, and it remains unclear which brain areas are involved in the effect of SOCS3 levels on energy balance. We investigated the role of SOCS3 expressed in the mediobasal hypothalamus (MBH) in the development of diet-induced obesity in adult rats. Socs3 mRNA was down-regulated by local injection of adeno-associated viral vectors expressing a short hairpin directed against Socs3, after which we determined the response to high-fat high-sucrose choice diet. In contrast to neuronal Socs3 knockout mice, rats with SOCS3 knockdown limited to the MBH showed increased body weight gain, larger amounts of white adipose tissue, and higher leptin concentrations at the end of the experiment. These effects were partly due to the decrease in locomotor activity, as 24 h food intake was comparable with controls. In addition, rats with Socs3 knockdown in the MBH showed alterations in their meal patterns: average meal size in the light period was increased and was accompanied by a compensatory decrease in meal frequency in the dark phase. In addition, neuropeptide Y (Npy) mRNA levels were significantly increased in the arcuate nucleus of Socs3 knockdown rats. Since leptin is known to stimulate Npy transcription in the absence of Socs3, these data suggest that knockdown of Socs3 mRNA limited to the MBH increases Npy mRNA levels, which subsequently decreases locomotor activity and alters feeding patterns.

A free-choice high-fat high-sugar diet induces changes in arcuate neuropeptide expression that support hyperphagia.

OBJECTIVES: The mechanisms for how saturated fat and sugar-based beverages contribute to human obesity are poorly understood. This paper describes a series of experiments developed to examine the response of hypothalamic neuropeptides to diets rich in sugar and fat, using three different diets: a high-fat high-sugar (HFHS) choice diet with access to chow, saturated fat and a 30% sugar solution; a high-fat (HF) choice diet with access to chow and saturated fat; or to a high-sugar (HS) choice diet with access to chow and a sugar solution. METHOD: We first studied caloric intake, body weight gain, hormonal alterations and hypothalamic neuropeptide expression when male Wistar rats were subjected to an HFHS choice, an HF choice or an HS choice diet for 1 week. Next, we studied caloric intake and body weight gain when rats were subjected to the choice diets for 5 weeks. Finally, we measured neuropeptide expression in hepatic vagotomized rats subjected to an HFHS choice, an HF choice or an HS choice diet for 1 week. RESULTS: In rats on an HF choice diet, plasma leptin concentrations and proopiomelanocortin (POMC) mRNA increased and neuropeptide Y (NPY) mRNA decreased. Rats on an HFHS choice diet showed identical plasma leptin concentrations as rats on an HF choice diet. However, NPY mRNA increased and POMC mRNA decreased. An HS choice diet for 1 week did not alter hypothalamic neuropeptide expression or plasma leptin concentrations. As hormonal changes did not explain the differences in hypothalamic neuropeptide expression between rats on the choice diets, we addressed whether neuronal feedback signals mediated the hypothalamic neuropeptide response. The POMC mRNA response to different diets depended on an intact innervation of liver and upper intestinal tract. CONCLUSION: Our data suggest that the specific combination of saturated fat and a 30% sugar solution results in hyperphagia-induced obesity and alters hypothalamic neuropeptide expression, and that the response of the melanocortin system is mediated by the hepatic vagus.

B-50/GAP-43 potentiates cytoskeletal reorganization in raft domains.

B-50 (GAP-43) is a neural, membrane-associated protein that has been implicated in neurite outgrowth and guidance. Following stable transfection of Rat1 fibroblasts with B-50 cDNA we observed a dispersed distribution of B-50 immunoreactivity in flattened resting cells. In contrast, motile cells exhibited high concentrations of B-50 at the leading edge of ruffling membranes, coinciding with actin polymerization. Time-lapse studies on Rat1 fibroblasts transiently transfected with B-50/EGFP revealed that large vesicles originated from the ruffling membranes. These large vesicles (pinocytes) were found positive for Thy-1, a GPI-anchored protein, but negative for rab-5, an early endosome marker. In primary hippocampal neurons B-50 also colocalized completely with the raft marker Thy-1. Antibody-mediated cross-linking of Thy-1 in hippocampal neurons resulted in a redistribution of the intracellular protein B-50 to Thy-1-immunopositive membrane patches, whereas syntaxin was mainly excluded from the patches, showing that B-50 is associated with rafts. Academic Press.

N-terminal cysteines essential for Golgi sorting of B-50 (GAP-43) in PC12 cells.

We have examined the subcellular distribution of the growth-associated protein B-50 (GAP-43) in pheochromocytoma (PC12) cells, using confocal microscopy. Proliferating PC12 cells contained very low levels of B-50 in the cytosol. Enhanced expression of B-50 in these cells, evoked by either nerve growth factor (NGF) treatment or transient transfection with rat B-50 cDNA, led to Golgi sorting and membrane targeting of the B-50 protein. Site directed mutagenesis of Cys3Cys4 to Ser3Gly4 in B-50 resulted in a cytosolic distribution. We conclude that Cys3, and Cys4 are essential for accumulation of B-50 both at the plasma membrane and in the Golgi apparatus of PC12 cells.

Expression levels of B-50/GAP-43 in PC12 cells are decisive for the complexity of their neurites and growth cones.

To study the role of the protein B-50/GAP-43 in NGF-induced neurite outgrowth, a number of stable PC12 subclones with either very low or considerably enhanced expression levels of the protein were selected. Cell bodies of subclones with suppressed B-50 expression (-B2, -B5, or -B12) possessed a relative small spherical shape and, on NGF-treatment for 7 d, developed processes that were virtually devoid of branches and that mostly bore short or blunt-ended growth cones. Cells of subclones with overexpression of B-50 (+B3, +B4, or +B11), on NGF treatment, acquired a flattened, spiky appearance with highly branched neurites possessing extended and complex growth cones. Confocal microscopy with immunofluorescence for B-50 and F-actin revealed that in neurites and growth cones of the B-50-deficient subclone -B2, no detectable B-50 and reduced amounts of filamentous F-actin were present, whereas in overexpressing +B3 cells, cell membranes, neurites, and complex growth cones were intensively stained for B-50 and exhibited numerous spikes, in which B-50 was strikingly colocalized with F-actin. These data suggest that, under normal conditions of neuritogenesis, the expression level of B-50 in PC12 cells is decisive for the complexity of neurites and growth cones.

Phosphoprotein B-50: localization of proteolytic sites for S. aureus V8 protease using truncated cRNAs for cell-free translation.

B-50 (= GAP-43, F1, and P-57 or neuromodulin) is a nervous tissue-specific, growth-associated protein, localized in the presynaptic membrane. Phosphorylation by protein kinase C at Ser41 appears to play a role in B-50/calmodulin interaction and neurotransmitter release. Previous studies have shown that digestion of the phosphorylated protein with S. aureus V8 protease (SAP) resulted consecutively in 28- and 15-kDa phospho fragments, the latter containing all incorporated phosphate. These proteolytic products of digestion with SAP have frequently been used to identify B-50 in various systems. Therefore we were interested to find out the location of these fragments in the rat B-50 molecule. For this purpose, the rat cDNA for B-50 was used to generate full-length and truncated cRNAs for cell-free translation. B-50 and B-50 peptides were either N-terminally labeled with [35S]methionine (residues 1 and 5) as a tracer, or they were phosphorylated in vitro by protein kinase C. SAP digestion of the immunoprecipitated, 35S-labeled translation products produced similar 28- and 15-kDa fragments as were obtained from 32P-labeled B-50, indicating that these fragments are N-terminal. Relative mobilities of the N-terminal B-50 fragments of known length were used as internal standards for the calculation of the length of SAP and phospho fragments. Comparing the 35S- and 32P-labeled products, four SAP sites at Glu12, Glu28, Glu65, and Glu132 could be deduced. The latter two sites are in accordance with sequence data of C-terminal fragments from the literature. All available data could be fitted into one scheme.

Mutation of serine 41 in the neuron-specific protein B-50 (GAP-43) prohibits phosphorylation by protein kinase C.

The neuron-specific, calmodulin-binding protein B-50 (also known as GAP-43, F1, or neuromodulin) is an endogenous substrate of protein kinase C (PKC). PKC exclusively phosphorylates Ser residues in B-50. As potential phosphorylation sites for PKC, Ser41, Ser110, and Ser122 were indicated, of which Ser41 is contained in the sequence ASF, which matches with the sequence of a synthetic PKC substrate. N-terminally 35S-labeled B-50, produced from cDNA, was subjected to digestion with Staphylococcus aureus V8 protease (SAP). Consecutively, 35S-labeled 28- and 15-kDa fragments were formed, similar to those after digestion of 32P-labeled B-50. In a previous study, we showed that the 32P-labeled 15-kDa SAP fragment contains all 32P radioactivity. The present data indicate that it contains the N-terminus of B-50 as well. The 15-kDa fragment, with a calculated length ranging from amino acid residue 1 to 65, contains only one potential PKC phosphorylation site, at Ser41. Mutagenesis of Ser41 into Thr or Ala resulted in recombinant B-50 products with mobilities on two-dimensional electrophoresis similar to those of the nonmutated recombinant B-50 and the rat brain B-50. Only [Ser41]B-50 was phosphorylated by PKC, whereas [Thr41]- or [Ala41]B-50 did not show any phosphorylation at the positions indicated on the immunoblots. This leads us to the conclusion that Ser41 is the sole phosphorylation site for PKC in vitro.

Microheterogeneity of the growth-associated neuronal protein B-50 (GAP-43). Contribution of phosphorylation by protein kinase Ca.

The neuron-specific, growth-associated protein B-50, also known as GAP-43. F1 and neuromodulin, shows a striking heterogeneous behaviour in many chromatographic and electrophoretic systems. A modulatory function has been proposed for the protein in receptor-mediated processes in the presynaptic membrane. Fatty acid acylation, calmodulin binding and phosphorylation appear to be tools in this respect. At least three discrete isoforms were present in separations made by reversed-phase fast protein liquid chromatography (FPLC) of the phosphorylated protein. In anion-exchange FPLC chromatography a conglomerate of eight peaks was eluted, which migrated as eight parallel curves in electrophoretic mobility studies. After dephosphorylation of the protein this number was reduced to two. Under non-reducing conditions, the phosphoprotein was eluted from an FPLC gel filtration column at Mr = 270 kDa, i.e. 8-12 times the size of the monomer (m = 23.6 kDa.) In sodium dodecyl sulphate polyacrylamide gel electrophoresis all isoforms showed only B-50 at Mr of 48 kDa and its breakdown product (Mr = 40 kDa) in a constant ratio. It was concluded that phosphorylation by protein kinase C of a single serine residue is only one factor in the microheterogeneity of B-50. Multimeric forms may also add to the heterogeneous behaviour of phosphorylated B-50.

Diet, lipoproteins and cholesteryl ester transfer activity in hyperlipidaemic subjects.

In order to study the response of cholesteryl ester transfer activity (CETA) to alteration in diet in humans we carried out a longitudinal study in hyperlipidaemic patients. Five subjects, all hyperlipidaemic, were first given a low fat diet for 7 to 15 days and then a diet high in fat for 7 to 21 days. In four out of five patients both diets were low in energy. The plasma lipoprotein levels and CETA were measured daily. All patients responded on the low fat diet by a decrease in total cholesterol and total triglycerides. No response on the high fat diet was noticed except in the patient for whom the high fat diet was also high in energy. In all patients the changes in CETA ran parallel to changes in (VLDL + LDL) cholesterol. It is concluded that diet induced changes in (VLDL + LDL) cholesterol are accompanied by similar changes in CETA in hyperlipidaemic subjects.

Muscarinic cholinergic receptors in peripheral lung tissue of normal subjects and of patients with chronic obstructive lung disease.

Muscarinic cholinergic receptors have been identified and characterized by radioligand binding studies in human peripheral lung tissue. The tissue was obtained at thoracotomy of 12 patients, of whom four had chronic obstructive lung disease. The radioligand 1-quinuclidinyl [phenyl-4-3H]benzilate (3H-QNB) was used to label the muscarinic cholinergic receptors. Binding was saturable, protein dependent and showed a high affinity and stereospecificity. Specific binding could be inhibited by agonists and antagonists; molar inhibition constants determined for the agents used were of the same order of magnitude as those reported for 3H-QNB inhibition in various tissues of laboratory animals. Inhibition experiments with agonists resulted in Hill slopes which were significantly different from unity, indicating multiple binding sites. The stable GTP analogue guanyl-5'-imidodiphosphate had no effect on the Hill slopes of agonists or antagonists. The number of binding sites was significantly less in lung tissue from patients with chronic obstructive lung disease.

Cholesteryl ester transfer activity. Localization and role in distribution of cholesteryl ester among lipoproteins in man.

The cholesteryl ester exchange/transfer protein is involved in the transport of cholesteryl ester from high density lipoproteins (HDL) to very low density lipoproteins (VLDL) and low density lipoproteins (LDL). Localization of cholesteryl ester transfer activity (CETA) in plasma was studied by measuring CETA in various delipidated fractions from a single step density ultracentrifugation gradient of plasma. CETA was measured in an in vitro system by calculating the exchange of cholesteryl ester in a standard mixture of [3H]CE-HDL and LDL. The method used for the delipidation of plasmas and fractions to be tested was critical. Optimal results were obtained by delipidation with diisopropylether-butanol (60: 40, v/v) at O degrees C. The bulk of CETA was detected in HDL3 (1.125 less than d less than 1.210 g/ml) when the lipoproteins were separated by single-step density gradient ultracentrifugation and in the 'lipoprotein-free' fraction (d greater than 1.250 g/ml) when the lipoproteins were separated by flotation ultracentrifugation including two washes. To determine whether CETA plays a role in the distribution of cholesteryl ester among the various lipoproteins, it was measured in whole plasma from normal and hyperlipidemic subjects. Plasma was delipidated before the assay in order to prevent bias due to variation of cholesterol content. CETA was higher in delipidated plasma of hyperlipidemic subjects (117.3 +/- 36.5 nmol CE/ml/h) than in delipidated plasma of normolipidemic controls (68.7 +/- 17.6 nmol CE/ml/h) (P less than 0.005). A positive correlation (r = 0.80, P less than 0.005) was found between CETA and (VLDL + LDL) cholesterol levels. A negative correlation (r = 0.57, P less than 0.05) existed between CETA and HDL cholesterol. This correlation was found both in the group as a whole and within the normal and the hyperlipidemic groups separately. The activity of the cholesteryl ester transfer appears to be a regulatory factor in the distribution of cholesteryl ester over the various lipoproteins.