Tissue and isoform-selective activation of protein kinase C in insulin-resistant obese Zucker rats - effects of feeding.

The mechanisms of insulin resistance in the obese Zucker rat have not been clearly established but increased diacylglycerol-protein kinase C (DAG-PKC) signalling has been associated with decreased glucose utilisation in states of insulin resistance and non-insulin-dependent diabetes mellitus. The purpose of this study was to characterise tissue- and isoform-selective differences in DAG-PKC signalling in insulin-sensitive tissues from obese Zucker rats, and to assess the effects of feeding on DAG-PKC pathways. Groups of male obese (fa/fa, n=24) and lean (fa/-, n=24) Zucker rats were studied after baseline measurements of fasting serum glucose, triglycerides, insulin and oral glucose tolerance tests. Liver, epididymal fat and soleus muscle samples were obtained from fed and overnight-fasted rats for measurements of DAG, PKC activity and individual PKC isoforms in cytosol and membrane fractions. Obese rats were heavier (488+/-7 vs 315+/-9 g) with fasting hyperglycaemia (10.5+/-0.8 vs 7.7+/-0.1 mM) and hyperinsulinaemia (7167+/-363 vs 251+/-62 pM) relative to lean controls. In fasted rats, PKC activity in the membrane fraction of liver was significantly higher in the obese group (174+/-16 vs 108+/-12 pmol/min/mg protein, P<0.05) but there were no differences in muscle and fat. The fed state was associated with increased DAG levels and threefold higher PKC activity in muscle tissue of obese rats, and increased expression of the major muscle isoforms, PKC-theta and PKC-epsilon: e.g. PKC activity in the membrane fraction of muscle from obese animals was 283+/-42 (fed) vs 107+/-20 pmol/min/mg protein (fasting) compared with 197+/-27 (fed) and 154+/-21 pmol/min/mg protein (fasting) in lean rats. In conclusion, hepatic PKC activity is higher in obese rats under basal fasting conditions and feeding-induced activation of DAG-PKC signalling occurs selectively in muscle of obese (fa/fa) rats due to increased DAG-mediated activation and/or synthesis of PKC-theta and PKC-epsilon. These changes in PKC are likely to exacerbate the hyperglycaemia and hypertriglyceridaemia associated with obesity-induced diabetes.     

Galanin-like peptide mRNA alterations in arcuate nucleus and neural lobe of streptozotocin-diabetic and obese zucker rats. Further evidence for leptin-dependent and independent regulation

Galanin-like peptide (GALP) is a 60-amino-acid peptide with structural similarities to galanin and a high affinity for galanin receptors. GALP is expressed by a discrete population of neurons in the arcuate nucleus (ARC) and median eminence of the hypothalamus of several species, including the rat. GALP neurons express leptin receptors and GALP mRNA levels are decreased slightly in fasted rats and stimulated significantly by acute leptin treatment in combination with fasting. In studies to further explore the leptin dependence of GALP expression, we examined GALP mRNA levels in the hypothalamus of obese Zucker and streptozotocin-induced diabetic (STZ-DM) rats. In leptin receptor-deficient obese Zucker rats, with 75% higher body weight than lean littermates, GALP mRNA levels in the ARC were decreased by 75%, while neuropeptide Y (NPY) mRNA levels were increased 7-fold (n = 5, p < 0.001), consistent with earlier reports. In hypoleptinemic diabetic rats with 4.5-fold higher blood glucose and 15% lower body weight than controls, GALP mRNA levels in the ARC were decreased by 90%, while NPY mRNA levels were increased 9-fold (n = 5, p < 0.001). GALP is also expressed by pituicytes in the neural lobe of the rat pituitary gland and GALP expression is increased by osmotic stimulation such as dehydration and salt loading. Thus, in STZ-DM rats that are in a hyperosmotic state with elevated plasma vasopressin levels, GALP mRNA levels were increased by approximately 20-fold in the neural lobe relative to control (n = 4, p < 0.001). The current findings are consistent with a strong tonic influence of leptin receptor signalling on hypothalamic GALP expression under normal conditions, and possible abnormalities in GALP neuronal signalling and their putative targets, thyrotropin-releasing hormone and gonadotropin hormone-releasing hormone neurons, under pathophysiological conditions such as diabetes and obesity. Our data in STZ-DM rats also clearly demonstrate that GALP gene expression is differentially regulated in neurons and pituicytes.     

Proopiomelanocortin-derived peptides in rat cerebrospinal fluid and hypothalamic extracts: evidence that secretion is regulated with respect to energy balance

Regulation of proopiomelanocortin (POMC) is an important means of controlling the central melanocortin system. It has never been established whether the spectrum of POMC-derived peptides synthesized and secreted from the hypothalamus is altered in response to changes in energy homeostasis in vivo. To monitor secretion, we analyzed peptide content of rat cerebrospinal fluid. Strikingly, both the POMC precursor and ACTH were readily detected. Moreover, levels of both were lower in samples from obese Zucker rats (fa/fa) vs. lean Zucker rats (+/+, fa/+) and from fasted vs. fed rats, whereas alpha MSH could not be detected. POMC levels were also decreased in hypothalamic extracts from obese and fasted animals. In contrast, despite being the most predominant peptide in extracts, alpha MSH levels were not significantly changed in any of the rat models. The ratio of precursor to derived peptides in cerebrospinal fluid was significantly higher in obese vs. lean and fed vs. fasted rats, indicating that secretion of POMC-derived peptides is differentially down-regulated during negative energy balance. In contrast to peptide analysis, we found that POMC gene expression was not significantly decreased in fasted rat hypothalami. We conclude that regulation of peptide secretion is an important mechanism by which the POMC system is controlled.     

Correlation between pancreatic islet uncoupling protein-2 (UCP2) mRNA concentration and insulin status in rats

Hypothesizing that UCP2 may influence insulin secretion by modifying the ATP/ADP ratio within pancreatic islets, we have investigated the expression of intraislet UCP2 gene in rats showing insulin oversecretion (non-diabetic Zucker fa/fa obese rats, glucose-infused Wistar rats) or insulin undersecretion (fasting and mildly diabetic rats). We found that in Zucker fa/fa obese rats, hyperinsulinemia (1222+/-98 pmol/l vs. 128+/-22 pmol/l in lean Zucker rats) was accompanied by a significant increase in UCP2 mRNA levels. In rat submitted to a 5 day infusion with glucose, hyperinsulinemia (1126+/-101 pmol/l vs. 215+/-25 pmol/l in Wistar control rats), coincided with an enhanced intraislet UCP2 gene expression, whereas a 8h or a 2 day-infusion did not induce significant changes in UCP2 mRNA expression. In rats made hypoinsulinemic and mildly diabetic by the injection of a low dose of streptozotocin, and in 4-day-fasting rats (plasma insulin 28+/-5 pmol/l) UCP2 gene expression was sharply decreased. A 3-day-fast was ineffective. The data show the existence of a time-dependent correlation between islet mRNA UCP2 and insulin that may be interpreted as an adaptative response to prolonged insulin excess.     

Physiological and anatomical circuitry between Agouti-related protein and leptin signaling

Agouti-related protein (AGRP) is an orexigenic neuropeptide that acts via central melanocortin receptors, and whose messenger RNA (mRNA) levels are elevated in leptin-deficient mice. Fasting associated with a decline in circulating leptin normally causes a 15-fold elevation of hypothalamic Agrp mRNA levels but has no effect in leptin-deficient mice. Chronic hyperleptinemia associated with the tubby and Cpe(fat) mutations has no effect on Agrp mRNA levels, but short term leptin administration causes a 17% reduction of Agrp mRNA levels in nonmutant mice and a 700% reduction in leptin-deficient mice. In young nonobese animals, melanocortin receptor blockade associated with the Ay mutation causes complete resistance to leptin-induced weight loss. Dual in situ hybridization reveals that Agrp-expressing neurons in the medial portion of the arcuate nucleus constitute a subpopulation different from Pomc-expressing neurons, and that a significant proportion of Agrp-expressing neurons (10-25%) coexpresses the leptin receptor, Lepr-b. Immunocytochemistry confirms distinct locations of AGRP- and POMC-expressing cell bodies, but reveals an overlapping distribution of their terminal fields in the arcuate nucleus, the paraventricular hypothalamus, and the dorsomedial hypothalamus. These results suggest that in the fed state, AGRP is normally suppressed by leptin, and that release of this suppression during fasting leads to increased ingestive behavior.     

Functional characterization of alpha-adrenoceptors on pancreatic islets of fa/fa Zucker rats.

Recently, a defect in pertussis toxin-independent actions of epinephrine on pancreatic B-cells of fa/fa Zucker rats was reported (Cawthorn and Chan (1991) Mol. Cell. Endocrinol. 75, 197-204). We now report studies of islet alpha 2-adrenoceptor function of fa/fa rats. Insulin and cAMP production by islets of obese rats were both inhibited by the alpha 2-adrenoceptor agonist clonidine. Calculated pD2 values for clonidine were 9.57 +/- 0.59 and 9.43 +/- 0.33 for lean and fa/fa rat islets, respectively. Yohimbine reversed clonidine effects equipotently in lean and obese rat islets (pA2 values of 7.48 +/- 0.57 vs 7.43 +/- 0.58). Unexpectedly, the alpha 1-antagonist prazosin stimulated insulin secretion from islets of obese but not lean rats. Functional characteristics of the alpha-adrenoceptors on fa/fa islets are thus similar to those recently designated alpha 2B. Altered expression of alpha-adrenoceptors on pancreatic islets of fa/fa rats may contribute to changes in the pertussis toxin-independent pathway of epinephrine action previously observed.     

Mice lacking pro-opiomelanocortin are sensitive to high-fat feeding but respond normally to the acute anorectic effects of peptide-YY(3-36)

Inactivating mutations of the pro-opiomelanocortin (POMC) gene in both mice and humans leads to hyperphagia and obesity. To further examine the mechanisms whereby POMC-deficiency leads to disordered energy homeostasis, we have generated mice lacking all POMC-derived peptides. Consistent with a previously reported model, Pomc(-/-) mice were obese and hyperphagic. They also showed reduced resting oxygen consumption associated with lowered serum levels of thyroxine. Hypothalami from Pomc(-/-) mice showed markedly increased expression of melanin-concentrating hormone mRNA in the lateral hypothalamus, but expression of neuropeptide Y mRNA in the arcuate nucleus was not altered. Provision of a 45% fat diet increased energy intake and body weight in both Pomc(-/-) and Pomc(+/-) mice. The effects of leptin on food intake and body weight were blunted in obese Pomc(-/-) mice whereas nonobese Pomc(-/-) mice were sensitive to leptin. Surprisingly, we found that Pomc(-/-) mice maintained their acute anorectic response to peptide-YY(3-36) (PYY(3-36)). However, 7 days of PYY(3-36) administration had no effect on cumulative food intake or body weight in wild-type or Pomc(-/-) mice. Thus, POMC peptides seem to be necessary for the normal response of energy balance to high-fat feeding, but not for the acute anorectic effect of PYY(3-36) or full effects of leptin on feeding. The finding that the loss of only one copy of the Pomc gene is sufficient to render mice susceptible to the effects of high fat feeding emphasizes the potential importance of this locus as a site for gene-environment interactions predisposing to obesity.     

Physiological difference between obese (fa/fa) Zucker rats and lean Zucker rats concerning adiponectin

Obese (fa/fa) Zucker rat is a spontaneous genetic obesity model and, by comparison with lean Zucker rat, exhibits hyperphagia, hyperinsulinemia, and hyperlipidemia. The aim of this study was to examine the physiological difference concerning adiponectin between obese (fa/fa) Zucker rats and control lean Zucker rats. We therefore measured plasma adiponectin level and analyzed adiponectin and adiponectin receptor 1 mRNA expression in retroperitoneal white adipose tissue (RT WAT), brown adipose tissue (BAT), liver, and soleus muscle. We also examined the tissue mRNA expression of peroxisome proliferator-activated receptor alpha (PPAR alpha), PPAR delta, and PPAR gamma, which regulate adiponectin expression sensitivity to a PPAR gamma agonist shown by brown adipocytes from obese (fa/fa) Zucker rats and lean Zucker rats, by measuring adiponectin release from these cells. Plasma adiponectin levels of obese (fa/fa) Zucker rats were significantly higher than those of lean Zucker rats. Adiponectin mRNA expression levels in RT WAT were lower in obese (fa/fa) Zucker rats than in lean Zucker rats, but those in BAT were higher. Adiponectin receptor 1 expression levels in RT WAT, BAT, and liver of obese (fa/fa) Zucker rats were lower than in lean Zucker rats. The expression level of PPAR alpha, PPAR delta, and PPAR gamma in BAT was lower in obese (fa/fa) Zucker rats than in lean Zucker rats. Moreover, the PPAR gamma agonist increased adiponectin release only from the brown adipocytes isolated from lean Zucker rats. It is the conclusive difference between obese (fa/fa) Zucker rats and lean Zucker rats that plasma adiponectin levels of obese (fa/fa) Zucker rats are significantly higher than those of lean Zucker rats. Moreover, we clarified that mRNA expression level of adiponectin receptor 1 in RT WAT, BAT, and liver of obese (fa/fa) Zucker rats is low despite high plasma adiponectin level, and low expression of PPARs in BAT leads to less sensibility of adiponectin release from brown adipocytes to a PPAR gamma agonist in obese (fa/fa) Zucker rats.     

Changes in hypothalamic expression levels of galanin-like peptide in rat and mouse models support that it is a leptin-target peptide

Galanin-like peptide (GALP) is a novel peptide that has been isolated from the porcine hypothalamus. The expression of GALP mRNA is localized to the hypothalamic arcuate nucleus and is thought to be under the regulation of leptin. First, we confirmed by real-time PCR analysis that sc administration of leptin to Wistar rats under food-deprived conditions resulted in a 1.5-fold increase in hypothalamic GALP mRNA levels. Next, GALP mRNA levels were found to be reduced by 50% in 11-wk-old male Zucker obese rats compared with age-matched Zucker lean rats, whereas neuropeptide Y mRNA levels were increased by 55% and proopiomelanocortin mRNA levels were reduced by 53% in Zucker obese rats. Analysis using a two-site enzyme immunoassay revealed a lower level of hypothalamic GALP immunoreactivity in 11-wk-old Zucker obese rats (5.9 fmol/mg protein) than in age-matched Zucker lean rats (19.6 fmol/mg protein). Immunohistochemical studies demonstrated that Zucker obese rats (11 wk old) had a reduced number of GALP immunoreactivity-positive cells (29.4 cells/3 slices) in the arcuate nucleus compared with age-matched Zucker lean rats (115 cells/3 slices). Furthermore, Zucker obese rats showed increased sensitivity to intracerebroventricularly administered GALP compared with Zucker lean rats, in that a lower dose of GALP increased plasma LH levels in male Zucker obese rats, but not in male Zucker lean rats. In addition, a reduction in the level of hypothalamic GALP mRNA was found in db/db and ob/ob mice. The result supports the hypothesis that the hypothalamic GALP gene expression is controlled by leptin signals and suggests possible involvement of GALP in the reproductive abnormalities of the Zucker obese rat.     

A functional leptin system is essential for sodium tungstate antiobesity action

Sodium tungstate is a novel agent in the treatment of obesity. In diet-induced obese rats, it is able to reduce body weight gain by increasing energy expenditure. This study evaluated the role of leptin, a key regulator of energy homeostasis, in the tungstate antiobesity effect. Leptin receptor-deficient Zucker fa/fa rats and leptin-deficient ob/ob mice were treated with tungstate. In lean animals, tungstate administration reduced body weight gain and food intake and increased energy expenditure. However, in animals with deficiencies in the leptin system, treatment did not modify these parameters. In ob/ob mice in which leptin deficiency was restored through adipose tissue transplantation, treatment restored the tungstate-induced body weight gain and food intake reduction as well as energy expenditure increase. Furthermore, in animals in which tungstate administration increased energy expenditure, changes in the expression of key genes involved in brown adipose tissue thermogenesis were detected. Finally, the gene expression of the hypothalamic neuropeptides, Npy, Agrp, and Cart, involved in the leptin regulation of energy homeostasis, was also modified by tungstate in a leptin-dependent manner. In summary, the results indicate that the effectiveness of tungstate in reducing body weight gain is completely dependent on a functional leptin system.     

Plasma concentrations of alpha-MSH, AgRP and leptin in lean and obese men and their relationship to differing states of energy balance perturbation

OBJECTIVE: A great deal of attention has focused on the central role of alpha melanocyte-stimulating hormone (alpha-MSH) and its antagonism at the melanocortin-4 receptor (MC4R) by agouti related protein (AgRP) in the regulation of energy balance. However, very little is known regarding the function of circulating AgRP and alpha-MSH in humans. We aimed to determine whether circulating alpha-MSH and AgRP are responsive to long-term perturbations in energy balance, in a manner consistent with their central putative functions. DESIGN AND MEASUREMENTS: Circulating alpha-MSH, AgRP and leptin were measured in both lean (n = 11) and obese (n = 18) male volunteers, some of whom (lean n = 11, obese n = 12) were then allocated one of two weight-loss dietary strategies to achieve about 5% weight loss. This was achieved by either total starvation (for 4-6 days) for rapid weight loss or a very low calorie diet (VLCD, 2.6 MJ/day) (11-12 days) for less rapid weight loss, in both the lean and obese volunteers. RESULTS: At baseline, prior to any weight loss both plasma alpha-MSH (15.8 +/- 1.2 vs. 5.8 +/- 1.0 pmol/l +/- SEM; P < 0.001) and AgRP (49.4 +/- 2.4 vs. 10.1 +/- 0.9 pg/ml +/- SEM; P < 0.001) were elevated in obese subjects compared with lean. In both cases this correlated closely with fat mass (P < 0.001), percentage body fat (P < 0.001) and leptin (P < 0.05). Plasma AgRP increased significantly during a 6-day fast in lean individuals (11.1 +/- 1.6 vs. 21.6 +/- 3.1 pg/ml +/- SEM; P < 0.05) but not in the VLCD subjects or in the obese, while alpha-MSH was not affected by any changes in energy balance in either the lean or the obese volunteers. CONCLUSION: We show a difference in alpha-MSH and AgRP in lean and obese subjects that correlates closely with body fat at baseline. We demonstrate an increase in plasma AgRP during a 6-day fast in lean individuals that is coincident with a decrease in plasma leptin. This increase in AgRP was not due to weight loss per se as there was no change in AgRP as a result of the same weight loss in the VLCD intervention in lean individuals. The source of the increase in plasma AgRP and its physiological function in the periphery remains to be elucidated but we suggest that the dynamics of the change in plasma leptin may determine the elevation in fasting plasma AgRP in lean subjects.     

Insulin resistance in the heart: studies on isolated cardiocytes of genetically obese Zucker rats

Isolated cardiac myocytes from lean and genetically obese (fa/fa) Zucker rats were used to study cellular alterations related to the obesity syndrome in this tissue. Scatchard analysis of insulin binding data suggested a reduction in the number of low affinity sites in cells from obese rats; in contrast, an unaltered high affinity segment with Kd values of 5.7 +/- 0.6 and 4.5 +/- 0.7 X 10(-10) mol/liter (n = 4) in lean and obese rats, respectively, has been observed. Insulin internalization, as estimated from the amount of increased cell-associated radioactivity in chloroquine-treated cells, was decreased by 70% from 12.8 fmol insulin/10(6) cells X 120 min in lean rats to 3.8 fmol/10(6) cells X 120 min in obese rats. Determinations of initial velocities of 3-O-methylglucose influx were used for assessing glucose transport activity. Basal activity of the glucose transport system was reduced in cells from obese animals. This was found to be due to a decreased maximum velocity of the carrier with corresponding values of 69.8 +/- 5.2 and 38.3 +/- 3.2 nmol/10 sec X 10(6) cells (n = 3) in cardiocytes from lean and obese rats, respectively. Glucose transport exhibited an unaltered sensitivity toward stimulation by insulin, but an impaired responsiveness in cardiocytes from obese rats. The data suggest involvement of both receptor and postreceptor defects in the development of an insulin-resistant state in cardiac muscle.     

Energetic efficiency and brown adipose tissue uncoupling protein of obese Zucker rats fed high-carbohydrate and high-fat diets: the effects of adrenalectomy

The influence of diet on the response of lean and obese fa/fa rats to adrenalectomy has been studied. Adrenalectomized and sham-operated rats were fed either a semi-synthetic high-carbohydrate (HC) or high-fat (HF) diet for 13 days. Energetic efficiency, calculated for measurements of energy storage and energy intake, was increased in obese rats fed both HC and HF diets and reduced close to values of lean rats after adrenalectomy. Brown adipose tissue mitochondrial GDP binding and uncoupling protein concentration were reduced in control obese rats fed both HC and HF diets. After adrenalectomy the level of GDP binding and uncoupling protein concentration were increased to levels of lean rats. Molar ratios of GDP binding to uncoupling protein were similar in lean and obese rats, were unaffected by adrenalectomy, but were elevated in rats fed the HC diet (0.40 +/- 0.02 vs 0.28 +/- 0.03). The data suggests that diet, but not obese genotype, may influence the masking of mitochondrial uncoupling protein.     

Decreased insulin binding to pancreatic islets of genetically obese (fa/fa) rats

Binding of insulin and insulin secretion were studied in isolated pancreatic islets of homozygous obese fa/fa rats, their lean littermates (Fa/?) and Wistar rats. Despite normoglycemia fa/fa rats exhibit hyperinsulinemia. Glucose-induced insulin secretion from pancreatic islets in vitro was increased by more than 50% in fa/fa rats compared with islets of lean littermates and normal Wistar rats when calculated per microgram islet protein. Exogenous insulin inhibited glucose (16.7 mM)-induced insulin secretion in islets of either of these rats, and maximum inhibition was rather the same (secretion was reduced by 62.3-65.6%). However, the EC50 (half-maximal effective concentration) for inhibition was increased in fa/fa rats being 1.4 +/- 0.1 nM compared with 0.6 +/- 0.2 and 0.5 +/- 0.2 nM in lean littermates and Wistar rats, respectively (P less than 0.05 vs. fa/fa rats). Islets of fa/fa rats found 24% less [125I]insulin (P less than 0.01) than islets of lean littermates and of Wistar rats. Scatchard analysis of data of displacement of [125I]insulin binding by native insulin showed 2 binding sites; a decrease in the number of high affinity insulin binding sites (Bmax) from 4.2 +/- 1.3 and 4.7 +/- 1.6 fmol/mg protein to 2.6 +/- 0.7 fmol/mg protein was calculated when islets of lean littermates and normal Wistar rats were compared to islets of fa/fa rats. The Kd of the high affinity binding site was not changed (0.77 +/- 0.06, 0.78 +/- 0.11 and 0.61 +/- 0.14 nM, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

Glucocorticoid receptor numbers in the brain and liver of the obese Zucker rat.

Scatchard analysis of ligand binding activity has been used to investigate type I and type II glucocorticoid receptor subtypes in the hippocampus, hypothalamus and liver of lean and genetically obese fa/fa rats. Despite normal levels of corticosterone in the hypothalamus and hippocampus of fa/fa rats, maximum binding to both type I and type II receptors was increased in the hippocampus and hypothalamus. The KD value of type I receptors was normal in fa/fa rats (lean 0.13 nM, obese 0.15 nM) whereas the KD for corticosterone binding to type II receptors was increased (lean 0.48nM, obese 1.02nM). Adrenalectomy increased Bmax of type II receptors in lean rats in a time dependent manner but had no effect on binding to receptors of obese rats after the initial clearance of endogenous bound ligand. KD values were not altered by adrenalectomy in either genotype. There were no differences in binding of corticosterone to hepatic receptors of lean and obese rats seven days after adrenalectomy. The data suggests that glucocorticoid binding to type I and type II receptors is abnormally regulated in the brains of fa/fa rats.