Modifications in GPR21 and GPR82 genes expression as a consequence of metabolic syndrome etiology.

Metabolic syndrome (MS) has been related with alterations in expression levels of orphan G protein coupled receptors (GPCRs) such as GPR21 and GPR82, which could be involved in some of the elements that characterizes the metabolic syndrome. The aim of this work was to evaluate changes in GPR21 and GPR82 receptors expression in two models of metabolic syndrome: one genetic (Zucker rats), and the other based on a diet (70% fructose for 9 weeks). GPR21 and GPR82 gene expressions were evaluated in brain, heart, aorta, liver and kidney by RT-qPCR. Rats with a high fructose diet, as well as obese Zucker rats, showed initial stages of pancreatic damage and alterations in some biochemical parameters related to the model consistent with the classification of MS. GPR21 and GPR82 receptors expressed in all tissues. The expression of GPR21 decreased in heart, aorta and kidney, but in liver the expression was different: decreased in diet model and increased in genetic model. In contrast, GPR82 expression depended of tissue and metabolic syndrome model. The results highlight the possible role of GPR21 and GPR82 receptors in the development MS. We conclude that the expression of GPR21 and GPR82 in different tissues is related with MS and depend of the origin of the syndrome, so they could be a therapeutic target for that syndrome.

Activation of peroxisome proliferator-activated receptor-alpha inhibits the injurious effects of adiponectin in rat steatotic liver undergoing ischemia-reperfusion.

UNLABELLED: Hepatic steatosis is a major risk factor in ischemia-reperfusion (I/R). Adiponectin acts as an antiobesity and anti-inflammatory hormone. Adiponectin activates peroxisome proliferator-activated receptor-alpha (PPAR-alpha), a transcription factor that regulates inflammation in liver disease. Ischemic preconditioning (PC) based on brief periods of I/R protects steatotic livers against subsequent sustained I/R injury, but just how this is achieved is poorly understood. This study explains the role of PPAR-alpha and adiponectin in the vulnerability shown by steatotic livers to I/R and the benefits of PC in this situation. PPAR-alpha and adiponectin levels in nonsteatotic livers undergoing I/R were similar to those found in the sham group. However, reduced PPAR-alpha and increased adiponectin levels, particularly the high molecular weight isoform, were observed in steatotic livers as a consequence of I/R. Our results suggest that mitogen-activated protein kinases (MAPKs) may be positive regulators of adiponectin accumulation in steatotic livers. The addition of adiponectin small interfering RNA (siRNA) before I/R protected steatotic livers against oxidative stress and hepatic injury. The induction of PC before I/R increased PPAR-alpha and reduced adiponectin levels in steatotic livers. PC, which increased PPAR-alpha, as well as PPAR-alpha agonist pretreatment reduced MAPK expression, adiponectin, oxidative stress, and hepatic injury that follows I/R. In addition, the administration of a PPAR-alpha antagonist in preconditioned steatotic livers eliminated the beneficial effects of PC on MAPKs, adiponectin, oxidative stress, and hepatic injury. CONCLUSION: Steatotic livers are more predisposed to down-regulate PPAR-alpha and overexpress adiponectin when subjected to I/R. PPAR-alpha agonists and adiponectin siRNA are promising candidates to protect steatotic livers. PPAR-alpha agonists as well as PC, through PPAR-alpha, inhibited MAPK expression following I/R. This in turn inhibited adiponectin accumulation in steatotic livers and adiponectin-worsening effects on oxidative stress and hepatic injury.

Bromocriptine administration reduces hyperphagia and adiposity and differentially affects dopamine D2 receptor and transporter binding in leptin-receptor-deficient Zucker rats and rats with diet-induced obesity.

BACKGROUND: The dopamine (DA) D(2) receptor (D2R) agonist bromocriptine (BC) decreases body fat in animal and human models and increases lean muscle mass, improves glucose intolerance and insulin resistance, and reduces triglycerides and free fatty acids. We have previously shown a negative correlation between D2R and body weight in obese individuals and in rodents, and that chronic food restriction increases D2R binding in genetically obese rats. The purpose of this study was to assess whether the antiobesity and metabolic effects of BC are related to changes in midbrain DA and D2R activity by measuring D2R and DA transporter (DAT) binding in a genetic (leptin-receptor-deficient) and environmental (diet-induced) rodent obesity model. METHODS: Obese (fa/fa) (leptin-receptor-deficient), lean (FA/FA) Zucker rats and rats with diet-induced obesity (DIO) were treated with 10 mg/kg BC for 4 weeks. Body weight, food intake, locomotor activity and blood glucose levels were measured along with D2R- and DAT-binding levels using in vitro receptor autoradiography. RESULTS: BC decreased food intake and body fat and increased locomotor activity in both the (fa/fa) and DIO rats. Furthermore, BC increased D2R binding in (fa/fa) but not in DIO rats. Finally, BC increased DAT binding in DIO rats but not in the (fa/fa) rats. CONCLUSION: These observations are all consistent with the existence of unique leptin-DA interactions and the hypothesis that there is hyposensitivity of the DA system in obesity.

Molecular cloning of rat obese cDNA and augmented gene expression in genetically obese Zucker fatty (fa/fa) rats.

The obese (ob) gene has recently been isolated through a positional cloning approach, the mutation of which causes a marked hereditary obesity and diabetes mellitus in mice. In the present study, we isolated rat ob cDNA and examined the tissue distribution of the ob gene expression in rats. We also studied the gene expression in genetically obese Zucker fatty (fa/fa) rats. The rat ob gene product, a 167 amino acid protein with a putative signal sequence, was 96 and 83% homologous to the mouse and human ob proteins, respectively. Northern blot analysis using the rat ob cDNA probe identified a single mRNA species of 4.5 kb in size in the adipose tissue, while no significant amount of ob mRNA was present in other tissues in rats. The ob gene was expressed in the adipose tissue with region specificities. The rank order of the ob mRNA level in the adipose tissue was epididymal, retroperitoneal, and pericardial white adipose tissue > mesenteric and subcutaneous white adipose tissue > or = interscapular brown adipose tissue. The ob gene expression occurred in mature adipocytes rather than in stromalvascular cells isolated from the rat adipose tissue. Expression of the ob gene was markedly augmented in all the adipose tissue examined in Zucker fatty (fa/fa) rats at the stage of established obesity. The present study leads to the better understanding of the physiologic and pathophysiologic roles of the ob gene.

Altered regulation of apolipoprotein A-IV gene expression in the liver of the genetically obese Zucker rat.

Apolipoprotein (apo) A-IV, a structural component of chylomicrons and high-density lipoproteins, may play a role in the catabolism of triglyceride-rich lipoproteins and in reverse cholesterol transport. To study the regulation of apoA-IV gene expression by genetic and nutritional factors, we determined the effect of a fish oil-rich and a sucrose-rich diet on apoA-IV gene transcription and nuclear and total cellular apoA-IV mRNA abundance in livers of genetically obese, hyperlipoproteinemic (fa/fa) Zucker rats and their lean (Fa/-) littermates. In obese rats fed chow, hepatic apoA-IV gene expression was more than twofold higher than in lean rats because of a post-transcriptional mechanism. apoA-I gene expression and apoC-III mRNA levels, studied as controls, were similar in both groups. The fish oil-rich diet reduced total cellular apoA-IV mRNA abundance transcriptionally to 34 +/- 4% of basal values in lean rats, but did not alter apoA-IV gene expression in obese rats. In contrast, this diet reduced apoA-I gene expression in both lean and obese animals. The sucrose-rich diet increased apoA-IV gene expression twofold in both lean and obese rats. Thus, genetic obesity alters the response of hepatic apoA-IV gene expression to a lipid-lowering diet rich in fish oil by a mechanism affecting transcriptional regulation.

Functional polymorphisms of the Lss and Fdft1 genes in laboratory rats.

We previously identified mutant alleles of the lanosterol synthase (Lss) and farnesyl diphosphate farnesyl transferase 1 (Fdft1) genes, which function in the cholesterol biosynthesis pathway, as determinants for hereditary cataracts in the SCR rat strain. Lss(S) and Fdft1(S) were established as hypomorphic alleles with missense nucleotide substitutions, while Lss(l) is a null allele with nucleotide deletion/insertion mutations. Here we report a more detailed characterization of the rat Lss and Fdft1 genes. Screening of various laboratory rat strains revealed that the hypomorphic Lss(S) and Fdft1(S) alleles are not specific to the SCR strain, but are widely prevalent in other laboratory rat strains. Meanwhile, Lss(l) was not found in any rat strains examined. It was also found that functional inter-strain polymorphisms are present in the Lss upstream regulatory region. The BN strain had a higher potential for expression of Lss transcripts than ACI and SCR under conditions where cholesterol synthesis is necessary. SCR was less efficient than BN and ACI in suppressing Lss transcription in circumstances when cholesterol synthesis should be halted. These findings not only imply that there is a genetic polymorphism for cholesterol homeostasis in laboratory rats, but also point to the possibility that rat strains with different Lss alleles exhibit different responses to measures intervening in cholesterol metabolism.

Genetic profiling of two phenotypically distinct outbred rats derived from a colony of the Zucker fatty rats maintained at Tokyo Medical University.

The Zucker fatty (ZF) rat is an outbred rat and a well-known model of obesity without diabetes, harboring a missense mutation (fatty, abbreviated as fa) in the leptin receptor gene (Lepr). Slc:Zucker (Slc:ZF) outbred rats exhibit obesity while Hos:ZFDM-Leprfa (Hos:ZFDM) outbred rats exhibit obesity and type 2 diabetes. Both outbred rats have been derived from an outbred ZF rat colony maintained at Tokyo Medical University. So far, genetic profiles of these outbred rats remain unknown. Here, we applied a simple genotyping method using Ampdirect reagents and FTA cards (Amp-FTA) in combination with simple sequence length polymorphisms (SSLP) markers to determine genetic profiles of Slc:ZF and Hos:ZFDM rats. Among 27 SSLP marker loci, 24 loci (89%) were fixed for specific allele at each locus in Slc:ZF rats and 26 loci (96%) were fixed in Hos:ZFDM rats, respectively. This indicates the low genetic heterogeneity in both colonies of outbred rats. Nine loci (33%) showed different alleles between the two outbred rats, suggesting considerably different genetic profiles between the two outbred rats in spite of the same origin. Additional analysis using 72 SSLP markers further supported these results and clarified the profiles in detail. This study revealed that genetic profiles of the Slc:ZF and Hos:ZFDM outbred rats are different for about 30% of the SSLP marker loci, which is the underlying basis for the phenotypic difference between the two outbred rats.

Modulation by protein kinase C of the hormonal responsiveness of hepatocytes from lean (Fa/fa?) and obese (fa/fa) Zucker rats.

The effect of phorbol myristate acetate (PMA) on the hormonal responsiveness of hepatocytes from lean and obese Zucker rats was studied. Phenylephrine-stimulated phosphatydylinositol labeling and phosphorylase activation were antagonized by PMA in cells from obese and lean animals; bigger residual effects were observed in cells from obese animals even at high PMA concentrations. Cyclic AMP accumulation induced by isoproterenol, glucagon, forskolin and cholera toxin was higher in cells from lean animals than in those from obese rats. PMA diminished glucagon- and cholera toxin-induced cyclic AMP accumulation; cells from lean animals were more sensitive to PMA. Two groups of isoforms of protein kinase C (PKC) were observed in hepatocytes from Zucker rats using DEAE-cellulose column chromatography: PKC 1 and PKC 2. The PKC 1 isozymes were separated into four peaks using hydroxylapatite: aa, 1a (PKC-beta), 1b (PKC-alpha) and 1c. Short treatment with PMA decreased the activity of PKC 1 (peaks 1b (PKC-alpha) and 1c) and to a lesser extent of PKC 2; cells from lean animals were more sensitive to PMA than those obtained from obese rats. Our results indicate that cells from genetically obese Zucker rats are in general less sensitive to this activator of protein kinase C than those from their lean littermates. The possibility that alterations in the phosphorylation/dephosphorylation cycles, that control metabolism and hormonal responsiveness, may contribute to this obese state is suggested.

A new genetically obese-hyperglycemic rat (Wistar fatty).

The fa-gene was transferred from the Zucker rat (13 M strain) to the Wistar Kyoto (WKY) rat. The survey, performed at the 10th generation of backcrossing, showed that Wistar fatty rats (fa/fa), a congenic strain of WKY, developed obesity and obesity-related features, such as hyperinsulinemia and hyperlipemia, in the same manner as Zucker fatty rats. Males, but not females, showed hyperglycemia, glucosuria, and polyuria as early as 8 wk of age. Tolerance and insulin response to oral glucose were decreased with advancing age in males. The diabetic changes appeared to be caused by an interaction between predisposition to develop diabetes in the WKY rat and fa-induced obesity. This is because WKY rats were found to be less sensitive to insulin than Zucker rats by both the glucose tolerance test and the steady-state blood glucose method which estimates overall insulin sensitivity.

Identification of a major defect in insulin-resistant tissues of genetically obese (fa/fa) rats. Impaired protein kinase C.

In perfused lean rat hearts, the activator of protein kinase C phorbol myristate acetate (PMA), when present alone, stimulates glucose transport but inhibits the insulin stimulation of this transport. PMA also inactivates glycogen synthase in hepatocytes. In contrast, none of these effects are observed in hearts and hepatocytes of obese animals, indicating an impaired protein kinase C activation in these tissues, which are insulin resistant. Direct measurements of protein kinase C activity in lean rat hearts revealed that PMA provokes a translocation of the enzyme from a soluble to a particulate fraction. In obese rat hearts, the basal distribution of protein kinase C is altered (more activity is found in the soluble and less in the particulate fraction), and the translocation induced by PMA is impaired. Pretreatment of lean rats with PMA in vivo, aimed at downregulating protein kinase C, induces the same defects (i.e., insulin resistance and unresponsiveness to PMA) as those observed in hearts of untreated obese animals. The results indicate that part of the insulin resistance might be the consequence of altered modulation of insulin action by protein kinase C.

A genetic defect in beta-cell gene expression segregates independently from the fa locus in the ZDF rat.

Type 2 diabetes is a strongly genetic disorder resulting from inadequate compensatory insulin secretion in the face of insulin resistance. The Zucker diabetic fatty (ZDF) rat is a model of type 2 diabetes and, like the human disease, has both insulin resistance (from a mutant leptin receptor causing obesity) and inadequate beta-cell compensation. To test for an independently inherited beta-cell defect, we examined beta-cell function in fetuses of ZDF-lean rats, which have wild-type leptin receptors. beta-Cell number and insulin content do not differ among wild-type, heterozygous, and homozygous ZDF-lean fetuses. However, insulin promoter activity is reduced 30-50% in homozygous ZDF-lean fetal islets, and insulin mRNA levels are similarly reduced by 45%. This is not a generalized defect in gene expression nor an altered transfection efficiency, because the islet amyloid polypeptide promoter and viral promoters are unaffected. Insulin promoter mapping studies suggest that the defect involves the critical A2-C1-E1 region. This study demonstrates that the ZDF rat carries a genetic defect in beta-cell transcription that is inherited independently from the leptin receptor mutation and insulin resistance. The genetic reduction in beta-cell gene transcription in homozygous animals likely contributes to the development of diabetes in the setting of insulin resistance.

Antecedent hyperglycemia, not hyperlipidemia, is associated with increased islet triacylglycerol content and decreased insulin gene mRNA level in Zucker diabetic fatty rats.

Type 2 diabetes is caused by a combination of beta-cell dysfunction and insulin resistance. Over time, hyperglycemia worsens, a phenomenon that has been attributed to deleterious effects of chronic hyperglycemia (glucotoxicity) or chronic hyperlipidemia (lipotoxicity) on beta-cell function and is often accompanied by increased islet triacylglycerol (TAG) content and decreased insulin gene expression. To examine these two potentially pathogenic forces, we studied Zucker rats (leptin receptor wild type, +/+; heterozygous, +/-; and mutant, -/-). First, +/+ and +/- Zucker rats were compared metabolically. At 6 weeks of age, the +/- rats had a lower level of islet insulin mRNA compared with +/+. At 12 weeks of age, differences were found in body weight and islet TAG content; however, levels of insulin mRNA were equivalent. Second, we examined whether worsening of the diabetic state in the homozygous mutant (-/-) Zucker diabetic fatty (ZDF) rat is related more to chronic hyperglycemia or to hyperlipidemia. The ZDF rats were treated for 6 weeks with either bezafibrate, a lipid-lowering drug that does not affect plasma glucose levels, or phlorizin, a drug that reduces plasma glucose without lowering lipid levels. Bezafibrate treatment lessened the rise in plasma TAG observed in nontreated rats (239 +/- 16 vs. 388 +/- 36 mg/dl, treated versus nontreated; P < 0.0001) but did not prevent the rise in fasting plasma glucose. Despite lowering plasma TAG, bezafibrate was not effective in preventing an increased islet TAG content and did not prevent the associated decrease in insulin mRNA levels. Phlorizin treatment prevented hyperglycemia (61 +/- 2 vs. 145 +/- 7 mg/dl, treated versus nontreated; P < 0.0001) and lowered islet TAG content (32.7 +/- 0.7 vs. 47.8 +/- 2.7 ng/islet, treated versus nontreated; P < 0.0001) and preserved insulin mRNA levels without preventing hypertriglyceridemia. Plasma free fatty acid level did not correlate with changes in islet TAG or insulin mRNA levels. We conclude that antecedent elevated plasma glucose levels, not plasma lipid levels, are associated with elevated islet TAG content and decreased insulin mRNA levels in ZDF animals.

Developmental regulation of amylin and insulin-gene expression in lean (Fa/Fa) and obese (fa/fa) Zucker rats.

Obese individuals are hyperinsulinemic and insulin resistant. Because amylin is cosecreted with insulin and may contribute to the insulin resistance of obesity, this study tested the hypothesis that insulin and amylin genes are coordinately regulated by obesity and carbohydrate feeding. Insulin and amylin gene expression were measured during the suckling/weaning transition in lean (Fa/Fa) and obese (fa/fa) Zucker rats, a period associated with marked changes in tissue insulin sensitivity. There was a decline in insulin mRNA (-90 +/- 15%, P less than 0.01) and amylin mRNA (-72 +/- 21%, P less than 0.01) content in pancreases of lean rats maintained on a high-fat diet from days 15 to 30, probably reflecting the relative increase in exocrine/endocrine development during this neonatal period and the effects of fat feeding. Weaning on high-carbohydrate versus high-fat diets resulted in enhanced expression of both insulin (P less than 0.05) and amylin (P less than 0.05) mRNAs. In contrast to the decline in pancreatic insulin and amylin mRNA content observed in lean rats, there was an increase in insulin mRNA (421.3 +/- 57.5%, P less than 0.05) and no change in amylin mRNA in obese rats maintained on a high-fat diet from days 15 to 30. There was no enhancement of insulin or amylin gene expression in obese rats with high carbohydrate relative to high-fat feeding, perhaps reflecting maximum rates of transcription in these obese insulin-resistant rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Intramyocellular lipid and insulin resistance: a longitudinal in vivo 1H-spectroscopic study in Zucker diabetic fatty rats.

Insulin resistance plays an important role in the pathogenesis of human type 2 diabetes. In humans, a negative correlation between insulin sensitivity and intramyocellular lipid (IMCL) content has been shown; thus, IMCL becomes a marker for insulin resistance. Recently, magnetic resonance spectroscopy (MRS) has been established as a dependable method for selective detection and quantification of IMCL in humans. To validate the interrelation between insulin sensitivity and IMCL in an animal model of type 2 diabetes, we established volume selective (1)H-MRS at 7 Tesla to noninvasively assess IMCL in the rat. In male obese Zucker Diabetic Fatty rats and their lean littermates, IMCL levels were determined repeatedly over 4 months, and insulin sensitivity was measured by the euglycemic-hyperinsulinemic clamp method at 6-7 and at 22-24 weeks of age. A distinct relation between IMCL and insulin sensitivity was demonstrated as well as age dependence for both parameters. Rosiglitazone treatment caused a clear reduction of IMCL and hepatic fat despite increased body weight, and a marked improvement of insulin sensitivity. Thus, the insulin sensitizing properties of rosiglitazone were consistent with a redistribution of lipids from nonadipocytic (skeletal muscle, liver) back into fat tissue.

Sex-differences in renal expression of selected transporters and transcription factors in lean and obese Zucker spontaneously hypertensive fatty rats.

The aim of this study was to identify sex-dependent expression of renal transporter mRNA in lean and obese Zucker spontaneously hypertensive fatty (ZSF1) rats and to investigate the interaction of the most altered transporter, organic anion transporter 2 (Oat2), with diabetes-relevant metabolites and drugs. Higher incidence of glomerulosclerosis, tubulointerstitial fibrosis, and protein casts in Bowman's space and tubular lumen was detected by PAS staining in obese male compared to female ZSF1 rats. Real-time PCR on RNA isolated from kidney cortex revealed that Sglt1-2, Oat1-3, and Oct1 were higher expressed in kidneys of lean females. Oct2 and Mrp2 were higher expressed in obese males. Renal mRNA levels of transporters were reduced with diabetic nephropathy in females and the expression of transcription factors Hnf1ß and Hnf4α in both sexes. The highest difference between lean and obese ZSF1 rats was found for Oat2. Therefore, we have tested the interaction of human OAT2 with various substances using tritium-labeled cGMP. Human OAT2 showed no interaction with diabetes-related metabolites, diabetic drugs, and ACE-inhibitors. However, OAT2-dependent uptake of cGMP was inhibited by furosemide. The strongly decreased expression of Oat2 and other transporters in female diabetic ZSF1 rats could possibly impair renal drug excretion, for example, of furosemide.

Growth hormone secretion in genetic large (LL) and small (SS) rats.

We investigated whether genetic selection for growth influences pituitary GH secretion in two strains of rats, LL (large) and SS (small). Animals were bled every 15 min for 6 h via an indwelling atrial Silastic catheter, and GH levels were determined by RIA. LL and SS males displayed a low frequency, high amplitude episodic pattern of GH secretion, with surges of GH occurring at 3- to 4-h intervals, separated by trough periods of approximately 60-120 min. In contrast, LL females showed a high frequency, low amplitude pattern of GH secretion, with GH pulses occurring every 1-2 h. The number of GH pulses in SS females was lower than that in LL females. SS males and SS females displayed lower peak amplitudes and lower baseline levels and, therefore, lower mean plasma GH levels compared to LL animals. The anterior pituitary GH content was not significantly different in LL and SS animals of either sex. Thus, the reduction of GH levels in SS animals is most likely the result of reduced release of GH-releasing factor from the hypothalamus or an attenuated pituitary sensitivity to GH-releasing factor.

Brain and liver insulin binding is decreased in Zucker rats carrying the 'fa' gene.

Insulin binding was measured in membrane particles prepared from the liver and several brain regions of 4-month-old female Zucker fa/fa (obese), Fa/fa (heterozygous), and Fa/Fa (lean) rats. High affinity insulin binding was decreased in the olfactory bulb of fatty (0.23 pmol bound/mg protein) and heterozygous (0.16 pmol/mg) rats compared with that in the lean controls (0.64 pmol/mg). Total binding was not changed in the cerebral cortex or hypothalamus. High affinity insulin binding was also decreased in the liver of both fatty (0.44 +/- 0.22 pmol/mg; P less than 0.01) and heterozygous (0.75 +/- 0.35 pmol/mg) animals compared with that in the lean rats (2.10 +/- 1.55 pmol/mg). This decreased binding is probably not due to down-regulation of receptors in the heterozygous rats, as they do not exhibit the hyperinsulinemia observed in the fatty rats. Rather, our findings suggest that there is a gene-related alteration in insulin binding in the Zucker rat, as low binding was observed in rats carrying either one (Fa/fa) or two (fa/fa) doses of the gene. We postulate that this central defect in insulin binding may contribute to inadequate perception of a central insulin feedback signal and to the hyperphagia observed in the obese rats.

Altered triiodothyronine metabolism in Zucker fatty rats.

Genetically obese Zucker fatty rats require two autosomal recessive genes (fa/fa) to express the obese phenotype. The obese Zucker rat (fa/fa) has decreased total and free serum T3 concentrations, but normal serum T4 concentrations, compared to those in their lean littermates. To elucidate the mechanism of these differences, we measured the MCR and production rate (PR) of T4 and T3 in the three genotypes of 4-month-old male Zucker rats (Fa/Fa, Fa/fa, and fa/fa). In addition, 5'-deiodinase activity in liver, kidney, and brown adipose tissue homogenates was determined. T4 MCRs were equivalent in all three genotypes, but a decreased T3 MCR was seen in Fa/fa and fa/fa rats. An additive effect of the fa gene was noted with respect to the decrease in T3 MCR (Fa/Fa, 42.0 +/- 1.5; Fa/fa, 38.7 +/- 2.4; fa/fa, 34.7 +/- 3.4 ml/h; P less than 0.05). Whole body T4 PRs were equal in all three genotypes, but the T3 PR was decreased in the fa/fa rat by 25% compared to that in the homozygous lean rats (15.7 +/- 2.1 vs. 21.2 +/- 2.4 ng/h; P less than 0.005). Liver and kidney 5'-deiodinase activities were decreased in the fa/fa rat by 34% (P less than 0.005) and 20% (P less than 0.01), respectively. Brown adipose tissue and pituitary 5'-deiodinase activity were similar in all three genotypes. These results show a reduction in T3, but not T4, MCR in obese Zucker rats. Whole body T3 production and type I 5'-deiodinase activity were decreased in the obese (fa/fa) rats. These results suggest that decreased T4 to T3 conversion is responsible for the decreased T3 production rate in the fatty rat and may contribute to its obesity.

Increased hypothalamic content of preproneuropeptide Y messenger ribonucleic acid in genetically obese Zucker rats and its regulation by food deprivation.

Neuropeptide Y (NPY) is a potent orexigenic agent capable of producing hyperphagia and obesity. NPY-containing neurons project from the hypothalmic arcuate nucleus to the paraventricular nucleus, an area known to be sensitive to the orexigenic effects of NPY. In this study we investigated the possibility that preproNPY messenger RNA (mRNA) content may be altered in obese Zucker rats compared to that of their lean littermates. Total RNA was isolated from hypothalamic dissections from male and female, obese and lean Zucker rats. RNA was also isolated from dissections of: olfactory bulb, entorhinal cortex, hippocampus, and striatum of female obese and lean rats. PreproNPY mRNA content was determined by solution hybridization-RNase protection analysis. The results revealed a 2- to 3-fold increase in preproNPY mRNA levels in the hypothalamus of obese animals compared to lean. The increase was observed in both sexes and was specific to the hypothalamus. In situ hybridization localized this increase to the arcuate nucleus. An additional RNase protection study was pursued to investigate the effects of 72 h food deprivation on hypothalamic preproNPY mRNA levels in lean and obese animals. Lean animals displayed an approximate 2-fold increase in preproNPY mRNA content, whereas obese animals showed no significant increase after food deprivation. These data are consistent with the hypothesis that NPY projections within the hypothalamus are involved in regulating feeding behavior and weight gain, and that disturbed regulation of hypothalamic NPY expression may play a role in the etiology of obesity in the genetically obese Zucker rat.