Weight gain induced by an energy-dense diet is hypothesized to arise in part from defects in the neuronal response to circulating adiposity negative feedback signals, such as insulin. Peripheral tissue insulin resistance involves cellular inflammatory responses thought to be invoked by excess lipid. Therefore, we sought to determine whether similar signaling pathways are activated in the brain of rats fed a high-fat (HF) diet. The ability of intracerebroventricular (icv) insulin to reduce food intake and activate hypothalamic signal transduction is attenuated in HF-fed compared with low-fat (LF)-fed rats. This effect was accompanied by both hypothalamic accumulation of palmitoyl- and stearoyl-CoA and activation of a marker of inflammatory signaling, inhibitor of kappaB kinase-beta (IKKbeta). Hypothalamic insulin resistance and inflammation were observed with icv palmitate infusion or HF feeding independent of excess caloric intake. Last, we observed that central IKKbeta inhibition reduced food intake and was associated with increased hypothalamic insulin sensitivity in rats fed a HF but not a LF diet. These data collectively support a model of diet-induced obesity whereby dietary fat, not excess calories, induces hypothalamic insulin resistance by increasing the content of saturated acyl-CoA species and activating local inflammatory signals, which result in a failure to appropriately regulate food intake.

Synopsis The Slc30a8 gene encodes the islet-specific zinc transporter ZnT-8, which provides zinc for insulin-hexamer formation. Polymorphic variants in amino acid 325 of human ZnT-8 are associated with altered susceptibility to type 2 diabetes and ZnT-8 autoantibody epitope specificity changes in type 1 diabetes. To assess the physiological importance of ZnT-8, mice carrying a Slc30a8 exon 3 deletion were analyzed histologically and phenotyped for energy metabolism and pancreatic hormone secretion. No gross anatomical or behavioral changes or differences in body weight were observed between wild type and ZnT-8 −/− mice and ZnT-8 −/− mouse islets were indistinguishable from wild type in terms of their numbers, size and cellular composition. However, total zinc content was markedly reduced in ZnT-8 −/− mouse islets, as evaluated both by Timm’s histochemical staining of pancreatic sections and direct measurements in isolated islets. Blood glucose levels were unchanged in 16 week old, 6 hr fasted animals of either gender, however, plasma insulin concentrations were reduced in both female (~31%) and male (~47%) ZnT-8 −/− mice. Intraperitoneal glucose tolerance tests demonstrated no impairment in glucose clearance in male ZnT-8 −/− mice but glucose-stimulated insulin secretion from isolated islets was reduced ~33% relative to wild type littermates. In summary, Slc30a8 gene deletion is accompanied by a modest impairment in insulin secretion without major alterations in glucose metabolism.

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The glucokinase gene is 15.5-kilobases long, appears to be present as a single copy, and contains 10 exons that range in size from 96 to 977 base pairs. The transcription start site was located 127 nucleotides upstream from the translation initiation codon. The 5' flanking DNA contains several regions similar to dermed promoter elements. These include a probable "TATA box," an Spl binding site, and several elements related to liver-specific gene expression. In addition, we determined that transcription of the glucokinase gene increased at least 20-fold when diabetic rats were treated with insulin for 2 hr.Glucokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) plays a key role in the regulation of glucose homeostasis by catalyzing the first step in glycolysis (1). Expression of the enzyme is limited to hepatocytes and pancreatic ,B cells (2, 3), and it is regulated differently in these two tissues. The hepatic enzyme is induced by insulin and repressed by cAMP (4) whereas in the p cell glucokinase activity is increased by glucose (5). The glucokinase gene is, therefore, of interest both because of its tissue-specific expression and because of the several regulatory processes that can be analyzed. Before the cis-acting DNA elements responsible for the tissuespecific expression and hormonal regulation of this enzyme can be identified and studied, the structure of the glucokinase gene, the transcription unit in each tissue, and the sequence of its 5' flanking DNA must be determined.Glucokinase is thought to be a member of a family of hexokinases that have a common evolutionary origin (6). This concept was based on indirect evidence because none of the complete structures of the mammalian hexokinases were available. We recently deduced the structure of rat liver glucokinase and found that it shares 33% and 53% amino acid sequence identity with yeast hexokinase and the carboxylterminal portion of rat brain hexokinase I, respectively (7). We now have determined the structure of the glucokinase genet as an initial step toward answering how the various hexokinase isozymes are related to each other. Primer-Extension Analysis. A 36-base oligonucleotide (5'-ATGTTCCTGACTCCTGAGGCCACCTGTTGCAGGTGA-3') complementary to sequences near the 5' end of the glucokinase mRNA was synthesized and 5'-end-labeled with [_y-32P]ATP (>5000 Ci/mmol; 1 Ci = 37 GBq) and T4 polynucleotide kinase. The primer (3 x 10s cpm) was annealed in a total volume of 20 gl to 20 pug to poly(A)+ RNA; the annealing buffer contained 20 mM Tris-HCl (pH 7.5), 250 mM NaCl, and 1 mM EDTA. After hybridization for 1 hr at 60TC the reaction mixtures, containing the annealed primer and RNA, were diluted with 130 1A ofa solution containing 50 mM Tris-HCl (pH 7.5), 40 mM KCl, 10 mM dithiothreitol, 3 mM MgCl2, actinomycin D (75 ,ug/ml), deoxyribonucleotides at 0.5 mM each, and 2 units of avian myeloma virus reverse transcriptase (Promega Biotec) and then incubated at 37TC for 1 hr. The products of the reactions were size-fractionated on a 5% polyacrylamide/7 ...

In addition to increasing insulin sensitivity and adipogenesis, peroxisome proliferator-activated receptor (PPAR)-gamma agonists cause weight gain and hyperphagia. Given the central role of the brain in the control of energy homeostasis, we sought to determine whether PPARgamma is expressed in key brain areas involved in metabolic regulation. Using immunohistochemistry, PPARgamma distribution and its colocalization with neuron-specific protein markers were investigated in rat and mouse brain sections spanning the hypothalamus, the ventral tegmental area, and the nucleus tractus solitarius. In several brain areas, nuclear PPARgamma immunoreactivity was detected in cells that costained for neuronal nuclei, a neuronal marker. In the hypothalamus, PPARgamma immunoreactivity was observed in a majority of neurons in the arcuate (including both agouti related protein and alpha-MSH containing cells) and ventromedial hypothalamic nuclei and was also present in the hypothalamic paraventricular nucleus, the lateral hypothalamic area, and tyrosine hydroxylase-containing neurons in the ventral tegmental area but was not expressed in the nucleus tractus solitarius. To validate and extend these histochemical findings, we generated mice with neuron-specific PPARgamma deletion using nestin cre-LoxP technology. Compared with littermate controls, neuron-specific PPARgamma knockout mice exhibited dramatic reductions of both hypothalamic PPARgamma mRNA levels and PPARgamma immunoreactivity but showed no differences in food intake or body weight over a 4-wk study period. We conclude that: 1) PPARgamma mRNA and protein are expressed in the hypothalamus, 2) neurons are the predominant source of PPARgamma in the central nervous system, although it is likely expressed by nonneuronal cell types as well, and 3) arcuate nucleus neurons that control energy homeostasis and glucose metabolism are among those in which PPARgamma is expressed.