Specificity of leptin action on elevated blood glucose levels and hypothalamic neuropeptide Y gene expression in ob/ob mice.

Correction of the obese state induced by genetic leptin deficiency reduces elevated levels of both blood glucose and hypothalamic neuropeptide Y (NPY) mRNA in ob/ob mice. To determine whether these responses are due to a specific action of leptin or to the reversal of the obese state, we investigated the specificity of the effect of systemic leptin administration to ob/ob mice (n = 8) on levels of plasma glucose and insulin and on hypothalamic expression of NPY mRNA. Saline-treated controls were either fed ad libitum (n = 8) or pair-fed to the intake of the leptin-treated group (n = 8) to control for changes of food intake induced by leptin. The specificity of the effect of leptin was further assessed by 1) measuring NPY gene expression in db/db mice (n = 6) that are resistant to leptin, 2) measuring NPY gene expression in brain areas outside the hypothalamus, and 3) measuring the effect of leptin administration on hypothalamic expression of corticotropin-releasing hormone (CRH) mRNA. Five daily intraperitoneal injections of recombinant mouse leptin (150 micrograms) in ob/ob mice lowered food intake by 56% (P < 0.05), body weight by 4.1% (P < 0.05), and levels of NPY mRNA in the hypothalamic arcuate nucleus by 42.3% (P < 0.05) as compared with saline-treated controls. Pair-feeding of ob/ob mice to the intake of leptin-treated animals produced equivalent weight loss, but did not alter expression of NPY mRNA in the arcuate nucleus. Leptin administration was also without effect on food intake, body weight, or NPY mRNA levels in the arcuate nucleus of db/db mice. In ob/ob mice, leptin did not alter NPY mRNA levels in cerebral cortex or hippocampus or the expression of CRH mRNA in the hypothalamic paraventricular nucleus (PVN). Leptin administration to ob/ob mice also markedly reduced serum glucose (8.3 +/- 1.2 vs. 24.5 +/- 3.8 mmol/l; P < 0.01) and insulin levels (7,263 +/- 1,309 vs. 3,150 +/- 780 pmol/l), but was ineffective in db/db mice. Pair-fed mice experienced reductions of glucose and insulin levels that were < 60% of the reduction induced by leptin. The results suggest that in ob/ob mice, systemic administration of leptin inhibits NPY gene overexpression through a specific action in the arcuate nucleus and exerts a hypoglycemic action that is partly independent of its weight-reducing effects. Furthermore, both effects occur before reversal of the obesity syndrome. Defective leptin signaling due to either leptin deficiency (in ob/ob mice) or leptin resistance (in db/db mice) therefore leads directly to hyperglycemia and the overexpression of hypothalamic NPY that is implicated in the pathogenesis of the obesity syndrome.

Recombinant ob protein reduces feeding and body weight in the ob/ob mouse.

To determine whether the product of the recently cloned ob gene functions as an adipose-related satiety factor, recombinant murine ob protein was administered intraperitoneally to ob/ob mice. Monomeric ob protein given as single morning injections to groups of three animals at seven doses ranging from 5 to 100 micrograms reduced 24-h chow consumption in a dose-dependent manner from values of 81 +/- 6.8% of control (10-micrograms dose, P = 0.04) to 29 +/- 7.7% of control (100-micrograms dose, P < 0.0001). Daily injections of 80 micrograms of ob protein into six ob/ob mice for 2 wk led to an 11 +/- 1.6% decrease in body weight (P = 0.0009) and suppressed feeding to 26 +/- 4.9% of baseline (P < 0.0001), with significant reduction of serum insulin and glucose levels. The effect of recombinant ob protein on feeding was not augmented by cofactors secreted by adipose tissue, nor did exposure of adipose tissue to ob protein affect intracellular ob mRNA levels. Posttranslational modification of ob protein was not required for activity; however, addition of a hexahistidine tag to the amino terminus of the mature ob protein resulted in prolonged suppression of feeding after injection into ob/ob mice. These results demonstrate a direct effect of the ob protein to suppress feeding in the ob/ob mouse and suggest that this molecule plays a critical role in regulating total body fat content.

Different Rates of Synthesis and Degradation of Two Chloroplastic Ammonium-Inducible NADP-Specific Glutamate Dehydrogenase Isoenzymes during Induction and Deinduction in Chlorella sorokiniana Cells.

The kinetics of accumulation (per milliliter of culture) of the alpha- and beta- subunits, associated with chloroplast-localized ammonium inducible nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase (NADP-GDH) isoenzymes, were measured during a 3 hour induction of synchronized daughter cells of Chlorella sorokiniana in 29 millimolar ammonium medium under photoautotrophic conditions. The beta-subunit holoenzyme(s) accumulated in a linear manner for 3 hours without an apparent induction lag. A 40 minute induction lag preceded the accumulation of the alpha-subunit holoenzyme(s). After 120 minutes, the alpha-subunit ceased accumulating and thereafter remained at a constant level (i.e. steady state between synthesis and degradation). From pulsechase experiments, using (35)SO(4) and immunochemical procedures, the rate of synthesis of the alpha-subunit was shown to be greater than the beta-subunit during the first 80 minutes of induction. The alpha- and beta-subunits had different rates of degradation during the induction period (t((1/2)) = 50 versus 150 minutes, respectively) and during the deinduction period (t((1/2)) = 5 versus 13.5 minutes) after removal of ammonium from the culture. During deinduction, total NADP-GDH activity decreased with a half-time of 9 minutes. Cycloheximide completely inhibited the synthesis and degradation of both subunits. A model for regulation of expression of the NADP-GDH gene was proposed.

Evidence for Chloroplastic Localization of an Ammonium-Inducible Glutamate Dehydrogenase and Synthesis of Its Subunit from a Cytosolic Precursor-Protein in Chlorella sorokiniana.

Chlorella sorokiniana cells, cultured for 12 hours in 30 millimolar ammonium medium, contained an ammonium inducible nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase (NADP-GDH) isoenzyme with subunits having a molecular weight of 53,000. In vitro translation of total cellular poly(A)(+) RNA, isolated from fully induced cells, resulted in synthesis of an NADP-GDH antigen with a molecular weight of 58,500. The 58,500 dalton antigen was processed in vitro, with a 100,000g supernatant prepared from broken fully induced Chlorella cells, to a protein with a molecular weight of 53,000. These data support the inference that the NADP-GDH subunit (M(r) = 53,000) is initially synthesized as a larger precursor protein (M(r) = 58,500). By use of a cytochemical staining procedure, dependent upon NADP-GDH catalytic activity, the holoenzyme was shown to be chloroplast-localized. An immunoelectron microscopy procedure, employing anti-NADP-GDH immunoglobulin G and Protein A-gold complex, showed that NADP-GDH antigen was absent from the nucleus but present in both the chloroplast and cytosol. Since synthesis of the enzyme can be inhibited by cycloheximide, the detection of NADP-GDH antigen in the cytosol was probably due to binding of the NADP-GDH antibody to nascent polypeptide chains of the precursor-protein being synthesized on cytosolic 80S ribosomes.

Effect of Different Carbon Sources on the Ammonium Induction of Different Forms of NADP-Specific Glutamate Dehydrogenase in Chlorella sorokiniana Cells Cultured in the Light and Dark.

The ammonium induction of the chloroplast-localized NADP-specific glutamate dehydrogenase (NADP-GDH) was shown not to be a light-dependent process per se in Chlorella sorokiniana. In the dark without exogenous organic substrates, the cells synthesized low levels of fully active NADP-GDH, provided endogenous starch reserves had not been depleted. When cells were supplied with exogenous acetate, the rate of induction of NADP-GDH activity per milliliter of culture in the dark was equal to or slightly greater than the rate observed under photosynthetic conditions without an organic carbon source. Glucose supported only a low rate of induction of NADP-GDH activity in the dark. Both acetate and glucose inhibited induction of enzyme activity in the light. The NADP-GDH holoenzyme had at least 7 different electrophoretic forms. These forms differed in net charge and/or molecular weight. Their difference in molecular weight was due to the presence of 2 subunits with similar antigenic properties but different molecular weights (M(r) = 55,500 and 53,000; alpha-and beta-subunits, respectively). Depending upon the cultural conditions and length of the induction period, a wide variation was observed in the alpha:beta subunit ratio and in the numbers and sizes of the NADP-GDH holoenzymes.