Effect of acute and antecedent hypoglycemia on sympathetic neural activity and catecholamine responsiveness in normal rats.

Adrenergic responsiveness to acute hypoglycemia is impaired after prior episodes of hypoglycemia. Although circulating epinephrine responses are blunted, associated alterations in adrenal sympathetic nerve activity (SNA) have not been reported. We examined adrenal nerve traffic in normal conscious rats exposed to acute insulin-induced hypoglycemia compared with insulin with (clamped) euglycemia. We also examined adrenal SNA and catecholamine responses to insulin-induced hypoglycemia in normal conscious rats after two antecedent episodes of hypoglycemia (days -2 and -1) compared with prior episodes of sham treatment. Acute insulin-induced hypoglycemia increased adrenal sympathetic nerve traffic compared with insulin administration with clamped euglycemia (165 +/- 12 vs. 118 +/- 21 spikes/s [P < 0.05]; or to 138 +/- 8 vs. 114 +/- 10% of baseline [P < 0.05]). In additional experiments, 2 days of antecedent hypoglycemia (days -2 and -1) compared with sham treatment significantly enhanced baseline adrenal SNA measured immediately before subsequent acute hypoglycemia on day 0 (180 +/- 11 vs. 130 +/- 12 spikes/s, respectively; P < 0.005) and during subsequent acute hypoglycemia (229 +/- 17 vs. 171 +/- 16 spikes/s; P < 0.05). However, antecedent hypoglycemia resulted in a nonsignificant reduction in hypoglycemic responsiveness of adrenal SNA when expressed as percent increase over baseline (127 +/- 5% vs. 140 +/- 14% of baseline). Antecedent hypoglycemia, compared with sham treatment, resulted in diminished epinephrine responsiveness to subsequent hypoglycemia. Norepinephrine responses to hypoglycemia were not significantly altered by antecedent hypoglycemia. In summary, prior hypoglycemia in normal rats increased adrenal sympathetic tone, but impaired epinephrine responsiveness to acute hypoglycemia. Hence, these data raise the intriguing possibility that increased sympathetic tone resulting from antecedent hypoglycemia downregulates subsequent epinephrine responsiveness to hypoglycemia. Alternatively, it is possible that the decrease in epinephrine responsiveness after antecedent hypoglycemia could be the result of reduced adrenal sympathetic nerve responsiveness.

Fasting and leptin modulate adipose and muscle uncoupling protein: divergent effects between messenger ribonucleic acid and protein expression.

Leptin is believed to act through hypothalamic centers to decrease appetite and increase energy utilization, in part through enhanced thermogenesis. In this study, we examined the effects of fasting for 2 days and exogenous s.c. leptin, 200 microg every 8 h for 2 days, on the regulation of uncoupling protein (UCP) subtypes in brown adipose tissue (BAT) and gastrocnemius muscle. Northern blot analysis (UCP-1) and ribonuclease protection (UCP-2 and 3) were used for quantitative messenger RNA (mRNA) analysis, and specific antibodies were used to measure UCP-1 and UCP-3 total protein expression. Leptin, compared with vehicle, did not alter BAT UCP-1 or UCP-3 mRNA or protein expression when administered to normal ad libitum fed rats. Fasting significantly decreased BAT UCP-1 and UCP-3 mRNA expression, to 31% and 30% of ad libitum fed controls, respectively, effects which were prevented by administration of leptin to fasted rats. Fasting also significantly decreased BAT UCP-1 protein expression, to 67% of control; however, that effect was not prevented by leptin treatment. Fasting also decreased BAT UCP-3 protein, to 85% of control, an effect that was not statistically significant. Fasting, with or without leptin administration, did not affect BAT UCP-2 mRNA; however, leptin administration to ad libitum fed rats significantly increased BAT UCP-2 mRNA, to 138% of control. Fasting significantly enhanced gastrocnemius muscle UCP-3 mRNA (411% of control) and protein expression (168% of control), whereas leptin administration to fasted rats did not alter either of these effects. In summary, UCP subtype mRNA and protein are regulated in tissue- and subtype-specific fashion by leptin and food restriction. Under certain conditions, the effects of these perturbations on UCP mRNA and protein are discordant.

Effects of adenoviral overexpression of uncoupling protein-2 and -3 on mitochondrial respiration in insulinoma cells.

The brown adipose tissue uncoupling protein 1 (UCP1) catalyzes proton reentry without ATP synthesis, thereby dissipating energy as heat. In contrast, the function(s) of the recently described homologs, UCP2 and UCP3, are less clear. The aim of the present study was to determine whether overexpressed UCP subtypes affect mitochondrial respiration and substrate oxidation in cultured insulin-secreting INS-1 insulinoma cells. Adenoviral overexpression of UCP2 significantly decreased the ADP/O ratio by 31% and 39% in comparison to beta-galactosidase (beta-gal) or the mitochondrial protein manganese superoxide dismutase (MnSOD), respectively, and increased state 4 respiration in the presence of succinate and oligomycin by 52% and 59% in comparison to beta-gal or MnSOD, respectively. Adenoviral overexpression of UCP3 also decreased the ADP/O ratio by 18% (nonsignificant) and increased state 4 respiration by 24% (nonsignificant) in comparison to ss-gal and significantly decreased the ADP/O ratio by 32% and increased state 4 respiration by 35% in comparison to MnSOD. Both UCP2 and UCP3 expression significantly increased whole cell lipid oxidation by 34% (P < 0.01) and 30% (P < 0.05), respectively, compared with cells expressing Ad5CMVlacZ. However, glucose oxidation was not significantly altered by UCP2 or UCP3 expression. Adenoviral UCP2 expression, but not UCP3 (compared with beta-gal), significantly inhibited insulin secretion in the presence of 15 mM glucose [6.17 +/- 0.42 ng/mg cell protein for beta-gal compared with 4.69 +/- 0.39 for UCP2 (P < 0.05) and 5.51 +/- 0.50 for UCP3]. Both overexpressed UCPs significantly reduced INS-1 cell ATP content. Within certain limitations, which are discussed, these data are the first to demonstrate increased respiration and impaired coupling of oxidative phosphorylation as a result of UCP homolog expression in isolated mammalian mitochondria. Our results also suggest an important role for UCP in lipid metabolism and, possibly, insulin secretion.

Expression of glia maturation factor beta mRNA and protein in rat organs and cells.

Rat glia maturation factor beta (GMF-beta) cDNA was obtained by reverse transcription of rat brain mRNA followed by polymerase chain reaction amplification, using primers from the human sequence. The deduced amino acid sequence of rat GMF-beta differed from the human counterpart in only three places: His27 in place of Asn, Val51 in place of Ile, and Leu93 in place of Val. The high degree of evolutionary conservation suggests that GMF-beta plays an essential role in animal cell physiology. The expression of GMF-beta mRNA in the rat was studied by the northern blot technique, using a rat cRNA probe corresponding to the entire coding region. GMF-beta mRNA was predominantly expressed in the brain and spinal cord, although trace levels were found in other organs, including testis and ovary. In the brain GMF-beta mRNA was detectable at as early as embryonic day 10, and persisted through as late as postnatal month 14, with minor variations in between. On the other hand, GMF-beta protein exhibited more obvious developmental changes, with its level increasing slowly prenatally and plateauing at 1 week after birth. GMF-beta mRNA and protein were also observed in several cultured cells. Some cells of neural origin contained higher levels of GMF-beta protein compared with cells derived from other sources. Through demonstration of mRNA and confirmation by immunoblotting, we conclude that GMF-beta is synthesized by rat organs and that GMF-beta is predominantly a brain protein.

Evidence that tyrosine phosphorylation regulates N-cadherin turnover during retinal development.

N-cadherin, a member of the cadherin family of calcium-dependent cell adhesion molecules, mediates adhesive and signaling interactions between cells during development. N-Cadherin undergoes dynamic spatiotemporal changes in expression which correlate with morphogenetic movements of cells during organogenesis and histogenesis. We have previously shown that N-cadherin expression during development is regulated by several mechanisms, including mRNA expression, cytokine modulation, and proteolytically mediated turnover, yielding the NCAD90 protein. The present study was directed at determining the extent to which N-cadherin in primary embryonic cells is the target of endogenous kinases and phosphatases, as well as the effects of modulation of these enzymes on NCAD90 expression. The results of phosphoamino acid analyses, peptide mapping, and measurements of N-cadherin and NCAD90 expression in embryonic tissues indicate that N-cadherin is indeed the target of endogenous kinase and phosphatase action, and that modulation of different classes of these enzymes can result in either stimulation or inhibition of NCAD90 production. These results provide a mechanistic explanation for observations that cadherin function is downregulated following expression of exogenously introduced viral tyrosine kinases and provide a function for the tyrosine phosphatases recently found in association with cadherins. The results indicate that N-cadherin expression during retinal development is possibly regulated in part by modulation of its phosphorylation state, the balance of which may determine whether N-cadherin remains stably expressed or is targeted for proteolytically mediated turnover to produce NCAD90.

Sympathetic inhibition, leptin, and uncoupling protein subtype expression in normal fasting rats.

To further investigate neural effects on leptin and uncoupling proteins (UCPs), we studied in vivo perturbations intended to block adrenergic input to peripheral tissues. We examined plasma leptin, leptin mRNA, and adipose and muscle UCP subtype mRNA in rats treated with alpha-methyl-p-tyrosine methyl ester (AMPT-ME), which inhibits catecholamine synthesis and 6-hydroxydopamine (6HDA), which is toxic to catecholinergic nerve terminals but, unlike AMPT-ME, does not enter the central nervous system. Intraperitoneal AMPT-ME, 250 mg/kg, was administered at 1800 and 0700 the following day, and rats were killed at 1200-1400. All rats were fasted with free access to water during this time. Intraperitoneal AMPT-ME increased plasma leptin by 15-fold, increased interscapular brown adipose tissue (IBAT) and epididymal fat leptin mRNA by 2- to 2.5-fold, and also increased plasma insulin and glucose concentrations. Intraperitoneal AMPT-ME decreased IBAT UCP-3 mRNA to 40% of control, while it increased epididymal adipose UCP-3 mRNA approximately twofold. Intravenous AMPT-ME, 250 mg/kg, administered to conscious rats for 5 h decreased lumbar sympathetic nerve activity, increased plasma leptin (5.89 +/- 1.43 compared with 2.75 +/- 0.31 ng/ml in vehicle-treated rats, n = 7, P < 0.05), and decreased cardiac rate with no sustained change in blood pressure. Intraperitoneal 6HDA, 100 mg/kg, as a single dose at 1800, increased plasma leptin approximately twofold after 18-20 h, increased IBAT (but not epididymal fat) leptin mRNA by two- to threefold, and decreased IBAT UCP-3 mRNA to 30-40% of control. Neither AMPT-ME nor 6HDA significantly altered mRNA encoding gastrocnemius muscle UCP-3, IBAT UCP-1, or IBAT and epididymal UCP-2. In summary, AMPT-ME and 6HDA increased plasma leptin and upregulated leptin mRNA expression. AMPT-ME also resulted in complex tissue and subtype-specific modulation of adipose UCP mRNA. These data are consistent with interaction between leptin and sympathetic nerve activity (SNA) in regulation of fat cell energy utilization. However, the in vivo modulation of leptin and UCPs appears complex and, beyond a causal effect of SNA per se, may depend on concurrent changes in plasma insulin, glucose, and circulatory hemodynamics.