Francine M. Gregoire

T3 stimulates resting metabolism and UCP-2 and UCP-3 mRNA but not nonphosphorylating mitochondrial respiration in mice

The molecular basis for variations in resting metabolic rate (RMR) within a species is unknown. One possibility is that variations in RMR occur because of variations in uncoupling protein 2 (UCP-2) and uncoupling protein 3 (UCP-3) expression, resulting in mitochondrial proton leak differences. We tested the hypothesis that UCP-2 and -3 mRNAs positively correlate with RMR and proton leak. We treated thyroidectomized and sham-operated mice with triiodothyronine (T(3)) or vehicle and measured RMR, liver, and skeletal muscle mitochondrial nonphosphorylating respiration and UCP-2 and -3 mRNAs. T(3) stimulated RMR and liver UCP-2 and gastrocnemius UCP-2 and -3 expression. Mitochondrial respiration was not affected by T(3) and did not correlate with UCP-2 and -3 mRNAs. Gastrocnemius UCP-2 and -3 expression did correlate with RMR. We conclude 1) T(3) did not influence intrinsic mitochondrial properties such as membrane structure and composition, and 2) variations in UCP-2 and -3 expression may partly explain variations in RMR. One possible explanation for these data is that T(3) stimulates the leak in vivo but not in vitro because a posttranslational regulator of UCP-2 and -3 is not retained in the mitochondrial fraction.The molecular basis for variations in resting metabolic rate (RMR) within a species is unknown. One possibility is that variations in RMR occur because of variations in uncoupling protein 2 (UCP-2) and uncoupling protein 3 (UCP-3) expression, resulting in mitochondrial proton leak differences. We tested the hypothesis that UCP-2 and -3 mRNAs positively correlate with RMR and proton leak. We treated thyroidectomized and sham-operated mice with triiodothyronine (T3) or vehicle and measured RMR, liver, and skeletal muscle mitochondrial nonphosphorylating respiration and UCP-2 and -3 mRNAs. T3 stimulated RMR and liver UCP-2 and gastrocnemius UCP-2 and -3 expression. Mitochondrial respiration was not affected by T3 and did not correlate with UCP-2 and -3 mRNAs. Gastrocnemius UCP-2 and -3 expression did correlate with RMR. We conclude 1) T3 did not influence intrinsic mitochondrial properties such as membrane structure and composition, and 2) variations in UCP-2 and -3 expression may partly explain variations in RMR. One possible explanation for these data is that T3 stimulates the leak in vivo but not in vitro because a posttranslational regulator of UCP-2 and -3 is not retained in the mitochondrial fraction.

Effects of tumor necrosis factor alpha on leptin secretion and gene expression: Relationship to changes of glucose metabolism in isolated rat adipocytes

OBJECTIVE: Our objective was to determine the effects of prolonged exposure to tumor necrosis factor-alpha (TNF-α) on leptin secretion from and leptin (OB) gene expression in isolated adipocytes. Because glucose uptake and the metabolism of glucose beyond lactate are important determinants of leptin production in adipocytes, we examined the effects of TNF-α on glucose uptake and lactate production and their relationship to leptin secretion.DESIGN AND METHODS: Isolated rat adipocytes were anchored in a defined matrix of basement membrane components and cultured with media containing 5u2005mM glucose, 0.16u2005nM insulin and several concentrations of TNF-α. Leptin secretion, steady-state levels of leptin mRNA levels, glucose uptake, and lactate production were assessed over 96u2005h.RESULTS: TNF-α at concentrations of 0.024, 0.24, 2.4 and 24u2005ng/ml did not affect leptin secretion over 24u2005h. TNF-α at concentrations of 0.24 to 24u2005ng/ml significantly inhibited leptin secretion over 96u2005h by 19–60%. TNF-α at concentrations of 0.024 to 24u2005ng/ml significantly decreased steady-state levels of leptin mRNA after 96u2005h by 32–95%. In addition, TNF-α at concentrations of 2.4 and 24u2005ng/ml significantly increased glucose uptake and lactate production over 96u2005h by 30–57%. TNF-α at a concentration of 0.024u2005ng/ml did not affect leptin secretion, glucose uptake or lactate production. Overall, for the TNF-α concentrations tested, leptin secretion was inversely related to the percent of glucose carbon released as lactate; however, TNF-α did not induce a proportional increase of lactate production from glucose.CONCLUSION: Short-term (24u2005h) exposure of isolated adipocytes to TNF-α does not affect leptin secretion. Prolonged exposure to TNF-α produces a concentration-dependent inhibition of leptin secretion and gene expression. This suggests that the acute effect of TNF-α to increase circulating leptin levels in vivo may be indirect. TNF-α at higher concentrations increases glucose uptake, but does not increase the conversion of glucose to lactate. Therefore, TNF-α appears to induce a dissociation between adipocyte glucose metabolism and leptin production.

Effects of inhibiting transcription and protein synthesis on basal and insulin-stimulated leptin gene expression and leptin secretion in cultured rat adipocytes

We have previously reported that glucose metabolism mediates the effects of insulin to increase leptin gene expression and leptin secretion by isolated adipocytes. The aim of the present study was to investigate the role of transcription and translation in the regulation of basal and insulin-stimulated leptin production. The short-term (4 h) and long-term (24-48 h) effects of actinomycin D, a transcriptional inhibitor, and cycloheximide, an inhibitor of protein synthesis, on leptin gene expression and leptin secretion by isolated adipocytes were determined. Actinomycin D (5 microg/ml) increased both basal and insulin-stimulated (1.6 nM) leptin secretion at 4 and 24h (193+/-14.9% and 153.8+/-10.4% of respective controls at 24h, both p<0.001). Similar effects of actinomycin D were observed on basal and insulin-stimulated leptin mRNA levels. 5,6-dichlororibofuranosyl benzimidazole (DRB), another inhibitor of transcription, also increased basal (175.4+/-18.2% of control; p<0.01) and insulin-stimulated leptin secretion (141.0+/-11.1% of insulin-treated cells; p<0.05) at 24 h. The effect of actinomycin D and DRB to increase basal leptin secretion observed at 4 and 24 h was not present at 48 h when actinomycin D and DRB both markedly inhibited insulin-stimulated leptin secretion (to 36+/-16%, p<0.05 and 21.9+/-5.6% of control, for actinomycin D and DRB, respectively, both p<0.001). Neither actinomycin D nor DRB had any effect on adipocyte glucose utilization between 24 and 48 h. The observed effects of inhibitors of transcription on leptin gene expression and leptin secretion are consistent with a long-term transcriptional mechanism for insulin-stimulated glucose metabolism to increase leptin production. Cycloheximide treatment (10 microg/ml) abolished the effects of insulin to stimulate leptin secretion (29+/-11% of control, p<0.01) during the first 4 h of treatment and at all later time points, which indicate that de novo protein synthesis is required for insulin-mediated glucose metabolism to increase leptin secretion.