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Earlier studies demonstrated that deprivation of growth hormone (GH) for >/=3 h decreased basal and maximally stimulated cytosolic Ca2+ in rat adipocytes and suggested that membrane Ca2+ channels might be decreased. Measurement of L-type Ca2+ channels in purified plasma membranes by immunoassay or dihydropyridine binding indicated a two- to fourfold decrease after 3 h of incubation without GH. No such decrease was seen in unfractionated adipocyte membrane preparations. The decrease in plasma membrane channel content was largely accounted for by redistribution of channels to a light microsomal membrane fraction. Immunoassay of alpha1-, alpha2/delta-, and beta-channel subunits in membrane fractions indicated that the channels redistributed as intact complexes. Addition of GH during the 1st h of incubation prevented channel redistribution, and addition of GH after 3 h restored channel distribution to the GH-replete state of freshly isolated adipocytes. The studies suggest that GH may regulate the abundance of Ca2+ channels in the adipocyte plasma membrane and thereby modulate sensitivity to signals, the expression of which is Ca2+ dependent.

In freshly isolated individual rat adipocytes, cytosolic free Ca2+ concentration ([Ca2+]i) as measured with fura 2 slowly declined during incubation but was sustained, or even somewhat increased, by brief treatment with growth hormone (GH) at the beginning of a 3-h incubation period. GH-treated adipocytes were more permeable to Ca2+ than GH-deprived cells as indicated, using Mn2- as a surrogate and monitoring influx by the rate of quenching of fura 2 fluorescence. Blockage of Ca2- channels with 100 nM nimodipine lowered [Ca2+]i in GH-treated cells to the level seen in GH-deprived cells. Increases in [Ca2+]i or the rate of Mn2+ entry were twofold greater in GH-treated than in GH-deprived cells when extracellular K+ was increased to 30 mM. Similarly, the Ca2+ channel agonist BAY K 5552 or the diacylglycerol analogue 1,2-dioctanoyl-sn-glycerol increased [Ca2+]i more in GH-treated than in GH-deprived adipocytes. Ca(2+)-ATPase activity was two times higher in plasma membranes isolated from GH-treated than from GH-deprived cells. Continued synthesis of Ca(2+)-ATPase may depend on [Ca2+]i, since the effects of GH on [Ca2+]i and Ca(2+)-ATPase were blocked by a cycloheximide or verapamil. We suggest that voltage-sensitive L-type Ca2+ channels regulate steady-state [Ca2+]i in rat adipocytes and that GH maintains the number or functional integrity of these channels.

Growth hormone (GH; 500 ng/ml) rapidly doubled cytosolic free Ca2+ concentration ([Ca2+]i) in rat adipocytes as determined with the Ca2+ indicator fura 2. No response was seen in Ca(2+)-free medium, suggesting that the increase in [Ca2+]i was due to Ca2+ influx. GH also doubled the influx of Mn2- as inferred from the rate of fluorescence quenching. Depolarization with 30 mMK+ also increased [Ca2+]i, and the increase in [Ca2+]i due to either GH or 30 mMK+ was blocked by 100 nM nimodipine, suggesting that GH increases [Ca2+]i by activating voltage-sensitive L-type Ca2+ channels. GH increased [Ca2+]i even when K+ channels were blocked, suggesting that activation of Ca2+ uptake was not secondary to closure of K+ channels and consequent depolarization. A diacylglycerol (PAG) analogue, 1,2-dioctanoyl-sn-glycerol (50 microM), duplicated, and the protein kinase C(PKC) inhibitors calphostin C (100 nM), chelerythrine (1 microM), and bis-indolylmaleimide (250 nM) inhibited the effects of GH on [Ca2+]i. Xanthogenate tricyclodecan-9-yl (D609), a specific inhibitor of phospholipase C(PLC), abolished the increase in [Ca2+]i due to GH but not to DAG. The results suggest that GH increases [Ca2+]i by activation of PLC, release of DAG, and activation of a Ca(2+)-independent isoform of PKC. PKC-catalyzed phosphorylation of either the Ca2+ channels or a protein that regulates them may account for the influx of Ca2+ produced by GH.

Hypophysectomy doubled the rate of oxidation of L-[1-14C]leucine to 14CO2 by segments of rat epididymal adipose tissue. Thyroidectomy, but not adrenalectomy, produced identical results. Acceleration of leucine oxidation occurred even in the presence of glucose and saturating concentrations of insulin and leucine, suggesting that thyroidectomy increased the capacity to degrade leucine. Treatment of thyroidectomized rats with triiodothyronine (T3) decreased leucine oxidation, but at least 4 days were required. Treatment of hypophysectomized rats with T3 for 6 days was ineffective unless growth hormone was also given. A similar acceleration was also seen in the rate of oxidation of alpha-keto[1-14C]isocaproate, the deaminated analogue of leucine, but neither hypophysectomy nor thyroidectomy accelerated the rate of oxidation of isovalerate, the next metabolite in the degradative sequence. These observations suggested that hypothyroidism, whether primary or secondary, might increase the activity of the mitochondrial reaction responsible for the decarboxylation of alpha-ketoisocaproate. Because thyroidectomy failed to modify the rate of oxidation of [1-14C]pyruvate that occurs by an analogue reaction and requires the same cofactors, an effect of thyroidectomy on cofactor availability was ruled out. Direct assay in a cell-free homogenate revealed a nearly twofold increase in the activity of the alpha-ketoisocaproate dehydrogenase enzyme complex. The findings support the conclusion that hypothyroidism increases the amount or activity of the mitochondrial enzyme complex responsible for decarboxylation of branched-chain alpha-keto acids.