Characterization of the short isoform of the growth hormone receptor synthesized by rat adipocytes.

Two mRNA transcripts that are believed to be alternately spliced products of the GH receptor gene have been reported in a variety of rat tissues. The smaller (1.2 kilobases) transcript was cloned from an adipocyte library, sequenced, and found to encode a protein identical to the soluble GH-binding protein (GHBP) in plasma. An assay that is specific for the short isoform of the GH receptor, often referred to as the GHBP, has been developed using a rabbit antiserum that recognizes the unique amino acid sequence at its carboxyl end. The assay depends upon immunoprecipitation of a complex consisting of [125I]human GH, the binding protein, antiserum, and protein-A cross-linked to agarose beads. To validate the assay, samples of rat plasma were analyzed and found to contain sufficient binding protein to bind 1.46 pmol (32 ng) GH/ml, with an affinity of 2.7 x 10(9) M-1. In adipocyte extracts, binding protein activity was sufficient to bind 61 fmol GH/g tissue, with an affinity of 2.3 x 10(9) M-1. The binding protein was found primarily in the particulate fraction of adipocytes, and it is estimated that adipocytes contain approximately 7000 copies of the binding protein/cell. Only 10% of the binding activity was present in the high speed supernatant of adipocyte homogenates, and soluble binding protein did not appear to be released into the incubation medium when adipocytes were incubated in vitro. A 50-kilodalton (kDa) 35S-labeled protein that may be a glycosylated form of the binding protein was immunoprecipitated from both the soluble and particulate fractions of adipocyte extracts by the antiserum, and addition of the synthetic peptide antigen blocked immunoprecipitation of this protein. A 150-kDa protein in the high speed supernatant fraction was also specifically immunoprecipitated by the antiserum. Although it is unlikely to be a glycosylated form of the binding protein, it may cross-react with the antiserum or perhaps be coprecipitated, because it interacts with the binding protein. In addition, 38- and 42-kDa bands were specifically immunoprecipitated from the detergent-treated particulate fraction of adipocyte extracts that were enriched for the binding protein by adsorption to immobilized GH. We conclude that 1) adipocytes synthesize the short isoform of the GH receptor, and that this protein is primarily associated with a membrane fraction of the cells; and 2) the GHBP expressed in adipocytes is not released into the incubation medium and differs in size from the GHBPs in rat plasma.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of hypophysectomy on growth hormone receptor gene expression in rat tissues.

Two cDNA probes derived from the nucleic acid sequence for the rabbit GH receptor were used to study RNA samples from normal and hypophysectomized (hypox) rat tissues by Northern analysis. Results obtained with a probe that contained a nucleotide sequence corresponding to part of the extracellular domain of the GH receptor indicated that rat liver, gastrocnemius muscle, and epididymal fat each contain a 4.4-kilobase (kb) message and one or more shorter messages that appear to be homologous to the rabbit GH receptor message. The other probe, which contained a nucleotide sequence that corresponds to the intracellular domain of the GH receptor, detected only one 4.4-kb message in these rat tissues. These results suggest that rat tissues may synthesize several forms of the GH receptor, but only one form that contains a region homologous to the intracellular domain of the rabbit liver GH receptor. Hypophysectomy increased the abundance of the 4.4-kb message 5-fold in muscle and reduced it by a factor of 2 in adipose tissue. No significant difference was seen between GH receptor message levels of normal and hypox rat liver when the results were expressed as a fraction of the total RNA. The level of the beta-actin message was also measured in liver, muscle, and fat from normal and hypox rats. No significant differences were found when the message levels in normal rats were compared to those for the corresponding tissue in hypox rats. When normalized to the beta-actin message levels, a significant increase was seen in the relative amount of the GH receptor mRNA in muscle and liver of hypox rats. The increased levels of the GH receptor message in muscle and liver and the simultaneous decreased level in fat suggest that GH receptor synthesis may be regulated selectively in these tissues by hormonal factors that are altered by hypophysectomy.

Subcellular distribution of the long and short isoforms of the growth hormone (GH) receptor in rat adipocytes: both isoforms participate in specific binding of GH.

In rodents the GH receptor gene encodes long and short isoforms of the receptor that arise from alternate splicing of the mRNA transcript. To determine whether the long and short isoforms reside on the plasma membrane, we compared their sensitivity to mild digestion of adipocytes with trypsin. Immunofunctional assays were used to measure each isoform in adipocyte extracts. The GH-binding capacity of the short isoform in cell extracts was more than 5 times that of the long. Digestion of freshly isolated adipocytes with trypsin decreased the specific binding of [125I]human GH by 77%. The GH-binding capacities of the long and short isoforms in cell extracts were diminished by 68% and 18%, respectively. The relative insensitivity of the short isoform to digestion with trypsin suggests that much of it may be inaccessible to trypsin because it is located within the cell. When cells were allowed to recover for 2 h after digestion with trypsin, GH binding was restored. Analysis of cell extracts indicated net synthesis of the long isoform and further loss of the short. Adipocytes lost more than half of the short isoform during a 2-h incubation in vitro regardless of whether the cells were first digested with trypsin. Loss of the short isoform during this period appeared to be limited to the intracellular component, as the amount of the trypsin-sensitive component remained constant. The abundance of RNA transcripts that encode the long and short isoforms of the GH receptor remained unchanged when adipocytes were incubated in vitro for 3 h in the presence or absence of actinomycin-D. Therefore, the acute changes seen in these experiments do not reflect regulation of GH receptor gene transcription. Adipocytes appear to maintain a fixed ratio of the long and short isoforms of the GH receptor on their surface. Each isoform accounts for approximately half of the cell's GH-binding capacity.

Hepatic growth hormone signaling in the late gestation fetal rat.

The role of GH in the developing fetus is poorly understood. Several studies have demonstrated a limited role for GH in late fetal life. In fact, few data are available regarding GH signal transduction in the late gestation fetus. We therefore focused on a comparison of hepatic GH signaling in near-term fetal rats [embryonic day 19 (E19)] and adult rats using a combination of in vitro studies employing hepatocytes in primary culture and in vivo studies. We found that GH receptor (GHr) binding was comparable in fetal liver and adult liver. The long isoform of the GHr underwent tyrosine phosphorylation in response to GH stimulation of E19 fetal hepatocytes in a manner similar to that seen in cultured adult hepatocytes. Furthermore, downstream signaling via the Janus kinase-2 tyrosine kinase, STAT1 (signal transducer and activator of transcription), and STAT5 was also intact in both, as demonstrated by the tyrosine phosphorylation of these signaling proteins. To confirm the relevance of these findings to the in vivo situation, GH was directly administered by ip injection to E 19 fetal and adult rats. In both cases, tyrosine phosphorylation of STAT5 was markedly and rapidly induced. Finally, transfection of E19 fetal hepatocytes with GH-responsive reporter elements [Spi2.1(-275/+85)-CAT and 8xGHRE-TKCAT] demonstrated intact transcriptional regulation. Our data indicate that GHr abundance and activity as well as downstream GH signaling are similar in the late gestation fetal rat and in the adult and that these mechanisms appear capable of supporting physiological GH functions in the developing liver.

Tissue distribution, turnover, and glycosylation of the long and short growth hormone receptor isoforms in rat tissues.

Two isoforms of the GH receptor, the full-length receptor (GHRL) and a short isoform (GHRS) that lacks the transmembrane and intracellular domains of GHRL, have been analyzed in rat tissue extracts by Western blotting and immunoprecipitation. Although quantitative estimates of GHRS and GHRL based on coprecipitation of [125I]GH indicated similar amounts of both isoforms in tissue extracts, the 110 kDa band corresponding to GHRL was generally not detected on Western blots without enrichment by immunoprecipitation. Two bands with electrophoretic mobilities corresponding to 38 and 42 kDa were present in extracts prepared from liver, muscle, and adipocytes. Western blots of the GH binding protein in rat serum also revealed two bands, but these had electrophoretic mobilities corresponding to 44 and 52 kDa. After digestion by endoglycosidase F, a single band with an electrophoretic mobility corresponding to 31 kDa was detected in samples from adipocytes, liver or serum, indicating that GHRS retained in tissues is glycosylated less extensively than that in rat serum. Digestion with neuraminidase indicated that the smaller glycoproteins in tissue extracts lack sialic acid residues that are present in serum samples. Furthermore, endoglycosidase H degraded GHRS in liver extracts to a 31 kDa band but did not degrade serum samples, suggesting that tissues retain a high mannose form of GHRS. The abundance of GHRS or GHRL in tissues from male, virgin female, and pregnant rats was estimated from the amount of 125I-GH that was bound to each isoform after immunoprecipitation. Liver contained more than 10 times as much GHRS per gram of tissue as fat or muscle. In liver, muscle, and fat, the amount of GHRS exceeded that of GHRL, sometimes by as much as 6-fold. GHBP levels in serum of females exceeded those in males, and rose even higher in pregnant females. The abundance of GHRS in all tissue extracts paralleled serum levels. In muscle and fat, the levels of GHRL did not differ in male, female and pregnant rats, whereas in liver, the pattern was similar to the GHRS pattern. In all tissues, pools of GHRS exceeded those of GHRL by a factor that grew larger as tissue and serum levels increased. The half life of GHBP in serum was estimated to be 2.4 h in rats treated with cycloheximide, whereas that of GHRS was 20 min in liver and 8.5 h in fat. These results suggest that GHRS is synthesized in liver 8 times faster than it is released into serum, whereas synthesis in fat is less than 30% of the rate at which it is released into serum by all tissues. Therefore, liver appears to be the major source of GHBP in serum. Although secretion into the circulatory system accounts for little or perhaps none of its turnover in some tissues, GHRS pools in tissues do appear to be regulated, suggesting that GHRS may function primarily in the cells in which it is synthesized.

Insulin produces a growth hormone-like increase in intracellular free calcium concentration in okadaic acid-treated adipocytes.

In vivo, GH and insulin usually produce opposing effects on carbohydrate and lipid metabolism in adipocytes, even though their signal transduction pathways overlap. However, when added to rat adipocytes that have been made GH deficient, GH briefly produces responses that are qualitatively like those of insulin. Subsequently, GH induces refractoriness to this acute insulin-like response, in a sense restricting its effects to a unique subset of possible physiological actions. Okadaic acid is an inhibitor of type I and IIa phosphoprotein phosphatases and affects glucose metabolism in fat cells in a manner that is reminiscent of GH. Okadaic acid initially mimics the actions of insulin, and subsequently, even after it has been removed by thorough washing, blunts the ability of adipocytes to accelerate glucose metabolism in response to insulin or GH. Because refractoriness to the insulin-like effect of GH is associated with GH-induced increases in intracellular free calcium concentrations ([Ca2+]i), we examined the effects of insulin on [Ca2+]i in okadaic acid-treated adipocytes. Adipocytes were incubated with 0.25 microM okadaic acid for 1 h, washed, and reincubated without okadaic acid for 2 h before measurement of [Ca2+]i using fura-2 as a calcium indicator. Neither GH (500 ng/ml) nor insulin (100 microU/ml) affected [Ca2+]i in cells in which glucose metabolism was stimulated, but both hormones rapidly increased [Ca2+]i in adipocytes that were refractory to insulin-like stimulation. The characteristics of the increase in [Ca2+]i produced by insulin were identical to those previously reported for GH. The effect of insulin was mimicked by the dihydropyridine calcium channel activator BayK 5552 or depolarization of the cell membrane with 30 mM KCl and was blocked by the dihydropyridine calcium channel blocker, nimodipine (100 nM), implicating activation of voltage-sensitive L-type Ca2+ channels. The increase in [Ca2+]i was also mimicked by sn-1,2-dioctanoylglycerol and blocked by inhibitors of protein kinase C (staurosporine, chelerythrine chloride, and calphostin), and D609, an inhibitor of phospholipase C, as reported for GH. Acquisition of the ability to increase [Ca2+]i in response to insulin required a lag period of at least 2 h after removal of okadaic acid and was prevented by inhibitors of RNA and protein synthesis. Adipocytes that were incubated with inhibitors of protein kinase A (KT-5720), or protein kinase C (staurosporine) along with okadaic acid also failed to increase [Ca2+]i in response to insulin. Conversely, adipocytes that were incubated with dibutyryl cAMP, methylisobutyl xanthine, or phorbol ester instead of okadaic acid increased [Ca2+]i when treated with insulin 2 h later. These results suggest that phosphorylated substrates of protein kinases A and C may mediate the transcriptional event(s) that enable adipocytes to activate L-type Ca2+ channels in response to insulin. Blockade of protein kinases A or C or removal of calcium from the incubation medium did not restore the ability of okadaic acid-treated adipocytes to increase glucose metabolism in response to insulin, nor did pretreatment of adipocytes with dibutyryl cAMP or phorbol ester decrease insulin-induced stimulation of glucose metabolism. The failure of insulin to increase glucose metabolism in okadaic acid-treated adipocytes thus cannot be ascribed to the increase in [Ca2+]i. These findings indicate that just as GH can produce an insulin-like response, so too can insulin produce a GH-like response, and highlight the need to understand how specificity of hormone action is achieved in cells that respond to different hormones that share elements of their transduction pathways.

Endotoxin-induced inhibition of growth hormone receptor signaling in rat liver in vivo.

The bacterial lipopolysaccharide endotoxin induces a catabolic response characterized by resistance to multiple anabolic hormones. The objective of this study was to determine the effects of endotoxin on the GH signaling pathway in rat liver in vivo. After the iv injection of Escherichia coli endotoxin (1 mg/kg), there was a progressive decrease in liver STAT5 (signal transducer and activator of transcription-5) tyrosine phosphorylation in response to GH (40% decrease 6 h after endotoxin), which occurred in the absence of a change in abundance of the STAT5 protein. Endotoxin resulted in a rapid 40-fold increase in liver Janus family kinase-2 (JAK2) messenger RNA, followed by a 2-fold increase in JAK2 protein abundance. This was associated with a 50% decrease in phosphorylated/total JAK2 after GH stimulation. GH receptor abundance was unchanged, suggesting a postreceptor site of endotoxin-induced GH resistance. Rat complementary DNAs for three members of the suppressor of cytokine signaling gene family were cloned [cytokine-inducible sequence (CIS), suppressor of cytokine signaling-2 (SOCS-2), and SOCS-3] and, using these probes, messenger RNAs for SOCS-3 and CIS were shown to be increased 10- and 4-fold above control values, respectively, 2 h after endotoxin infusion. The finding of endotoxin inhibition of in vivo STAT5 tyrosine phosphorylation in response to a supramaximal dose of GH in the absence of a change in GH receptor abundance or total GH-stimulated JAK2 tyrosine phosphorylation provides the first demonstration of acquired postreceptor GH resistance. We hypothesize that this may occur through a specificity-spillover mechanism involving the induction of SOCS genes by cytokines released in response to endotoxin and subsequent SOCS inhibition of GH signaling.

Cellular effects of growth hormone on adipocytes.

Adipocytes are physiological targets for GH in both growing and nongrowing individuals. In adipocytes that have been deprived of GH for at least 3 h, GH initially produces a response that is characterized by increased metabolism of glucose and inhibition of the lipolytic effects of catecholamines. This insulin-like effect disappears within 2-3 h despite continued stimulation and cannot be elicited again unless cells are deprived of GH for at least 3 h. Despite refractoriness to the insulin-like action of GH, the lipolytic effect of GH is evident at this time. Although termination of the insulin-like response and induction of both refractoriness and lipolysis all depend upon synthesis of RNA and proteins, these 3 effects of GH appear to be neither temporally nor causally related. Scatchard analysis of ligand binding data suggests that these various effects are produced by interaction of GH with a single class of receptors. However, since modification of either the hormone or the carbohydrate moiety of the receptor can selectively attenuate either the insulin-like or the lipolytic response, more than one hormone receptor interaction is likely. Northern analysis indicates the presence of at least 2 alternately spliced mRNA transcripts for the GH receptor, and at least 3 different complexes are seen after GH is covalently crosslinked to intact adipocytes. Refractoriness does not result from changes in either the number or affinity of GH receptors, but may result from increased cytosolic calcium. Although the protein kinase C activator phorbol myristate acetate mimics both the insulin-like and lipolytic actions of GH, increased activity of protein kinase C probably does not mediate either action of GH. The intracellular mediators of the diverse actions of GH are unknown at this time.

Insulin regulation of the activity and phosphorylation of branched-chain 2-oxo acid dehydrogenase in adipose tissue.

The activity of the intramitochondrial branched-chain 2-oxo acid dehydrogenase (BCDH), like that of pyruvate dehydrogenase, is regulated, at least in part, by interconversion between the active dephosphorylated enzyme and its inactive phosphorylated form. The stimulatory effect of insulin on BCDH activity was compared with its effect on phosphorylation of the enzyme. Intact tissues were incubated in the presence or the absence of insulin, and then mitochondria were isolated and disrupted before assaying for enzyme activity or estimating the extent of enzyme phosphorylation. Tissues were incubated in either the presence or the absence of leucine, which also stimulated BCDH activity up to 10-fold. Insulin (1 munit/ml) doubled the activity of BCDH in the absence and in the presence of leucine. Together, 1 mM-leucine and insulin appeared to stimulate BCDH activity fully. Phosphorylation of BCDH was estimated indirectly by measuring the incorporation of 32P into phosphorylation sites that remained unesterified after preparing mitochondrial extracts under conditions that preserved the effect of insulin on BCDH activity. Increased incorporation of 32P in these experiments implies decreased phosphorylation in situ when tissues were incubated with insulin and leucine. In the absence of leucine, little incorporation of 32P into BCDH was detected. In the presence of leucine, however, incorporation of 32P into BCDH was markedly increased, and insulin increased 32P incorporation still further. The results support the hypothesis that leucine and insulin both stimulate the activity of BCDH by promoting its dephosphorylation.

Studies of 5'-nucleotidase in the perfused rat heart. Including measurements of the enzyme in perfused skeletal muscle and liver.

Perfused rat hearts catalyze the hydrolysis of AMP added to the perfusion fluid at a rate of 35 mumol/g dry weight/min. The activity is specific for 5'-nucleoside monophosphates, little activity being observed with 2' and 3'-AMP. The enzyme exhibits Michaelis-Menten kinetics in situ and is inhibited competitively by adenosine-5'-alpha, beta-methylene diphosphonate (Ki = 13 muM). This, as well as the nucleotide specificity, confirms that the hydrolysis is catalyzed by 5'-nucleotidase. The maximum activity of 5'-nucleotidase in perfused hearts is equal to or greater than that found in heart homogenates; thus, all of the enzyme is accessible to AMP added externally. Hydrolysis of endogenous AMP was studied in the perfused heart. Under aerobic conditions hearts contain very low amounts of purine nucleosides, and little or no nucleoside is found in the effluent perfusate. Under anaerobic conditions hearts accumulate adenosine, inosine, and hypoxanthine and release all three substances into the perfusate. Hydrolysis of externally added AMP was also observed in perfused skeletal muscle and liver, at rates of 10 and 17 mumol/g dry weight/min, respectively.

Effects of growth hormone on the oxidation of [1-14C]-pyruvate in adipose tissue of hypophysectomized rats.

The acute effects of growth hormone on the oxidation of [1-14C]-pyruvate to 14CO2 were studied in epididymal adipose tissue obtained from hypophysectomized rats. At concentrations ranging from 10 ng/ml to 1 microgram/ml, growth hormone increased the rate of pyruvate oxidation by 20-60%. A lag period of up to 30 min was required for the full effect of the hormone to develop. Addition of fructose to the incubation medium increased the rate of pyruvate oxidation in response to either growth hormone or insulin. The effects of 1 microgram/ml growth hormone were comparable in magnitude to those of 1 mU/ml insulin, and pyruvate oxidation in the presence of both agents was no greater than in the presence of either on its own. The enhancement of pyruvate oxidation by growth hormone, like that caused by insulin, probably results from activation of pyruvate dehydrogenase. Increased activity of pyruvate dehydrogenase was found in cell-free extracts of adipose tissue that had been exposed to either growth hormone or insulin. The response of tissue segments to growth hormone followed the same pattern as observed for other acute insulin-like effects of the hormones; it was transient and disappeared within 3 hours despite continued presence of the hormone. Previous exposure of the tissues to growth hormone made them refractory to the hormone upon reexposure.

Insulin regulation of branched chain alpha-keto acid dehydrogenase in adipose tissue.

The enzyme which oxidizes alpha-keto[1-14C]isocaproate to 14CO2 is activated by incubation of adipose tissue segments with insulin. A 3-fold reduction in the apparent Km of the enzyme for alpha-ketoisocaproate was observed when homogenates of adipose tissue segments treated with insulin were compared to homogenates of control tissues. The enzyme was assayed at various times after homogenization of adipose tissue segments. Relatively small changes were observed in the activity from control or insulin-treated tissues for 30 min after homogenization. The persistence of the insulin effect after homogenization suggests that insulin may cause a covalent modification of the enzyme. The possibility that alpha-ketoisocaproate is oxidized by pyruvate dehydrogenase, which is also stimulated by insulin, is unlikely since the enzyme responsible for oxidation of 14C-labeled branched chain alpha-keto acids can be inactivated by heat at a rate distinct from that of pyruvate dehydrogenase. Moreover, unlabeled branched chain alpha-keto acids inhibit the oxidation of alpha-keto[1-14C]isocaproate but not that of [1-14C]pyruvate. Branched chain alpha-keto acid hydrogenase can be activated by incubation of adipose tissue homogenates in the presence of magnesium chloride and in the absence of ATP. The addition of ATP plus an ATP-regenerating system reverses the activation of the enzyme. The apparent Km of the enzyme is reduced and the Vmax is increased by incubation of tissue extracts under appropriate conditions.

Vectorial production of adenosine by 5'-nucleotidase in the perfused rat heart.

Rat hearts were perfused simultaneously with [8-3H] AMP and [8-14C]adenosine. [8-3H] AMP was hydrolzyed by 5'-nucleotidase to produce intra- and extracellular [8-3H] adenosine. Comparison of the specific activities of [3H]- and [14C]adenosine in the heart cells with the specific activities of [3H]- and [14C]adenosine in the effluent perfusate showed that much more [3H]adenosine accumulated in the tissue than would be expected if extracellular adenosine were the immediate precursor of intracellular adenosine. Conversely, perfusion of rat hearts with [8-14C]AMP and [8-3H]adenosine led to a much greater accumulation of intracellular [14C]adenosine than would be expected from an uptake of adenosine from the perfusate. These results are interpreted to be due to hydrolysis of extracellular AMP by 5'-nucleotidase, located in the plasma membrane, and release of the resulting adenosine inside the cell. Measurements of the specific activities of 3H and 14C in ATP, ADP, AMP, and inosine support this interpretation.

Transamination and oxidation of leucine and valine in rat adipose tissue.

Leucine was oxidized by rat adipose tissue at a rate which was not limited by the activity of branched chain amino acid transaminase since high concentrations (10 mM) of [1-14C]leucine and its transamination product, alpha-keto[1-14C]isocaproate, were oxidized at similar rates. Despite the apparent abundance of transaminase activity, however, [1-14C]valine was oxidized at only 10 to 25% of the rate of its transamination product, alpha-keto[1-14C]isovalerate. The net rate at which [1-14C] valine was transaminated by intact tissues was estimated as the sum of the rates of 14CO2 production and alpha-ketoiso[1-14C]valerate release into the medium. Transamination did not limit the rate of valine oxidation since valine was transaminated 3 times as fast as it was oxidized. The rate of valine transamination increased 18-fold when its concentration was raised 100-fold, but the fraction of [1-14C]valine oxidized to 14CO2 remained constant over the range of incubation conditions studied. The oxidation/transamination ratio for leucine was also constant and exceeded the oxidation/transamination ratio for valine unless valine oxidation was stimulated, either by the addition of glucose or leucine. Stimulation of valine oxidation did not increase its transamination but reduced the rate at which alpha-ketoisovalerate was released from the tissue. The faster oxidation of alpha-ketoisocaproate than of alpha-ketoisovalerate may be due to the activation of branched chain alpha-keto acid dehydrogenase by alpha-ketoisocaproate, but the alpha-keto acid oxidation rates do not fully account for the faster transamination of leucine than of valine.

The significance of the short form of the growth hormone receptor in rat adipocytes.

An isoform of the growth hormone (GH) receptor that lacks the transmembrane and intracellular domains is formed in adipocytes by alternate splicing of mRNA. This isoform is identical to the circulating GH-binding protein. The short isoform is about six times as abundant as the long isoform in rat adipocytes. It is located largely in the cytosolic compartment in association with intracellular membranes, but about 20% is present on the adipocyte surface as judged by the susceptibility to digestion by trypsin. In contrast, about 80% of the long isoform of the receptor is present on the cell surface. The two isoforms turn over at different rates and appear to be independently regulated. Both appear to contribute equally to GH binding. In preliminary studies, immunoneutralization of the short isoform decreased the magnitude of the effect of GH on glucose metabolism, suggesting that the short isoform may mediate some of the responses to GH.

Overexpression of the short form of the growth hormone receptor in 3T3-L1 mouse preadipocytes.

In rodents, the gene for the growth hormone receptor (GHR) gives rise to two mRNA transcripts encoding two proteins: a larger membrane spanning receptor (GHRL) and a smaller isoform, GHRs that consists of the extracellular domain and a unique hydrophilic carboxyl terminus. We examined the hypothesis that GHRs may contribute to cellular binding of GH and play a role in growth hormone (GH) signaling. Rat cDNA encoding GHRs was ligated into the mammalian expression vector pcDNA-I/neo and stably transfected into mouse 3T3-L1 preadipocytes which have endogenous GH receptors and, when differentiated into adipocytes, have the biochemical machinery to express the various GH effects. Sixteen of 24 neomycin resistant clones secreted at least twice as much GHRs in the growth medium as cells transfected with the vector alone, and in nine of these, GH binding was increased 2- to 4-fold. The amount of GHRL in extracts of these cells was unchanged, indicating that increased binding could not be accounted for by effects on formation or degradation of GHRL. The transfected cDNA for GHRs directs the synthesis of a 50 kDa protein. Cross-linking of [125I]hGH to transfected 3T3-L1 cells indicated a 3.5-fold increase in a 72 kDa GHRs[125I]hGH complex. In the presence of 10% newborn calf serum, incorporation of [35S]methionine into cellular proteins was similar in transfected clones and control cells. Deprivation of serum for 2 hr decreased protein synthesis by approximately 70% in control cells, but in the transfected cells, protein synthesis was reduced only by approximately 50% or 30% in cells exhibiting 2x or 3x increases in GH binding. In all cells, 1 nM IGF-1 restored protein synthesis to the serum replete level. Similarly, although 3H-2-deoxy-D-glucose (2DG) uptake was comparable in all cells after 2 hr of serum deprivation, 18 hr of serum deprivation decreased uptake by approximately 70% in control cells, but only by approximately 30% in cells with increased GH binding. One nanomolar IGF-1 restored 2DG uptake to levels seen after 2 hr or serum deprivation. IGF-1 had no effect after only 2 hr of serum deprivation. Measurement of IGF-1 secreted into the medium, revealed that clones which overexpress GHRs produce greater amounts of IGF-1 than control cells or transfected clones that failed to overexpress GHRs. We conclude that GHRs contributes to GH binding and may therefore be a functional receptor. In addition, overexpression of GHRs in 3T3-L1 cells altered cell function in the absence of GH.

Growth hormone regulates the distribution of L-type calcium channels in rat adipocyte membranes.

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.

L-Leucine activates branched chain alpha-keto acid dehydrogenase in rat adipose tissue.

The activity of branched chain alpha-keto acid dehydrogenase in extracts of adipose tissue was elevated after homogenization of tissue segments which had been incubated in buffer containing 0.3 mM leucine. A maximum increase (4-fold) was observed in extracts of tissues incubated in buffer containing 2.5 mM leucine, alpha-Ketoisocaproate and leucine caused maximum increases which were of similar magnitude and which required the same length of incubation of the tissue segments (5 to 15 min). The effect of leucine on branched chain alpha-keto acid dehydrogenase activity was observed both in the presence and absence of insulin, which also increased the activity of the enzyme in tissue extracts. Intact adipose tissue segments oxidized [I-14C]leucine at a maximum rate approximately 4 times that of [1-(14)C]valine. The rate of valine oxidation by intact tissue segments was doubled by addition of 0.2 to 0.5 mM unlabeled leucine, but not isoleucine, to medium containing 2 mM [1-(14)C]valine. Leucine, but not valine, also stimulated the rate of oxidation of 2 mM [U-14C]isoleucine by intact tissue segments. These results suggest that branched chain alpha-keto acid dehydrogenase activity, which is thought to limit the rate of branched chain amino acid oxidation in adipose tissue, may be sensitive to changes in the concentration of leucine in rat blood.

Effects of hypophysectomy and thyroidectomy on leucine metabolism in adipose tissue.

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.

Growth hormone stimulates tyrosine phosphorylation of insulin receptor substrate-1.

Growth hormone (GH) produces insulin-like effects in rat adipocytes that have been deprived of GH for at least 3 h. The effect of a saturating concentration of GH is qualitatively and quantitatively similar to that produced by 2-4 ng/ml insulin but differs from that of insulin in the respect that adipocytes become refractory to prolonged or repeated stimulation with GH. Since activation of tyrosine kinase is an early event in the action of both hormones, we investigated the possibility that GH stimulation of tyrosine phosphorylation of some protein in the insulin transduction cascade might result in the similar effect of the two hormones. Adipocytes were preincubated for 3 h in the absence of hormones and then reincubated without or with 500 ng/ml GH or 4-400 ng/ml insulin for 10 min. The cells were lysed with an equal volume of buffer containing 1% SDS and preheated to 100 degrees C. Proteins were separated by electrophoresis on 7.5% polyacrylamide gels and transferred to nitrocellulose membranes, and tyrosine-phosphorylated proteins were detected using anti-phosphotyrosine antiserum coupled to horseradish peroxidase and reagents to produce chemiluminescence. The faint band seen at 185 kDa in control lanes was increased by GH treatment in five independent experiments. Insulin produced a similar effect at a concentration of 4 ng/ml, and phosphorylation increased in a dose-related manner in cells treated with higher concentrations of insulin. A prominent approximately 95-kDa band that is probably not the beta subunit of the insulin receptor was also seen in GH-treated cells. The beta subunit of the insulin receptor has similar electrophoretic mobility to the 95-kDa protein, but was not phosphorylated to an extent that allowed detection when insulin was added at concentrations below 400 ng/ml. Phosphorylation of the 185- and 95-kDa bands was evident within 1 min after addition of GH, persisted for at least 30 min, and was equally prominent in sensitive and refractory cells. Antiserum to IRS-1 immunoprecipitated the tyrosine-phosphorylated 185-kDa protein. The data suggest that IRS-1 is a substrate for a GH-activated tyrosine kinase, possibly JAK-2, which may account for the insulin-like effects of GH. The data further suggest that refractoriness to insulin-like stimulation by GH may result from an additional GH-dependent action that is distinct from phosphorylation of IRS-1.