Activation of glucose transport in muscle by prolonged exposure to insulin. Effects of glucose and insulin concentrations

Glucose transport activity was found to increase over 5 h in rat epitrochlearis muscle in response to a moderate concentration (50-100 microunits/ml) of insulin. This process was examined using 3-methylglucose. The increase in permeability to 3-methylglucose was 2- to 4-fold greater after 5 h than after 1 h in muscles incubated with 50 microunits/ml of insulin and 1 or 8 mM glucose. The increase in permeability to 3-methylglucose during the period between 1 and 5 h of exposure to 50 microunits/ml of insulin and 1 mM glucose was due to an increase in the apparent Vmax of sugar transport. There were two components to this activation of glucose transport. One, which was not influenced by inhibition of protein synthesis, resulted in activation of sugar transport to the same extent by 50 microunits/ml as by 20,000 microunits/ml of insulin; however, this activation took approximately 20 times longer with 50 microunits/ml insulin. The other, which was blocked by cycloheximide, resulted in a further activation of sugar transport to a level higher than that attained in response to 20,000 microunits/ml of insulin. Glucose had no effect on activation of sugar transport during the first hour, but a high concentration (20-36 mM) of glucose prevented the further activation of glucose transport during prolonged treatment with 50 microunits/ml of insulin. It appears from these results that prolonged exposure to a moderate concentration of insulin has previously unrecognized effects that include: a progressive activation of glucose transport over a long time that eventually results in as great a response as a "supramaximal" insulin concentration, and in the presence of low glucose concentration, further activation of glucose transport by an additional, protein synthesis-dependent mechanism. The results also show that a high concentration of glucose can, under some conditions, inhibit stimulation of its own transport.     

Differential modulation of ACTH-stimulated cortisol and androstenedione secretion by insulin

Results of previous clinical studies suggested counter regulatory actions between insulin and DHEA(S). The present studies were performed using primary monolayer cultures of bovine fasciculata-reticularis cells to test the hypothesis that insulin directly affects adrenal androgen secretion. Although having no independent effect, insulin exhibited complex time- and concentration-specific actions on ACTH-stimulated secretion of both C21 (cortisol) and C19 (androstenedione) corticosteroids. In the presence of low concentrations (0.05-0.1 nM) of ACTH, cortisol secretion during a 2 h incubation was about 2-fold greater in the presence than in the absence of insulin (0.01-100 ng/ml). In the presence of a maximal concentration (10 nM) of ACTH, on the other hand, cortisol secretion was not affected by insulin at concentrations less than or equal to 0.1 ng/ml, but was decreased at higher insulin concentrations. ACTH-stimulated androstenedione secretion was not significantly affected by insulin during a short-term (2 h) incubation. During a prolonged (24 h) incubation, insulin produced a concentration-dependent inhibition of ACTH-stimulated cortisol secretion. At an insulin concentration of 100 ng/ml, ACTH (10 nM)-stimulated cortisol secretion declined to a level only 30% of that produced by ACTH alone. In contrast, insulin exhibited biphasic effects on the secretion of androstenedione by cells maintained in the presence of ACTH for 24 h; an effect that was most dramatic in the presence of a maximal concentration of ACTH. At an insulin concentration of 0.1 ng/ml, androstenedione secretion by cells maintained in the presence of 10 nM ACTH was increased approximately 2.5-fold. At higher concentrations of insulin, ACTH-stimulated androstenedione secretion was inhibited to an extent comparable to that in cortisol secretion. The effects of insulin on ACTH-stimulated cortisol and androstenedione secretion could not be accounted for by changes in steroid degradation or a loss in 11 beta-hydroxylase activity. These results indicate that insulin interacts with ACTH to modulate the secretion of both C21 and C19 corticosteroids and that physiological concentrations (less than or equal to 1 ng/ml) of insulin may have a long-term effect to enhance selectively adrenal androgen secretion. These data are consistent with a servo mechanism between insulin and DHEA(S) in vivo and indicate that the correlations observed clinically result, at least in part, from a direct action of insulin to modulate the rate of adrenal androgen production.     

Effect of insulin and adrenaline on the respiration and glycolysis of the skeletal muscle of frog]

It was shown in the experiments on isolated sartorius muscles of the frog that insulin increased endogenous respiration by 10 mm3 O2 hr per gramm of wet weight. The effect of insulin and epinephrine on respiration is additive. Insulin in middle (5--50 milliunits/ml) and low, physiological, (50 microunits/ml) concentration does not induce acceleration of glycolyses. The conclusion was confirmed that the increase of the endogenous respiration by insulin cannot be accounted for by the mobilization of the substrate of glycolysis. In the presence of epinephrine, the low concentration (50 microunits/ml) of insulin induces the decrease of the content of glucose-6-phosphate. It is probably connected with the influence of insulin on the concentration level of the cAMP.     

Mechanisms of insulin inhibition of ACTH-stimulated steroid secretion by cultured bovine adrenocortical cells.

Results of previous studies indicated that insulin at levels comparable to those in humans during hyperinsulinemia decreased ACTH-stimulated cortisol and androstenedione secretion by bovine adrenal fasciculata-reticularis cells in primary culture. In the present studies this inhibitory action was examined further by comparing the effects of insulin on ACTH-stimulated corticosteroid secretion with its effects on 8-(4-chlorophenylthio)-cAMP (cpt-cAMP), forskolin- and [5val]angiotensin II (Ang II)-stimulated corticosteroid secretion. Effects on corticosteroid secretion were correlated with effects on cAMP accumulation and rates of cAMP production. Monolayers were incubated for 24 h in the absence or presence of each agonist alone or in combination with insulin. Insulin (1.7 x 10(-9) or 17.5 x 10(-9) M) caused about a 50% decrease in cortisol and androstenedione secretion in response to ACTH (10(-11) or 10(-8) M). Insulin also decreased ACTH-stimulated aldosterone secretion by cultured glomerulosa cells. Cpt-cAMP (10(-4) or 10(-3) M)-stimulated increases in cortisol and androstenedione secretion were inhibited by insulin, but to a lesser extent than those in response to ACTH. The inhibition of cpt-cAMP-stimulated steroid secretion was not related to increased degradation of the cyclic nucleotide. Increases in cortisol and androstenedione secretion caused by a submaximal concentration (10(-6) M) of forskolin were decreased 50-70% by insulin. In contrast, insulin failed to significantly affect cortisol or androstenedione secretion caused by a maximal concentration (10(-5) M) of forskolin. The secretory responses to Ang II (10(-8) M) were also unaffected by insulin. The effect of insulin to inhibit ACTH-stimulated steroid secretion was accompanied by a reduction in cAMP accumulation as well as an apparent inhibition of adenylate cyclase activation. These data indicate that the effect of insulin to attenuate ACTH-stimulated corticosteroid secretion results from both an inhibition of ACTH-stimulated adenylate cyclase activity and an antagonism of the intracellular actions of cAMP.     

The effects of insulin and hyperglycemia on surfactant phospholipid synthesis in organotypic cultures of type II pneumocytes

Organotypic cultures of fetal type II epithelial cells were incubated in media containing insulin at concentrations ranging from 10 to 400 microunits/ml. Exposure to insulin resulted in increased glucose uptake from the media and in the rate of glucose conversion to CO2. Furthermore, both glucose uptake and CO2 production were dependent on the glucose concentration in the media. Surfactant and residual phosphatidylcholine fractions were isolated from the organotypic cultures by sucrose density centrifugation. The presence of low doses of insulin (10-25 microunits/ml) caused a significant increase in the incorporation of glucose into both surfactant and residual phosphatidylcholine. Insulin at levels of 100 microunits/ml or higher resulted in a significant decrease in glucose incorporation into both phosphatidylcholine fractions. Increasing the media glucose concentration from 5.6 to 20 mM caused a 2- to 2.5-fold increase in glucose utilization for surfactant and residual phospholipid synthesis, but did not produce any significant changes in choline incorporation into either surfactant or residual phosphatidylcholine. The addition of 400 microunits/ml of insulin to media containing 20 mM glucose, however, resulted in a 20% decrease in choline incorporation into surfactant phosphatidylcholine but had no effect on choline incorporation into residual phosphatidylcholine. These results suggest that insulin is an important hormone regulating fetal lung maturation and that hyperinsulinemia may be responsible for the delayed lung development in infants of diabetic mothers.     

Activation of rat adipocyte glycogen synthase by insulins.

Incubation of fat cells with insulin increased glycogen synthase I activity without changing total synthase activity. This effect of insulin was dependent upon the particular lot of albumin present in the medium and was abolished by incubating cells with trypsin. Half-maximal activation of glycogen synthase was obtained with 8 microunits/ml of insulin, a concentration very similar to that which half-maximally stimulated 3-O-methylglucose uptake. The basal percentage of phosphorylase a activity was not detectably altered by insulin, although it was decreased by incubating cells with 5 mM glucose. Insulin (50 microunits/ml) markedly opposed actions of epinephrine (0.05 to 10 muM) to increase phosphorylase a activity and decrease glycogen synthase I activity, effects which were observed without glucose. Partial activation of glycogen synthase by insulin was seen after 1 min and complete activation after 4 min. Glucose alone produced a transient increase in synthase I activity. When cells were incubated with insulin plus glucose for 4 min, the increase in the percent synthase I activity was much greater than the additive effects of insulin and glucose alone. This potentiation of the effect of insulin on glucogen synthase I activity depended on the time of incubation with glucose and on the concentration of the hexose. If cells were incubated with cytochalasin B before insulin plus glucose, the effect of glucose was abolished. These results suggest that there are at least two mechanisms by which insulin can increase fat cell glycogen synthase I activity. One requires glucose and activation occurs secondary to an increase in glucose transport; where another mechanism(s) is operative even in the absence of glucose.     

Triacylglycerol synthesis in isolated fat cells. An effect of insulin on microsomal fatty acid coenzyme A ligase activity.

Fatty acid CoA ligase (AMP) (EC 6.2.1.3) specific activity was increased approximately 2-fold in microsomes prepared from isolated rat fat cells incubated with 400 microunits of insulin/ml (2.9 nM) for 45 to 60 min compared to paired controls using an assay based on the conversion of [3H]oleic acid to [3H]oleoyl-CoA. Similar insulin-dependent increases in microsomal fatty acid CoA ligase specific activities were observed using an assay based on the conversion of [3H]CoA to fatty acyl-[3H]CoA. Fatty acid CoA ligase activity was predominately (about 80%) associated with the microsomal fraction. The insulin-dependent increase in microsomal fatty acid CoA ligase specific activity was maximal in 2 to 5 min at 400 microunits/ml. At 10 min, 80 to 100 microunits of insulin/ml caused a maximal increase in fatty acid CoA ligase specific activity. Similar apparent Km values for ATP, CoA, and fatty acid were observed for fatty acid CoA ligase activity in microsomal preparations from control and insulin-exposed cells. These data suggest that fatty acid CoA ligase activity is regulated in adipose tissue by insulin. Such regulation may serve to promote the capture of fatty acid and thereby, triacylglycerol synthesis in adipose tissue.     

Effect of the bonito insulin on the insulin release from monolayer culture of rat pancreas.

The effect of bonito insulin on insulin release was examined in the monolayer culture of rat pancreatic beta-cells. The beta-cells were preincubated for 5 to 20 hr with or without a small dose (100 microunits/ml) of bonito insulin in the medium containing 100 mg% glucose. And then, they were incubated in 300 mg% glucose alone or together with bonito insulin for 5 hr. There was no significant difference between the IRI release from these beta-cells with or without bonito insulin. The concentration of bonito insulin was augmented from 100 microunits/ml to 500, 1,000 and 2,000 microunits/ml. A significant inhibitory effect on the glucose-induced insulin release was observed only after the preincubation for 20 hr with 2,000 microunits/ml of bonito insulin.     

Rounding and steroidogenesis of enzyme-and ACTH-treated Y-1 mouse adrenal tumor cells

Cultured Y-1 mouse adrenal tumor cells treated with ACTH (0.5 U/ml) rounded, formed filopodia and numerous thin microvilli, and produced steroids. Rounding, filopodia and bleb formation occurred for trypsin (0.01%), and hyaluronidase (0.1%), treated cells; but neither affected control or ACTH-stimulated steroidogenesis. Neuraminidase treatment (20 mU/ml) caused rounding, thin microvilli, bleb formation, slightly increased steroid production and prevented subsequent ACTH effects. Neuraminidase appeared to alter a carbohydrate-containing ACTH receptor preventing ACTH binding. We conclude rounding and steroidogenesis are not always associated.     

Insulin sensitivity and responsiveness of epitrochlearis and soleus muscles from fed and starved rats. Recognition of differential changes in insulin sensitivities of protein synthesis and glucose incorporation into glycogen.

The insulin sensitivity of protein synthesis and glucose incorporation into glycogen by the soleus and epitrochlearis muscles from fed rats and 24 h-starved rats was determined in vitro during the first and second hours of incubation after isolation of the muscles. Rates of protein synthesis by both muscles from fed rats in the first hour of incubation were 2-fold higher than in the second hour and were not increased by insulin. Rates of protein synthesis during the first hour in the presence of 6000 microunits of insulin/ml were increased in soleus, but not in epitrochlearis, muscles from starved rats. Rates of protein synthesis in both muscles from fed and starved rats were increased significantly by insulin during the second hour. High concentrations of insulin caused a marked stimulation of the rates of glucose incorporation by both muscles from fed and starved rats in both the first and second hours of incubation. The insulin sensitivity of glucose incorporation during the second hour, defined as the concentration of insulin causing half-maximal stimulation, was increased 10-fold for both muscle types from starved rats (soleus, 65 microunits/ml; epitrochlearis, 45 microunits/ml) relative to muscles from fed rats (soleus, 600 microunits/ml; epitrochlearis, 500 microunits/m). The insulin sensitivity of protein synthesis in the second hour was greater for soleus muscles from starved rats (65 microunits/ml) than from fed rats (500 microunits/ml). In contrast, the insulin sensitivity of protein synthesis in epitrochlearis muscles from starved rats was significantly decreased (225 microunits/ml) compared with fed rats (25 microunits/ml Maximal rates achieved by high concentrations of insulin were not different from those in the same muscle from fed rats. It is suggested that protein synthesis, in distinction to glucose utilization, may be resistant to insulin stimulation during periods of acute starvation in muscles with fibre compositions similar to the epitrochlearis, but not in muscles with fibre compositions similar to the soleus. Partial reversal of the resistance observed in vitro for epitrochlearis muscles from starved rats may be due to the loss of factors which suppress the effect of insulin in vivo.     

Signaling cascade of insulin-induced stimulation of L-dopa uptake in renal proximal tubule cells

The inward l-dihydroxyphenylalanine (L-dopa) transport supplies renal proximal tubule cells (PTCs) with the precursor for dopamine synthesis. We have previously described insulin-induced stimulation of L-dopa uptake into PTCs. In the present paper we examined insulin-related signaling pathways involved in the increase of l-dopa transport into isolated rat PTCs. Insulin (50-500 microU/ml) increased L-dopa uptake by PTCs, reaching the maximal increment (60% over the control) at 200 microU/ml. At this concentration, insulin also increased insulin receptor tyrosine phosphorylation. Both effects were abrogated by the tyrosine kinase inhibitor genistein (5 microM). In line, inhibition of the protein tyrosine phosphatase by pervanadate (0.2-100 microM) caused a concentration-dependent increase in both the uptake of L-dopa (up to 400%) and protein tyrosine phosphorylation. A synergistic effect between pervanadate and insulin on L-dopa uptake was observed only when threshold (0.2 microM), but not maximal (5 microM), concentrations of pervanadate were assayed. Insulin-induced stimulation of L-dopa uptake was also abolished by inhibition of phosphatidylinositol 3-kinase (PI3K; 100 nM wortmannin, and 25 microM LY-294002) and protein kinase C (PKC; 1 microM RO-318220). Insulin-induced activation of PKC-zeta was confirmed in vitro by its translocation from the cytosol to the membrane fraction, and in vivo by immunohistochemistry studies. Insulin caused a wortmannin-sensitive increase in Akt/protein kinase B (Akt/PKB) phosphorylation and a dose-dependent translocation of Akt/PKB to the membrane fraction. Our findings suggest that insulin activates PKC-zeta, and Akt/PKB downstream of PI3K, and that these pathways contribute to the insulin-induced increase of L-dopa uptake into PTCs.     

Insulin-induced insulin resistance of alpha-aminoisobutyric acid transport in cultured human skin fibroblasts.

Cultured fibroblasts derived from skin biopsies were used to develop a system for studying insulin resistance in human tissue in vitro. Uptake of alpha-aminoisobutyric acid by cultured human skin fibroblasts was found to occur by a combination of saturable and nonsaturable processes. Insulin stimulated uptake by decreasing the Km of the saturable transport system from 0.58 mM to 0.26 mM. The maximal velocity of saturable uptake was 16.6 nmol/10(7) cells/min in both the presence and absence of insulin. Uptake of alpha-aminoisobutyric acid at 0.2 mM was studied in human skin fibroblasts with and without chronic exposure to insulin for 4 days at an initial concentration of 10 micrograms/ml. Unstimulated uptake was increased from 17 to 20 nmol/10(8) cells/min, and the increase in uptake due to maximal stimulation by insulin was unchanged at 16 nmol/10(8) cells/min in the cells exposed chronically to insulin. The apparent Km for insulin was increased from 80 microunits/ml to 2400 microunits/ml in the insulin-exposed cells. Thus, chronic exposure to insulin induces resistance of alpha-aminoisobutyric acid uptake by decreasing the apparent affinity for insulin.     

Genistein differentially inhibits postreceptor effects of insulin in rat adipocytes without inhibiting the insulin receptor kinase.

Genistein, an isoflavone putative tyrosine kinase inhibitor, was used to investigate the coupling of insulin receptor tyrosine kinase activation to four metabolic effects of insulin in the isolated rat adipocyte. Genistein inhibited insulin-stimulated glucose oxidation in a concentration-dependent manner with an ID50 of 25 micrograms/ml and complete inhibition at 100 micrograms/ml. Genistein also prevented insulin's (10(-9) M) inhibition of isoproterenol-stimulated lipolysis with an ID50 of 15 micrograms/ml and a complete effect at 50 micrograms/ml. The effect of genistein (25 micrograms/ml) was not reversed by supraphysiological (10(-7) M) insulin levels. In contrast, genistein up to 100 micrograms/ml had no effect on insulin's (10(-9) M) stimulation of either pyruvate dehydrogenase or glycogen synthase activity. We determined whether genistein influenced insulin receptor beta-subunit autophosphorylation or tyrosine kinase substrate phosphorylation either in vivo or in vitro by anti-phosphotyrosine immunoblotting. Genistein at 100 micrograms/ml did not inhibit insulin's (10(-7) M) stimulation of insulin receptor tyrosine autophosphorylation or tyrosine phosphorylation of the cellular substrates pp185 and pp60. Also, genistein did not prevent insulin-stimulated autophosphorylation of partially purified human insulin receptors from NIH 3T3/HIR 3.5 cells or the phosphorylation of histones by the activated receptor tyrosine kinase. In control experiments using either NIH 3T3 fibroblasts or partially purified membranes from these cells, genistein did inhibit platelet-derived growth factor's stimulation of its receptor autophosphorylation. These findings indicate the following: (a) Genistein can inhibit certain responses to insulin without blocking insulin's stimulation of its receptor tyrosine autophosphorylation or of the receptor kinase substrate tyrosine phosphorylation. (b) In adipocytes genistein must block the stimulation of glucose oxidation and the antilipolytic effects of insulin at site(s) downstream from the insulin receptor tyrosine kinase. (c) The inhibitory effects of genistein on hormonal signal transduction cannot necessarily be attributed to inhibition of tyrosine kinase activity, unless specifically demonstrated.     

Sensitivity to insulin of glycolysis and glycogen synthesis of isolated soleus-muscle strips from sedentary, exercised and exercise-trained rats.

The half-maximal stimulation of the rates of glycolysis and glycogen synthesis in soleus-muscle strips from sedentary animals occurred at a concentration of insulin of about 100 microunits/ml. In soleus-muscle strips from exercise-trained rats (5 weeks of treadmill training), half-maximal stimulation of the rate of glycolysis occurred at about 10 microunits of insulin/ml, whereas that for glycogen synthesis occurred between 10 and 100 microunits of insulin/ml. The sensitivity of glycolysis to insulin after exercise training is similar to that of adipose tissue from sedentary animals. This finding suggests that, in sedentary animals, the effects of normal changes in insulin concentration may affect muscle primarily indirectly via the anti-lipolytic effect on adipose tissue, whereas after training insulin may effect the rate of glycolysis in muscle directly. A single period of exercise did not change the sensitivity of glycolysis in soleus muscle to insulin, nor probably that of glycogen synthesis. It is suggested that the improvement in insulin sensitivity of glycolysis in muscle caused by exercise-training could account, in part, for the well-established improvement in glucose tolerance and insulin sensitivity observed in man and rats after exercise-training.     

The insulin receptor and calmodulin. Calmodulin enhances insulin-mediated receptor kinase activity and insulin stimulates phosphorylation of calmodulin

Despite intensive research efforts, the functional role and regulation of the insulin receptor kinase remain enigmatic. In this investigation, we demonstrate that calmodulin enhances insulin-stimulated phosphorylation of the beta subunit of the insulin receptor and histone H2b and that insulin also stimulates phosphorylation of calmodulin. Using wheat germ lectin-enriched insulin receptor preparations obtained from rat adipocyte plasma membranes, calmodulin stimulated the rate and increased the amount of 32P incorporated predominantly into tyrosine residues of the beta subunit of the receptor when assayed in the presence of insulin. The stimulatory effect of calmodulin was both dose-dependent and saturable with half-maximal and maximal phosphorylation of the beta subunit occurring at 0.4 and 2.0 microM calmodulin, respectively. Ca2+ enhanced the ability of calmodulin to stimulate insulin-mediated phosphorylation of the beta subunit with an apparent K0.5 of approximately 0.6 microM. Calmodulin also induced an approximately 2-fold increase in both the rate and amount of insulin-mediated incorporation of 32P into histone H2b. The stimulatory effect of calmodulin was only observed in the presence of insulin and was concentration-dependent (K0.5 approximately 3.0 microM calmodulin), saturable (at 5 microM calmodulin), and Ca2+-dependent (K0.5 = 0.2 microM free Ca2+). Insulin also induced phosphorylation of a 17-kDa protein. On the basis of its molecular weight and purification via immunoadsorption with protein A-Sepharose-bound anti-calmodulin IgG, this phosphoprotein was identified as a phosphorylated form of calmodulin. Phosphorylation of calmodulin was only observed in the presence of insulin and was both Ca2+- and insulin concentration-dependent with half-maximal effects observed at 0.1 microM free Ca2+ and 350 microunits/ml insulin. Collectively, these results support the hypothesis that Ca2+ and calmodulin participate in the molecular mechanism whereby binding of insulin to its receptor is coupled to changes in cellular metabolism.     

Factors affecting the adrenocorticotropic hormone stimulation of rabbit adrenal 17α-hydroxylase activity

Adrenocorticotropic hormone (ACTH)-stimulated 17α-hydroxylase activity of rabbit adrenal tissue has been shown to be associated with the subcellular fractions sedimented from 0.25 M sucrose at 33 000 × g for 60 min and at 105 000 × g for 60 min. The fraction sedimenting at 9000 × g for 20 min (mitochondria) contained the majority of the 11β-hydroxylase activity but also had a significant amount of 17α-hydroxylase activity. All subcellular 17α-hydroxylase activity showed an apparent preference for pregnenolone over progesterone. A 1 : 1 mixture of wholehomogenates of adrenal tissue from control and ACTH-stimulated rabbits incubated with[4-¹⁴C]pregnenolone synthesized as much 17α-hydroxylated corticosteroids as homogenate from the ACTH-stimulated tissue alone. However, the mixed homogenate synthesized only 1/4th–1/5th as much 17-deoxycorticosteroids as control, non-stimulated tissue, suggesting that the control tissue contained no inhibitor of 17α-hydroxylation, whereas ACTH-stimulated tissue may contain an inhibitor of 17-deoxycorticoid formation. 24-h dialysis of whole homogenates and subcellular fractions of adrenal tissue from control and ACTH-stimulated animals showed that 17α-hydroxylation was not activated in control tissue and somewhat inactivated in ACTH-stimulated tissue by this treatment. On the other hand, dialysis activated 17-deoxycorticoid formation by whole homogenates, but not in subcellular fractions, of both ACTH-stimulated and control adrenal tissue. Injection of 5 mg/kg cycloheximide prior to the first of 2 daily ACTH injections caused an average of 270 g body weight loss while not affecting the increase in adrenal weight effected by the ACTH. Adrenal tissue homogenates from cycloheximide injected animals produced only 50% as much 17α-hydroxycorticosteroids as homogenates of tissue from animals injected with ACTH alone and produced an amount of17-deoxycorticoids intermediate between homogenates of control and ACTH-stimulated tissue, suggesting the requirement of protein synthesis for 17α-hydroxylation stimulating activity of ACTH.     

Stimulation of nitric oxide synthesis and protective role of insulin in acute thrombosis in vivo

Administration of physiologic amounts of insulin in mice (200 microunits/g body weight) resulted in 9 fold increase of basal nitric oxide level from 0.51+/-0.1224 nmol/ml (mean+/-SD, n=12) to 4.45+/-0.645 nmol/ml after 30min of the injection of the hormone. Since NO is a potent inhibitor of platelet aggregation both in vitro and in vivo, we tested the possibility whether the administration of the hormone would result in the in vivo inhibition of thrombosis through the increase of NO level in the circulation. It was found that administration of insulin (200 microunits/g body weight) in mice protected >90%(p<0.00001, n=500) of these animals from death due to thrombosis in the coronary arteries induced by ADP injection in the heart. This effect of insulin in vivo was found to be directly related to the hormone induced increase of NO level in the system. The thromboprotective effect of insulin could not be achieved by using either prostacyclin, a well known antithrombotic agent or its stable probe prostaglandin E1 instead of insulin. The efficacy of insulin was neither related to the blood glucose level nor was the consequence of the hypoglycemic effect of the hormone. In contrast, inhibition of insulin induced increase of NO level resulted in the complete loss of the thromboprotective effect of the hormone. These results suggest that insulin besides being a hypoglycemic hormone could also be a potent antithrombotic humoral factor.     

Microtubules, organelle transport, and steroidogenesis in cultured adrenocortical tumor cells. 1. An ultrastructural analysis of cells in which basal and ACTH-induced steroidogenesis was inhibited by taxol

In adrenocortical cells, the first step in the enzymatic processing of cholesterol to steroid end products occurs in the mitochondria. ACTH increases mitochondrial cholesterol and steroidogenesis. In cultured mouse adrenocortical tumor cells, microtubule-based organelle motility may increase the proximity of mitochondria to the SER, lipid droplets and endoscome-derived lysosomes, thereby facilitating the transfer of cholesterol from these organelles to the mitochondrial outer membrane. ACTH may increase opportunities for the transfer by promoting organelle motility and by increasing the number of lysosomes. Taxol, a microtubule polymerizer, inhibits basal and ACTH-induced steroidogenesis in these cells, presumably at the step where mitochondria obtain cholesterol. We examined the ultrastructure of taxol-treated, unstimulated and ACTH-stimulated cells, seeking alterations which conceivably could interefer with the proposed organelle transport and encounters, and thus correlate with taxol's inhibition of steroidogenesis. Primary cultured cells were incubated in serum-containing medium for 4 hr with and without ACTH (10 mU/ml), with 10 micrograms/ml and 50 micrograms/ml of taxol, and with ACTH and taxol 10 or taxol 50 simultaneously. Culture media were analyzed for the presence of secreted steroids at the end of 1, 2, and 4 hr of incubation. At the end of the fourth hour, unstimulated cells and cells treated with ACTH, taxol 50, and both agents simultaneously, were fixed and processed for EM. Taxol inhibited basal and ACTH-induced steroidogenesis in a dose-dependent fashion. In both unstimulated and ACTH-stimulated cells, taxol 50 formed numerous microtubule bundles, but did not markedly change the distribution of mitochondria and lipid droplets. SER tubules, and clusters of Golgi fragments, endosomes, and lysosomes appeared to be translocated towards the cell periphery along some of the microtubules. Taxol permitted an ACTH-induced cell rounding and microfilament rearrangement considered to facilitate organelle motility. Our data indicate that taxol disrupts the formation of lysosomes by these adrenal cells, but it seemed unlikely that taxol's ultrastructural effects could prevent organelle transport proposed to cause meetings between mitochondria and the SER or lipid droplets, or prevent ACTH-caused increases in these encounters. Taxol may instead prevent the transfer of lipid droplet or SER-contained cholesterol to adjacent mitochondria, by a means not detectable in our electron micrographs.     

gAd-globular head domain of adiponectin increases fatty acid oxidation in newborn rabbit hearts

Adiponectin is an adipocyte-derived hormone that has a number of metabolic effects in the body, including the control of both glucose and fatty acid metabolism. The globular head domain of adiponectin, gAd, has also been shown to increase fatty acid oxidation in skeletal muscle. Within days after birth, a rapid increase in fatty acid oxidation occurs in the heart. We examined whether adiponectin or gAd plays a role in this maturation of cardiac fatty acid oxidation. Plasma adiponectin increased in newborn rabbits following birth: 1.2 +/- 0.3 microg/ml in 1-day-old, 6.8 +/- 1.8 microg/ml in 7-day-old, and 45 +/- 5 microg/ml in 6-week-old rabbits. Because plasma insulin levels decrease and remain low throughout the suckling period, and because this decrease may contribute to the maturation of fatty acid oxidation, we examined the effects of adiponectin and gAd on fatty acid oxidation in isolated perfused 1-day-old rabbit hearts in the presence or absence of 100 microunits/ml insulin. Adiponectin (10 microg/ml) did not alter fatty acid oxidation in the presence of insulin. In the absence of insulin, the addition of recombinant gAd (1.5 microg/ml) increased fatty acid oxidation compared with control (129 +/- 18 versus 66 +/- 11 nmol.g dry weight(-1).min(-1), respectively (p < 0.05). In 7-day-old hearts, where fatty acid oxidation rates were 5-fold higher than 1-day-old hearts, gAd did not alter fatty acid oxidation rates. The increase in fatty acid oxidation in 1-day-old hearts occurred independently of changes in 5'-AMP-activated protein kinase, acetyl-CoA carboxylase, or malonyl-CoA. The effect of gAd on fatty acid oxidation was reversed in the presence of 100 microunits/ml insulin. These results suggest that a decrease in plasma insulin and increase in gAd are involved in the increase of cardiac fatty acid oxidation in the immediate newborn period.     

Changes in insulin secretion after secretin administration and the implications in diabetes mellitus

Secrepan (Eisai Co. Tokyo, Japan) was administered to 9 healthy volunteers and 36 patients with non-insulin dependent diabetes mellitus (NIDDM) to clarify the effect of secretin on the pancreatic B-cell, by determining the changes in blood of insulin (IRI). Whereas IRI in healthy subjects showed a monophasic change, reaching a peak (delta IRI = 43 +/- 7.3 microunits/ml, M +/- SE) 5 min after secretin loading and returning to the basal level in 15 min, NIDDM patients on diet therapy (delta IRI = 40.2 +/- 7.6 microunits/ml) showed no significant difference from the control group, but NIDDM patients on sulfonylurea (SU) (15.5 +/- 2.4 microunits/ml) and those on insulin therapy (5.3 +/- 1.4 microunits/ml), both showed a significant depression in responsiveness. Further, the changes in insulin secretion after atropine administration in healthy subjects and the changes in IRI response to Secrepan in vagotomized patients were also determined. As a result, data which preclude the possibility of association of the vagus nerve and cholinergic nerve with the stimulation of insulin secretion by secretin were obtained, and a direct action of secretin on the cell of islets of Langerhans was suggested. The maximum IRI response after a secretin load had a significant positive correlation with the IRI response after a 75-gm GTT and the content of C-peptide immunoreactivity in 24-hour urine. Therefore, insulin response to a secretin load can be useful in assessing endogenous insulin secretion and provides a pertinent clinical guide for the selection of an appropriate therapy for diabetes mellitus.