The conserved amino-terminal domain of hSRP1 alpha is essential for nuclear protein import

Transgenic mice that overexpressed IGFBP-1 are hyperinsulinemic in the first week of life and gradually develop fasting hyperglycemia. In adult transgenic mice, the hypoglycemic response to IGF-I but not insulin or des (1-3) IGF-I was attenuated (P < 0.05) compared with wild-type mice. Furthermore, in isolated adipocytes from transgenic mice, the stimulatory effect of IGF-I but not insulin on 2-deoxy-[3H]-glucose uptake was reduced (P < 0.02). In contrast, in isolated soleus muscle, the effects of both IGF-I and insulin on 2-deoxy-3H-glucose uptake and on [3H]-glucose incorporation into glycogen were significantly reduced compared to wild-type mice. The decline in specific activity of the 2-deoxy-3H-glucose, a measure of glucose appearance in the circulation, was more marked in transgenic animals (P < 0.05). In addition, tissue uptake of glucose was significantly higher in diaphragm, heart, intestine, liver, soleus muscle, and adipose tissue from fasting transgenic mice. Plasma concentrations of alanine, lysine, and methionine were also elevated in transgenic mice. These data suggest that overexpression of IGFBP-1 attenuates the hypoglycemic effect of endogenous IGF-I, which is initially compensated for by enhanced pancreatic insulin production. However, in adult mice pancreatic insulin content is reduced, insulin resistance is demonstrable in skeletal muscle and fasting hyperglycemia develops.     

Effect of soleus unweighting on stimulation of insulin-independent glucose transport activity

Unweighting of the rat soleus by tail-cast suspension results in increased insulin action on stimulation of glucose transport, which can be explained, at least in part, by increased insulin binding and enhanced glucose transporter protein levels. Glucose transport is also activated by an insulin-independent mechanism stimulated by in vitro muscle contractions or hypoxia. Therefore, the purpose of this study was to determine if soleus unweighting leads to an enhanced response of the insulin-independent pathway for stimulation of glucose transport. The hindlimbs of juvenile male Wistar rats were suspended by a tail-cast system for 3 or 6 days. Glucose transport activity in isolated soleus strips (approximately 18 mg) was then assessed by using 2-deoxy-[1,2-3H]glucose (2-DG) uptake. Insulin (2 mU/ml) had a progressively enhanced effect on 2-DG uptake after 3 and 6 days of unweighting (+44 and +72% vs. control, respectively; both P < 0.001). At these same times, there was no difference between groups for activation of 2-DG uptake by maximally effective treatments with contractions (10 tetanuses), hypoxia (60 min), or caffeine (5 mM). These results indicate that the enhanced capacity for stimulation of glucose transport after soleus unweighting is restricted to the insulin pathway, with no apparent enhancement of the insulin-independent pathway.     

Decreased basal, noninsulin-stimulated glucose uptake and metabolism by skeletal soleus muscle isolated from obese-hyperglycemic (ob/ob) mice

Insulin resistance of diaphragms of ob/ob mice has been repeatedly demonstrated previously both in vitro and in vivo. In the present study, transport and metabolism of glucose with and without insulin stimulation were compared in a skeletal muscle more likely than diaphragm or heart to be representative of the overall striated muscle mass, i.e. isolated soleus muscle. Compared with soleus muscle from lean controls, unstimulated lactate release in the presence of exogenous glucose was depressed from 16.2 to 12.3 nmol/60 min per mg wet wt in soleus from ob/ob mutants; glycolysis was decreased from 6.6 to 3.7 and [14C]glucose oxidation to 14CO2 from 0.90 to 0.33 nmol glucose/60 min per mg wet wt. Uptake of 2-deoxyglucose (2-DOG), both with and without insulin, was very much less for soleus from ob/ob than from lean mice, at 2-DOG concentrations ranging from 0.1 to 10 mM, and in mice of 6-15 wk. When 2-DOG concentration was 1 mM, its basal uptake was 0.53 nmol/30 min per mg wet wt for soleus of ob/ob as against 0.96 for soleus of lean mice. The absolute increment due to 1 mU/ml insulin was 0.49 in muscle of ob/ob as against 1.21 in that of lean mice. When the resistance to insulin action was decreased by pretreatment in vivo by either streptozotocin injection or fasting, the decreased basal 2-DOG uptake of subsequently isolated soleus muscle was not improved. Inhibition of endogenous oxidation of fatty acids by 2-bromostearate, while greatly increasing 14CO2 production from [14C]glucose, did not affect basal [5-3H]glucose metabolism or 2-DOG uptake. It is suggested that transport and/or phosphorylation of glucose under basal, unstimulated conditions are depressed in soleus muscle of ob/ob mice, whether or not resistance to insulin and hyperinsulinemia are also present. Although the origin of the decreased basal glucose uptake remains unknown it might be related to a similar decrease in basal glucose uptake by ventromedial hypothalamic cells, an event presumably resulting in a tendency to hyperphagia. Decreased basal glucose uptake by soleus muscle of ob/ob mice might explain the hyperglycemia, and hence partly the hyperinsulinemia and excessive fat deposition of those animals.     

Training effects on maternal and fetal glucose uptake following acute exercise in the rat

The purpose of the present study was to determine the effects of chronic maternal exercise on glucose uptake in maternal tissues after one bout of treadmill running during late gestation in the rat and to determine the effects on glucose accumulation in the fetus and placenta. Trained pregnant animals (PR) ran at 30 m.min-1, on a 10 degrees incline for 1 h on day 20 of gestation with a similarly treated trained nonpregnant group (NPR). Immediately after the run the animals were infused with a bolus of 1 g.kg-1 body wt as a 50% dextrose solution mixed with 2-deoxy-D-[1-3H] glucose through a carotid catheter. Sedentary pregnant (P) and nonpregnant animals (C) were also infused with the solution after no food and water for the same time frame. After 60 min, tissues were analyzed for radioactivity. Radioactive tracer was augmented in the red gastrocnemius and soleus of the PR group and the soleus of P rats. However, tracer accumulation in the fetus and placenta of the trained animals was not different than P animals. These results indicate that acute exercise in trained animals increased glucose uptake in maternal skeletal muscles without compromising conceptus glucose accumulation.     

Triterpenoid saponins from Ilex latifolia

Three new triterpenoid saponins, latifolosides F, G, H were isolated from the leaves of Ilex latifolia. Their structures were elucidated on the basis of chemical and spectral evidence. Latifoloside F was determined to be 3-O-[alpha-L-rhamnopyranosyl(1-2)]-[beta-D-glucopyranosyl(1-3)-]- alpha-L-arabinopyranosyl ilexgenin B 28-O-[alpha-L-rhamnopyranosyl(1-2)]-beta-D-glucopyranoside. Latifoloside G was 3-O-[alpha-L-rhamnopyranosyl(1-2)]-[beta-D-glucopyranosyl(1-3)-]- alpha-L-arabinopyranosyl pomolic acid 28-O-[alpha-L-rhamnopyranosyl(1-2)]-beta-D-glucopyranoside. Latifolioside H(3) was 3-O-[alpha-L-rhamnopyranosyl(1-2)]-[beta-D-glucopyranosyl(1-3)-]- alpha-L-arabinopyranosyl siaresinolic acid 28-O-[alpha-L-rhamnopyranosyl(1-2)]-beta-D-glucopyranoside.     

Disaccharide polyphosphates based upon adenophostin A activate hepatic D-myo-inositol 1,4,5-trisphosphate receptors

The glyconucleotides adenophostin A and B are the most potent known agonists at type 1 inositol trisphosphate [Ins(1,4,5)P3] receptors, although their stuctures differ markedly from that of Ins(1,4,5)P3. Equilibrium competition binding with [3H]Ins(1,4,5)P3 and unidirectional 45Ca2+ flux measurements were used to examine the effects of adenophostin A in hepatocytes, which express predominantly type 2 Ins(1,4,5)P3 receptors. Both Ins(1,4,5)P3 (Kd = 8.65 +/- 0.98 nM) and adenophostin A (Kd = 0.87 +/- 0.20 nM) bound to a single class of [3H]Ins(1,4,5)P3-binding site and each fully mobilized the same intracellular Ca2+ pool; although, adenophostin A (EC50 = 10.9 +/- 0.7 nM) was more potent than Ins(1,4,5)P3 (EC50 = 153 +/- 11 nM). Working on the assumption that it is the phosphorylated glucose component of the adenophostins that mimics the critical features of Ins(1,4,5)P3, we synthesized various phosphorylated disaccharide analogs containing this structure. The novel disaccharide-based analogs, sucrose 3,4,3'-trisphosphate [Sucr(3,4,3')P3], alpha,alpha'-trehalose 3,4,3',4'-tetrakisphosphate [Trehal(3,4,3',4')P4], alpha,alpha'-trehalose 2,4,3', 4'-tetrakisphosphate [Trehal(2,4,3',4')P4], and methyl 3-O-(alpha-d-glucopyranosyl)-beta-d-ribofuranoside 2,3', 4'-trisphosphate [Rib(2,3',4')P3], were all able to mobilize the same intracellular Ca2+ pool as Ins(1,4,5)P3 and adenophostin A; although, none was as potent as adenophostin A. The rank order of potency of the analogs, adenophostin A > Ins(1,4,5)P3 approximately Rib(2,3',4')P3 > Trehal(2,4,3',4')P4 > Glc(2',3,4)P3 approximately Trehal(3,4,3',4')P4 > Sucr(3,4,3')P3, was the same in radioligand binding and functional assays of hepatic Ins(1,4,5)P3 receptors. Both Rib(2,3',4')P3, which was as potent as Ins(1,4,5)P3, and Trehal(2,4,3',4')P4 bound with significantly higher affinity ( approximately 27 and approximately 3-fold, respectively) than the only active carbohydrate agonist of Ins(1,4,5)P3 receptors previously examined [Glc(2',3,4)P3]. We conclude that phosphorylated disaccharides provide novel means of developing high-affinity ligands of Ins(1,4,5)P3 receptors.     

The uptake of 3-deoxy-3-fluoro-D-glucose by synaptosomes from rat brain cortex

D-[3-3H]-3-deoxy-3-fluoroglucose was synthesized chemically and shown to be transported into rat brain synaptosomes by a saturable process with a Km 6.2 x 10(-4) M and a Vmax 2.8 nmole x mg protein-1. After an initial, rapid period of transport, further uptake of the fluorosugar is restricted by the rate of its phosphorylation. Both D-glucose and cytochalasin B are competitive inhibitors of 3-deoxy-3-fluoro-D-glucose transport with Ki values of 93 micron and 6.0 x 10(-7) M, respectively. Phloretin, N-ethylmaleimide and p-chloromercuribenzoate also inhibit the fluorosugar uptake, whereas ouabain and changes in K+, Na+, Mg2+ and Ca2+ ions have only a small effect. The recorded 3-deoxy-3-fluoro-D-glucose influx is slightly reduced by potassium cyanide, antimycin A, 2,4-dinitrophenol, and rotenone. The uptake reduction caused by these four reagents is relieved by the addition of exogenous ATP. The possible influence of hexokinse activity on the uptake process is discussed.     

Specific interaction of Golgi coatomer protein alpha-COP with phosphatidylinositol 3,4,5-trisphosphate

The phosphoinositide binding selectivity of Golgi coatomer COPI polypeptides was examined using photoaffinity analogs of the soluble inositol polyphosphates Ins(1,4,5)P3, Ins(1,3,4,5)P4, and InsP6, and of the polyphosphoinositides PtdIns(3,4,5)P3, PtdIns(4,5)P2, and PtdIns(3,4)P2. Highly selective Ins(1,3,4,5)P4-displaceable photocovalent modification of the alpha-COP subunit was observed with a p-benzoyldihydrocinnamide (BZDC)-containing probe, [3H]BZDC-Ins(1,3,4,5)P4. A more highly phosphorylated probe, [3H]BZDC-InsP6 probe labeled six of the seven subunits, with only beta, beta', delta, and epsilon-COP showing competitive displacement by excess InsP6. Importantly, [3H]BZDC-triester-PtdIns(3,4,5)P3, the lipid with the same phosphorylation pattern as Ins(1,3,4,5)P4, showed specific, PtdIns(3,4,5)P3-displaceable labeling of only alpha-COP. Labeling by the PtdIns(4,5)P2 and PtdIns(3,4)P2 photoaffinity probes was less intense and showed no discrimination based on PtdInsPn ligand. Thus, both the D-3 and D-5 phosphates are critical for the alpha-COP-PtdIns(3,4,5)P3 interaction, suggesting an important role for this polyphosphoinositide in vesicular trafficking.     

Clinical implications of penicillin and ceftriaxone resistance among children with pneumococcal bacteremia

BACKGROUND: The influence of non-ionic osmols on thermoregulation is unclear. HYPOTHESIS: Hyperglycemia will attenuate the rise in exercise core temperature. METHODS: Dehydrated by 4-h of water immersion (34.5 degrees C) to the neck, 6 men, (35+/-SD 7 yr) participated in each of three trials where 2.0 g x kg(-1) body wt of oral glucose (33.8% weight per volume) was consumed followed by 80 min supine rest (Glu/Rest), or 70 min supine cycle exercise at 62.8%+/-SE 0.5% (1.97+/-0.02 L x min(-1)) peak O2 uptake, followed by 10 min supine recovery with prior (Glu/Ex) or without glucose (No Glu/Ex) ingestion. Blood samples were taken periodically for measurement of Hb, Hct, Na+, K+, Osm, and glucose; mean skin (Tsk) and rectal (Tre) temperatures, and sweating rate (resistance hygrometry) and skin blood velocity (laser Doppler) were measured intermittently. RESULTS: Mean percent changes in plasma volume (p<0.05) for the exercise trials were not different: -12.3+/-2.2% (No Glu/Ex) and -12.1+/-2.1% (Glu/Ex). Mean (+/-SE) pre-exercise plasma [glucose] for Glu/Ex was higher than that of No Glu/Ex (108.4+/-3.9 vs. 85.6+/-1.6 mg x dL(-1), respectively, p<0.05). Glu/Ex vs. No Glu/Ex data, respectively, at the end of exercise indicated that: Tre was lower by 0.4 degrees C (38.2+/-0.2 vs. 38.6+/-0.1 degrees C, p<0.05), Tsk was lower (32.0+/-0.3 vs. 32.4+/-0.2 degrees C, p<0.05), forearm sweating rate was lower (0.94+/-0.09 vs. 1.05+/-0.07 mg x cm(-2) x min(-1), p<0.05); and head (temporal) skin blood velocity was not different (1.67+/-0.21 vs. 1.51+/-0.24 Hz x 10(3), NS). CONCLUSIONS: Elevation of plasma [glucose] prior to supine submaximal exercise in dehydrated men attenuates the increase of Tre without alteration of heat production, total body sweating, serum electrolytes and osmolality, or exercise-induced hypoglycemia: the mechanism may be enhanced peripheral blood flow that could enhance body heat loss.     

Leptin increases glucose transport and utilization in skeletal muscle in vitro

1. The present study examines the effect of leptin on glucose transport and metabolism in incubated soleus muscle from male lean albino rats. 2. Insulin (100 microU/ml) increased glucose uptake by twofold while the leptin group (100 nmol/l) reached 75% of the insulin response after 1 hr of incubation. However, leptin did not potentiate the insulin effect on glucose uptake in soleus muscle. 3. Leptin elicited a significant increase (27.7%) in total lactate production, accompanied by a three-fold increment in glycogen synthesis from [U-14C]D-glucose. 4. Insulin raised glycogen synthesis by sixfold. The leptin plus insulin group increased glycogen synthesis by eightfold, which is equivalent to the sum of the separated leptin and insulin groups. 5. Leptin per se exerts an insulin-like effect stimulating glucose uptake, glycogen synthesis, and lactate formation and also seems to potentiate the effect of insulin on glucose incorporation into glycogen in incubated soleus muscle.     

Profound underestimation of glucose uptake by [18F]2-deoxy-2-fluoroglucose in reperfused rat heart muscle

BACKGROUND: [18F]2-deoxy-2-fluoroglucose (FDG) is widely used as a tracer for glucose uptake in ischemic heart muscle. We tested the effects of low-flow ischemia and reperfusion on the ratio of tracer/tracee (lumped constant, LC). METHODS AND RESULTS: Isolated working rat hearts were perfused with Krebs-Henseleit buffer containing only glucose 5 mmol/L (group 1) or glucose 5 mmol/L plus oleate 0.4 mmol/L (group 2, fed; group 3, fasted). Dynamic glucose uptake was measured simultaneously with [2-3H]glucose and with FDG. After 20 minutes, coronary flow was reduced by 75% for 30 minutes before it was returned to control conditions for the final 20 minutes. Hexokinase activity in the cytosolic and mitochondrial fractions and tissue metabolites were determined. Rates of glucose uptake were highest when glucose was the only substrate. Glucose uptake, FDG uptake, and the LC increased during ischemia only in group 3. There was no change of these parameters during ischemia in groups 1 and 2. FDG uptake decreased significantly with reperfusion in groups 2 and 3, and there was a striking fall in the LC (from >1.0 to <0.2, P<.001). The fall in the LC was associated with a significant increase in intracellular free glucose. Neither ischemia nor reperfusion affected the kinetic properties of hexokinase. CONCLUSIONS: FDG profoundly underestimates glucose uptake during reperfusion in the presence of fatty acids. In the fasted state, however, FDG overestimates glucose uptake during ischemia. The results indicate limitations in the use of FDG to quantify myocardial glucose uptake in human heart.     

Acute effects of cell isolation on InsP profiles in adult rat cardiomyocytes

The isolation and culture of adult rat cardiomyocytes was shown to cause major changes in the contents of [3H]-labeled inositol phosphates and inositol phospholipids. Undigested heart tissue contained high levels of [3H]Ins(1,4,5)P3 (5364+/-800 ct/min/g tissue, 80+/-12 ct/min/mg protein) and mass content averaged 13.8 nmol/g tissue or 208+/-36 pmol/mg protein (mean+/-S.E.M., n=4). After collagenase digestion, [3H]Ins(1,4,5)P3 was undetectable and the mass content of Ins(1,4,5)P3 had decreased to 0.8+/-0.2 pmol/mg protein (mean+/-S.E.M., n=4, P<0.01). [3H]Ins(1,4)P2 was reduced by 80% and [3H]PtdIns(4,5)P2 by 90%. These profiles remained essentially unchanged when the isolated cells were maintained in culture for up to 24 h, even though the inositol phosphate response remained sensitive to norepinephrine. Similar to findings in intact tissue, the inositol phosphate response to norepinephrine in these cells was inhibited by neither U-73122 (5 microM) nor by neomycin (5 mM). By 48 h in culture, the relative levels of [3H]Ins(1,4,5)P3 and [3H]Ins(1,4)P2 had increased in relation to the total inositol phosphate content and responses appeared to better reflect intact tissue. However, while retaining insensitivity to neomycin, cells at 48 h were fully sensitive to U-73122 (5 microM). These data demonstrate that altered inositol phosphate responses are observed in adult cardiomyocytes from the time of isolation and that while the profiles change over time in culture, a pattern similar to that in intact heart is not re-established.     

Enantiomers of myo-inositol-1,3,4-trisphosphate and myo-inositol-1,4,6 -trisphosphate: stereospecific recognition by cerebellar and platelet myo-inositol-1,4,5-trisphosphate receptors

The naturally occurring tetrakisphosphate myo-inositol-1,3,4, 6-tetrakisphosphate [Ins(1,3,4,6)P4] was able to release Ca2+ from the intracellular stores of permeabilized rabbit platelets but was 40-fold less potent than D-myo-inositol-1,4,5-trisphosphate [Ins(1,4,5)P3]. The Ca2+ releasing activity of Ins(1,3,4,6)P4 was rationalized by envisaging two alternative receptor binding orientations in which the vicinal D-1,6-bisphosphate of Ins(1,3,4,6)P4 mimics the D-4,5-bisphosphate in the Ins(1,4,5)P3 binding conformation. This rationalization predicted that Ins(1,4,5)P3 regioisomers [i.e, D-myo-inositol -1,4,6-trisphosphate [D-Ins(1,4,6)P3] and D-myo-inositol-1,3,6 -trisphosphate [D-Ins(1,3,6)P3]] should also possess Ca(2+)-releasing activity. The unambiguous total synthesis of the enatiomers of Ins(1,4,6)P3 [i.e., D-Ins(1,4,6)P3 and D-Ins(3,4,6)P3] and the enatiomers of Ins(1,3,4)P3 [i.e., D-Ins(1,3,6)P3 and D-Ins(1,3,4)P3] allowed an examination of this prediction. D-Ins(1,4,6)P3 released Ca2+ from the intracellular stores of permeabilized platelets and was only 2-3-fold less potent than Ins(1,4,5)P3. D-Ins(1,3,6)P3 [alternative nomenclature, L-Ins(1,3,4)P3] also released Ca2+ but was 12-fold less potent than Ins(1,4,5)P3. Both D-Ins(1,4,6)P3 and D-Ins(1,3,6)P3 displaced specifically bound [3H]Ins(1,4,5)P3 from the Ins(1,4,5)P3 receptor on rat cerebellar membranes. In contrast, however, D-Ins(3,4,6)P3 [alternative nomenclature, L-Ins(1,4,6)P3] and D-Ins(1,3,4)P3 neither possessed Ca(2+)-releasing activity nor displaced [3H]Ins(1,4,5)P3. The ability of D-Ins(1,3,6)P3 to release Ca2+ in permeabilized platelets is in contrast to its apparent lack of Ca(2+)-mobilizing activity previously reported in rat basophilic leukemic cells. The possibility that this is a reflection of the different Ins(1,4,5)P3 receptor subtypes possessed by these two cell types is discussed.     

Growth factors increase pericellular proteoglycans independently of their mitogenic effects on A10 rat vascular smooth muscle cells

Several reports suggest that serotonin2A (5HT2A) receptors and this receptor-mediated phosphatidyl inositol (PI) hydrolysis signal transduction system are altered in platelets of depressed patients. Inositol 1,4,5-trisphosphate (Ins[1,4,5]P3), an important component of the PI signaling system, plays a crucial role in various physiological processes by releasing Ca2+ from intracellular stores after binding with Ins(1,4,5)P3 receptors. To examine the role of Ins(1,4,5)P3 receptors in depression, we determined [3H]Ins(1,4,5)P3 binding sites and expressed protein levels of Ins(1,4,5)P3 receptors in platelets of depressed patients (n=15) and normal control subjects (n=17). We observed that the mean Bmax of [3H]Ins(1,4,5)P3 binding to Ins(1,4,5)P3 receptors was significantly higher in platelets of depressed subjects compared with normal control subjects, whereas there was no significant difference in K(D) between these two groups. The immuno-detectable expressed level of Ins(1,4,5)P3 receptor protein was also significantly increased in depressed patients in contrast to the levels of normal control subjects. Moreover, a significant correlation was observed in Bmax and the protein level of Ins(1,4,5)P3 receptors. The increase in the number of [3H]Ins(1,4,5)P3 binding sites in platelets of depressed subjects appears to be due to an increase in the amount of Ins(1,4,5)P3 receptor proteins. These results suggest that Ins(1,4,5)P3 receptors may be involved in the pathophysiology of depression.     

Effect of theophylline on substrate metabolism during exercise

We tested the hypothesis that adenosine is involved in regulating substrate metabolism during exercise. Seven trained cyclists were studied during 30 minutes of exercise at approximately 75% maximal oxygen uptake (VO2max). Lipid metabolism was evaluated by infusing [2H5]glycerol and [1-13C]palmitate, and glucose kinetics were evaluated by infusing [6,6-2H]glucose. Fat and carbohydrate oxidation were also measured by indirect calorimetry. The same subjects performed two identical exercise tests, but in one trial theophylline, a potent adenosine receptor antagonist, was infused for 1 hour before and throughout exercise. Theophylline did not increase whole-body lipolysis (glycerol rate of appearance [Ra]) or free fatty acid (FFA) release during exercise, but fat oxidation was lower than control values (9.5 +/- 3.0 v 18.0 +/- 4.2 micromol x min(-1) x kg(-1), P < .01). Glucose Ra was not affected by theophylline infusion, but glucose uptake was lower (31.6 +/- 4.1 v 40.4 +/- 5.0 micromol x min(-1) x kg(-1), P < .05) and glucose concentration was higher (6.4 +/- 0.6 v 5.8 +/- 0.4 mmol/L, P < .05) than in the control trial. Total carbohydrate oxidation (302.3 +/- 26.2 v 265.5 +/- 11.7 micromol x min(-1) x kg(-1), P < .06), estimated muscle glycogenolysis (270.7 +/- 23.1 v 225.1 +/- 9.7 micromol x min(-1) x kg(-1), P < .05), and plasma lactate concentration (7.9 +/- 1.6 v 5.9 +/- 1.1 mmol/L, P < .001) were also higher during the theophylline trial. These data suggest that adenosine may play a role in stimulating glucose uptake and restraining glycogenolysis but not in limiting lipolysis during exercise.     

Chronic effects of glucose on insulin signaling in A-10 vascular smooth muscle cells

To examine the effects of hyperglycemia on insulin signaling in A-10 vascular smooth muscle cells, cells were treated with extracellular D-glucose and effects of insulin were studied on the diacylglycerol-protein kinase C signaling system. A-10 cells specifically bound 125I-insulin, and insulin-like growth factor-I did not displace the label. 125I-insulin binding was unaltered under hyperglycemic conditions. To determine if insulin receptors were coupled to other insulin-regulated processes, diacylglycerol, protein kinase C, and glucose transport were evaluated. Insulin increased cellular diacylglycerol (DAG) levels which were also increased following glucose treatment and not further stimulated by insulin. The uptake of 2-[3H]deoxy-D-glucose (2-DOG) was stimulated by insulin and 12-O-tetradecanoyl phorbol 13-acetate (TPA). Insulin- and TPA-stimulated 2-[3H]DOG uptake was inhibited by a protein kinase inhibitor, staurosporine. Preincubation of cells with 500 nM TPA overnight resulted in the inhibition of insulin- and TPA-stimulated 2-[3H]DOG uptake. Protein kinase C activity was translocated from cytosolic to membrane fractions following insulin treatment. Overnight glucose (25 mM) treatment resulted in a 50% decrease in protein kinase C enzyme activity and > 90% decrease in protein kinase C beta immunoreactive levels. Protein kinase C activity and levels were not affected by osmotic control media containing mannitol. A-10 cells express GLUT4-type glucose transporters. Neither insulin-regulatable glucose transporter (GLUT4) mRNA nor GLUT4 protein levels were diminished by glucose. Significant decreases in insulin- and TPA-stimulated 2-[3H]DOG uptake occurred, however, with glucose. The down-regulation of protein kinase C beta and resultant inhibition of 2-[3H]DOG uptake by chronic glucose suggests a biochemical link between hyperglycemia and DAG-protein kinase C signaling in vascular smooth muscle cells.     

Insulin attenuates atrophy of unweighted soleus muscle by amplified inhibition of protein degradation

Unweighting atrophy of immature soleus muscle occurs rapidly over the first several days, followed by slower atrophy coinciding with increased sensitivity to insulin of in vitro protein metabolism. This study determined whether this increased sensitivity might account for the diminution of atrophy after 3 days of tall-cast hindlimb suspension. The physiological significance of the increased response to insulin in unweighted muscle was evaluated by analyzing in vivo protein metabolism for day 3 (48 to 72 hours) and day 4 (72 to 96 hours) of unweighting in diabetic animals either injected with insulin or not treated. Soleus from nontreated diabetic animals showed a similar loss of protein during day 3 (-16.2%) and day 4 (-14.5%) of unweighting, whereas muscle from insulin-treated animals showed rapid atrophy (-14.5%) during day 3 only, declining to just -3.1% the next day. Since fractional protein synthesis was similar for both day 3 (8.6%/d) and day 4 (7.0%/d) of unweighting in insulin-treated animals, the reduction in protein loss must be accounted for by a slowing of protein degradation due to circulating insulin. Intramuscular (IM) injection of insulin (600 nmol/L) stimulated in situ protein synthesis similarly in 4-day unweighted (+56%) and weight-bearing (+90%) soleus, even though unweighted muscle showed a greater in situ response of 2-deoxy-[3H]glucose uptake to IM injection of either insulin (133 nmol/L) or insulin-like growth factor-I (IGF-I) (200 nmol/L) than control muscle. These findings suggest that unweighted muscle is selectively more responsive in vivo to insulin, and that the slower atrophy after 3 days of unweighting was due to an increased effect of insulin on inhibiting protein degradation.     

Effects of tyrosine kinase inhibitors on tyrosine phosphorylations and the insulin-like effects in response to human growth hormone in isolated rat adipocytes

Recent data suggest involvement of the Janus tyrosine kinase-2 (JAK2) in human GH-induced tyrosine phosphorylation of the GH receptor and the insulin receptor substrates 1 and 2 (IRS-1 and IRS-2), leading to activation of the phosphatidylinositol 3-kinase and the acute insulin-like effects in primary rat adipocytes. To investigate the functional role of this kinase, we screened a number of tyrosine kinase inhibitors for their ability to inhibit three rapid effects of GH on primary adipocytes: increased lipogenesis, inhibition of noradrenaline-induced lipolysis, and promotion of JAK2 tyrosine phosphorylation. Only staurosporine was found to inhibit all three effects. The inhibition of lipogenesis and antilipolysis exhibited the same staurosporine dose dependency (IC50, approximately 40 nM) as inhibition of JAK2 and IRS-1 tyrosine phosphorylation as well as binding of the p85 subunit of phosphatidylinositol 3-kinase to IRS-1 and IRS-2. The unidentified cytosolic tyrosine-phosphorylated protein pp95, in contrast, was not affected, suggesting that it is not phosphorylated primarily by JAK2. Protein kinase C does not seem to be directly involved in the insulin-like effects, because the selective protein kinase C inhibitor calphostin C had no effect at levels up to 100 nM above which unspecific cellular effects occurred. Methyl-2,5-dihydroxy cinnamate inhibited GH-induced lipogenesis from [3-3H]glucose and nonstimulated lipogenesis from [2-14C]-pyruvate and [3H]acetate, but was without effect on GH-induced 2-deoxy-D-[1-3H]glucose uptake, JAK2 phosphorylation and antilipolysis, suggesting unspecific effects on mitochondrial metabolism rather than a direct effect on the GH-mediated signal. Tyrphostin 25 and herbimycin A had no effect on any of the parameters studied, except for a slight increase in JAK2 phosphorylation in response to tyrphostin 25. In summary, these data support the role for JAK2 in mediating the insulin-like effects of GH in adipocytes.     

Non-insulin and non-exercise related increase of glucose utilization in rats and mice

The effects of high-energy phosphate contents in muscles on glucose tolerance and glucose uptake into tissues were studied in rats and mice. Enhanced glucose tolerance associated with depleted high-energy phosphates and elevated glycogen content in muscles and liver was observed in animals fed creatine analogue beta-guanidinopropionic acid (beta-GPA). Distribution of infused 2-[1-14C]deoxy-D-glucose in tissues especially in the soleus muscle, kidney, and brain was greater in mice fed beta-GPA than controls. The glucose uptake was decreased when the contents of ATP and glycogen were normalized following creatine supplementation. Plasma insulin in animals at rest was lower and its concentration after intraperitoneal glucose infusion tended to be less in animals fed beta-GPA than controls (p > 0.05), although the pattern of insulin response to glucose loading was similar to the control. The daily voluntary activity in beta-GPA fed mice was also less than controls. These results suggest that improved glucose tolerance is not related to elevated insulin concentration and/or decreased glycogen following exercise. Such improvement may be due to an increased mitochondrial energy metabolism caused by depletion of high-energy phosphates.     

Effect of increased blood glucose availability on glucose kinetics during exercise

This study examined the effect of increased blood glucose availability on glucose kinetics during exercise. Five trained men cycled for 40 min at 77 +/- 1% peak oxygen uptake on two occasions. During the second trial (Glu), glucose was infused at a rate equal to the average hepatic glucose production (HGP) measured during exercise in the control trial (Con). Glucose kinetics were measured by a primed continuous infusion of D-[3-3H]glucose. Plasma glucose increased during exercise in both trials and was significantly higher in Glu. HGP was similar at rest (Con, 11.4 +/- 1.2; Glu, 10.6 +/- 0.6 micromol . kg-1 . min-1). After 40 min of exercise, HGP reached a peak of 40.2 +/- 5.5 micromol . kg-1 . min-1 in Con; however, in Glu, there was complete inhibition of the increase in HGP during exercise that never rose above the preexercise level. The rate of glucose disappearance was greater (P < 0.05) during the last 15 min of exercise in Glu. These results indicate that an increase in glucose availability inhibits the rise in HGP during exercise, suggesting that metabolic feedback signals can override feed-forward activation of HGP during strenuous exercise.