Glucose metabolism in mouse pancreatic islets

1. Rates of glucose oxidation, lactate output and the intracellular concentration of glucose 6-phosphate were measured in mouse pancreatic islets incubated in vitro. 2. Glucose oxidation rate, measured as the formation of (14)CO(2) from [U-(14)C]glucose, was markedly dependent on extracellular glucose concentration. It was especially sensitive to glucose concentrations between 1 and 2mg/ml. Glucose oxidation was inhibited by mannoheptulose and glucosamine but not by phlorrhizin, 2-deoxyglucose or N-acetylglucosamine. Glucose oxidation was slightly stimulated by tolbutamide but was not significantly affected by adrenaline, diazoxide or absence of Ca(2+) (all of which may inhibit glucose-stimulated insulin release), by arginine or glucagon (which may stimulate insulin release) or by cycloheximide (which may inhibit insulin synthesis). 3. Rates of lactate formation were dependent on the extracellular glucose concentration and were decreased by glucosamine though not by mannoheptulose; tolbutamide increased the rate of lactate output. 4. Islet glucose 6-phosphate concentration was also markedly dependent on extracellular glucose concentration and was diminished by mannoheptulose or glucosamine; tolbutamide and glucagon were without significant effect. Mannose increased islet fructose 6-phosphate concentration but had little effect on islet glucose 6-phosphate concentration. Fructose increased islet glucose 6-phosphate concentration but to a much smaller extent than did glucose. 5. [1-(14)C]Mannose and [U-(14)C]fructose were also oxidized by islets but less rapidly than glucose. Conversion of [1-(14)C]mannose into [1-(14)C]glucose 6-phosphate or [1-(14)C]glucose could not be detected. It is concluded that metabolism of mannose is associated with poor equilibration between fructose 6-phosphate and glucose 6-phosphate. 6. These results are consistent with the idea that glucose utilization in mouse islets may be limited by the rate of glucose phosphorylation, that mannoheptulose and glucosamine may inhibit glucose phosphorylation and that effects of glucose on insulin release may be mediated through metabolism of the sugar.     

Insulin stimulation of glucose entry in cultured human fibroblasts.

The effect of insulin on glucose entry has been studied in monolayer cultures of human diploid fibroblastic cells. Influence of insulin on total cell glucose incorporation was evaluated using [14C] glucose. Glucose incorporation was increased up to two-fold in the presence of insulin. Insulin action occurred within 30 minutes and could be observed with insulin concentrations as low as 10(-10) M (10 microU)ml). The action of insulin was enhanced by preincubation in glucose-free medium. After glucose starvation the cells converted glucose primarily to glycogen and nucleotides, and the stimulation by insulin was observed equally in both fractions. Influence of insulin on the kinetics of hexose transport was studied using 2-deoxyglucose and 3-0-methyl glucose. A large diffusion component was corrected using rho-chloromercuribenzoic acid or phloridzin. Km for facilitated diffusion averaged 1.9 mM for 2-deoxyglucose and 5.3 mM for 3-O-methyl glucose, and Vmax ranged from 10-24 nmoles/min/mg cell protein. Insulin resulted in a 150% increase in Vmax with no significant change in Km. The data suggest that human diploid fibroblasts can be a useful system for the study of insulin's glucoregulatory action.     

Mechanisms of the ability of insulin to activate the glucose-transport system in rat adipocytes.

Isolated rat adipocytes were used to assess the mechanisms of the ability of insulin to accelerate glucose transport. Glucose transport was determined by measuring the initial rates of 2-deoxyglucose uptake, and at 24 degrees C insulin increased the Vmax. of transport from 7.3 +/- 1 to 23.1 +/- 2 nmol/min per 10(6) cells, but the Km value remained unchanged (2.5, cf. 2.4 mM). When the Vmax. of basal and insulin-stimulated transport was measured as a function of temperature (15-37 degrees C), parallel Arrhenius plots were obtained yielding equal activation energies of approx. 59kJ/mol. Since both processes have equal activation energies the data indicate that insulin increases Vmax. by increasing the number of available carriers rather than enhancing intrinsic activity of already functioning carriers. Since the ability of insulin to activate glucose transport did not decrease with temperature (whereas plasma-membrane fluidity declines), it is suggested that lateral diffusion of insulin receptors within the plasma-membrane bilayer is not a rat-determining step in insulin action.     

Regulation of glucose formation from lactate and pyruvate in isolated tubules of chicken kidney.

1. Isolated kidney tubules from chicken have been used to study the actions of ethanol, ouabain and aminooxyacetate on glucose formation from lactate and pyruvate. 2. In kidney tubules from well-fed chickens the rate of glucose production from lactate was higher than from pyruvate. Ethanol (10 mM) and ouabain (0.1 mM) were found to increase glucose formation from pyruvate but not from lactate. 3. It is concluded that in the presence of ethanol the fluxes of pyruvate through pyruvate dehydrogenase are in favour of the pyruvate carboxylase reaction restricted. 4. Glucose formation from lactate is decreased by aminooxyacetate (0.1 mM) and ouabain (0.1 mM). 5. Aminooxyacetate inhibited glucose formation from lactate, although chicken phosphoenolpyruvate carboxykinase is located intramitochondrially. 6. The results indicate that the effect of aminooxyacetate like that of ouabain is caused by the restricted formation of pyruvate.     

The metabolism of glucose in diaphragm muscle from normal rats, from streptozotocin-treated diabetic rats and from rats treated with anti-insulin serum

1. The metabolism of [U-¹⁴C]glucose by the isolated diaphragm muscle of normal rats, rats rendered diabetic with streptozotocin and rats with transitory insulin deficiency after an injection of anti-insulin serum was studied. 2. The incorporation of [¹⁴C]glucose into glycogen and oligosaccharides was significantly decreased in the diabetic diaphragm muscle and in the muscle from rats treated with anti-insulin serum. 3. Neither diabetes nor transitory insulin deficiency influenced the oxidation of glucose, or the formation of lactate and hexose phosphate esters from glucose. 4. Insulin fully restored the incorporation of glucose into glycogen and maltotetraose in the diabetic muscle, but the incorporation into oligosaccharides, although increased in the presence of insulin, was significantly lower than the values obtained with normal diaphragm in the presence of insulin.     

Effects of insulin and norepinephrine on glucose transport and metabolism in rat brown adipocytes. Potentiation by insulin of norepinephrine-induced glucose oxidation

Glucose is an important fuel for rat brown adipose tissue in vivo and its utilization is highly sensitive to insulin. In this study, the different glucose metabolic pathways and their regulation by insulin and norepinephrine were examined in isolated rat brown adipocytes, using [6-14C]glucose as a tracer. Glucose utilization was stimulated for insulin concentrations in the range of 40-1000 microU/ml. Furthermore, the addition of adenosine deaminase (200 mU/ml) or adenosine (10 microM) did not alter insulin sensitivity of glucose metabolism. The major effect of insulin (1 mU/ml) was a respective 7-fold and 5-fold stimulation of lipogenesis and lactate synthesis, whereas glucose oxidation remained very low. The 5-fold stimulation of total glucose metabolism by 1 mU/ml of insulin was accompanied by an 8-fold increase in glucose transport. In the presence of norepinephrine (8 microM), total glucose metabolism was increased 2-fold. This was linked to a 7-fold increase of glucose oxidation, whereas lipogenesis was greatly inhibited (by 72%). In addition, norepinephrine alone did not modify glucose transport. The addition of insulin to adipocytes incubated with norepinephrine, induced a potentiation of glucose oxidation, while lipogenesis remained very low. In conclusion, in the presence of insulin and norepinephrine glucose is a oxidative substrate for brown adipose tissue. However the quantitative importance of glucose as oxidative fuel remains to be determined.     

The influence of insulin on glucose and fatty acid metabolism in the isolated perfused rat hind quarter.

Glucose and fatty acid metabolism of resting skeletal muscle were studied by perfusion of the isolated rat hind leg with a hemoglobin-free medium. Tissue integrity was demonstrated by normal ATP, ADP and creatine phosphate levels, by a sufficient oxygen supply, and by a normal appearance of perfused muscle specimens under the electron microscope. The rates of glucose and fatty acid uptake, and of lactate, alanine, glycerol and fatty acid release were constant over a perfusion period of 60 min. Insulin (1 unit/l) caused a more than threefold increase in glucose uptake, a stimulation of lactate production, and a 20% increase in the muscular glycogen levels. Fatty acids and alanine release were significantly diminished by insulin, but glycerol release did not change. The uptake of oleate by the rat hind leg was dependent on the medium concentration in a range of 0.7-1.9mM oleate, and was stimulated by insulin. Glucose uptake was not influenced by oleate, whether sodium was present or not. When the leg was perfused with [1-14C]oleate, 75% of the incorporated fatty acids were found in muscle lipids, 10% were oxidized to CO2, and 5% were recovered in bone lipids. The absolute amount of oleate oxidation was not altered by insulin. In all experiments with and without glucose in the medium, 70-80% of the 14C label incorporated into muscle lipids was found in the triglyceride fraction. In the presence of glucose, insulin significantly increased the incorporation of [1-14C]oleate into muscle triglycerides, whereas no insulin effect, either on fatty acid uptake or on triglyceride formation, could be observed when glucose was omitted from the perfusate. The present results indicate that a "glucose-fatty acid cycle" as found in rat heart muscle does not operate in resting peripheral skeletal muscle tissue. They also demonstrate that the stimulating effect of insulin on muscular fatty acid uptake and triglyceride synthesis is dependent on glucose supply. This finding can be intrepreted as a stimulation of fatty acid esterification by sn-glycerol 3-phosphate derived from an increased glucose turnover, which is in turn due to insulin.     

Insulin regulation of glucose metabolism in HT29 colonic adenocarcinoma cells: activation of glycolysis without augmentation of glucose transport

The effects of insulin on glucose transport and metabolism were examined in cultured HT29 human colonic adenocarcinoma cells. The presence of glucose transporters was verified by D-glucose displaceable [3H]cytochalasin B binding. The Kd and Bmax values from cytochalasin B binding studies were 190 +/- 30 nM and 8.4 +/- 1.4 pmol/mg protein, respectively. Glucose transport determined with 3-O-methylglucose showed saturable kinetics with a Km of 5.8 +/- 0.4 mM and a Vmax of 0.047 +/- 0.003 mumol/mg protein per min at 25 degrees C. Moreover, in HT29 cells, two classes of insulin binding sites were detected in radioligand binding experiments. Although insulin failed to stimulate glucose transport, it was found to activate glycolysis in HT29 cells. Glucose consumption increased from 0.33 +/- 0.03 mumol/mg protein per h to 0.49 +/- 0.05 mumol/mg protein per h and lactate production was augmented from 0.67 +/- 0.04 mumol/mg protein per h to 0.87 +/- 0.06 mumol/mg protein per h in response to 10(-7) to 10(-5) M insulin. Insulin also enhanced mannose metabolism. Apart from these two hexoses, HT29 cells exhibited a surprisingly narrow substrate specificity. With the possible exception of glyceraldehyde, little lactate was produced from alternative substrates, including adenosine, inosine, ribose, deoxyribose, dihydroxyacetone, galactose and fructose either with or without insulin. Despite its limited utilization by the glycolytic pathway, adenosine was readily salvaged for de novo synthesis of adenine nucleotides. These findings suggest that insulin directly influences substrate utilization through the glycolytic pathway in HT29 cells without activating the glucose transport pathway.     

Glucagon secretion by dispersed alpha cell enriched islets from streptozotocin treated hamsters in perifusion

Streptozotocin (70 mg/kg) was administered intravenously to female Syrian hamsters. The hamsters received insulin (5U/animal/day). Insulin treatment was withdrawn 3 days before sacrifice in one group, while another group was maintained on insulin until sacrifice. Ten to 14 days following streptozotocin administration the animals were killed, and the pancreatic islets isolated and subsequently dispersed. Islet DNA content was decreased while the glucagon content was elevated by streptozotocin treatment. The glucagon secretory responsiveness of the dispersed alpha cells of control animals was stimulated by glucopenia and decreased by glucose. Alpha cells of streptozotocin hamsters were not only suppressed but were actually stimulated by high glucose concentrations. Treatment with insulin in vivo but not in vitro, resulted in a restoration of the alpha cells responsiveness to glucose suppression. Dispersed alpha cells from control and streptozotocin treated animals were stimulated by arginine. Basal and total glucagon secretion was greatest in dispersed alpha cells from streptozotocin treated animals. We concluded: that the paradoxical response of alpha cells to glucose noted in diabetes is not due to short term insulin deprivation or the lack of morphologic contact with beta cells; that the alpha cells require and insulin stimulated islet metabolite and extra islet materials to respond appropriately to glucose; and that the alpha cells response to arginine is mediated independently of glucose regulation.     

Effect of insulin, prostaglandin E1 and uptake inhibitors on glucose transport in the perfused guinea-pig placenta

The effects of insulin, prostaglandin E1 (PGE1) and uptake inhibitors on unidirectional D-glucose influx at brush border (maternal) and basal (fetal) sides of the guinea-pig syncytotrophoblast were investigated in the intact, perfused guinea-pig placenta by rapid, paired-tracer dilution. Experiments were performed in either an in situ preparation artificially perfused through the umbilical vessels (intact maternal circulation) or in the fully isolated dually-perfused placenta in which both interfaces were studied simultaneously. Kinetic characterization of unidirectional D-glucose influx gave apparent Km values (mean +/- SEM) at maternal and fetal sides of 70 +/- 6 and 87 +/- 16 mM respectively; corresponding Vmax values were 53 +/- 3 and 82 +/- 6 mumol min-1g-1. At the fetal side (singly-perfused placenta) cytochalasin B (50 microM), ethylidene-D-glucose (100 mM) and PGE1 (1 microM) partially inhibited D-glucose uptake whereas cortisol (50 microM) and progesterone (100 microM) had no effect. Abolition of the sodium gradient across the fetal interface did not modulate the kinetics of influx. In the presence of 150 mu units ml-1 insulin (dually-perfused placenta), unidirectional uptake into the trophoblast and transplacental D-[3H]glucose transfer were unaltered. In contrast, prostaglandin E1 (1 microM) markedly reduced the Km and Vmax for D-glucose at both interfaces and the inhibitory effect was reflected in a reduction in specific transplacental D-glucose transfer. Further experiments showed that the isolated placenta releases prostaglandins (PGE; PGF2 alpha) into both circulations. Bilateral insulin perfusion did not affect either lactate release by the placenta or rapid metabolism of D-[14C]glucose to [3H]lactate (usually less than 10% effluent [14C]lactate in 5 min). An asymmetric degradation of exogenous insulin was observed in the dually-perfused placenta: uterine venous samples contained 24 +/- 7 microunits ml-1 immunoreactive insulin when compared to the arterial concentration (151 +/- 3 microU ml-1 perfusate) while no change was measureable in the fetal circulation within the same time period (152 +/- 5 microU ml-1). This asymmetry was confirmed in experiments employing [125I]insulin. These results demonstrate that glucose transport in the intact guinea-pig placenta occurs by a sodium-independent, cytochalasin B-inhibitable system which is insulin-insensitive. Prostaglandin E1 appeared to be a potent transport inhibitor which suggests that prostaglandins may be involved in the 'down' regulation of placental glucose transport in vivo.     

Basolateral glucose transport in distal segments of the dog nephron

The transport of glucose by canine thick ascending limbs (TAL) and inner medullary collecting ducts (IMCD) was studied using tubule suspensions and membrane vesicles. The uptake of D-[14C(U)]glucose by a suspension of intact TAL tubules was reduced largely by phloretin (Pt), moderately by phlorizin (Pz), and completely suppressed by a combination of both agents. A selective effect of Pz on the transport of [14C]alpha-methyl-D-glucoside, but not on 2-[3H]deoxyglucose, was also observed in TAL tubules. In contrast, glucose transport was unaffected by Pz but entirely suppressed by Pt alone in IMCD tubules. The metabolism of glucose was largely suppressed by Pt but unaffected by Pz in both types of tubules. Membrane vesicles were prepared from the red medulla and the white papilla or from TAL and IMCD tubules isolated from these tissues. Vesicle preparations from both tissues demonstrated a predominant carrier-mediated, sodium-independent, Pt- and cytochalasin B-sensitive glucose transport. Following purification of basolateral membrane on a Percoll gradient, the sodium-insensitive D-[14C(U)]glucose transport activity copurified with the activity of the basolateral marker Na(+)-K+ ATPase in both tissues. However, a small sodium-dependent and Pz-sensitive component of glucose transport was found in membrane vesicles prepared from the red medulla or from thick ascending limb tubules but not from the papilla nor collecting duct tubules. The kinetic analysis of the major sodium-independent processes showed that the affinity of the transporter for glucose was greater in collecting ducts (Km = 2.3 mM) than in thick ascending limbs (Km = 4.9 mM). We conclude that glucose gains access into the cells largely through a basolateral facilitated diffusion process in both segments. However a small sodium-glucose cotransport is also detected in membranes of TAL tubules. The transport of glucose presents an axial differentiation in the affinity of glucose transporters in the renal medulla, ensuring an adequate supply of glucose to the glycolytic inner medullary structures.     

Effects of insulin on the pattern of glucose metabolism in the perfused working and Lagendorff heart of normal and insulin-deficient rats

1. The metabolic pattern of [U-(14)C]glucose in the isolated rat heart has been studied, with both retrograde aortic (Langendorff) and atrially (working) perfused preparations in the presence and absence of insulin, in normal animals, animals rendered insulin-deficient (by injection of anti-insulin serum 1hr. before excision of the heart) and animals rendered diabetic by streptozotocin injection 7 days before use. 2. Radioautochromatograms of heart extracts show that the pattern of glucose metabolism in heart muscle is more complex than in diaphragm muscle. In addition to (14)CO(2), glycogen, oligosaccharides, phosphorylated sugars and lactate (the main metabolites formed from [(14)C]glucose in diaphragm muscle), (14)C label from [(14)C]glucose appears in heart muscle in glutamate, glutamine, aspartate and alanine, and in tricarboxylic acid-cycle intermediates. 3. By a quantitative scanning technique of two-dimensional chromatograms it was found that a mechanical work load stimulates glucose metabolism, increasing by a factor of 2-3 incorporation of (14)C into all the metabolites mentioned above except lactate and phosphorylated sugars, into which (14)C incorporation is in fact diminished; (14)CO(2) production is equally stimulated. 4. Addition of insulin to the perfusion fluid of the working heart causes increases in (14)C incorporation, by a factor of about 1.5 into (14)CO(2), by a factor of about 3-5 into glycogen, lactate and phosphorylated sugars, by a factor of about 2-3 into glutamate and tricarboxylic acid-cycle intermediates and by a factor of about 0.5 into aspartate, whereas incorporation into alanine and glutamine is not affected. The effect of a work load on the pattern of glucose metabolism is thus different from that of insulin. 5. Increasing the concentration of glucose in the perfusion fluid from 1 to 20mm leads to changes of the pattern of glucose metabolism different from that brought about by insulin. (14)CO(2) production steadily increases whereas [(14)C]lactate and glycogen production levels off at 10mm-glucose, at values well below those reached in the presence of insulin. 6. In Langendorff hearts of animals rendered insulin-deficient by anti-insulin serum or streptozotocin, glucose uptake, formation of (14)CO(2) and [(14)C]lactate, and (14)C incorporation into glycogen and oligosaccharides are decreased. In insulin-deficient working hearts, however, glucose uptake and (14)CO(2) production are normal, whereas incorporation of (14)C into glycogen and [(14)C]lactate production are greatly decreased. 7. Insulin added to the perfusion fluid restores (14)C incorporation from glucose into (14)CO(2), glycogen and lactate in the Langendorff heart from animals rendered insulin-deficient by anti-insulin serum; in hearts from streptozotocin-diabetic animals addition of insulin restores (14)C incorporation into glycogen and lactate, but (14)CO(2) production remains about 50% below normal. 8. The bearing of these results on the problem of the mode of action of insulin is discussed.     

The effect of selenium-deficiency on rat fat-cell glucose oxidation.

When rats are fed a selenium-deficient diet, the glutathione peroxidase activity of epididymal fat-cells decreases to 5-9% of that of control rats fed the same diet supplemented with 0.5 p.p.m. of selenium as sodium selenite. [1-14C]Glucose oxidation in fat-cells from rats fed a selenium-deficient diet is unresponsive to the action of t-butyl hydroperoxide, which stimulates 14CO2 formation from [1-14C]glucose 4-fold in control rats. Insulin enhances [1-14C]glucose oxidation and incorporation into lipids in fat-cells from both groups of rats; however, the response elicited is reduced in fat-cells prepared from selenium-deficient animals. The 'C-1/C-6 ratio' (ratio of glucose C-1 to glucose C-6 oxidized) is enhanced by insulin to a similar degree in fat-cells from both groups of animals. The stimulatory action of Zn2+ and dithiothreitol on [1-14C]glucose oxidation observed in fat-cells from selenium-supplemented rats is greatly reduced in fat-cells from selenium-deficient rats. [1-14C]Glucose oxidation in fat-cells from both groups of animals is highly sensitive to the stimulatory action of adenosine. It is concluded that the enhanced formation and glutathione-linked destruction of H2O2 plays, at the most, only a minor role in the stimulation of the flux of glucose through the pentose phosphate pathway elicited by insulin, although elimination of glutathione peroxidase activity may influence the action of insulin on glucose oxidation. Production and subsequent destruction of H2O2 may play an important role in the stimulatory action of Zn2+ and dithiothreitol on fat-cell [1-14C]glucose oxidation.     

Sensitivity of the soleus muscle to insulin in resting and exercising rats with experimental hypo- and hyper-thyroidism.

1. The effects of hypothyroidism (caused by surgical thyroidectomy followed by treatment for 1 month with propylthiouracil) and of hyperthyroidism [induced by subcutaneous administration of L-tri-iodothyronine (T3)] on glucose tolerance and skeletal-muscle sensitivity to insulin were examined in rats. Glucose tolerance was estimated during 2 h after subcutaneous glucose injection (1 g/kg body wt.). The sensitivity of the soleus muscle to insulin was studied in vitro in sedentary and acutely exercised animals. 2. Glucose tolerance was impaired in both hypothyroid and hyperthyroid rats in comparison with euthyroid controls. 3. In the soleus muscle, responsiveness of the rate of lactate formation to insulin was abolished in hypothyroid rats, whereas the sensitivity of the rate of glycogen synthesis to insulin was unchanged. In hyperthyroid animals, opposite changes were found, i.e. responsiveness of the rate of glycogen synthesis was inhibited and the sensitivity of the rate of lactate production did not differ from that in control sedentary rats. 4. A single bout of exercise for 30 min potentiated the stimulatory effect of insulin on lactate formation in hyperthyroid rats and on glycogen synthesis in hypothyroid animals. 5. The data suggest that thyroid hormones exert an interactive effect with insulin in skeletal muscle. This is likely to be at the post-receptor level, inhibiting the effect of insulin on glycogen synthesis and stimulating oxidative glucose utilization.     

Modulation of basal glucose transporter Km in the adipocyte by insulin and other factors

We have previously described experimental conditions where basal methylglucose transport in adipocytes exhibited an apparent Km of approximately 35 mM. Under those conditions insulin stimulated transport predominantly by decreasing the transport Km (Whitesell, R. R., and Abumrad, N. A. (1985) J. Biol. Chem. 260, 2894-2899). Our findings were in contrast with earlier reports that the Km of basal glucose transport was low (3-5 mM) and similar to that of transport in insulin-treated cells. In this study we have investigated the effect of different experimental conditions on the kinetics of basal glucose transport in adipocytes. When transport was assayed at 37 degrees C, cell agitation for 10 min prior to the transport assay decreased the basal Km from 35 to 12 mM. Deprivation of metabolic substrate produced a further reduction down to 2 mM. Refeeding starved cells with 1 mM glucose returned the Km back up to 12 mM in agitated cells and to 40 mM in stabilized cells. The effects of agitation to lower and of glucose to raise the basal Km were prevented by preincubating cells with dinitrophenol. Cell agitation or substrate lack did not alter the Vmax of basal transport and were without effect on both Km and Vmax in insulin-treated cells. The temperature dependencies of the kinetics of basal and stimulated transport were studied. A decrease in the assay temperature from 37 to 23 degrees C caused both basal Km and Vmax to drop proportionately from 25 to 5 mM, and 13 to 3.6 nmol/(microliter X min), respectively. In insulin-stimulated cells, only the Vmax was decreased (Km went from 3.5 to 3 mM, Vmax from 45 to 17 nmol/(microliter X min]. The results support the concept that experimental conditions can produce large changes in the Km of basal glucose transporters. Furthermore they explain why, under certain assay conditions (with temperatures around 23 degrees C or with deprivation of metabolic substrate), the effect of insulin on transport Km is not observed. Our data also suggest that basal transport characteristics do not persist in insulin-treated cells. We would propose that one of the actions of insulin (in addition to raising Vmax) is to change the characteristics of basal transporters by overriding metabolic factors which keep the Km high. Alternatively, insulin could cause the disappearance of basal transporters as new and different ones are recruited from intracellular stores.     

Insulin stimulates glucose metabolism via the pentose phosphate pathway in Drosophila Kc cells

Drosophila melanogaster has become a prominent and convenient model for analysis of insulin action. However, to date very little is known regarding the effect of insulin on glucose uptake and metabolism in Drosophila. Here we show that, in contrast to effects seen in mammals, insulin did not alter [(3)H]2-deoxyglucose uptake and in fact decreased glycogen synthesis ( approximately 30%) in embryonic Drosophila Kc cells. Insulin significantly increased ( approximately 1.5-fold) the production of (14)CO(2) from D-[1-(14)C]glucose while the production of (14)CO(2) from D-[6-(14)C]glucose was not altered. Thus, insulin-stimulated glucose oxidation did not occur via increasing Krebs cycle activity but rather by stimulating the pentose phosphate pathway. Indeed, inhibition of the oxidative pentose phosphate pathway by 6-aminonicotinamide abolished the effect of insulin on (14)CO(2) from D-[U-(14)C]glucose. A corresponding increase in lactate production but no change in incorporation of D-[U-(14)C]glucose into total lipids was observed in response to insulin. Glucose metabolism via the pentose phosphate pathway may provide an important source of 5'-phosphate for DNA synthesis and cell replication. This novel observation correlates well with the fact that control of growth and development is the major role of insulin-like peptides in Drosophila. Thus, although intracellular signaling is well conserved, the metabolic effects of insulin are dramatically different between Drosophila and mammals.     

Differential modulation of glucose,lactate, and pyruvate oxidation by insulin and dichloroacetate in the rat heart

Despite the fact that lactate and pyruvate are potential substrates for energy production in vivo, our understanding of the control and regulation of carbohydrate metabolism is based principally on studies where glucose is the only available carbohydrate. Therefore, the purpose of this study was to determine the contributions of lactate, pyruvate, and glucose to energy production in the isolated, perfused rat heart over a range of insulin concentrations and after activation of pyruvate dehydrogenase with dichloroacetate (DCA). Hearts were perfused with physiological concentrations of [1-13C]glucose, [U-13C]lactate, [2-13C]pyruvate, and unlabeled palmitate for 45 min. Hearts were freeze clamped, and 13C NMR glutamate isotopomer analysis was performed on tissue extracts. Glucose, lactate, and pyruvate all contributed significantly to myocardial energy production; however, in the absence of insulin, glucose contributed only 25-30% of total pyruvate oxidation. Even under conditions where carbohydrates represented >95% of substrate entering the tricarboxylic acid (TCA) cycle, we found that glucose contributed at most 50-60% of total carbohydrate oxidation. Despite being present at only 0.1 mM, pyruvate contributed between approximately 10% and 30% of total acetyl-CoA entry into the TCA cycle. We also found that insulin and DCA not only increased glucose oxidation but also exogenous pyruvate oxidation; however, lactate oxidation was not increased. The differential effects of insulin and DCA on pyruvate and lactate oxidation provide further evidence for compartmentation of cardiac carbohydrate metabolism. These results may have important implications for understanding the mechanisms underlying the beneficial effects of increasing cardiac carbohydrate metabolism.     

Kinetics of utilization of organic substrates by Mycoplasma mycoides subsp. mycoides in a salts solution: a flow-microcalorimetric study

The metabolism of various organic substrates by suspensions of Mycoplasma mycoides subsp. mycoides in a salts solution was followed by microcalorimetry. Enthalpy changes associated with metabolism were in good agreement with theoretical values. Substrate utilization showed Michaelis kinetics, allowing saturation constants (Km) and maximum specific rates of substrate utilization (Vmax) to be determined. In cells grown on a complex medium containing glucose, Km values were: glucose, fructose, N-acetylglucosamine, glycerol and pyruvate, less than 5 microM; lactate, 20 microM; glucosamine, 130 microns, and mannose, 1 mM. Values of Vmax for glycerol, pyruvate and lactate were similar and approximately twice those for glucose, mannose, glucosamine and N-acetylglucosamine; Vmax for fructose was one-quarter of that for glucose. In cells grown on complex medium in which glucose was replaced by mannose, glucosamine or N-acetylglucosamine, Vmax and Km for the respective growth sugars and for glucose were not significantly affected. However, in cells grown in the presence of fructose, Vmax for fructose increased to the value observed for glucose. It is suggested that M. mycoides is adapted to, and is constitutive for, the utilization of a single sugar (glucose), and a single amino sugar (N-acetylglucosamine), but that in the presence of fructose a fructose-utilizing pathway is induced.     

Hormone secretion and glucose metabolism in islets of Langerhans of the isolated perfused pancreas from normal and streptozotocin diabetic rats.

The glucose responsiveness of alpha- and beta-cells of normal as well as untreated and insulin-treated streptozotocin diabetic rats was tested in the extracorporeal perfusion system. Also assessed was the possible in vitro effect of added insulin on the glucose sensitivity of islets from untreated diabetic animals. Insulin and glucose responsiveness of the two cell types. The rate of glucose entry islet tissue was estimated, and the effect of glucose on the tissue supply of ATP and lactate and the cyclic 3':5'-AMP level of islets was measured under the above in vitro conditions. It was demonstrated that beta-cells are more accessible to glucose than alpha-cells, that glucose entry into islet cells is not significantly modified by insulin and that glucose had no effect on ATP, lactate and cyclic 3':5'-AMP levels of islet tissue under any of the conditions investigated. High insulin in vitro elevated ATP levels of alpha-cell islets independent of extracellular glucose. Glucose caused insulin release from normal but not from diabetic islets and rapidly and efficiently suppressed stimulated glucagon secretion of the pancreas from normal and insulin treated diabetic rats. Glucose was less effective in inhibiting stimulated glucagon secretion by the pancreas from untreated diabetic rats whether insulin was added to the perfusion media or not. Therefore, profound differences of glucose responsiveness of alpha-cells fail to manifest themselves in alterations of basic parameters of glucose and energy metabolism in contrast to what had been postulated in the literature. It is however, apparent that the glucose responsiveness of alpha-cells is modified by insuling by an as yet undefined mechanism.     

3-O-methyl-D-glucose uptake in cultured bovine adrenal chromaffin cells

The membrane transport of glucose was studied in bovine adrenal chromaffin cell cultures by following the cell/medium distribution of the nonmetabolizable glucose analog, 3-O-methyl-D-glucose. Uptake of this sugar in day-1 cultures that are undergoing rapid morphological change and differentiation had a Vmax of 138 nmol/(mg protein.min) and Km of 15 mM, and was only slightly increased by 50 mU/mL insulin. In day-5 cultures where morphological changes were essentially completed, Vmax and Km decreased to 51 nmol/(mg protein.min) and 9.5 mM, respectively, and the response to insulin was restored to the level found in freshly isolated cells; this effect was abolished in the nominal absence of Ca2+. Thus, saturation kinetics and insulin and Ca2+ sensitivity of 3-methylglucose uptake observed in freshly isolated cells were maintained in culture. However, the insulin response was almost absent during the initial period of rapid morphological change when sugar transport was strongly stimulated. Culture of chromaffin cells in the presence of dexamethasone did not inhibit the formation of processes, but decreased 3-methylglucose uptake in day-5 cultures by an apparently competitive effect.