Perfused hearts from Type 2 diabetic (db/db) mice show metabolic responsiveness to insulin

Diabetic (db/db) mice provide an animal model of Type 2 diabetes characterized by marked in vivo insulin resistance. The effect of insulin on myocardial metabolism has not been fully elucidated in this diabetic model. In the present study we tested the hypothesis that the metabolic response to insulin in db/db hearts will be diminished due to cardiac insulin resistance. Insulin-induced changes in glucose oxidation (GLUox) and fatty acid (FA) oxidation (FAox) were measured in isolated hearts from control and diabetic mice, perfused with both low as well as high concentration of glucose and FA: 10 mM glucose/0.5 mM palmitate and 28 mM glucose/1.1 mM palmitate. Both in the absence and presence of insulin, diabetic hearts showed decreased rates of GLUox and elevated rates of FAox. However, the insulin-induced increment in GLUox, as well as the insulin-induced decrement in FAox, was similar or even more pronounced in diabetic that in control hearts. During elevated FA and glucose supply, however, the effect of insulin was blunted in db/db hearts with respect to both FAox and GLUox. Finally, insulin-stimulated deoxyglucose uptake was markedly reduced in isolated cardiomyocytes from db/db mice, whereas glucose uptake in isolated perfused db/db hearts was clearly responsive to insulin. These results show that, despite reduced insulin-stimulated glucose uptake in isolated cardiomyocytes, isolated perfused db/db hearts are responsive to metabolic actions of insulin. These results should advocate the use of insulin therapy (glucose-insulin-potassium) in diabetic patients undergoing cardiac surgery or during reperfusion after an ischemic insult.     

Phorbol esters imitate in rat fat-cells the full effect of insulin on glucose-carrier translocation, but not on 3-O-methylglucose-transport activity.

Tumour-promoting phorbol esters have insulin-like effects on glucose transport and lipogenesis in adipocytes and myocytes. It is believed that insulin activates the glucose-transport system through translocation of glucose transporters from subcellular membranes to the plasma membrane. The aim of the present study was to investigate if phorbol esters act through the same mechanism as insulin on glucose-transport activity of rat adipocytes. We compared the effects of the tumour-promoting phorbol ester tetradecanoylphorbol acetate (TPA) and of insulin on 3-O-methylglucose transport and on the distribution of D-glucose-inhibitable cytochalasin-B binding sites in isolated rat adipocytes. Insulin (100 mu units/ml) stimulated 3-O-methylglucose uptake 9-fold, whereas TPA (1 nM) stimulated the uptake only 3-fold (mean values of five experiments, given as percentage of equilibrium reached after 4 s: basal 7 +/- 1.3%, insulin 60 +/- 3.1%, TPA 22 +/- 2.3%). In contrast, both agents stimulated glucose-transporter translocation to the same extent [cytochalasin B-binding sites (pmol/mg of protein; n = 7): plasma membranes, basal 6.2 +/- 1.0, insulin 13.4 +/- 2.0, TPA 12.7 +/- 2.7; low-density membranes, basal 12.8 +/- 2.1, insulin 6.3 +/- 0.9, TPA 8.9 +/- 0.7; high-density membranes, 6.9 +/- 1.1; insulin 12.5 +/- 1.0, TPA 8.1 +/- 0.9]. We conclude from these data: (1) TPA stimulates glucose transport in fat-cells by stimulation of glucose-carrier translocation; (2) insulin and TPA stimulate the carrier translocation to the same extent, whereas the stimulation of glucose uptake is 3-fold higher with insulin, suggesting that the stimulatory effect of insulin on glucose-transport activity involves other mechanisms in addition to carrier translocation.     

Glucose-induced insulin resistance of skeletal-muscle glucose transport and uptake.

The ability of glucose and insulin to modify insulin-stimulated glucose transport and uptake was investigated in perfused skeletal muscle. Here we report that perfusion of isolated rat hindlimbs for 5 h with 12 mM-glucose and 20,000 microunits of insulin/ml leads to marked, rapidly developing, impairment of insulin action on muscle glucose transport and uptake. Thus maximal insulin-stimulated glucose uptake at 12 mM-glucose decreased from 34.8 +/- 1.9 to 11.5 +/- 1.1 mumol/h per g (mean +/- S.E.M., n = 10) during 5 h perfusion. This decrease in glucose uptake was accompanied by a similar change in muscle glucose transport as measured by uptake of 3-O-[14C]-methylglucose. Simultaneously, muscle glycogen stores increased to 2-3.5 times initial values, depending on fibre type. Perfusion for 5 h in the presence of glucose but in the absence of insulin decreased subsequent insulin action on glucose uptake by 80% of the effect of glucose with insulin, but without an increase in muscle glycogen concentration. Perfusion for 5 h with insulin but without glucose, and with subsequent addition of glucose back to the perfusate, revealed glucose uptake and transport similar to initial values obtained in the presence of glucose and insulin. The data indicate that exposure to a moderately increased glucose concentration (12 mM) leads to rapidly developing resistance of skeletal-muscle glucose transport and uptake to maximal insulin stimulation. The effect of glucose is enhanced by simultaneous insulin exposure, whereas exposure for 5 h to insulin itself does not cause measurable resistance to maximal insulin stimulation.     

Effects of chronic Akt activation on glucose uptake in the heart

Acute activation of the serine-threonine kinase Akt is cardioprotective and increases glucose uptake, at least in part, through enhanced expression of GLUT4 on the sarcolemma. The effects of chronic Akt activation on glucose uptake in the heart remain unclear. To address this issue, we examined the effects of chronic Akt activation on glucose uptake, glycogen storage, and relevant glucose transporters in the hearts of transgenic mice. We found that chronic cardiac activation of Akt led to a substantial increase in the rate of basal glucose uptake (P < 0.05) but blunted the response to insulin (1.9 vs. 18.1-fold increase compared with baseline) using NMR in ex vivo perfused heart. Basal glucose uptake was also increased in Akt transgenic mice in vivo (P < 0.005). These changes were associated with an increase on glycogen deposition, examined with histochemical staining, biochemical (>6-fold, P < 0.001) and in vivo radioactive (5-fold, P < 0.01) assays. Studies in chimeric hearts of female X-linked transgenic Akt mice suggested that increased glycogen deposition occurred as a cell autonomous effect of transgene expression. Interestingly, although sarcolemmal GLUT1 was not significantly altered, chronic Akt activation actually decreased plasma membrane GLUT4. Moreover, intracellular pools of GLUT1 were modestly reduced, whereas intracellular GLUT4 was substantially reduced. It seems likely that neither GLUT1 nor GLUT4 explains the increase in basal glucose uptake but that these reductions contribute to the loss of insulin responsiveness that we observed. These data demonstrate that chronic Akt activation increases basal glucose uptake and glycogen deposition while inhibiting the response to insulin.     

Contractile activity increases glucose uptake by muscle in severely diabetic rats

Muscle contractile activity is associated with an acceleration of glucose transport into muscle. It has been reported that the acceleration of glucose uptake by contractile activity in perfused rat muscles requires the presence of insulin in the perfusate. This claim was investigated using the perfused rat hindlimb preparation in the present study. Rats were made diabetic by injection of 125 mg/kg of streptozotocin and either studied 72 h later or maintained on insulin for 2 wk and then studied 3 days after cessation of insulin therapy. Only rats with plasma insulin levels too low to measure were used. The hindlimbs were washed out with 630 ml of medium over 75 min using a single flow-through washout before muscle stimulation. Despite the absence of insulin in the perfusion medium, stimulation of muscle contraction resulted in large increases in glucose uptake in both the diabetic and control rats. These findings do not support the claim that the stimulatory effect of muscle contraction on glucose uptake by perfused rat muscles requires the presence of insulin.     

AMPK activation restores the stimulation of glucose uptake in an in vitro model of insulin-resistant cardiomyocytes via the activation of protein kinase B

Diabetic hearts are known to be more susceptible to ischemic disease. Biguanides, like metformin, are known antidiabetic drugs that lower blood glucose concentrations by decreasing hepatic glucose production and increasing glucose disposal in muscle. Part of these metabolic effects is thought to be mediated by the activation of AMP-activated protein kinase (AMPK). In this work, we studied the relationship between AMPK activation and glucose uptake stimulation by biguanides and oligomycin, another AMPK activator, in both insulin-sensitive and insulin-resistant cardiomyocytes. In insulin-sensitive cardiomyocytes, insulin, biguanides and oligomycin were able to stimulate glucose uptake with the same efficiency. Stimulation of glucose uptake by insulin or biguanides was correlated to protein kinase B (PKB) or AMPK activation, respectively, and were additive. In insulin-resistant cardiomyocytes, where insulin stimulation of glucose uptake was greatly reduced, biguanides or oligomycin, in the absence of insulin, induced a higher stimulation of glucose uptake than that obtained in insulin-sensitive cells. This stimulation was correlated with the activation of both AMPK and PKB and was sensitive to the phosphatidylinositol-3-kinase/PKB pathway inhibitors. Finally, an adenoviral-mediated expression of a constitutively active form of AMPK increased both PKB phosphorylation and glucose uptake in insulin-resistant cardiomyocytes. We concluded that AMPK activators, like biguanides and oligomycin, are able to restore glucose uptake stimulation, in the absence of insulin, in insulin-resistant cardiomyocytes via the additive activation of AMPK and PKB. Our results suggest that AMPK activation could restore normal glucose metabolism in diabetic hearts and could be a potential therapeutic approach to treat insulin resistance.     

Contraction-induced translocation of the glucose transporter Glut4 in isolated ventricular cardiomyocytes.

Field stimulation of isolated adult ventricular cardiomyocytes was used to study the effect of contractile activity on 3-O-methylglucose transport and the subcellular distribution of Glut4. Cells contracting at a frequency of 1 Hz for 30 min exhibited unaltered basal and insulin-stimulated rates of glucose transport when compared to resting cells. However, at 5 Hz 3-O-methylglucose transport increased to 224% of control after 5 min. Under these conditions insulin was unable to produce a significant additional stimulation of glucose transport. Immunoblotting with an anti-Glut4 polyclonal antibody showed that both insulin and contraction (5 Hz) increased the amount of Glut4 in a plasma membrane fraction by about 8-fold with a parallel decrease in an intracellular membrane fraction by 60-65%. These data suggest the existence of an identical insulin- and contraction-recruitable Glut4 transporter pool in cardiomyocytes.     

Stimulation of cardiac glucose transport by thioctic acid and insulin.

Thioctic acid (alpha-lipoic acid) has been shown to improve insulin-regulated glucose disposal in animal models of insulin resistance and type 2 diabetic patients. In the present study, we have used isolated adult ventricular cardiomyocytes in order to analyze 1) direct effects of this compound on glucose uptake in a primary muscle cell, and 2) the interaction with the insulin signalling cascade. Both insulin and thioctic acid (2.5 mM) induced a rapid increase in 3-O-methylglucose transport to 322+/-43 and 385+/-58 (n = 5) percent of basal control, respectively. Combined stimulation did not result in an additional significant increase in the transport rate. Preincubation of cardiomyocytes with the phosphatidylinositol 3-kinase inhibitor wortmannin completely abolished the effects of insulin and thioctic acid, whereas gamma-linolenic acid selectively blocked the effect of this compound. These data show that thioctic acid mimics insulin action by activating the signalling cascade at or before the level of phosphatidylinositol 3-kinase.     

Oxfenicine diverts rat muscle metabolism from fatty acid to carbohydrate oxidation and protects the ischaemic rat heart

In isolated diaphragms from rats fed on a high-fat diet, oxfenicine (S-4-hydroxyphenylglycine) stimulated the depressed rates of pyruvate decarboxylation (2-fold) and glucose oxidation (5-fold). In diaphragms from normal-fed rats, oxfenicine had no effect on pyruvate decarboxylation but doubled the rate of glucose oxidation and inhibited the oxidation of palmitate. Treatment of fat-fed rats with oxfenicine restored the proportion of myocardial pyruvate dehydrogenase in the active form to that observed in normal-fed rats. In rat hearts perfused in the presence of glucose, insulin and palmitate, oxfenicine increased carbohydrate oxidation and stimulated cardiac performance with no increase in oxygen consumption - i.e. improved myocardial efficiency. Working rat hearts perfused with glucose, insulin and palmitate and subjected to 10 min global ischaemia recovered to 81% of their pre-ischaemic cardiac output after 30 min reperfusion, and released large amounts of lactate dehydrogenase into the perfusate. Hearts perfused with oxfenicine had slightly higher pre-ischaemic cardiac outputs and, on reperfusion, recovered more completely (to 96% of the pre-ischaemic value). Oxfenicine reduced the amount of lactate dehydrogenase released by 73%. We conclude that, in rat hearts with high rates of fatty acid oxidation, a relative increase in carbohydrate oxidation will improve myocardial efficiency, and preserve mechanical function and cellular integrity during acute ischaemia.     

Role of microtubules in insulin and glucagon stimulation of amino acid transport in isolated rat hepatocytes

The effects of the microtubule inhibitor, colchicine, on insulin or glucagon stimulation of alpha-amino[1-14C]-isobutyric acid (AIB) transport were investigated in isolated hepatocytes from normal fed rats. Under all conditions tested, AIB uptake appeared to occur through two components of transport: a low affinity (Km approximately 50 mM) component and a high affinity (Km approximately 1 mM) component. Within 2 h of incubation, insulin and glucagon, at maximal concentrations, increase AIB (0.1 mM) uptake by 2- to 3-fold and 4- to 6-fold, respectively. Colchicine, at the low concentration of 5 X 10(-7) M, slightly reduces basal AIB transport, decreases by 80% the simulatory effect of insulin, and diminishes by 40% the stimulatory effect of either glucagon or dibutyryl cAMP. Kinetic analysis of AIB influx indicates that the drug inhibits the increase in Vmax of a high affinity (Km approximately 1 mM) component of transport stimulated by insulin or glucagon, without affecting the kinetic parameters of a low affinity component of transport (Km approximately 50 mM). Various short term hormonal effects of insulin and glucagon (changes in glucose, urea, and lactate production) were found not to be modified by the drug. Vinblastine elicits similar changes as colchicine on AIB uptake. Lumicolchicine, a colchicine analogue that does not bind to tubulin, has no effect. The concentration of colchicine (10(-7) M) required for half-maximal inhibition of hormone-stimulated AIB transport is in the appropriate range for specific microtubule disruption. These data suggest that microtubules are involved in the regulation of the insulin or glucagon stimulation of AIB transport in isolated rat hepatocytes.     

Effect of insulin on the glucose utilization in isolated cardiac myocytes from adult rat

The acute effects of insulin on glucose utilization in isolated rat quiescent cardiac myocytes were studied. Insulin (80 nM) increased the rate of glucose clearance by 2-3 times in the presence of glucose ranging from 0.3 microM to 5.5 mM. Glucose transport, which was measured in terms of both D-glucose uptake in the presence of 0.3 microM D-glucose and initial rate of uptake of 3-O-methylglucose, was stimulated 3-fold in the presence of insulin. At higher glucose concentrations (greater than 100 microM), a decrease in glucose clearance rate due to a shift of the rate-limiting step from glucose transport to a post-transport step in the pathway of glucose metabolism was observed. At the physiological concentration of glucose (5.5 mM), about 73% of glucose was metabolized into lactate, about 10% was oxidized into CO2 and the rest (17%) remained inside the cells. The pentose phosphate pathway did not contribute to the glucose metabolism in these cells. Insulin (80 nM) significantly increased the uptake of glucose (112%), and the conversions of glucose into lactate (16%), glycogen (64%), and triglyceride (18%), but not into CO2 (3%). Insulin transiently increased the percentage of I-form of glycogen synthase by 16% above basal, but did not affect the percentage of a-form of glycogen phosphorylase. The content of glucose 6-phosphate in the cells was increased by 46% above the basal value in the presence of insulin. These results indicate that insulin has different acute stimulatory effects on various steps in the metabolic pathway of glucose in isolated quiescent cardiac myocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein synthesis by perfused hearts from normal and insulin-deficient rats. Effect of insulin in the presence of glucose and after depletion of glucose, glucose 6-phosphate and glycogen

In the absence of glucose, insulin stimulated the incorporation of (14)C-labelled amino acids into protein by perfused rat hearts that had been previously substantially depleted of endogenous glucose, glucose 6-phosphate and glycogen by substrate-free perfusion. This stimulation was also demonstrated in hearts perfused with buffer containing 2-deoxy-d-glucose, an inhibitor of glucose utilization. It is concluded that insulin exerts an effect on protein synthesis independent of its action on glucose metabolism. Streptozotocin-induced diabetes was found to have no effect either on (14)C-labelled amino acid incorporation by the perfused heart or on the polyribosome profile and amino acid-incorporating activity of polyribosomes prepared from the non-perfused hearts of these insulin-deficient rats, which show marked abnormalities in glucose metabolism. Protein synthesis was not diminished in the perfused hearts from rats treated with anti-insulin antiserum. The significance of these findings is discussed in relation to the reported effects of insulin deficiency on protein synthesis in skeletal muscle.     

Sarcolemmal glucose transport in Ca2+-tolerant myocytes from adult rat heart. Calcium dependence of insulin action

Cardiac myocytes were isolated from adult rat ventricles by a method which preserves their functional integrity, including long survival in physiological concentrations of Ca2+. Sarcolemmal glucose transport was assessed by measuring linear initial uptake rates of the nonmetabolized glucose analog 3-O-methyl-D-glucose. Transport was saturable and showed competition by D-glucose and other features of chemical and stereo-selectivity. Transport was stimulated by insulin in a dose-dependent manner, resulting in an almost 5-fold increase in Vmax, with little change in Km. Stimulation of 3-methylglucose transport by insulin was largely Ca2+-dependent. Omission of Ca2+ from the incubation medium caused a minor rise in basal 3-methylglucose uptake but the insulin-stimulated rise in Vmax was only 30%. The Ca2+ antagonist D600 also antagonized stimulation of hexose transport by insulin. In all the above respects, 3-methylglucose transport in myocytes is identical to that in intact heart muscle. In addition, the decrease in insulin response by Ca2+ omission was partially reversed by subsequent return to a Ca2+-containing medium. ATP levels remained stable in the absence of Ca2+, showing that the Ca2+ dependence did not reflect nonspecific cell damage.     

Effects of decavanadate and insulin enhancing vanadium compounds on glucose uptake in isolated rat adipocytes

The effects of different vanadium compounds namely pyridine-2,6-dicarboxylatedioxovanadium(V) (V5-dipic), bis(maltolato) oxovanadium(IV) (BMOV) and amavadine, and oligovanadates namely metavanadate and decavanadate were analysed on basal and insulin stimulated glucose uptake in rat adipocytes. Decavanadate (50 μM), manifest a higher increases (6-fold) on glucose uptake compared with basal, followed by BMOV (1 mM) and metavanadate (1 mM) solutions (3-fold) whereas V5 dipic and amavadine had no effect. Decavanadate (100 μM) also shows the highest insulin like activity when compared with the others compounds studied. In the presence of insulin (10 nM), only decavanadate increases (50%) the glucose uptake when compared with insulin stimulated glucose uptake whereas BMOV and metavanadate, had no effect and V5 dipic and amavadine prevent the stimulation to about half of the basal value. Decavanadate is also able to reduce or eradicate the suppressor effect caused by dexamethasone on glucose uptake at the level of the adipocytes. Altogether, vanadium compounds and oligovanadates with several structures and coordination spheres reveal different effects on glucose uptake in rat primary adipocytes.     

Infusion of a biotinylated bis-glucose photolabel: a new method to quantify cell surface GLUT4 in the intact mouse heart

Glucose uptake in the heart is mediated by specific glucose transporters (GLUTs) present on cardiomyocyte cell surface membranes. Metabolic stress and insulin both increase glucose transport by stimulating the translocation of glucose transporters from intracellular storage vesicles to the cell surface. Isolated perfused transgenic mouse hearts are commonly used to investigate the molecular regulation of heart metabolism; however, current methods to quantify cell surface glucose transporter content in intact mouse hearts are limited. Therefore, we developed a novel technique to directly assess the cell surface content of the cardiomyocyte glucose transporter GLUT4 in perfused mouse hearts, using a cell surface impermeant biotinylated bis-glucose photolabeling reagent (bio-LC-ATB-BGPA). Bio-LC-ATB-BGPA was infused through the aorta and cross-linked to cell surface GLUTs. Bio-LC-ATB-BGPA-labeled GLUT4 was recovered from cardiac membranes by streptavidin isolation and quantified by immunoblotting. Bio-LC-ATB-BGPA-labeling of GLUT4 was saturable and competitively inhibited by d-glucose. Stimulation of glucose uptake by insulin in the perfused heart was associated with parallel increases in bio-LC-ATB-BGPA-labeling of cell surface GLUT4. Bio-LC-ATB-BGPA also labeled cell surface GLUT1 in the perfused heart. Thus, photolabeling provides a novel approach to assess cell surface glucose transporter content in the isolated perfused mouse heart and may prove useful to investigate the mechanisms through which insulin, ischemia, and other stimuli regulate glucose metabolism in the heart and other perfused organs.     

Insulin-like action of catecholamines and Ca2+ to stimulate glucose transport and GLUT4 translocation in perfused rat heart

The uptake of 2-deoxyglucose by perfused rat hearts was compared to the distribution of the insulin-regulatable glucose transporter (GLUT4) in membrane preparations from the same hearts. The hearts were treated with the alpha-adrenergic combination of epinephrine + propranolol, the beta-adrenergic agonist isoproterenol, high (8 mM) Ca2+ concentrations, insulin and the alpha adrenergic combination or insulin alone. Epinephrine (1 microM) + propranolol (10 microM), isoproterenol (10 microM), high Ca2+, insulin (1 microM) + epinephrine (1 microM) + propranolol (10 microM) and insulin (1 microM) each led to an increase in 2-deoxyglucose uptake and a shift in the recovery of the GLUT4 from a high-speed pellet membrane fraction (putatively intracellular) to a low-speed pellet membrane fraction (putatively sarcolemmal). There were significant correlations (r = -0.673, P less than 0.001) between the stimulation of 2-deoxyglucose uptake and the loss of GLUT4 from the intracellular membrane fraction, or the increase in the sarcolemmal fraction. The data provide evidence that the GLUT4 is translocated by agents that stimulate glucose transport in heart, and therefore this mechanism is not restricted to insulin.     

Sulfonylureas Stimulate Glucose Uptake through GLUT4 Transporter Translocation in Rat Skeletal Muscle

We studied the effect of gliclazide on glucose uptake and GLUT4 translocation in skeletal muscle. Rat hindquarters were perfused in the absence or presence of gliclazide (300 μg/ml), insulin or both drugs. The basal glucose uptake was increased 2.7-fold by gliclazide (p<0.05). Gastrocnemius muscles perfused with gliclazide had a significant increase (2.4-fold) in the GLUT4 content in plasma membranes compared to basal conditions (p<0.05). The stimulations produced by 1 nM insulin on muscle glucose uptake (2.8-fold) and on GLUT4 level in plasma membranes (2.5-fold) were similar to those produced by gliclazide. The effect of insulin on the glucose uptake and on the GLUT 4 translocation was significantly enhanced by gliclazide (3.4-fold and 3.7-fold vs basal, respectively). These data show that sulfonylureas stimulate glucose uptake in skeletal muscle by promoting the movement of GLUT4 to the plasma membrane.     

Phosphorylation of cardiac protein kinase B is regulated by palmitate

In this study isolated perfused working rat hearts were used to investigate the role of palmitate-regulated protein kinase B (PKB) phosphorylation on glucose metabolism. Rat hearts were perfused aerobically in working mode with 11 mM glucose and either 100 microU/ml insulin or 100 microU/ml insulin and 1.2 mM palmitate. PKB activity and phosphorylation state were reduced in the presence of 1.2 mM palmitate, which correlates with a decrease in glycolysis (47%), glucose oxidation (84%), and glucose uptake (43%). In contrast to skeletal muscle, neither p38 nor ERK underwent changes in their phosphorylation states in response to insulin or insulin and palmitate. Moreover, pharmacological restoration of glucose oxidation rates in hearts perfused with 1.2 mM palmitate demonstrated no increase in PKB phosphorylation state. In cultured mouse cardiac muscle HL-1 cells, insulin markedly increased PKB phosphorylation, which was blunted by pre- and cotreatment with 1.2 mM palmitate. However, neither palmitate nor C(2)-ceramide treatment of insulin-stimulated cells was able to accelerate PKB dephosphorylation beyond that observed following the removal of insulin alone. Taken together, these experiments show the control of PKB phosphorylation by palmitate is independent of ceramide and suggest that this signaling event may be an important regulator of myocardial glucose uptake and oxidation.     

Accumulation of 2-deoxy-D-glucose-6-phosphate as a measure of glucose uptake in the isolated perfused heart: a 31P NMR study

The accumulation of 2-deoxy-D-glucose-6-phosphate (2DG6P), detected using 31P NMR spectroscopy, has been used as a measure of the rate of glucose uptake, yet the accuracy of this measurement has not been verified. In this study, isolated rat hearts were perfused with different substrates or isoproterenol for 30 min before measurement of either 2DG6P accumulation or [2-3H]glucose uptake, without and with insulin. Basal contractile function and metabolite concentrations were the same for all hearts. The basal rates of 2DG6P accumulation differed significantly, depending on the preceding perfusion protocol, and were 38-60% of the [2-3H]glucose uptake rates, whereas insulin-stimulated 2DG6P accumulation was the same or 71% higher than the [2-3H]glucose uptake rates. Therefore the ratio of 2DG6P accumulation/[2-3H]glucose uptake rates varied from 0.38 to 1.71, depending on the prior perfusion conditions or the presence of insulin. The rates of 2DG6P hydrolysis were found to be proportional to the intracellular 2DG6P concentrations, with a K(m) of 17.5mM and V(max) of 1.4 micromol/g dry weight/min. We conclude that the rates of 2DG6P accumulation do not accurately reflect glucose uptake rates under all physiological conditions in the isolated heart and should be used with caution.     

Mouse and human resistins impair glucose transport in primary mouse cardiomyocytes, and oligomerization is required for this biological action

The adipocytokine resistin impairs glucose tolerance and insulin sensitivity in rodents. Here, we examined the effect of resistin on glucose uptake in isolated adult mouse cardiomyocytes. Murine resistin reduced insulin-stimulated glucose uptake, establishing the heart as a resistin target tissue. Notably, human resistin also impaired insulin action in mouse cardiomyocytes, providing the first evidence that human and mouse resistin homologs have similar functions. Resistin is a cysteine-rich molecule that circulates as a multimer of a dimeric form dependent upon a single intermolecular disulfide bond, which, in the mouse, involves Cys26; mutation of this residue to alanine (C26A) produces a monomeric molecule that appears to be bioactive in the liver. Remarkably, unlike native resistin, monomeric C26A resistin had no effect on basal or insulin-stimulated glucose uptake in mouse cardiomyocytes. Resistin impairs glucose uptake in cardiomyocytes by mechanisms that involve altered vesicle trafficking. Thus, in cardiomyocytes, both mouse and human resistins directly impair glucose transport; and in contrast to effects on the liver, these actions of resistin require oligomerization.