Glucose transport and metabolism in the perfused hindquarters of lean and obese-hyperglecemic (db/db) mice effects of insulin and electrical stimulation

Changes in glucose transport and metabolism in skeletal muscles of the obese-diabetic mice (db/db) was characterized using the perfused mouse hindquarter preparation. Metabolism of [5-³H]glucose, uptake of 3-O-[methyl-³H]glucose (methylglucose) and [2-¹⁴C]deoxyglucose (deoxyglucose) was studied under resting, electrically stimulated contracting, and insulin-stimulated conditions. Basal rate of methylglucose uptake was 255 ± 18 and 180 ± 9 μl/15 min per ml intracellular fluid space for lean and db/db mice, respectively. The V_? of methylglucose transport was decreased with no change in K_m in the db/db mice. Both electrical stimulation and insulin (1/mU/ml) increased methylglucose uptake rate 2-fold in both lean and obese mice. We observed no significant change in insulin sensitivity in the db/db mice in stimulating methylglucose uptake which was subnormal under all conditions. Similar results were obtained using deoxyglucose. Likewise, uptake of glucose and ³H₂O production from [5-³H]glucose were significantly reduced, both at rest and during electrically stimulated contraction in the db/db mouse. However, lactate production in the electrically stimulated db/db mouse preparations was not significantly different from that in the lean mice. These data suggest a major contribution from an impaired glucose transport activity to the reduction in glucose metabolism in the db/db mouse skeletal muscle.     

Metabolic effects of insulin on cardiomyocytes from control and diabetic db/db mouse hearts

Diabetic db/db mice exhibit profound insulin resistance in vivo, but the specific degree of cardiac insensitivity to insulin has not been assessed. Therefore, the effect of insulin on cardiomyocytes from db/db hearts was assessed by measuring two metabolic responses (deoxyglucose uptake and fatty acid oxidation) and the phosphorylation of two enzymes in the insulin-signaling cascade [Akt and AMP-activated protein kinase (AMPK)]. Maximal insulin-stimulated deoxyglucose transport was reduced to 58 and 40% of control in cardiomyocytes from db/db mice at two ages (6 and 12 wk). Insulin-stimulated deoxyglucose uptake was also reduced in myocytes from transgenic db/db mice overexpressing the insulin-sensitive glucose transporter (db/db-hGLUT4). Treatment of db/db mice for 1 wk with an insulin-sensitizing peroxisome proliferator-activated receptor-gamma agonist (COOH) completely normalized insulin-stimulated deoxyglucose uptake. Insulin had no direct effect on palmitate oxidation by either control or db/db cardiomyocytes, but the combination of insulin and glucose reduced palmitate oxidation, likely an indirect effect secondary to increased glucose uptake. Insulin had no effect on AMPK phosphorylation from either control or db/db cardiomyocytes. Insulin increased the phosphorylation of Akt in all cardiomyocyte preparations (control, db/db, COOH-treated db/db) to the same extent. Thus insulin has selective metabolic actions in mouse cardiomyocytes; deoxyglucose uptake and Akt phosphorylation are increased, but fatty acid oxidation and AMPK phosphorylation are unchanged. Insulin resistance in db/db cardiomyocytes is manifested by reduced insulin-stimulated deoxyglucose uptake.     

Dietary zinc supplementation attenuates hyperglycemia in db/db mice

Although zinc (Zn) deficiency has been associated with insulin resistance, and altered Zn metabolism (e.g., hyperzincuria, low-normal plasma Zn concentrations) may be present in diabetes, the potential effects of Zn on modulation of insulin action in Type II diabetes have not been established. The objective of this study was to compare the effects of dietary Zn deficiency and Zn supplementation on glycemic control in db/db mice. Weanling db/db mice and lean littermate controls were fed Zn-deficient (3 ppm Zn; dbZD and InZD groups), Zn-adequate control (30 ppm Zn; dbC and InC groups) or Zn-supplemented (300 ppm Zn; dbZS and InZS groups) diets for 6 weeks. Mice were assessed for Zn status, serum and urinary indices of diabetes, and gastrocnemius insulin receptor concentration and tyrosine kinase activity. Fasting serum glucose concentrations were significantly lower in the dbZS group compared with the dbZD group (19.3 +/- 2.9 and 27.9 +/- 4.1 mM, respectively), whereas the dbC mice had an intermediate value. There was a negative correlation between femur Zn and serum glucose concentrations (r = -0.59 for lean mice, P = 0.007). The dbZS group had higher pancreatic Zn and lower circulating insulin concentrations than dbZC mice. Insulin-stimulated tyrosine kinase activity in gastrocnemius muscle was higher in the db/db genotype, and insulin receptor concentration was not altered. In summary, dietary Zn supplementation attenuated hyperglycemia and hyperinsulinemia in db/db mice, suggesting that the roles of Zn in pancreatic function and peripheral tissue glucose uptake need to be further investigated.     

Insulin binding and effects of insulin on glucose uptake and metabolism in cultured rabbit coronary microvessel endothelium

Microvascular endothelial cells were isolated from rabbit cardiac tissue, and cultivated by standard tissue culture techniques. The conversion of [U-14C]glucose to CO2 and lipids was significantly enhanced by insulin treatment. Insulin stimulated uptake of 2-[3H]deoxyglucose and enhanced the transport of 3-O[3H]methyl-D-glucose. Specific binding of [125I]insulin to RCME cells, displaceable by cold insulin, was also observed. These data demonstrate that insulin is capable of regulating metabolic activities in coronary microvascular endothelium.     

Altered metabolism causes cardiac dysfunction in perfused hearts from diabetic (db/db) mice

Contractile function and substrate metabolism were characterized in perfused hearts from genetically diabetic C57BL/KsJ-lepr(db)/lepr(db) (db/db) mice and their non-diabetic lean littermates. Contractility was assessed in working hearts by measuring left ventricular pressures and cardiac power. Rates of glycolysis, glucose oxidation, and fatty acid oxidation were measured using radiolabeled substrates ([5-(3)H]glucose, [U-(14)C]glucose, and [9,10-(3)H]palmitate) in the perfusate. Contractile dysfunction in db/db hearts was evident, with increased left ventricular end diastolic pressure and decreased left ventricular developed pressure, cardiac output, and cardiac power. The rate of glycolysis from exogenous glucose in diabetic hearts was 48% of control, whereas glucose oxidation was depressed to only 16% of control. In contrast, palmitate oxidation was increased twofold in db/db hearts. The hypothesis that altered metabolism plays a causative role in diabetes-induced contractile dysfunction was tested using perfused hearts from transgenic db/db mice that overexpress GLUT-4 glucose transporters. Both glucose metabolism and palmitate metabolism were normalized in hearts from db/db-human insulin-regulatable glucose transporter (hGLUT-4) hearts, as was contractile function. These findings strongly support a causative role of impaired metabolism in the cardiomyopathy observed in db/db diabetic hearts.     

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.     

Cardiac metabolism in mice: tracer method developments and in vivo application revealing profound metabolic inflexibility in diabetes

Studies of cardiac fuel metabolism in mice have been almost exclusively conducted ex vivo. The major aim of this study was to assess in vivo plasma FFA and glucose utilization by the hearts of healthy control (db/+) and diabetic (db/db) mice, based on cardiac uptake of (R)-2-[9,10-(3)H]bromopalmitate ([3H]R-BrP) and 2-deoxy-D-[U-14C]glucose tracers. To obtain quantitative information about the evaluation of cardiac FFA utilization with [3H]R-BrP, simultaneous comparisons of [3H]R-BrP and [14C]palmitate ([14C]P) uptake were first made in isolated perfused working hearts from db/+ mice. It was found that [3H]R-BrP uptake was closely correlated with [14C]P oxidation (r2 = 0.94, P < 0.001). Then, methods for in vivo application of [3H]R-BrP and [14C]2-DG previously developed for application in the rat were specially adapted for use in the mouse. The method yields indexes of cardiac FFA utilization (R(f)*) and clearance (K(f)*), as well as glucose utilization (R(g)'). Finally, in the main part of the study, the ability of the heart to switch between FFA and glucose fuels (metabolic flexibility) was investigated by studying anesthetized, 8-h-fasted control and db/db mice in either the basal state or during glucose infusion. In control mice, glucose infusion raised plasma levels of glucose and insulin, raised R(g)' (+58%), and lowered plasma FFA level (-48%), K(f)* (-45%), and R(f)* (-70%). This apparent reciprocal regulation of glucose and FFA utilization by control hearts illustrates metabolic flexibility for substrate use. By contrast, in the db/db mice, glucose infusion raised glucose levels with no apparent influence on cardiac FFA or glucose utilization. In conclusion, tracer methodology for assessing in vivo tissue-specific plasma FFA and glucose utilization has been adapted for use in mice and reveals a profound loss of metabolic flexibility in the diabetic db/db heart, suggesting a fixed level of FFA oxidation in fasted and glucose-infused states.     

Insulin receptor tyrosine kinase activity is unaltered in ob/ob and db/db mouse skeletal muscle membranes

Insulin binding and insulin receptor tyrosine kinase activity were examined in two rodent models with genetic insulin resistance using partially-purified skeletal muscle membrane preparations. Insulin binding activity was decreased about 50% in both 12-week (219 +/- 184 vs 1255 +/- 158 fmoles/mg, p less than 0.01) and 24-week old (2120 +/- 60 vs 1081 +/- 60 fmoles/mg, p less than 0.01) ob/ob mice. In contrast, insulin binding to membrane derived from 24-week old db/db mice was not significantly different from lean controls (1371 +/- 212 vs 1253 +/- 247 fmoles/mg). Insulin-associated tyrosine kinase activity of membranes from ob/ob skeletal muscle was decreased, compared to its normal lean littermate, when compared on a per mg of protein basis in both 12-week (37 +/- 3 vs 21 +/- 3 pmoles/min/mg, p less than 0.05) and 24-week old (71 +/- 5 vs 37 +/- 6 pmoles/min/mg, p less than 0.01) mice. However, no significant differences in kinase activities were observed when the data were normalized and compared on a per fmole of insulin-binding activity basis for the 12-week (12 +/- 1 vs 11 +/- 2) and 24-week (27 +/- 2 vs 20 +/- 3) age groups. Insulin receptor tyrosine kinase activity of db/db skeletal muscle membranes was not different than its normal lean littermate whether expressed on a protein (34 +/- 7 vs 30 +/- 3) or fmole of insulin-binding activity (21 +/- 4 vs 18 +/- 4) basis. These data suggest that insulin receptor tyrosine kinase is not associated with the insulin resistance observed in ob/ob and db/db mice and demonstrate differences in receptor regulation between both animal models.     

Effects of okadaic acid, an inhibitor of protein phosphatases-1 and -2A, on glucose transport and metabolism in skeletal muscle.

The effect of okadaic acid, an inhibitor of protein phosphatases-1 and -2A, was studied on glucose transport and metabolism in soleus muscles isolated from lean and insulin-resistant obese mice. In muscles from lean mice, the uptake of 2-deoxyglucose, an index of glucose transport and phosphorylation, was increased by okadaic acid in a concentration-dependent manner. At 5 microM, okadaic acid was as efficient as a maximally effective insulin concentration. Glucose metabolism (glycolysis and glycogen synthesis) was also measured. Whereas glycolysis was stimulated by okadaic acid, glycogen synthesis was unchanged. When okadaic acid and insulin were added together in the incubation medium, the rates of glucose transport, glycolysis, and glycogen synthesis were similar to those obtained with insulin alone, whether maximal or submaximal insulin concentrations were used. Furthermore, okadaic acid did not activate the kinase activity of the insulin receptor studied in an acellular system or in intact muscles. These results indicate that a step in the insulin-induced stimulation of muscle glucose transport involves a serine/threonine phosphorylation event that is regulated by protein phosphatases-1 and/or -2A. In muscles of insulin-resistant obese mice, the absolute values of deoxyglucose uptake stimulated by okadaic acid were lower than in muscles from lean mice. However, the okadaic acid effect, expressed as a fold stimulation, was normal. These observations suggest that in the insulin-resistant state linked to obesity, the serine/threonine phosphorylation event is likely occurring normally, but a defect at the level of the glucose transporter itself would prevent a normal response to insulin or okadaic acid.     

Renal and glycemic effects of high-dose chromium picolinate in db/db mice: assessment of DNA damage

This study examined renal and glycemic effects of chromium picolinate [Cr(pic)3] supplementation in the context of its purported potential for DNA damage. In preventional protocol, male obese diabetic db/db mice were fed diets either lacking or containing 5, 10 or 100 mg/kg chromium as Cr(pic)3 from 6 to 24 weeks of age; male lean nondiabetic db/m mice served as controls. Untreated db/db mice displayed increased plasma glucose and insulin, hemoglobin A1c, renal tissue advanced glycation end products, albuminuria, glomerular mesangial expansion, urinary 8-hydroxydeoxyguanosine (an index of oxidative DNA damage) and renal tissue immunostaining for γH2AX (a marker of double-strand DNA breaks) compared to db/m controls. Creatinine clearance was lower in untreated db/db mice than their db/m controls, while blood pressure was similar. High Cr(pic)3 intake (i.e., 100-mg/kg diet) mildly improved glycemic status and albuminuria without affecting blood pressure or creatinine clearance. Treatment with Cr(pic)3 did not increase DNA damage despite marked renal accumulation of chromium. In interventional protocol, effects of diets containing 0, 100 and 250 mg/kg supplemental chromium, from 12 to 24 weeks of age, were examined in db/db mice. The results generally revealed similar effects to those of the 100-mg/kg diet of the preventional protocol. In conclusion, the severely hyperglycemic db/db mouse displays renal structural and functional abnormalities in association with DNA damage. High-dose Cr(pic)3 treatment mildly improves glycemic control, and it causes moderate reduction in albuminuria, without affecting the histopathological appearance of the kidney and increasing the risk for DNA damage.     

Bile acid sequestration reduces plasma glucose levels in db/db mice by increasing its metabolic clearance rate

Aims/Hypothesis

Bile acid sequestrants (BAS) reduce plasma glucose levels in type II diabetics and in murine models of diabetes but the mechanism herein is unknown. We hypothesized that sequestrant-induced changes in hepatic glucose metabolism would underlie reduced plasma glucose levels. Therefore, in vivo glucose metabolism was assessed in db/db mice on and off BAS using tracer methodology.

Methods

Lean and diabetic db/db mice were treated with 2% (wt/wt in diet) Colesevelam HCl (BAS) for 2 weeks. Parameters of in vivo glucose metabolism were assessed by infusing [U-¹³C]-glucose, [2-¹³C]-glycerol, [1-²H]-galactose and paracetamol for 6 hours, followed by mass isotopologue distribution analysis, and related to metabolic parameters as well as gene expression patterns.

Results

Compared to lean mice, db/db mice displayed an almost 3-fold lower metabolic clearance rate of glucose (p = 0.0001), a ∼300% increased glucokinase flux (p = 0.001) and a ∼200% increased total hepatic glucose production rate (p = 0.0002). BAS treatment increased glucose metabolic clearance rate by ∼37% but had no effects on glucokinase flux nor total hepatic or endogenous glucose production. Strikingly, BAS-treated db/db mice displayed reduced long-chain acylcarnitine content in skeletal muscle (p = 0.0317) but not in liver (p = 0.189). Unexpectedly, BAS treatment increased hepatic FGF21 mRNA expression 2-fold in lean mice (p = 0.030) and 3-fold in db/db mice (p = 0.002).

Conclusions/Interpretation

BAS induced plasma glucose lowering in db/db mice by increasing metabolic clearance rate of glucose in peripheral tissues, which coincided with decreased skeletal muscle long-chain acylcarnitine content.     

Contraction-activated glucose uptake is normal in insulin-resistant muscle of the obese Zucker rat.

The rates of muscle glucose uptake of lean and obese Zucker rats were assessed via hindlimb perfusion under basal conditions (no insulin), in the presence of a maximal insulin concentration (10 mU/ml), and after electrically stimulated muscle contraction in the absence of insulin. The perfusate contained 28 mM glucose and 7.5 microCi/mmol of 2-deoxy-D-[3H-(G)]glucose. Glucose uptake rates in the soleus (slow-twitch oxidative fibers), red gastrocnemius (fast-twitch oxidative-glycolytic fibers), and white gastrocnemius (fast-twitch glycolytic fibers) under basal conditions and after electrically stimulated muscle contraction were not significantly different between the lean and obese rats. However, the rate of glucose uptake during insulin stimulation was significantly lower for obese than for lean rats in all three fiber types. Significant correlations were found for insulin-stimulated glucose uptake and glucose transporter protein isoform (GLUT-4) content of soleus, red gastrocnemius, and white gastrocnemius of lean (r = 0.79) and obese (r = 0.65) rats. In contrast, the relationships between contraction-stimulated glucose uptake and muscle GLUT-4 content of lean and obese rats were negligible because of inordinately low contraction-stimulated glucose uptakes by the solei. These results suggest that maximal skeletal muscle glucose uptake of obese Zucker rats is resistant to stimulation by insulin but not to contractile activity. In addition, the relationship between contraction-stimulated glucose uptake and GLUT-4 content appears to be fiber-type specific.     

Insulin-stimulated glucose transport in muscle. Evidence for a protein-kinase-C-dependent component which is unaltered in insulin-resistant mice.

The aim of our work was to investigate a possible role of protein kinase C (PKC) in insulin-stimulated glucose uptake in mouse skeletal muscle, and to search for a defect in PKC activation in insulin resistance found in obesity. In isolated soleus muscle of lean mice, insulin (100 nM) and 12-O-tetradecanoylphorbol 13-acetate (TPA) (1 microM) acutely stimulated glucose uptake 3- and 2-fold respectively. The effects of insulin and TPA were not additive. When PKC activity was down-regulated by long-term (24 h) TPA pretreatment, before measurement of glucose transport, the TPA effect was abolished, but in addition insulin-stimulated glucose transport returned to basal values. Furthermore, polymyxin B, which inhibits PKC in muscle extracts, prevented insulin-stimulated glucose uptake in muscle. In muscle of obese insulin-resistant mice, glucose uptake evoked by insulin was decreased, whereas the TPA effect, expressed as a fold increase, was unaltered. Thus both agents stimulated glucose transport to the same extent. Furthermore, no difference was observed when PKC activation by TPA was measured in muscle from lean and obese mice. These results suggest that: (1) PKC is involved in the insulin effect on glucose transport in muscle; (2) PKC activation explains only part of the insulin stimulation of glucose transport; (3) the defect in insulin response in obese mice does not appear to be due to an alteration in the PKC-dependent component of glucose transport. We propose that insulin stimulation of glucose uptake occurs by a sequential two-step mechanism, with first translocation of transporters to the plasma membrane, which is PKC dependent, and second, activation of the glucose transporters. In obesity only the activation step was decreased, whereas the translocation step was unaltered.     

Glucose metabolism in perfused skeletal muscle. Demonstration of insulin resistance in the obese Zucker rat.

1. The effect of insulin (0.5, 10 and 50 munits/ml of perfusate) on glucose uptake and disposal in skeletal muscle was studied in the isolated perfused hindquarter of obese (fa/fa) and lean (Fa/Fa) Zucker rats and Osborne-Mendel rats. 2. A concentration of 0.5 munit of insulin/ml induced a significant increase in glucose uptake (approx. 2.5 mumol/min per 30 g of muscle) in lean Zucker rats and in Osborne-Mendel rats, and 10 munits of insulin/ml caused a further increase to approx. 6 mumol/min per 30 g of muscle; but 50 munits of insulin/ml had no additional stimulatory effect. In contrast, in obese Zucker rats only 10 and 50 munits of insulin/ml had a stimulatory effect on glucose uptake, the magnitude of which was decreased by 50-70% when compared with either lean control group. Since under no experimental condition tested was an accumulation of free glucose in muscle-cell water observed, the data suggest an impairment of insulin-stimulated glucose transport across the muscle-cell membrane in obese Zucker rats. 3. The intracellular disposal of glucose in skeletal muscle of obese Zucker rats was also insulin-insensitive: even at insulin concentrations that clearly stimulated glucose uptake, no effect of insulin on lactate oxidation (nor an inhibitory effect on alanine release) was observed; [14C]glucose incorporation into skeletal-muscle lipids was stimulated by 50 munits of insulin/ml, but the rate was still only 10% of that observed in lean Zucker rats. 4. The data indicate that the skeletal muscle of obese Zucker rats is insulin-resistant with respect to both glucose-transport mechanisms and intracellular pathways of glucose metabolism, such as lactate oxidation. The excessive degree of insulin-insensitivity in skeletal muscle of obese Zucker rats may represent a causal factor in the development of the glucose intolerance in this species.     

Glucose uptake in vivo in skeletal muscles of insulin-injected chicks

Glucose uptake across the plasma membrane in animal cells plays a crucial role in whole-body glucose homeostasis. Insulin-stimulated glucose transport activity in vivo in several tissues was estimated using the 2-deoxy-D-[1-(3)H]glucose ([(3)H]2DG) uptake determination method. A tracer dose of [(3)H]2DG was injected intravenously into 8-day-old chicks (Gallus gallus) administered simultaneously or previously with porcine insulin (40 microg/kg BW). After 10 or 20 min, several major tissues, including skeletal and cardiac muscle, were sampled and their 2-deoxy-D-[1-(3)H]glucose 6-phosphate content analyzed. Plasma glucose concentration and [(3)H]2DG radioactivity were lowered by insulin within 20 min of [(3)H]2DG administration, while the plasma [(3)H]2DG/glucose ratio was not significantly different between chicks injected with insulin and their control counterparts. A marked uptake of 2DG was observed in cardiac tissue and brain, followed by kidney and skeletal muscles. In skeletal muscles, insulin increased the 2DG uptake in soleus, extensor digitorum longus and pectoralis superficialis muscles. On the other hand, no significant increases in insulin-induced 2DG uptake were detected in cardiac muscle or adipose tissue compared to controls. The results show that glucose transport across the plasma membrane in vivo in most skeletal muscles tested, but not cardiac muscle, was increased by insulin administration to chicks. These findings suggest that an insulin-responsive glucose transport mechanism is present in chickens, even though they intrinsically lack GLUT4 homologous gene, the insulin-responsive glucose transporter in mammals.     

Rate-limiting steps of 2-deoxyglucose uptake in rat adipocytes

2-Deoxy[1-¹⁴C]glucose uptake in rat adipocytes was measured as a function of time in the absence and presence of unlabelled glucose or 2-deoxyglucose. Uptake of tracer alone was linear from 2 s to 6 min. At 37°C the rate of uptake in insulin-stimulated cells decreased markedly after a few seconds in the presence of glucose (0.5–10 mM) and after 0.5–2 min in the presence of deoxyglucose (2–10 mM). Similar data were obtained at 22°C. With 10 mM glucose (37°C, 30 s) approx. 80% of the intracellular radioactivity was non-phosphorylated deoxyglucose and with 10 mM deoxyglucose approx. 40% was non-phosphorylated. The results show that deoxy[¹⁴C]glucose uptake after a few minutes is mainly limited by hexokinase in the presence of glucose and at least partially in the presence of deoxyglucose. The data suggest caution in using deoxyglucose uptake as a measure of transport, especially in complex kinetic studies.In addition, the initial velocity of tracer ¹$\text{3-O-}\text{methylglucose}$ was found to be approx. 2-fold higher than that of tracer deoxyglucose even though both sugars inhibited the initial velocity of labelled methylglucose half-maximally at a concentration of 5 mM. These data suggest a fundamental difference between deoxyglucose and methylglucose transport.     

Effect of oral treatment with a new hypoglycemic agent, AS-6, on the metabolic activities of adipocytes in db/db mice: a comparative study

The mechanism of a new hypoglycemic agent, AS-6, was comparatively studied using the adipocytes from AS-6 treated and untreated genetically obese diabetic mice, db/db. the db/db mice were treated for 1 week with a diet admixture of AS-6 (0.1%). The treatment resulted in the following alterations in metabolic activities; AS-6 treatment increased 125I-insulin binding by 1.4-3.3 fold over the insulin range of 1-1000 microU/ml, the treatment increased the basal activities in 2-deoxyglucose uptake, and in CO2 generation and lipogenesis from U-(14C)-glucose compared with the db/db controls, the treatment partially restored insulin responsiveness in 2-DG uptake and CO2 generation, and 1 mU/ml of insulin greatly stimulated lipogenesis by 5.6 fold above the basal in the control adipocytes while AS-6 treatment changed the lipogenic response less stimulative to the insulin. The results suggest that AS-6 treatment significantly increases insulin binding to the adipocytes associating with an enhancement in glucose metabolism under basal and physiological concentrations of insulin.     

Impaired insulin receptor phosphorylation in skeletal muscle membranes of db/db mice: the use of a novel skeletal muscle plasma membrane preparation to compare insulin binding and stimulation of receptor phosphorylation

A method has been developed to isolate skeletal muscle plasma membranes from mice in good yield without harsh extraction procedures. The method involves perfusion of mouse hindquarters with a calcium-deficient buffer containing collagenase and hyaluronidase. This is followed by gentle disruption, filtration, and differential centrifugations. The entire procedure takes about six hours and the yield is approximately 4 mg. protein from 10 g. equivalent of hindquarter muscle. The preparation contained predominantly plasma membranes based on specific activities of marker enzymes, electron microscopic data, and specific binding sites for insulin and a -adrenergic ligand. Studies using such preparations from lean, 4-5 week old and 12-20 week old db/db mice showed marked reduction in the phosphorylation of the 95 kDa subunit of the insulin receptor of the obese mice with no change in insulin binding. In addition, there was a progressive reduction in insulin sensitivity in stimulating receptor phosphorylation in the db/db mice.     

Abnormal function and glucose metabolism in the type-2 diabetic db/db mouse heart

This study examined cardiac function and glucose metabolism in the 6-month-old db/db mouse, a model of type-2 diabetes. Cine magnetic resonance spectroscopy (MRI) was used to measure cardiac function in vivo. The db/db mice had decreased heart rates (17%, p<0.01) and stroke volumes (21%, p<0.05) that resulted in lower cardiac output (35%, p<0.01) than controls. Although there was no difference in ejection fraction between the 2 groups, db/db mouse hearts had a 35% lower maximum rate of ejection (p<0.01) than controls. In a protocol designed to assess maximal insulin-independent glucose uptake, hearts were isolated and perfused in Langendorff mode and subjected to 0.75 mL.min(-1).(g wet mass)(-1) low flow ischemia for 32 min. Glucose uptake during ischemia was 21% lower than in controls, and post-ischemic recovery of cardiac function was decreased by 30% in db/db mouse hearts (p<0.05). Total cardiac GLUT 4 protein was 56% lower (p<0.01) in db/db mice than in controls. In summary, the db/db mouse has abnormal left ventricular function in vivo, with impaired glucose uptake during ischemia, leading to increased myocardial damage.     

Effects of rexinoids on glucose transport and insulin-mediated signaling in skeletal muscles of diabetic (db/db) mice

Rexinoids and thiazolidinediones (TZDs) are two classes of nuclear receptor ligands that induce insulin sensitization in diabetic rodents. TZDs are peroxisome proliferator-activated receptor gamma (PPARgamma) activators, whereas rexinoids are selective ligands for the retinoid X receptors (RXRs). Activation of both the insulin receptor substrates (IRSs)/Akt and the c-Cbl-associated protein (CAP)/c-Cbl pathways are important in regulating insulin-stimulated glucose transport. We have compared the effects of a rexinoid (LG268) and a TZD (rosiglitazone) on these two signal pathways in skeletal muscle of diabetic (db/db) mice. The results we have obtained show that treatment of db/db mice with either LG268 or rosiglitazone for 2 weeks results in a significant increase in insulin-stimulated glucose transport activity in skeletal muscle. Treatment with LG268 increases insulin-stimulated IRS-1 tyrosine phosphorylation and Akt phosphorylation in skeletal muscle without affecting the activity of the CAP/c-Cbl pathway. In contrast, rosiglitazone increases the levels of CAP expression and insulin-stimulated c-Cbl phosphorylation without affecting the IRS-1/Akt pathway. The effects of LG268 on the IRS-1/Akt pathway were associated with a decrease in the level of IRS-1 Ser(307) phosphorylation. Taken together, these data suggest that rexinoids improve insulin sensitivity via changes in skeletal muscle metabolism that are distinct from those induced by TZDs. Rexinoids represent a novel class of insulin sensitizers with potential applications in the treatment of insulin resistance.