Insulin-independent Glucose Transport Research Articles

Glucose autoregulates its uptake in skeletal muscle: involvement of AMP-activated protein kinase.

Preexposure to a low concentration of glucose upregulates glucose transport into skeletal muscle, whereas exposure to a high concentration of glucose has the opposite effect. This autoregulatory process occurs independently of insulin, and the mechanism by which it operates is incompletely understood. Activation of the energy-sensing enzyme AMP-activated protein kinase (AMPK) has been shown to increase insulin-independent glucose transport into skeletal muscle in response to such stimuli as exercise and hypoxia. In the present study, we examined whether AMPK could also mediate glucose autoregulation. The activity of the alpha2 isoform of AMPK and 2-deoxyglucose uptake were assessed in incubated rat extensor digitorum longus muscle after preincubation for 4 h in media containing 0, 3, 6, or 25 mmol/l glucose. The principal findings were as follows. First, AMPK activity was highest in muscles incubated with no added glucose, and it decreased as the concentration of glucose was increased. In keeping with these findings, the concentration of malonyl CoA was increased, and acetyl CoA carboxylase phosphorylation at serine 79 was decreased as the medium glucose concentration was raised. Second, decreases in AMPK activity at the higher glucose concentrations correlated closely with decreases in glucose transport (2-deoxyglucose uptake), measured during a subsequent 20-min incubation at 6 mmol/l glucose (r(2) = 0.93, P < 0.001). Third, the decrease in AMPK activity at the higher glucose concentrations was not associated with changes in whole-tissue concentrations of creatine phosphate or adenine nucleotides; however, it did correlate with increases in the rate of glycolysis, as estimated by lactate release. The results suggest that glucose autoregulates its own transport into skeletal muscle by a mechanism involving AMPK. They also suggest that this autoregulatory mechanism is not paralleled by changes in whole-tissue concentrations of creatine phosphate ATP, or AMP, but they leave open the possibility that alterations in a cytosolic pool of these compounds play a regulatory role.

Sphingomyelin/cholesterol ratio: an important determinant of glucose transport mediated by GLUT-1 in 3T3-L1 preadipocytes

Sphingomyelin pathway has been linked with insulin signaling through insulin-dependent GLUT-4 glucose transporter, but a relationship between sphingomyelin and the GLUT-1 transporter responsible for the basal (insulin-independent) glucose transport has not been clearly established. As GLUT-1 is mainly distributed to the cell surface, we explored the effects of changes in membrane sphingomyelin content on glucose transport through GLUT-1. The addition of exogenous sphingomyelin or glutathione (an inhibitor of endogenous sphingomyelinase) to the culture medium increased membrane sphingomyelin and cholesterol contents. Basal glucose uptake was enhanced and positively correlated to sphingomyelin (SM), cholesterol (CL) and SM/CL ratio. The exposure of 3T3-L1 preadipocytes to sphingomyelinase (SMase) significantly increased basal glucose uptake, membrane fluidity and decreased membrane sphingomyelin and cholesterol contents 60 min after SMase addition. There was no significant change in the abundance of GLUT-1 at the cell surface. The membrane sphingomyelin and cholesterol contents, fluidity and basal glucose transport returned to baseline levels within 2 h. The basal glucose uptake was negatively correlated with cholesterol contents and positively with SM/CL ratio. The SM/CL ratio might represent an important parameter controlling basal glucose uptake and a mechanism by which insulin resistance might be induced.

Cardiac-specific overexpression of GLUT1 prevents the development of heart failure attributable to pressure overload in mice.

Increased rates of glucose uptake and glycolysis have been repeatedly observed in cardiac hypertrophy and failure. Although these changes have been considered part of the fetal gene reactivation program, the functional significance of increased glucose utilization in hypertrophied and failing myocardium is poorly understood. We generated transgenic (TG) mice with cardiac-specific overexpression of insulin-independent glucose transporter GLUT1 to recapitulate the increases in basal glucose uptake rate observed in hypertrophied hearts. Isolated perfused TG hearts showed a greater rate of basal glucose uptake and glycolysis than hearts isolated from wild-type littermates, which persisted after pressure overload by ascending aortic constriction (AAC). The in vivo cardiac function in TG mice, assessed by echocardiography, was unaltered. When subjected to AAC, wild-type mice exhibited a progressive decline in left ventricular (LV) fractional shortening accompanied by ventricular dilation and decreased phosphocreatine to ATP ratio and reached a mortality rate of 40% at 8 weeks. In contrast, TG-AAC mice maintained LV function and phosphocreatine to ATP ratio and had <10% mortality. We found that increasing insulin-independent glucose uptake and glycolysis in adult hearts does not compromise cardiac function. Furthermore, we demonstrate that increasing glucose utilization in hypertrophied hearts protects against contractile dysfunction and LV dilation after chronic pressure overload.

Effects of nandrolone treatment on recovery in horses after strenuous physical exercise.

To test the effect of nandrolone on their recovery, six adult half-bred riding horses performed a competition exercise test (CET) and a standardized exercise test (SET) on consecutive days before and after a 2-week treatment with the anabolic steroid nandrolone laurate. Blood samples were collected during and between these tests for the determination of red cell volume and concentrations of blood lactate, plasma glucose, non-esterified fatty acids, glycerol, triglycrides, erythropoietin, cortisol, insulin, and glucagon. Muscle biopsy specimens were taken immediately after the CET and before the SET for analysis of glycogen content, citrate synthase, and 3-hydroxyacyl CoA dehvdrogenase activity. Nandrolone administration increased the rate of muscle glycogen repletion after exercise, an increase that may be explained by increased glucose output by the liver, higher plasma insulin concentration, and increased insulin-independent glucose transport, but not by better availability of lipid fuels during recovery.

Glucose Homeostasis in the Teleost Fish Tilapia: Insights from Brockmann Body Xenotransplantation Studies

Certain teleost fish have large anatomically discrete islet organs called Brockmann bodies (BBs). When transplanted into streptozotocin diabetic athymic nude mice, tilapia BBs provide long-term normoglycemia. This has afforded us the opportunity to examine tilapia islet in vivo function in a non-piscine environment and compare this with in vivo function in the donor species. As expected, fasting and non-fasting glycemic levels in long-term murine recipients of tilapia BBs were analogous to corresponding values in donor tilapia, but, surprisingly, tilapia BB grafts provided mammalian-like glucose tolerance profiles. Teleost fish, in general, are severely glucose intolerant. When glucose tolerance tests were performed in tilapia, the mean glucose disappearance rates were very low; however, diabetic nude mice bearing long-term tilapia BB grafts were extremely glucose responsive. This suggested a severe or absolute peripheral resistance to the glucostatic effects of insulin. Using Western blotting with polyclonal antibodies and then confirmed by Northern analysis, tilapia peripheral tissues appear to be devoid of GLUT-4, the insulin-sensitive glucose transporter responsible for the hypoglycemic effect of insulin in mammals, but not GLUT-1, the insulin independent glucose transporter. This may explain why tilapia, and possibly other teleost fish, are severely glucose intolerant after pharmacologic glucose-loading. Because tilapia do not tend to consume large quantities of glucose in the wild, it is not surprising that they have evolved without a mechanism to move glucose rapidly from the bloodstream into muscle and fat. Nevertheless, insulin still appears to play an important role in maintaining normoglycemia in tilapia; however, this is mostly likely a result of its effect on glucose uptake in the liver. We also present comparative data on tilapia beta cell function, quantification of islet cell numbers and types, islet products, insulin gene structure and expression, and beta cell sensitivity to the diabetogenic drug streptozotocin.

Glucose Homeostasis in the Teleost Fish Tilapia: Insights from Brockmann Body Xenotransplantation Studies1

Certain teleost fish have large anatomically discrete islet organs called Brockmann bodies (BBs). When transplanted into streptozotocin diabetic athymic nude mice, tilapia BBs provide long-term normoglycemia. This has afforded us the opportunity to examine tilapia islet in vivo function in a non-piscine environment and compare this with in vivo function in the donor species. As expected, fasting and non-fasting glycemic levels in long-term murine recipients of tilapia BBs were analogous to corresponding values in donor tilapia, but, surprisingly, tilapia BB grafts provided mammalian-like glucose tolerance profiles. Teleost fish, in general, are severely glucose intolerant. When glucose tolerance tests were performed in tilapia, the mean glucose disappearance rates were very low; however, diabetic nude mice bearing long-term tilapia BB grafts were extremely glucose responsive. This suggested a severe or absolute peripheral resistance to the glucostatic effects of insulin. Using Western blotting with polyclonal antibodies and then confirmed by Northern analysis, tilapia peripheral tissues appear to be devoid of GLUT-4, the insulin-sensitive glucose transporter responsible for the hypoglycemic effect of insulin in mammals, but not GLUT-1, the insulin independent glucose transporter. This may explain why tilapia, and possibly other teleost fish, are severely glucose intolerant after pharmacologic glucose-loading. Because tilapia do not tend to consume large quantities of glucose in the wild, it is not surprising that they have evolved without a mechanism to move glucose rapidly from the bloodstream into muscle and fat. Nevertheless, insulin still appears to play an important role in maintaining normoglycemia in tilapia; however, this is mostly likely a result of its effect on glucose uptake in the liver. We also present comparative data on tilapia beta cell function, quantification of islet cell numbers and types, islet products, insulin gene structure and expression, and beta cell sensitivity to the diabetogenic drug streptozotocin.

Metabolic stress and altered glucose transport: activation of AMP-activated protein kinase as a unifying coupling mechanism.

5'AMP-activated protein kinase (AMPK) can be activated in response to cellular fuel depletion and leads to switching off ATP-consuming pathways and switching on ATP-regenerating pathways in many cell types. We have hypothesized that AMPK is a central mediator of insulin-independent glucose transport, which enables fuel-depleted muscle cells to take up glucose for ATP regeneration under conditions of metabolic stress. To test this hypothesis, rat epitrochlearis muscles were isolated and incubated in vitro under several conditions that evoke metabolic stress accompanied by intracellular fuel depletion. Rates of glucose transport in the isolated muscles were increased by all of these conditions, including contraction (5-fold above basal), hypoxia (8-fold), 2,4-dinotrophenol (11-fold), rotenone (7-fold), and hyperosmolarity (8-fold). All of these stimuli simultaneously increased both alpha1 and alpha2 isoform-specific AMPK activity. There was close correlation between alpha1 (r2 = 0.72) and alpha2 (r2 = 0.67) AMPK activities and the rate of glucose transport, irrespective of the metabolic stress used, all of which compromised muscle fuel status as judged by ATP, phosphocreatine, and glycogen content. 5-Aminoimidazole-4-carboxamide ribonucleoside, a pharmacological AMPK activator that is metabolized to an AMP-mimetic ZMP, also increased both glucose transport and AMPK activity but did not change fuel status. Insulin stimulated glucose transport by 6.5-fold above basal but did not affect AMPK activity. These results suggest that the activation of AMPK may be a common mechanism leading to insulin-independent glucose transport in skeletal muscle under conditions of metabolic stress.

GLUT-4 Deficiency and Severe Peripheral Resistance to Insulin in the Teleost Fish Tilapia

Teleost fish, in general, are glucose intolerant; this trait has been attributed to piscine islets secreting insulin primarily in response to amino acid secretogogues rather than glucose. However, pancreatic islets from the teleost fish tilapia, when transplanted into diabetic nude mice, were glucose responsive even though tilapia were severely glucose intolerant. This suggested a strong peripheral resistance to the glucostatic effects of insulin. Using Western blotting with polyclonal antibodies as well as Northern analysis for mRNA, tilapia tissues were found to be devoid of GLUT-4, the insulin-sensitive glucose transporter responsible for the hypoglycemic effect of insulin in mammals. The absence of GLUT-4 in peripheral tissues may explain why tilapia, and possibly other teleost fish, are severely glucose intolerant. This suggests that tilapia islets have evolved along mammalian lines to be glucose sensitive while tilapia peripheral tissue have diverged widely. Using the same methods, tilapia were found to have a very limited tissue distribution of the insulin-independent glucose transporter, GLUT-1, which is responsible for basal glucose transport in mammalian cells. It is suggested that tilapia provide a naturally occurring GLUT-4 knockout model.

Role of glucose transport in glycogen supercompensation in reweighted rat skeletal muscle.

Hindlimb weight bearing after a 3-day period of hindlimb suspension (reweighting) of juvenile rats results in a marked transient elevation in soleus glycogen concentration that cannot be explained on the basis of the activities of glycogen synthase and phosphorylase. We have hypothesized that enhanced glucose transport activity could underlie this response. We directly tested this hypothesis by assessing the response of insulin-dependent and insulin-independent glucose transport activity (in vitro 2-[1,2-3H]deoxy-D-glucose uptake) as well as glucose transporter (GLUT-4) protein levels during a 48-h reweighting period. After a net glycogen loss (from 29 +/- 2 to 16 +/- 1 nmol/mg muscle; P < 0.05) during the first 2 h of reweighting, glycogen accumulated at an average rate of 1.4 nmol.mg-1.h-1 up to 18 h, reaching an apex of 38 +/- 1 nmol/mg. During this same reweighting period, insulin-independent, but not insulin-dependent, glucose transport activity was significantly enhanced (P < 0.05 vs. weight-bearing control values) and was associated with an elevated level of GLUT-4 protein and the specific activity of total hexokinase. The specific activity of citrate synthase was also increased. By 24 h of reweighting, although insulin-independent glucose transport activity and GLUT-4 protein remained elevated, glycogen accumulation had ceased, likely due to enhanced phosphorylase activity at this time point. These results are consistent with the interpretation that the glycogen supercompensation seen during reweighting of the rat soleus may be regulated in part by an enhanced glucose flux arising from an increase in insulin-independent glucose transport activity and hexokinase activity.

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

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

Reversal of enhanced muscle glucose transport after exercise: roles of insulin and glucose

Exercise stimulates insulin-independent glucose transport in skeletal muscle and also increases the sensitivity of the glucose transport process in muscle to insulin. A previous study [D. A. Young, H. Wallberg-Henriksson, M. D. Sleeper, and J. O. Holloszy. Am. J. Physiol. 253 (Endocrinol. Metab. 16): E331-E335, 1987] showed that the exercise-induced increase in glucose transport activity disappears rapidly when rat epitrochlearis muscles are incubated for 3 h in vitro in the absence of insulin and that 7.5 microU/ml insulin in the incubation medium apparently slowed the loss of enhanced sugar transport. We examined whether addition of insulin several hours after exercise increases glucose transport to the same extent as continuous insulin exposure. Addition of 7.5 microU/ml insulin 2.5 h after exercise (when glucose transport has returned to basal levels) increased sugar transport to the same level as that which resulted from continuous insulin exposure. This finding provides evidence for an increase in insulin sensitivity rather than a slowing of reversal of the exercise-induced increase in insulin-independent glucose transport activity. Glucose transport was enhanced only at submaximal, not at maximal, insulin concentrations. Exposure to a high concentration of glucose and a low insulin concentration reduced the exercise-induced increase in insulin-sensitive glucose transport. Incubation with a high concentration of 2-deoxy-D-glucose (2-DG) did not alter the increase in insulin sensitivity, even though a large amount of 2-DG entered the muscle and was phosphorylated.(ABSTRACT TRUNCATED AT 250 WORDS)