Effects of the HIV Protease Inhibitor Ritonavir on GLUT4 Knock-out Mice

HIV protease inhibitors acutely block glucose transporters (GLUTs) in vitro, and this may contribute to altered glucose homeostasis in vivo. However, several GLUT-independent mechanisms have been postulated. To determine the contribution of GLUT blockade to protease inhibitor-mediated glucose dysregulation, the effects of ritonavir were investigated in mice lacking the insulin-sensitive glucose transporter GLUT4 (G4KO). G4KO and control C57BL/6J mice were administered ritonavir or vehicle at the start of an intraperitoneal glucose tolerance test and during hyperinsulinemic-euglycemic clamps. G4KO mice exhibited elevated fasting blood glucose compared with C57BL/6J mice. Ritonavir impaired glucose tolerance in control mice but did not exacerbate glucose intolerance in G4KO mice. Similarly, ritonavir reduced peripheral insulin sensitivity in control mice but not in G4KO mice. Serum insulin levels were reduced in vivo in ritonavir-treated mice. Ritonavir reduced serum leptin levels in C57BL/6J mice but had no effect on serum adiponectin. No change in these adipokines was observed following ritonavir treatment of G4KO mice. These data confirm that a primary effect of ritonavir on peripheral glucose disposal is mediated through direct inhibition of GLUT4 activity in vivo. The ability of GLUT4 blockade to contribute to derangements in the other molecular pathways that influence insulin sensitivity remains to be determined.     

GLUT4: a key player regulating glucose homeostasis? Insights from transgenic and knockout mice (review).

Studies in which GLUT4 has been overexpressed in transgenic mice provide definitive evidence that glucose transport is rate limiting for muscle glucose disposal. Transgenic overexpression of GLUT4 selectively in skeletal muscle results in increased whole body glucose uptake and improves glucose homeostasis. These studies strengthen the hypothesis that the level of muscle GLUT4 affects the rate of whole body glucose disposal, and underscore the importance of GLUT4 in skeletal muscle for maintaining whole body glucose homeostasis. Studies in which GLUT4 has been ablated or 'knocked-out' provide proof that GLUT4 is the primary effector for mediating glucose transport in skeletal muscle and adipose tissue. Genetic ablation of GLUT4 results in impaired insulin tolerance and defects in glucose metabolism in skeletal muscle and adipose tissue. Because impaired muscle glucose transport leads to reduced whole body glucose uptake and hyperglycaemia, understanding the molecular regulation of glucose transport in skeletal muscle is important to develop effective strategies to prevent or reduce the incidence of Type II diabetes mellitus. In patients with Type II diabetes mellitus, reduced glucose transport in skeletal muscle is a major factor responsible for reduced whole body glucose uptake. Overexpression of GLUT4 in skeletal muscle improves glucose homeostasis in animal models of diabetes mellitus and protects against the development of diabetes mellitus. Thus, GLUT4 is an attractive target for pharmacological intervention strategies to control glucose homeostasis. This review will focus on the current understanding of the role of GLUT4 in regulating cellular glucose uptake and whole body glucose homeostasis.     

Tissue-specific alterations of glucose transport and molecular mechanisms of intertissue communication in obesity and type 2 diabetes.

Insulin resistance plays a major role in the pathogenesis of type 2 diabetes. Insulin regulates blood glucose levels primarily by promoting glucose uptake from the blood into multiple tissues and by suppressing glucose production from the liver. The glucose transporter, GLUT4, mediates insulin-stimulated glucose uptake in muscle and adipose tissue. Decreased GLUT4 expression in adipose tissue is a common feature of many insulin resistant states. GLUT4 expression is preserved in skeletal muscle in many insulin resistant states. However, functional defects in the intracellular trafficking and plasma membrane translocation of GLUT4 result in impaired insulin-stimulated glucose uptake in muscle. Tissue-specific genetic knockout of GLUT4 expression in adipose tissue or muscle of mice has provided new insights into the pathogenesis of insulin resistance. We recently determined that the expression of serum retinol binding protein (RBP4) is induced in adipose tissue as a consequence of decreased GLUT4 expression. We found that RBP4 is elevated in the serum of insulin resistant humans and mice. Furthermore, we found that increasing serum RBP4 levels by transgenic overexpression or by injection of purified RBP4 protein into normal mice causes insulin resistance. Therefore, RBP4 appears to play an important role in mediating adipose tissue communication with other insulin target tissues in insulin resistant states.     

Galpha i2 enhances in vivo activation of and insulin signaling to GLUT4.

Heterotrimeric G-proteins, including Galpha(i2), have been implicated in modulating glucose disposal and insulin signaling. This cross-talk between G-protein-coupled and tyrosine kinase-coupled signaling pathways is a focal point for the study of integration of cell signaling. Herein we study the role of Galpha(i2) in modulating glucose transport, focusing upon linkages to insulin signaling. Utilizing mice harboring a transgene that directs the expression of a constitutively activated, GTPase-deficient mutant of Galpha(i2) (Q205L) in adipose tissue, skeletal muscle, and liver, we demonstrate that Galpha(i2) regulates the translocation of the insulin-sensitive GLUT4 glucose transporter in skeletal muscle and adipose tissue. The expression of Q205L Galpha(i2) increased glucose transport and translocation of GLUT4 to the plasma membrane in vivo in the absence of insulin stimulation. Adipocytes from the Q205L Galpha(i2) mice displayed enhanced insulin-stimulated glucose transport and GLUT4 translocation to the plasma membrane to levels nearly twice that of those from littermate controls. Phosphatidylinositol 3-kinase and Akt activities were constitutively activated in tissues expressing the Q205L Galpha(i2). Studies of adipocytes from wild-type mice displayed short term activation of phosphatidylinositol 3-kinase, Akt, and GLUT4 translocation in response to activation of Galpha(i2) by lysophosphatidic acid, a response sensitive to pertussis toxin. These data provide an explanation for the marked glucose tolerance of the Q205L Galpha(i2) mice and demonstrate a linkage between Galpha(i2) and GLUT4 translocation.     

Altered GLUT4 translocation in skeletal muscle of 12/15-lipoxygenase knockout mice.

We have recently shown that 12(S)-hydroxyeicosatetraenoic acid plays a role in the organization of actin microfilaments in rat cardiomyocytes, and that inhibition of 12-lipoxygenase abrogates insulin-stimulated GLUT4 translocation in these cells. In the present study, we used mice that were null for the leukocyte 12/15-lipoxygenase to explore the implications of this enzyme for insulin action under IN VIVO conditions. Insulin induced a profound reduction in blood glucose in both control and knockout mice. However, significantly higher serum insulin levels were observed in these animals. GLUT4 expression in heart and skeletal muscle was unaffected in KO mice. Insulin-regulated serine phosphorylation of Akt and GSK3alpha and GSK3beta was unaltered in heart and skeletal muscle of knockout mice, suggesting unaltered insulin signaling. Fractionation of hind limb muscles showed that insulin had induced a prominent translocation of GLUT4 to skeletal muscle plasma membranes in control mice. However, this response was largely reduced in knockout animals. Our data show that the lack of leukocyte 12/15-lipoxygenase does not lead to the development of an insulin-resistant phenotype. However, perturbation of GLUT4 translocation in skeletal muscle of knockout mice may indicate latent insulin resistance, and supports our hypothesis that eicosanoids are involved in insulin-mediated regulation of muscle glucose transport.     

Targeted disruption of the glucose transporter 4 selectively in muscle causes insulin resistance and glucose intolerance

The prevalence of type 2 diabetes mellitus is growing worldwide. By the year 2020, 250 million people will be afflicted. Most forms of type 2 diabetes are polygenic with complex inheritance patterns, and penetrance is strongly influenced by environmental factors. The specific genes involved are not yet known, but impaired glucose uptake in skeletal muscle is an early, genetically determined defect that is present in non-diabetic relatives of diabetic subjects. The rate-limiting step in muscle glucose use is the transmembrane transport of glucose mediated by glucose transporter (GLUT) 4 (ref. 4), which is expressed mainly in skeletal muscle, heart and adipose tissue. GLUT4 mediates glucose transport stimulated by insulin and contraction/exercise. The importance of GLUT4 and glucose uptake in muscle, however, was challenged by two recent observations. Whereas heterozygous GLUT4 knockout mice show moderate glucose intolerance, homozygous whole-body GLUT4 knockout (GLUT4-null) mice have only mild perturbations in glucose homeostasis and have growth retardation, depletion of fat stores, cardiac hypertrophy and failure, and a shortened life span. Moreover, muscle-specific inactivation of the insulin receptor results in minimal, if any, change in glucose tolerance. To determine the importance of glucose uptake into muscle for glucose homeostasis, we disrupted GLUT4 selectively in mouse muscles. A profound reduction in basal glucose transport and near-absence of stimulation by insulin or contraction resulted. These mice showed severe insulin resistance and glucose intolerance from an early age. Thus, GLUT4-mediated glucose transport in muscle is essential to the maintenance of normal glucose homeostasis.     

Impaired glucose homeostasis in insulin-like growth factor-binding protein-3-transgenic mice

Glucose homeostasis was examined in male transgenic (Tg) mice that overexpressed the human insulin-like growth factor (IGF)-binding protein (IGFBP)-3 cDNA, driven by either the cytomegalovirus (CMV) or the phosphoglycerate kinase (PGK) promoter. The Tg mice of both lineages demonstrated increased serum levels of human (h) IGFBP-3 and total IGF-I compared with wild-type (Wt) mice. Fasting blood glucose levels were significantly elevated in 8-wk-old CMV-binding protein (CMVBP)-3- and PGK binding protein (PGKBP)-3-Tg mice compared with Wt mice: 6.35 +/- 0.22 and 5.22 +/- 0.39 vs. 3.99 +/- 0.26 mmol/l, respectively. Plasma insulin was significantly elevated only in CMVBP-3-Tg mice. The responses to a glucose challenge were significantly increased in both Tg strains: area under the glucose curve = 1,824 +/- 65 and 1,910 +/- 115 vs. 1,590 +/- 67 mmol. l(-1). min for CMVBP-3, PGKBP-3, and Wt mice, respectively. The hypoglycemic effects of insulin and IGF-I were significantly attenuated in Tg mice compared with Wt mice. There were no differences in adipose tissue resistin, retinoid X receptor-alpha, or peroxisome proliferator-activated receptor-gamma mRNA levels between Tg and Wt mice. Uptake of 2-deoxyglucose was reduced in muscle and adipose tissue from Tg mice compared with Wt mice. These data demonstrate that overexpression of hIGFBP-3 results in fasting hyperglycemia, impaired glucose tolerance, and insulin resistance.     

Calpain system regulates muscle mass and glucose transporter GLUT4 turnover

The experiments in this study were undertaken to determine whether inhibition of calpain activity in skeletal muscle is associated with alterations in muscle metabolism. Transgenic mice that overexpress human calpastatin, an endogenous calpain inhibitor, in skeletal muscle were produced. Compared with wild type controls, muscle calpastatin mice demonstrated normal glucose tolerance. Levels of the glucose transporter GLUT4 were increased more than 3-fold in the transgenic mice by Western blotting while mRNA levels for GLUT4 and myocyte enhancer factors, MEF 2A and MEF 2D, protein levels were decreased. We found that GLUT4 can be degraded by calpain-2, suggesting that diminished degradation is responsible for the increase in muscle GLUT4 in the calpastatin transgenic mice. Despite the increase in GLUT4, glucose transport into isolated muscles from transgenic mice was not increased in response to insulin. The expression of protein kinase B was decreased by approximately 60% in calpastatin transgenic muscle. This decrease could play a role in accounting for the insulin resistance relative to GLUT4 content of calpastatin transgenic muscle. The muscle weights of transgenic animals were substantially increased compared with controls. These results are consistent with the conclusion that calpain-mediated pathways play an important role in the regulation of GLUT4 degradation in muscle and in the regulation of muscle mass. Inhibition of calpain activity in muscle by overexpression of calpastatin is associated with an increase in GLUT4 protein without a proportional increase in insulin-stimulated glucose transport. These findings provide evidence for a physiological role for calpains in the regulation of muscle glucose metabolism and muscle mass.     

GLUT4: a key player regulating glucose homeostasis? Insights from transgenic and knockout mice

Studies in which GLUT4 has been overexpressed in transgenic mice provide definitive evidence that glucose transport is rate limiting for muscle glucose disposal. Transgenic overexpression of GLUT4 selectively in skeletal muscle results in increased whole body glucose uptake and improves glucose homeostasis. These studies strengthen the hypothesis that the level of muscle GLUT4 affects the rate of whole body glucose disposal, and underscore the importance of GLUT4 in skeletal muscle for maintaining whole body glucose homeostasis. Studies in which GLUT4 has been ablated or 'knocked-out' provide proof that GLUT4 is the primary effector for mediating glucose transport in skeletal muscle and adipose tissue. Genetic ablation of GLUT4 results in impaired insulin tolerance and defects in glucose metabolism in skeletal muscle and adipose tissue. Because impaired muscle glucose transport leads to reduced whole body glucose uptake and hyperglycaemia, understanding the molecular regulation of glucose transport in skeletal muscle is important to develop effective strategies to prevent or reduce the incidence of Type II diabetes mellitus. In patients with Type II diabetes mellitus, reduced glucose transport in skeletal muscle is a major factor responsible for reduced whole body glucose uptake. Overexpression of GLUT4 in skeletal muscle improves glucose homeostasis in animal models of diabetes mellitus and protects against the development of diabetes mellitus. Thus, GLUT4 is an attractive target for pharmacological intervention strategies to control glucose homeostasis. This review will focus on the current understanding of the role of GLUT4 in regulating cellular glucose uptake and whole body glucose homeostasis.     

PID1 regulates insulin-dependent glucose uptake by controlling intracellular sorting of GLUT4-storage vesicles

The phosphotyrosine interacting domain-containing protein 1 (PID1) serves as a cytosolic adaptor protein of the LDL receptor-related protein 1 (LRP1). By regulating its intracellular trafficking, PID1 controls the hepatic, LRP1-dependent clearance of pro-atherogenic lipoproteins. In adipose and muscle tissues, LRP1 is present in endosomal storage vesicles containing the insulin-responsive glucose transporter 4 (GLUT4). This prompted us to investigate whether PID1 modulates GLUT4 translocation and function via its interaction with the LRP1 cytosolic domain. We initially evaluated this in primary brown adipocytes as we observed an inverse correlation between brown adipose tissue glucose uptake and expression of LRP1 and PID1. Insulin stimulation in wild type brown adipocytes induced LRP1 and GLUT4 translocation from endosomal storage vesicles to the cell surface. Loss of PID1 expression in brown adipocytes prompted LRP1 and GLUT4 sorting to the plasma membrane independent of insulin signaling. When placed on a diabetogenic high fat diet, systemic and adipocyte-specific PID1-deficient mice presented with improved hyperglycemia and glucose tolerance as well as reduced basal plasma insulin levels compared to wild type control mice. Moreover, the improvements in glucose parameters associated with increased glucose uptake in adipose and muscle tissues from PID1-deficient mice. The data provide evidence that PID1 serves as an insulin-regulated retention adaptor protein controlling translocation of LRP1 in conjunction with GLUT4 to the plasma membrane of adipocytes. Notably, loss of PID1 corrects for insulin resistance-associated hyperglycemia emphasizing its pivotal role and therapeutic potential in the regulation of glucose homeostasis.     

Postexercise glucose uptake and glycogen synthesis in skeletal muscle from GLUT4-deficient mice.

To determine the role of GLUT4 on postexercise glucose transport and glycogen resynthesis in skeletal muscle, GLUT4-deficient and wild-type mice were studied after a 3 h swim exercise. In wild-type mice, insulin and swimming each increased 2-deoxyglucose uptake by twofold in extensor digitorum longus muscle. In contrast, insulin did not increase 2-deoxyglucose glucose uptake in muscle from GLUT4-null mice. Swimming increased glucose transport twofold in muscle from fed GLUT4-null mice, with no effect noted in fasted GLUT4-null mice. This exercise-associated 2-deoxyglucose glucose uptake was not accompanied by increased cell surface GLUT1 content. Glucose transport in GLUT4-null muscle was increased 1.6-fold over basal levels after electrical stimulation. Contraction-induced glucose transport activity was fourfold greater in wild-type vs. GLUT4-null muscle. Glycogen content in gastrocnemius muscle was similar between wild-type and GLUT4-null mice and was reduced approximately 50% after exercise. After 5 h carbohydrate refeeding, muscle glycogen content was fully restored in wild-type, with no change in GLUT4-null mice. After 24 h carbohydrate refeeding, muscle glycogen in GLUT4-null mice was restored to fed levels. In conclusion, GLUT4 is the major transporter responsible for exercise-induced glucose transport. Also, postexercise glycogen resynthesis in muscle was greatly delayed; unlike wild-type mice, glycogen supercompensation was not found. GLUT4 it is not essential for glycogen repletion since muscle glycogen levels in previously exercised GLUT4-null mice were totally restored after 24 h carbohydrate refeeding.-Ryder, J. W., Kawano, Y., Galuska, D., Fahlman, R., Wallberg-Henriksson, H., Charron, M. J., Zierath, J. R. Postexercise glucose uptake and glycogen synthesis in skeletal muscle from GLUT4-deficient mice.     

Diet-induced insulin resistance in mice lacking adiponectin/ACRP30

Here we investigated the biological functions of adiponectin/ACRP30, a fat-derived hormone, by disrupting the gene that encodes it in mice. Adiponectin/ACRP30-knockout (KO) mice showed delayed clearance of free fatty acid in plasma, low levels of fatty-acid transport protein 1 (FATP-1) mRNA in muscle, high levels of tumor necrosis factor-alpha (TNF-alpha) mRNA in adipose tissue and high plasma TNF-alpha concentrations. The KO mice exhibited severe diet-induced insulin resistance with reduced insulin-receptor substrate 1 (IRS-1)-associated phosphatidylinositol 3 kinase (PI3-kinase) activity in muscle. Viral mediated adiponectin/ACRP30 expression in KO mice reversed the reduction of FATP-1 mRNA, the increase of adipose TNF-alpha mRNA and the diet-induced insulin resistance. In cultured myocytes, TNF-alpha decreased FATP-1 mRNA, IRS-1-associated PI3-kinase activity and glucose uptake, whereas adiponectin increased these parameters. Our results indicate that adiponectin/ACRP30 deficiency and high TNF-alpha levels in KO mice reduced muscle FATP-1 mRNA and IRS-1-mediated insulin signaling, resulting in severe diet-induced insulin resistance.     

Hepatic response to restoration of GLUT4 in skeletal muscle of GLUT4 null mice

Expression of GLUT4 in fast-twitch skeletal muscle fibers of GLUT4 null mice (G4-MO) normalized glucose uptake in muscle and restored peripheral insulin sensitivity. GLUT4 null mice exhibit altered carbohydrate and lipid metabolism in liver and skeletal muscle. To test the hypothesis that increased glucose utilization by G4-MO muscle would normalize the changes seen in the GLUT4 null liver, serum metabolites and hepatic metabolism were compared in control, GLUT4 null, and G4-MO mice. The fed serum glucose and triglyceride levels of G4-MO mice were similar to those of control mice. In addition, the alternations in liver metabolism seen in GLUT4 nulls including increased GLUT2 expression and fatty acid synthesis accompanied by an increase in the oxidative arm of the pentose phosphate pathway were absent in G4-MO mice. The transgene used for GLUT4 restoration in muscle was specific for fast-twitch muscle fibers. The mitochondria hypertrophy/hyperplasia in all GLUT4 null skeletal muscles was absent in transgene-positive extensor digitorum longus muscle but present in transgene-negative soleus muscle of G4-MO mice. Results of this study suggest that the level of muscle GLUT4 expression influences mitochondrial biogenesis. These studies also demonstrate that the type and amount of substrate that muscle takes up and metabolizes, determined in part by GLUT4 expression levels, play a major role in directing hepatic carbohydrate and lipid metabolism.     

GLUT4 ablation in mice results in redistribution of IRAP to the plasma membrane

Glucose transporter (GLUT) 4 is the insulin responsive glucose transporter in adipose tissue, skeletal muscle, and heart. Insulin elicits increased glucose uptake by recruiting GLUT4 from a specialized intracellular storage site to the cell surface. Expression of various proteins that colocalize with GLUT4 and/or are involved in insulin-stimulated GLUT4 translocation was examined in adipocytes as well as skeletal and cardiac muscles from GLUT4 null mice. Our data demonstrate that expression of insulin-regulated aminopeptidase (IRAP) is divergently regulated in GLUT4 null tissues, e.g., upregulated 1.6-fold in GLUT4 null adipocytes and downregulated in GLUT4 null skeletal muscle (40%) and heart (60%). IRAP exhibited abnormal subcellular distribution and impaired insulin-stimulated translocation in GLUT4-deficient tissues. We propose the compartment containing IRAP and proteins normally associated with GLUT4 vesicle traffics constitutively to the cell surface in GLUT4 null adipocytes and skeletal muscle.     

Mice deficient in the insulin-regulated membrane aminopeptidase show substantial decreases in glucose transporter GLUT4 levels but maintain normal glucose homeostasis

The insulin-regulated aminopeptidase (IRAP) is a zinc-dependent membrane aminopeptidase. It is the homologue of the human placental leucine aminopeptidase. In fat and muscle cells, IRAP colocalizes with the insulin-responsive glucose transporter GLUT4 in intracellular vesicles and redistributes to the cell surface in response to insulin, as GLUT4 does. To address the question of the physiological function of IRAP, we generated mice with a targeted disruption of the IRAP gene (IRAP-/-). Herein, we describe the characterization of these mice with regard to glucose homeostasis and regulation of GLUT4. Fed and fasted blood glucose and insulin levels in the IRAP-/- mice were normal. Whereas IRAP-/- mice responded to glucose administration like control mice, they exhibited an impaired response to insulin. Basal and insulin-stimulated glucose uptake in extensor digitorum longus muscle, and adipocytes isolated from IRAP-/- mice were decreased by 30-60% but were normal for soleus muscle from male IRAP-/- mice. Total GLUT4 levels were diminished by 40-85% in the IRAP-/- mice in the different muscles and in adipocytes. The relative distribution of GLUT4 in subcellular fractions of basal and insulin-stimulated IRAP-/- adipocytes was the same as in control cells. We conclude that IRAP-/- mice maintain normal glucose homeostasis despite decreased glucose uptake into muscle and fat cells. The absence of IRAP does not affect the subcellular distribution of GLUT4 in adipocytes. However, it leads to substantial decreases in GLUT4 expression.     

Adipose-specific deletion of stearoyl-CoA desaturase 1 up-regulates the glucose transporter GLUT1 in adipose tissue

Stearoyl-CoA desaturase 1 (SCD1) deficiency protects mice from diet-induced obesity and insulin resistance. To understand the tissue-specific role of SCD1 in energy homeostasis, we have generated mice with an adipose-specific knockout of Scd1 (AKO), and report here that SCD1 deficiency increases GLUT1 expression in adipose tissue of AKO mice, but not global SCD1 knockout (GKO) mice. In 3T3-L1 adipocytes treated with an SCD inhibitor, basal glucose uptake and the cellular expression of GLUT1 were significantly increased while GLUT4 expression remained unchanged. Consistently, adipose-specific SCD1 knockout (AKO) mice had significantly elevated GLUT1 expression, but not GLUT4, in white adipose tissue compared to Lox counterparts. Concurrently, adiponectin expression was significantly diminished, whereas TNF-α expression was elevated. In contrast, in adipose tissue of GKO mice, GLUT4 and adiponectin expression were significantly elevated with lowered TNF-α expression and little change in GLUT1 expression, suggesting a differential responsiveness of adipose tissue to global- or adipose-specific SCD1 deletion. Taken together, these results indicate that adipose-specific deletion of SCD1 induces GLUT1 up-regulation in adipose tissue, associated with decreased adiponectin and increased TNF-α production, and suggest that GLUT1 may play a critical role in controlling glucose homeostasis of adipose tissue in adipose-specific SCD1-deficient conditions.     

Adipose Overexpression of Heme Oxygenase-1 Does Not Protect against High Fat Diet-Induced Insulin Resistance in Mice

Heme oxygenase-1 (HO-1) is a stress-responsive enzyme with potent anti-oxidant and anti-inflammatory activities. Previous studies have shown that systemic induction of HO-1 by chemical inducers reduces adiposity and improves insulin sensitivity. To dissect the specific function of HO-1 in adipose tissue, we generated transgenic mice with adipose HO-1 overexpression using the adipocyte-specific aP2 promoter. The transgenic (Tg) mice exhibit similar metabolic phenotype as wild type (WT) control under chow-fed condition. High fat diet (HFD) challenge significantly increased the body weights of WT and Tg mice to a similar extent. Likewise, HFD-induced glucose intolerance and insulin resistance were not much different between WT and Tg mice. Analysis of the adipose tissue gene expression revealed that the mRNA levels of adiponectin and interleukin-10 were significantly higher in chow diet-fed Tg mice as compared to WT counterparts, whereas HFD induced downregulation of adiponectin gene expression in both Tg and WT mice to a similar level. HFD-induced proinflammatory cytokine expression in adipose tissues were comparable between WT and transgenic mice. Nevertheless, immunohistochemistry and gene expression analysis showed that the number of infiltrating macrophages with preferential expression of M2 markers was significantly higher in the adipose tissue of obese Tg mice than WT mice. Further experiment demonstrated that myeloid cells from Tg mice expressed higher level of HO-1 and exhibited greater migration response toward chemoattractant in vitro. Collectively, these data indicate that HO-1 overexpression in adipocytes does not protect against HFD-induced obesity and the development of insulin resistance in mice.     

Cyclic AMP acutely stimulates translocation of the major insulin-regulatable glucose transporter GLUT4.

Facilitated glucose transport across plasma membranes is mediated by a family of transporters (GLUT1-GLUT5) that have different tissue distributions and Km values for transport. It has been shown that insulin stimulates glucose transport in fat and muscle tissues by causing the redistribution of one of these proteins (GLUT4) from inside the cell to the plasma membrane. Previous studies have shown that agents that change cAMP levels are able to modulate glucose transport in fat cells. The aim of this study was to investigate the mechanisms responsible for modulation of glucose transport by cAMP. 2-Deoxyglucose transport and insulin-regulatable glucose transporter (GLUT4) immunoreactivity in plasma and low density microsomal membranes were measured in adipocytes incubated for 30 min with insulin or dibutyryl-cAMP (Bt2cAMP). Low concentrations of Bt2cAMP (10 microM) increased 2-deoxyglucose uptake by translocating GLUT4 from low density microsomal membranes to the plasma membranes. Bt2cAMP at 1000 microM inhibited glucose transport below basal but further increased translocation of GLUT4. The effect of Bt2cAMP on translocation was additive to that of 7 nM insulin. We conclude that in rat adipocytes, Bt2cAMP acutely translocates GLUT4 but inhibits its activity to transport glucose.     

Effects of the presence, absence, and overexpression of uncoupling protein-3 on adiposity and fuel metabolism in congenic mice

Uncoupling protein-3 (UCP3) is a poorly understood mitochondrial inner membrane protein expressed predominantly in skeletal muscle. The aim of this study was to examine the effects of the absence or constitutive physiological overexpression of UCP3 on whole body energy metabolism, glucose tolerance, and muscle triglyceride content. Congenic male UCP3 knockout mice (Ucp3-/-), wild-type, and transgenic UCP3 overexpressing (UCP3Tg) mice were fed a 10% fat diet for 4 or 8 mo after they were weaned. UCP3Tg mice had lower body weights and were less metabolically efficient than wild-type or Ucp3-/- mice, but they were not hyperphagic. UCP3Tg mice had smaller epididymal white adipose tissue and brown adipose tissue (BAT) depots; however, there were no differences in muscle weights. Glucose and insulin tolerance tests revealed that both UCP3Tg and Ucp3-/- mice were protected from development of impaired glucose tolerance and were more sensitive to insulin. 2-Deoxy-D-[1-3H]glucose tracer studies showed increased uptake of glucose into BAT and increased storage of liver glycogen in Ucp3-/- mice. Assessments of intramuscular triglyceride (IMTG) revealed decreases in quadriceps of UCP3Tg mice compared with wild-type and Ucp3-/- mice. When challenged with a 45% fat diet, Ucp3-/- mice showed increased accumulation of IMTG compared with wild-type mice, which in turn had greater IMTG than UCP3Tg mice. Results are consistent with a role for UCP3 in preventing accumulation of triglyceride in both adipose tissue and muscle.     

Calorie restriction improves whole-body glucose disposal and insulin resistance in association with the increased adipocyte-specific GLUT4 expression in Otsuka Long-Evans Tokushima fatty rats

Calorie restriction (CR) has been shown to improve peripheral insulin resistance and type 2 diabetes in animal models. However, the exact mechanism of CR on GLUT4 expression and translocation in insulin-sensitive tissues has not been well elucidated. In the present study, we examine the effect of CR on the expression of glucose transporter 4 (GLUT4), GLUT4 translocation, and glucose transport activity in adipose tissue from Otsuka Long-Evans Tokushima Fatty (OLETF) rat and control (LETO) rats. CR (70% of satiated group) ameliorated hyperglycemia and improved impaired glucose tolerance (IGT) in OLETF rats. In skeletal muscle, the expression levels of GLUT4 and GLUT1 were not significantly different between LETO and OLETF rats, and were not affected by CR. By contrast, the expression level of GLUT4 was markedly decreased in the adipose tissue of OLETF rats, but was dramatically increased by CR. The GLUT4 recruitment stimulated by insulin was also improved in OLETF rat adipocytes by CR. The insulin-stimulated 2-deoxyglucose (2DG) uptake was significantly increased in adipocytes from the CR OLETF rats, as compared with the satiated OLETF rats. Taken together, these results suggest that CR improves whole body glucose disposal and insulin resistance in OLETF rats, and that these effects may associate with the increased adipocyte-specific GLUT4 expression.