Ellen B. Katz

Enhanced Insulin Action Due to Targeted GLUT4 Overexpression Exclusively in Muscle

Dysregulation of GLUT4, the insulin-responsive glucose transporter, is associated with insulin resistance in skeletal muscle. Although skeletal muscle is the major target of insulin action, muscle GLUT4 has not been linked causally to whole-body insulin sensitivity and regulation of glucose homeostasis. To address this, we generated a line of transgenic mice that overexpresses GLUT4 in skeletal muscle. We demonstrate that restricted overexpression of GLUT4 in fast-twitch skeletal muscles of myosin light chain (MLC)–GLUT4 transgenic mice induces a 2.5-fold increase in insulin-stimulated 2-deoxyglucose uptake in transgene-overexpressing cells. Consequently, glycogen content is increased in the fast-twitch skeletal muscles under insulin action (5.75 ± 1.02 vs. 3.24 ± 0.26 mg/g). This indicates that insulin-stimulated glucose transport is partly rate-limiting for glycogen synthesis. At the whole-body level, insulin-stimulated glucose turnover is increased 2.5-fold in unconscious MLC-GLUT4 mice. Plasma glucose and insulin levels in MLC-GLUT4 mice are altered as a result of increased insulin action. In 2- to 3-month-old MLC-GLUT4 mice, fasting insulin levels are decreased (0.43 ± 0.05 vs. 0.74 ± 0.10 microgram/l), whereas normal fasting glycemia is maintained. Conversely, 7- to 9-month-old MLC-GLUT4 mice exhibit decreased fasting glycemia (5.75 ± 0.73 vs. 8.11 ± 0.57 mmol/l) with normal insulin levels. Fasting plasma lactate levels are elevated in both age groups (50–100%). Additionally lipid metabolism is affected by skeletal muscle GLUT4 overexpression. This is indicated by changes in plasma free fatty acid and β-hydroxybutyrate levels. These studies underscore the importance of GLUT4 in the regulation of glucose homeostasis and its interaction with lipid metabolism.

Localization and regulation of GLUTx1 glucose transporter in the hippocampus of streptozotocin diabetic rats

We describe the localization of the recently identified glucose transporter GLUTx1 and the regulation of GLUTx1 in the hippocampus of diabetic and control rats. GLUTx1 mRNA and protein exhibit a unique distribution when compared with other glucose transporter isoforms expressed in the rat hippocampus. In particular, GLUTx1 mRNA was detected in hippocampal pyramidal neurons and granule neurons of the dentate gyrus as well as in nonprincipal neurons. With immunohistochemistry, GLUTx1 protein expression is limited to neuronal cell bodies and the most proximal dendrites, unlike GLUT3 expression that is observed throughout the neuropil. Immunoblot analysis of hippocampal membrane fractions revealed that GLUTx1 protein expression is primarily localized to the intracellular compartment and exhibits limited association with the plasma membrane. In streptozotocin diabetic rats compared with vehicle-treated controls, quantitative autoradiography showed increased GLUTx1 mRNA levels in pyramidal neurons and granule neurons; up-regulation of GLUTx1 mRNA also was found in nonprincipal cells, as shown by single-cell emulsion autoradiography. In contrast, diabetic and control rats expressed similar levels of hippocampal GLUTx1 protein. These results indicate that GLUTx1 mRNA and protein have a unique expression pattern in rat hippocampus and suggest that streptozotocin diabetes increases steady-state mRNA levels in the absence of concomitant increases in GLUTx1 protein expression.

Peripheral glucose administration stimulates the translocation of GLUT8 glucose transporter to the endoplasmic reticulum in the rat hippocampus

The expression and localization of glucose transporter isoforms play essential roles in the glucoregulatory activities of the hippocampus and ultimately contribute to cognitive status in physiological and pathophysiological settings. The recently identified glucose transporter GLUT8 is uniquely expressed in neuronal cell bodies in the rat hippocampus and therefore may contribute to hippocampal glucoregulatory activities. We show here that GLUT8 has a novel intracellular distribution in hippocampal neurons and is translocated to intracellular membranes following glucose challenge. Immunoblot analysis revealed that GLUT8 is expressed in high‐density microsomes (HDM), suggesting that GLUT8 is associated with intracellular organelles under basal conditions. Immunogold electron microscopic analysis confirmed this observation, in that GLUT8 immunogold particles were associated with the rough endoplasmic reticulum (ER) and cytoplasm. Peripheral glucose administration produced a rapid twofold increase in GLUT8 levels in the HDM fraction while decreasing GLUT8 levels in low‐density microsomes. Similarly, peripheral glucose administration significantly increased GLUT8 association with the rough ER in the hippocampus. Conversely, under hyperglycemic/insulinopenic conditions, namely, in streptozotocin (STZ) diabetes, hippocampal GLUT8 protein levels were decreased in the HDM fraction. These results demonstrate that GLUT8 undergoes rapid translocation to the rough ER in the rat hippocampus following peripheral glucose administration, trafficking that is impaired in STZ diabetes, suggesting that insulin serves as a stimulus for GLUT8 translocation in hippocampal neurons. Because glucose is liberated from oligosaccharides during N‐linked glycosylation events in the rough ER, we propose that GLUT8 may serve to transport glucose out of the rough ER into the cytosol and in this manner contribute to glucose homeostasis in hippocampal neurons. J. Comp. Neurol. 452:103–114, 2002.

The metabolic consequences of altered glucose transporter expression in transgenic mice

Abstract Glucose transporters are a family of membrane proteins which mediate glucose uptake across the cell membrane. The facilitative glucose transporter proteins are products of unique genes and are expressed in a tissue-specific manner. They are very similar structurally, containing 12 putative membrane spanning domains. Functionally they vary in their affinity for glucose and sensitivity to hormones such as insulin. Glucose homeostasis depends mainly on controlled changes in glucose transport in insulin-responsive tissues such as skeletal muscle and adipose cells where both glucose transporter 1 and glucose transporter 4 are expressed. Glucose transporter 4 is the major glucose transporter in these tissues and translocates from an intracellular vesicle to the cell membrane in response to insulin. Alterations of the level of expression of these glucose transporters should result in changes in insulin sensitivity and modification of whole-body metabolism. To test these hypotheses transgenic mouse models have been generated which overexpress glucose transporters in specific tissues or in the whole body. Glucose transporter 1 and glucose transporter 4 have been overexpressed specifically in skeletal muscle and glucose transporter 4 specifically in adipose tissue. Mice have also been made which overexpress glucose transporter 4 in the whole body. Using homologous recombination technology to disrupt the glucose transporter 4 gene, a ”knockout” mouse has been created which expresses no glucose transporter 4. The metabolic consequences of these genetic manipulations on the level of expression of glucose transporters in the mouse are reviewed. The future applications of transgenic mouse technology in creating models which mimic human diseases are also discussed.

Reduced glucose uptake precedes insulin signaling defects in adipocytes from heterozygous GLUT4 knockout mice

Decreased GLUT4 expression, impaired insulin receptor (IR), IRS‐1, and pp60/IRS‐3 tyrosine phosphorylation are characteristics of adi‐pocytes from insulin‐resistant animal models and obese NIDDM humans. However, the sequence of events leading to the development of insulin signaling defects and the significance of decreased GLUT4 expression in causing adipocyte insulin resistance are unknown. The present study used male heterozygous GLUT4 knockout mice (GLUT4(+/–)) as a novel model of diabetes to study the development of insulin signaling defects in adipo‐cytes with the progression of whole body insulin resistance and diabetes. Male GLUT4(+/–) mice with normal fed glycemia and insulinemia (N/N), normal fed glycemia and hyperinsulinemia (N/H), and fed hyperglycemia with hyperinsulinemia (H/H) exist at all ages. The expression of GLUT4 protein and the maximal insulin‐stimulated glucose transport was 50% decreased in adipocytes from all three groups. Insulin signaling was normal in N/N adipose cells. From 35 to 70% reductions in insulin‐stimulated tyrosine phosphorylation of IR, IRS‐1, and pp60/IRS‐3 were noted with no changes in the cellular content of IR, IRS‐1, and p85 in N/H adipocytes. Insulin‐stimulated protein tyrosine phos‐phorylation was further decreased to 12–23% in H/H adipose cells accompanied by 42% decreased IR and 80% increased p85 expression. Insulin‐stimulated, IRS‐1‐associated PI3 kinase activity was decreased by 20% in N/H and 68% reduced in H/H GLUT4(+/–) adipocytes. However, total insulin‐stimulated PI3 kinase activity was normal in H/H GLUT4(+/–) adipocytes. Taken together, these results strongly suggest that hyperinsulinemia triggers a reduction of IR tyrosine kinase activity that is further exacerbated by the appearance of hypergly‐cemia. However, the insulin signaling cascade has sufficient plasticity to accommodate significant changes in specific components without further reducing glucose uptake. Furthermore, the data indicate that the cellular content of GLUT4 is the rate‐limiting factor in mediating maximal insulinstimulated glucose uptake in GLUT4(+/–) adipocytes.—Li, J., Houseknecht, K. L., Stenbit, A. E., Katz, E. B., Charron, M. J. Reduced glucose uptake precedes insulin signaling defects in adipocytes from heterozygous GLUT4 knockout mice. FASEB J. 14, 1117–1125 (2000)

Muscle-specific transgenic complementation of GLUT4-deficient mice. Effects on glucose but not lipid metabolism.

We have taken the approach of introducing the muscle-specific myosin light chain (MLC)-GLUT4 transgene into the GLUT4-null background to assess the relative role of muscle and adipose tissue GLUT4 in the etiology of the GLUT4-null phenotype. The resulting MLC-GLUT4-null mice express GLUT4 predominantly in the fast-twitch extensor digitorum longus (EDL) muscle. GLUT4 is nearly absent in female white adipose tissue (WAT) and slow-twitch soleus muscle of both sexes of MLC-GLUT4-null mice. GLUT4 content in male MLC-GLUT4-null WAT is 20% of that in control mice. In transgenically complemented EDL muscle, 2-deoxyglucose (2-DOG) uptake was restored to normal (male) or above normal (female) levels. In contrast, 2-DOG uptake in slow-twitch soleus muscle of MLC-GLUT4-null mice was not normalized. With the normalization of glucose uptake in fast-twitch skeletal muscle, whole body insulin action was restored in MLC-GLUT4-null mice, as shown by the results of the insulin tolerance test. These results demonstrate that skeletal muscle GLUT4 is a major regulator of skeletal muscle and whole body glucose metabolism. Despite normal skeletal muscle glucose uptake and insulin action, the MLC-GLUT4-null mice exhibited decreased adipose tissue deposits, adipocyte size, and fed plasma FFA levels that are characteristic of GLUT4-null mice. Together these results indicate that the defects in skeletal muscle and whole body glucose metabolism and adipose tissue in GLUT4-null mice arise independently.

Metabolic and therapeutic lessons from genetic manipulation of GLUT4

This review focuses on the effects of varying levels of GLUT4, the insulin-sensitive glucose transporter, on insulin sensitivity and whole body glucose homeostasis. Three mouse models are discussed including MLC-GLUT4 mice which overexpress GLUT4 specifically in skeletal muscle, GLUT4 null mice which express no GLUT4, and the MLC-GLUT4 null mice which express GLUT4 only in skeletal muscle. Overexpressing GLUT4 specifically in the skeletal muscle results in increased insulin sensitivity in the MLC-GLUT4 mice. In contrast, the GLUT4 null mice exhibit insulin intolerance accompanied by abnormalities in glucose and lipid metabolism. Restoring GLUT4 expression in skeletal muscle in the MLC-GLUT4 null mice results in normal glucose metabolism but continued abnormal lipid metabolism. The results of experiments using these mouse models demonstrates that modifying the expression of GLUT4 profoundly affects whole body insulin action and consequently glucose and lipid metabolism.

High-Fat Intake During Pregnancy and Lactation Exacerbates High-Fat Diet-Induced Complications in Male Offspring in Mice

Altered fetal environments, such as a high-fat milieu, induce metabolic abnormalities in offspring. Different postnatal environments reveal the predisposition for adult diseases that occur during the fetal period. This study investigates the ability of a maternal high-fat diet (HFD) to program metabolic responses to HFD reexposure in offspring after consuming normal chow for 23 weeks after weaning. Wild-type CD1 females were fed a HFD (H) or control (C) chow during pregnancy and lactation. At 26 weeks of age, offspring were either reexposed (H-C-H) or newly exposed (C-C-H) to the HFD for 19 weeks. Body weight was measured weekly, and glucose and insulin tolerance were measured after 10 and 18 weeks on the HFD. The metabolic profile of offspring on a HFD or C diet during pregnancy and lactation and weaned onto a low-fat diet was similar at 26 weeks. H-C-H offspring gained more weight and developed larger adipocytes after being reintroduced to the HFD later in life than C-C-H. H-C-H mice were glucose and insulin intolerant and showed reduced gene expression of cox6a2 and atp5i in muscle, indicating mitochondrial dysfunction. In adipocytes, the expression of slc2a4, srebf1, and adipoq genes was decreased in H-C-H mice compared with C-C-C, indicating insulin resistance. H-C-H showed extensive hepatosteatosis, accompanied by increased gene expression for cd36 and serpin1, compared with C-C-H. Perinatal exposure to a HFD programs a more deleterious response to a HFD challenge later in life even after an interval of normal diet in mice.

Shared Effects of Genetic and Intrauterine and Perinatal Environment on the Development of Metabolic Syndrome

Genetic and environmental factors, including the in utero environment, contribute to Metabolic Syndrome. Exposure to high fat diet exposure in utero and lactation increases incidence of Metabolic Syndrome in offspring. Using GLUT4 heterozygous (G4+/−) mice, genetically predisposed to Type 2 Diabetes Mellitus, and wild-type littermates we demonstrate genotype specific differences to high fat in utero and lactation. High fat in utero and lactation increased adiposity and impaired insulin and glucose tolerance in both genotypes. High fat wild type offspring had increased serum glucose and PAI-1 levels and decreased adiponectin at 6 wks of age compared to control wild type. High fat G4+/− offspring had increased systolic blood pressure at 13 wks of age compared to all other groups. Potential fetal origins of adult Metabolic Syndrome were investigated. Regardless of genotype, high fat in utero decreased fetal weight and crown rump length at embryonic day 18.5 compared to control. Hepatic expression of genes involved in glycolysis, gluconeogenesis, oxidative stress and inflammation were increased with high fat in utero. Fetal serum glucose levels were decreased in high fat G4+/− compared to high fat wild type fetuses. High fat G4+/−, but not high fat wild type fetuses, had increased levels of serum cytokines (IFN-γ, MCP-1, RANTES and M-CSF) compared to control. This data demonstrates that high fat during pregnancy and lactation increases Metabolic Syndrome male offspring and that heterozygous deletion of GLUT4 augments susceptibility to increased systolic blood pressure. Fetal adaptations to high fat in utero that may predispose to Metabolic Syndrome in adulthood include changes in fetal hepatic gene expression and alterations in circulating cytokines. These results suggest that the interaction between in utero-perinatal environment and genotype plays a critical role in the developmental origin of health and disease.

Use of GLUT-4 null mice to study skeletal muscle glucose uptake.

1. The present review focuses on the effects of varying levels of GLUT‐4, the insulin‐senstive glucose transporter, on insulin sensitivity and whole body glucose homeostasis.