Antine E. Stenbit

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.

Pulmonary exacerbations in cystic fibrosis.

Purpose of review The chronic infection and inflammation of cystic fibrosis (CF) lung disease causes a progressive decline of lung function resulting in daily symptoms such as cough and sputum production. There are intermittent episodes of acute worsening of symptoms, more commonly referred to as pulmonary exacerbations. Despite this being a common event, there is still no standardized definition of an exacerbation. A recent set of guidelines from the CF Foundation Pulmonary Therapies Committee on the treatment of exacerbations noted the paucity of data supporting commonly used therapies. This review describes our current understanding of pulmonary exacerbations and the therapies used to treat them. Recent findings The treatment of an exacerbation is intended to resolve the worsened symptoms and to restore the lung function that is commonly lost in the acute presentation. A most striking finding is the observation that for many patients there is no restoration of lung function, suggesting we either need better therapies to prevent exacerbations or better treatment of exacerbations. Summary We have established recommendations on specific treatment of a pulmonary exacerbation and have outlined the areas where we need better information on appropriate therapies. Once we have a standardized definition of an exacerbation, we can proceed with clinical trials of therapies specific for its treatment.

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.

Forebrain endothelium expresses GLUT4, the insulin-responsive glucose transporter.

The presence of GLUT4, the insulin-responsive glucose transporter, in microvascular endothelium and the responsiveness of glucose transport at the blood-brain barrier to insulin have been matters of controversy. To address these issues, we examined GLUT4 mRNA and protein expression in isolated brain microvessels and in cultured calf vascular cells derived from brain microvessels and aorta. We report here that GLUT4 mRNA can be detected in rat forebrain and its microvasculature using high stringency hybridization of poly(A)+ RNA isolated from these sources. This mRNA is identical to that found in adipose cells from rat. Immunoblot analysis of isolate brain microvessels reveals that GLUT4 protein is also present. Peptide preadsorption studies and absence of our antibody reaction to human red cells suggest these findings are specific. Immunohistochemical staining of cultured calf vascular cells reveals that GLUT4 is expressed in brain endothelial cells but not pericytes, nor in aortic endothelium or smooth muscle cells. The sensitivity of the methods required to detect GLUT4 in brain and comparison to its abundance in low density microsomes from rat adipose cells indicate that GLUT4 is expressed in relatively low abundance in brain microvascular endothelium. No significant differences are observed in steady state levels of GLUT4 mRNA in brain from streptozotocin diabetic compared to control rats. This last finding supports the concept of tissue-specific regulation of GLUT4. We conclude that brain microvascular endothelium specifically expressed GLUT4 while other vascular cells do not.

Restoration of Hypoxia-stimulated Glucose Uptake in GLUT4-deficient Muscles by Muscle-specific GLUT4 Transgenic Complementation

To investigate whether GLUT4 is required for exercise/hypoxia-induced glucose uptake, we assessed glucose uptake under hypoxia and normoxia in extensor digitorum longus (EDL) and soleus muscles from GLUT4-deficient mice. In EDL and soleus from wild type control mice, hypoxia increased 2-deoxyglucose uptake 2–3-fold. Conversely, hypoxia did not alter 2-deoxyglucose uptake in either EDL or soleus from either male or female GLUT4-null mice. Next we introduced the fast-twitch skeletal muscle-specific MLC-GLUT4 transgene into GLUT4-null mice to determine whether changes in the metabolic milieu accounted for the lack of hypoxia-mediated glucose transport. Transgenic complementation of GLUT4 in EDL was sufficient to restore hypoxia-mediated glucose uptake. Soleus muscles from MLC-GLUT4-null mice were transgene-negative, and hypoxia-stimulated 2-deoxyglucose uptake was not restored. Although ablation of GLUT4 in EDL did not affect normoxic glycogen levels, restoration of GLUT4 to EDL led to an increase in glycogen under hypoxic conditions. Male GLUT4-null soleus displayed reduced normoxic glycogen stores, but female null soleus contained significantly more glycogen under normoxia and hypoxia. Reduced normoxic levels of ATP and phosphocreatine were measured in male GLUT4-null soleus but not in EDL. However, transgenic complementation of GLUT4 prevented the decrease in hypoxic ATP and phosphocreatine levels noted in male GLUT4-null and control EDL. In conclusion, we have demonstrated that GLUT4 plays an essential role in the regulation of muscle glucose uptake in response to hypoxia. Because hypoxia is a useful model for exercise, our results suggest that stimulation of glucose transport in response to exercise in skeletal muscle is totally dependent upon GLUT4. Furthermore, the compensatory glucose transport system that exists in GLUT4-null soleus muscle is not sensitive to hypoxia/muscle contraction.