Ann Louise Olson

Myocyte enhancer factor 2 (MEF2)-binding site is required for GLUT4 gene expression in transgenic mice. Regulation of MEF2 DNA binding activity in insulin-deficient diabetes.

We have previously demonstrated that important regulatory elements responsible for regulated expression of the humanGLUT4 promoter are located between −1154 and −412 relative to transcription initiation (Olson, A. L., and Pessin, J. E. (1995) J. Biol. Chem. 270, 23491–23495). Through further analysis of this promoter regulatory region, we have identified a perfectly conserved myocyte enhancer factor 2 (MEF2)-binding domain (-CTAAAAATAG-) that is necessary, but not sufficient, to support tissue-specific expression of a chloramphenicol acetyltransferase reporter gene in transgenic mice. Biochemical analysis of this DNA element demonstrated the formation of a specific DNA-protein complex using nuclear extracts isolated from heart, hindquarter skeletal muscle, and adipose tissue but not from liver. DNA binding studies indicated that this element functionally interacted with the MEF2A and/or MEF2C MADS family of DNA binding transcription factors. MEF2 DNA binding activity was substantially reduced in nuclear extracts isolated from both heart and skeletal muscle of diabetic mice, which correlated with decreased transcription rate of theGLUT4 gene. MEF2 binding activity completely recovered to control levels following insulin treatment. Together these data demonstrated that MEF2 binding activity is necessary for regulation of the GLUT4 gene promoter in muscle and adipose tissue.

GLUT4 Gene Regulation and Manipulation

A decade has passed since the cloning of the insulin-responsive glucose transporter, GLUT4. Numerous studies have demonstrated the complex hormonal and metabolic regulation of GLUT4 gene expression in adipose tissue and muscle. Careful dissection of the regulatory elements in the GLUT4 promoter has provided insight into the intricate control of this central gene of glucose homeostasis. Genetic manipulation of mice has provided further insight into the role of GLUT4 in carbohydrate and lipid metabolism at the whole body and tissue-specific levels. Analysis of GLUT41/2, GLUT4 null, and muscle-complemented GLUT4 knockout mice has furthered our understanding of peripheral insulin sensitivity. Additional studies on GLUT4 gene regulation and GLUT4 knockout models are likely to lead to novel therapies for type II diabetes and other diseases of insulin resistance.

Identification of a 30-base pair regulatory element and novel DNA binding protein that regulates the human GLUT4 promoter in transgenic mice

We have previously demonstrated that the important cis-acting elements regulating transcription of the human GLUT4 gene reside within 895 base pairs (bp) upstream of the transcription initiation site (Thai, M. V., Guruswamy, S., Cao, K. T., Pessin, J. E., and Olson, A. L. (1998) J. Biol. Chem. 273, 14285–14292). Our studies demonstrated that an MEF2 binding site within this region was necessary, but not sufficient, for GLUT4 promoter function in transgenic mice. We have identified a second regulatory element (Domain I) that functions cooperatively with the MEF2 domain in regulating GLUT4 transcription. Using a yeast-one hybrid screen, we obtained a partial cDNA and generated an antibody directed against a protein binding specifically to Domain I. Sequence analysis of the partial cDNA indicates that the protein binding to Domain I is a novel protein. The antibody specifically labels two proteins of approximately 70 and 50 kDa in Western blot analysis. These molecular masses correspond to Domain I binding proteins identified by UV-cross-linking nuclear extracts to a Domain I probe. The antibody raised against the Domain I binding protein inhibited formation of a Domain I-protein complex in electrophoretic mobility shift assays. We conclude that we have identified an authentic, novel, Domain I binding protein required for transcriptional regulation of the human GLUT4 promoter.

Moderate GLUT4 Overexpression Improves Insulin Sensitivity and Fasting Triglyceridemia in High-Fat Diet–Fed Transgenic Mice

The GLUT4 facilitative glucose transporter mediates insulin-dependent glucose uptake. We tested the hypothesis that moderate overexpression of human GLUT4 in mice, under the regulation of the human GLUT4 promoter, can prevent the hyperinsulinemia that results from obesity. Transgenic mice engineered to express the human GLUT4 gene and promoter (hGLUT4 TG) and their nontransgenic counterparts (NT) were fed either a control diet (CD) or a high-fat diet (HFD) for up to 10 weeks. Homeostasis model assessment of insulin resistance scores revealed that hGLUT4 TG mice fed an HFD remained highly insulin sensitive. The presence of the GLUT4 transgene did not completely prevent the metabolic adaptations to HFD. For example, HFD resulted in loss of dynamic regulation of the expression of several metabolic genes in the livers of fasted and refed NT and hGLUT4 TG mice. The hGLUT4 TG mice fed a CD showed no feeding-dependent regulation of SREBP-1c and fatty acid synthase (FAS) mRNA expression in the transition from the fasted to the fed state. Similarly, HFD altered the response of SREBP-1c and FAS mRNA expression to feeding in both strains. These changes in hepatic gene expression were accompanied by increased nuclear phospho-CREB in refed mice. Taken together, a moderate increase in expression of GLUT4 is a good target for treatment of insulin resistance.

Expression of a Prenylation-Deficient Rab4 Interferes with Propagation of Insulin Signaling through Insulin Receptor Substrate-11

Rab proteins are small GTP-binding proteins of the Ras superfamily that function in the regulation of vesicle transport processes. The Rab4 isoform has been implicated in insulin action. For instance, overexpression of a prenylation-deficient form of Rab4 has been shown to inhibit insulin-dependent GLUT4 translocation. Other steps affected by Rab4 in the cascade of events resulting from insulin receptor activation have not been elucidated. In the present studies, we measured effects on insulin-signaling proteins in 3T3-L1 adipocytes transiently expressing cytoplasmic forms of Rab4 and Rab5. Expression of a mutant Rab4 lacking a prenylation site resulted in reduced insulin-dependent phosphorylation of cytoplasmic and internal membrane-associated insulin receptor substrate-1, leading to decreased insulin receptor substrate-1-associated phosphatidylinositol 3′-OH kinase activation and decreased Akt activation. These effects were not observed upon introduction of a similar mutant form of Rab5. These data indi...

Regulation of GLUT4 and Insulin-Dependent Glucose Flux

GLUT4 has long been known to be an insulin responsive glucose transporter. Regulation of GLUT4 has been a major focus of research on the cause and prevention of type 2 diabetes. Understanding how insulin signaling alters the intracellular trafficking of GLUT4 as well as understanding the fate of glucose transported into the cell by GLUT4 will be critically important for seeking solutions to the current rise in diabetes and metabolic disease.

Rab5 Activity Regulates GLUT4 Sorting Into Insulin-Responsive and Non-Insulin-Responsive Endosomal Compartments: A Potential Mechanism for Development of Insulin Resistance

Glucose transporter isoform 4 (GLUT4) is the insulin-responsive glucose transporter mediating glucose uptake in adipose and skeletal muscle. Reduced GLUT4 translocation from intracellular storage compartments to the plasma membrane is a cause of peripheral insulin resistance. Using a chronic hyperinsulinemia (CHI)-induced cell model of insulin resistance and Rab5 mutant overexpression, we determined these manipulations altered endosomal sorting of GLUT4, thus contributing to the development of insulin resistance. We found that CHI induced insulin resistance in 3T3-L1 adipocytes by retaining GLUT4 in a Rab5-activity-dependent compartment that is unable to equilibrate with the cell surface in response to insulin. Furthermore, CHI-mediated retention of GLUT4 in this non-insulin-responsive compartment impaired filling of the transferrin receptor (TfR)-positive and TfR-negative insulin-responsive storage compartments. Our data suggest that hyperinsulinemia may inhibit GLUT4 by chronically maintaining GLUT4 in the Rab5 activity-dependent endosomal pathway and impairing formation of the TfR-negative and TfR-positive insulin-responsive GLUT4 pools. This model suggests that an early event in the development of insulin-resistant glucose transport in adipose tissue is to alter the intracellular localization of GLUT4 to a compartment that does not efficiently equilibrate with the cell surface when insulin levels are elevated for prolonged periods of time.

Increased Skeletal Muscle GLUT4 Expression in Obese Mice After Voluntary Wheel Running Exercise Is Posttranscriptional.

Exercise promotes glucose clearance by increasing skeletal muscle GLUT4-mediated glucose uptake. Importantly, exercise upregulates muscle GLUT4 expression in an insulin-independent manner under conditions of insulin resistance, such as with type 2 diabetes. However, the insulin-independent mechanism responsible for rescued muscle GLUT4 expression is poorly understood. We used voluntary wheel running (VWR) in mice to test the prevailing hypothesis that insulin-independent upregulation of skeletal muscle GLUT4 protein expression with exercise is through increased Glut4 transcription. We demonstrate that 4 weeks of VWR exercise in obese mice rescued high-fat diet–induced decreased muscle GLUT4 protein and improved both fasting plasma insulin and hepatic triacylglyceride levels, but did not rescue muscle Glut4 mRNA. Persistent reduction in Glut4 mRNA suggests that a posttranscriptional mechanism regulated insulin-independent muscle GLUT4 protein expression in response to exercise in lean and obese mice. Reduction of GLUT4 protein in sedentary animals upon treatment with rapamycin revealed mTORC1-dependent GLUT4 regulation. However, no difference in GLUT4 protein expression was observed in VWR-exercised mice treated with either rapamycin or Torin 1, indicating that exercise-dependent regulation on GLUT4 was mTOR independent. The findings provide new insight into the mechanisms responsible for exercise-dependent regulation of GLUT4 in muscle.

RalA signaling may reveal the true nature of 3T3-L1 adipocytes as a model for thermogenic adipocytes

In PNAS, Skorobogatko et al. (1) report that RalA signaling regulates glucose homeostasis in mice by regulating GLUT4 translocation to the plasma membrane and glucose uptake in brown adipose tissue, but not in white adipose tissue. Interestingly, the foundational work leading to this paper was carried out in 3T3-L1 adipocytes. Quite to their surprise, manipulation of RalA signaling in mice impacted glucose transport in brown adipose tissue exactly as predicted from the 3T3-L1 model. However, RalA signaling in white adipose tissue did not affect glucose uptake (Fig. 1). This was unexpected given the fact that 3T3-L1 cells are frequently used as a model to predict the function of white adipose tissue, not brown adipose tissue.nnnnFig. 1. nWhat type of adipocyte is modeled by the 3T3-L1 adipocyte? Based on lipid droplet morphology and Ral-dependent glucose uptake, the 3T3-L1 adipocyte models a thermogenic adipocyte.nnnnFor decades, 3T3-L1 adipocytes have served as a workhorse for studying mechanisms of adipocyte differentiation, adipocyte gene expression, triglyceride synthesis, insulin and beta-adrenergic signal transduction, and insulin-dependent glucose uptake as a model for white adipose tissue. Howard Green established the 3T3-derived adipocyte lines (3T3-L1) in the mid-1970s using clonal selection of 3T3 mouse fibroblast lines derived from disaggregated Swiss mouse embryo (2). In that first report, Green and Meuth (2) speculated that the 3T3-L1 model most resembled a brown adipose cell, or possibly an immature white adipose cell. This was largely due to the fact that the differentiation cells displayed multilocular … nn[↵][1]1Email: ann-olson{at}ouhsc.edu.nn [1]: #xref-corresp-1-1

Translocation and Redistribution of GLUT4 Using a Dual-Labeled Reporter Assay

It is crucial to determine the regulation of GLUT4 translocation and redistribution to the plasma membrane. The HA-GLUT4-GFP dual-reporter construct has become an important tool in the assessment of GLUT4 recycling in cultured adipocytes and myocytes. Through the use of light microscopy, this reporter construct allows for visualization of GLUT4 specifically at the cell surface or GLUT4 that has recycled from the cell surface while simultaneously marking the total GLUT4 pool. Here, we discuss and outline the general application of this reporter construct and its use in evaluating GLUT4 translocation within cultured adipocytes.