PFKFB3-dependent glucose metabolism regulates 3T3-L1 adipocyte development.

Proliferation and differentiation of preadipocytes, and other cell types, is accompanied by an increase in glucose uptake. Previous work showed that a pulse of high glucose was required during the first 3 days of differentiation in vitro, but was not required after that. The specific glucose metabolism pathways required for adipocyte differentiation are unknown. Herein, we used 3T3-L1 adipocytes as a model system to study glucose metabolism and expansion of the adipocyte metabolome during the first 3 days of differentiation. Our primary outcome measures were GLUT4 and adiponectin, key proteins associated with healthy adipocytes. Using complete media with 0 or 5 mM glucose, we distinguished between developmental features that were dependent on the differentiation cocktail of dexamethasone, insulin, and isobutylmethylxanthine alone or the cocktail plus glucose. Cocktail alone was sufficient to activate the capacity for 2-deoxglucose uptake and glycolysis, but was unable to support the expression of GLUT4 and adiponectin in mature adipocytes. In contrast, 5 mM glucose in the media promoted a transient increase in glucose uptake and glycolysis as well as a significant expansion of the adipocyte metabolome and proteome. Using genetic and pharmacologic approaches, we found that the positive effects of 5 mM glucose on adipocyte differentiation were specifically due to increased expression of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3), a key regulator of glycolysis and the ancillary glucose metabolic pathways. Our data reveal a critical role for PFKFB3 activity in regulating the cellular metabolic remodeling required for adipocyte differentiation and maturation.

Glucose availability controls adipogenesis in mouse 3T3-L1 adipocytes via up-regulation of nicotinamide metabolism.

Expansion of adipose tissue in response to a positive energy balance underlies obesity and occurs through both hypertrophy of existing cells and increased differentiation of adipocyte precursors (hyperplasia). To better understand the nutrient signals that promote adipocyte differentiation, we investigated the role of glucose availability in regulating adipocyte differentiation and maturation. 3T3-L1 preadipocytes were grown and differentiated in medium containing a standard differentiation hormone mixture and either 4 or 25 mm glucose. Adipocyte maturation at day 9 post-differentiation was determined by key adipocyte markers, including glucose transporter 4 (GLUT4) and adiponectin expression and Oil Red O staining of neutral lipids. We found that adipocyte differentiation and maturation required a pulse of 25 mm glucose only during the first 3 days of differentiation. Importantly, fatty acids were unable to substitute for the 25 mm glucose pulse during this period. The 25 mm glucose pulse increased adiponectin and GLUT4 expression and accumulation of neutral lipids via distinct mechanisms. Adiponectin expression and other early markers of differentiation required an increase in the intracellular pool of total NAD/P. In contrast, GLUT4 protein expression was only partially restored by increased NAD/P levels. Furthermore, GLUT4 mRNA expression was mediated by glucose-dependent activation of GLUT4 gene transcription through the cis-acting GLUT4-liver X receptor element (LXRE) promoter element. In summary, this study supports the conclusion that high glucose promotes adipocyte differentiation via distinct metabolic pathways and independently of fatty acids. This may partly explain the mechanism underlying adipocyte hyperplasia that occurs much later than adipocyte hypertrophy in the development of obesity.

Weight Loss Results in Increased Expression of Anti-Inflammatory Protein CRISPLD2 in Mouse Adipose Tissue.

OBJECTIVE: Obesity is a major risk factor for cardiovascular disease, metabolic syndrome, and type 2 diabetes mellitus, whereas weight loss is associated with improved health outcomes. It is therefore important to learn how adipose contraction during weight loss contributes to improved health. It was hypothesized that adipose tissue undergoing weight loss would have a unique transcriptomic profile, expressing specific genes that might improve health. METHODS: This study conducted an RNA-sequencing analysis of the epididymal adipose tissue of mice fed either a high-fat diet (HFD) or a regular rodent chow diet (RD) ad libitum for 10 weeks versus a cohort of mice fed HFD for the first 5 weeks before being swapped to an RD for the remainder of the study (swapped diet [SWAP]). RESULTS: The swapped diet resulted in weight loss, with a parallel improvement in insulin sensitivity. RNA sequencing revealed several transcriptomic signatures distinct to adipose tissue in SWAP mice, distinguished from both RD and HFD adipose tissue. The analysis found a unique upregulated mRNA that encodes a secreted lipopolysaccharide-binding glycoprotein (CRISPLD2) in adipose tissue. Whereas cellular CRISPLD2 protein levels were unchanged, plasma CRIPSLD2 levels increased in SWAP mice following weight loss and could correlate with insulin sensitivity. CONCLUSIONS: Taken together, these data demonstrate that CRISPLD2 is a circulating adipokine that may regulate adipocyte remodeling during weight loss.

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.

Enhanced GLUT4-Dependent Glucose Transport Relieves Nutrient Stress in Obese Mice Through Changes in Lipid and Amino Acid Metabolism.

Impaired GLUT4-dependent glucose uptake is a contributing factor in the development of whole-body insulin resistance in obese patients and obese animal models. Previously, we demonstrated that transgenic mice engineered to express the human GLUT4 gene under the control of the human GLUT4 promoter (i.e., transgenic [TG] mice) are resistant to obesity-induced insulin resistance. A likely mechanism underlying increased insulin sensitivity is increased glucose uptake in skeletal muscle. The purpose of this study was to investigate the broader metabolic consequences of enhanced glucose uptake into muscle. We observed that the expression of several nuclear and mitochondrially encoded mitochondrial enzymes was decreased in TG mice but that mitochondrial number, size, and fatty acid respiration rates were unchanged. Interestingly, both pyruvate and glutamate respiration rates were decreased in TG mice. Metabolomics analyses of skeletal muscle samples revealed that increased GLUT4 transgene expression was associated with decreased levels of some tricarboxylic acid intermediates and amino acids, whereas the levels of several glucogenic amino acids were elevated. Furthermore, fasting acyl carnitines in obese TG mice were decreased, indicating that increased GLUT4-dependent glucose flux decreases nutrient stress by altering lipid and amino acid metabolism in skeletal muscle.

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.

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.

Class II histone deacetylases downregulate GLUT4 transcription in response to increased cAMP signaling in cultured adipocytes and fasting mice.

Insulin-mediated glucose uptake is highly sensitive to the levels of the facilitative glucose transporter protein, GLUT4. Repression of GLUT4 expression is correlated with insulin resistance in adipose tissue. We have shown that differentiation-dependent GLUT4 transcription was under control of class II histone deacetylases (HDACs). We hypothesized that HDACs may regulate gene expression in adipocytes as a result of adrenergic activation. To test this hypothesis, we activated cAMP signaling in 3T3-L1 adipocytes and in mice after an overnight fast. Chromatin immunoprecipitation experiments showed the association of HDAC4/5 with the GLUT4 promoter in vivo and in vitro in response to elevated cAMP. Knockdown of HDACs by small interfering RNA in cultured adipocytes prevented the cAMP-dependent decrease in GLUT4 transcription. HDAC4/5 recruitment to the GLUT4 promoter was dependent on the GLUT4 liver X receptor (LXR) binding site. Treatment of cells with an LXR agonist prevented the cAMP-dependent decrease in GLUT4 transcription. A loss of function mutation in the LXR response element was required for cAMP-dependent downregulation of GLUT4 expression in vitro, in fasted mice, and in mice subjected to diet-induced obesity. This suggests that activation of LXR signaling can prevent loss of GLUT4 expression in diabetes and obesity.

Acute inhibition of fatty acid import inhibits GLUT4 transcription in adipose tissue, but not skeletal or cardiac muscle tissue, partly through liver X receptor (LXR) signaling.

OBJECTIVE: Insulin-mediated glucose uptake is highly sensitive to the levels of the facilitative GLUT protein GLUT4. Transcription of the GLUT4 gene is repressed in states of insulin deficiency and insulin resistance and can be induced by states of enhanced energy output, such as exercise. The cellular signals that regulate GLUT4 transcription are not well understood. We hypothesized that changes in energy substrate flux regulate GLUT4 transcription. RESEARCH DESIGN AND METHODS: To test this hypothesis, we used transgenic mice in which expression of the chloramphenicol acetyltransferase (CAT) gene is driven by a functional 895-bp fragment of the human GLUT4 promoter, thereby acting as a reporter for transcriptional activity. Mice were treated with a single dose of etomoxir, which inhibits the transport of long-chain fatty acids into mitochondria and increases basal, but not insulin-mediated, glucose flux. GLUT4 and transgenic CAT mRNA were measured. RESULTS: Etomoxir treatment significantly reduced CAT and GLUT4 mRNA transcription in adipose tissue, but did not change transcription in heart and skeletal muscle. Downregulation of GLUT4 transcription was cell autonomous, since etomoxir treatment of 3T3-L1 adipocytes resulted in a similar downregulation of GLUT4 mRNA. GLUT4 transcriptional downregulation required the putative liver X receptor (LXR) binding site in the human GLUT4 gene promoter in adipose tissue and 3T3-L1 adipocytes. Treatment of 3T3-L1 adipocytes with the LXR agonist, TO901317, partially restored GLUT4 expression in etomoxir-treated cells. CONCLUSIONS: Our data suggest that long-chain fatty acid import into mitochondria in adipose tissue may produce ligands that regulate expression of metabolic genes.

GLUT4 enhancer factor (GEF) interacts with MEF2A and HDAC5 to regulate the GLUT4 promoter in adipocytes.

The insulin-responsive glucose transporter, GLUT4, is regulated in various physiologic states at the transcriptional level. When expressed in transgenic mice, the human GLUT4 promoter is governed by two cis-acting sequences: an MEF2 binding domain and Domain I, that function both as positive and negative regulators depending on the physiologic state. MEF2 proteins and GLUT4 enhancer factor (GEF) are known ligands for these cis-acting elements, but their mechanism of action is unclear. To begin to understand this important process, we have characterized GEF structural domains and its interactions with the MEF2A isoform. We find that the C terminus of GEF comprises its DNA-binding domain, but does not contribute to GEF homo-oligomerization. We also have found that GEF dimerizes with increased affinity to a hypophosphorylated form of MEF2A. Furthermore, we demonstrated that MEF2A binding to its cognate binding site can increase the DNA binding activity of GEF to Domain I, suggesting a novel mechanism for MEF2A transcriptional activation. Finally, we have demonstrated that the transcriptional co-repressor HDAC5 can interact with GEF in the absence of MEF2 proteins and specifically inhibit GLUT4 promoter activity. These findings lead to the hypothesis that GEF and the MEF2 proteins form a complex on the GLUT4 promoter that allows for recruitment of transcriptional co-regulators (repressors and/or activators) to control GLUT4 promoter activity.

Hyperphosphorylation of MEF2A in primary adipocytes correlates with downregulation of human GLUT4 gene promoter activity.

GLUT4 promoter activity is regulated by hormonal, metabolic, and tissue-specific controls. This complicates the study of GLUT4 gene transcription, as no cell culture model adequately recapitulates these extracellular regulators. While investigating cultured primary adipocytes as a model system for GLUT4 transcription, we observed that GLUT4 mRNA was specifically and rapidly downregulated upon tissue dispersal. Downregulation of GLUT4 mRNA was mediated in part by loss of regulatory control by the trans-acting factors that control GLUT4 transcriptional activity [the myocyte enhancer factor 2 (MEF2) transcription factor family and the GLUT4 enhancer factor] and their cognate DNA binding sites in transgenic mice. The differences in GLUT4 transcription when whole adipose tissue and cell culture model systems are compared can be correlated to a posttranslational phosphorylation of the transcription factor MEF2A. The difference in the MEF2A phosphorylation state in whole tissue vs. isolated cells may provide a further basis for the development of an in vitro system that could recapitulate fully regulated GLUT4 promoter activity. Development of an in vitro system to reconstitute GLUT4 transcriptional regulation will further efforts to discern the molecular mechanisms that underlie GLUT4 expression.

Regulation of the human GLUT4 gene promoter: interaction between a transcriptional activator and myocyte enhancer factor 2A.

The GLUT4 gene is subject to complex tissue-specific and metabolic regulation, with a profound impact on insulin-mediated glucose disposal. We have shown, by using transgenic mice, that the human GLUT4 promoter is regulated through the cooperative function of two distinct regulatory elements, domain 1 and the myocyte enhancer factor 2 (MEF2) domain. The MEF2 domain binds transcription factors MEF2A and MEF2D in vivo. Domain I binds a transcription factor, GLUT4 enhancer factor (GEF). In this report, we show a restricted pattern of GEF expression in human tissues, which overlaps with MEF2A only in tissues expressing high levels of GLUT4, suggesting the hypothesis that GEF and MEF2A function together to activate GLUT4 transcription. Data obtained from transiently transfected cells support this hypothesis. Neither GEF nor MEF2A alone significantly activated GLUT4 promoter activity, but increased promoter activity 4- to 5-fold when expressed together. Deletion of the GEF-binding domain (domain I) and the MEF2-binding domain prevented activation, strengthening the conclusion that promoter regulation occurs through these elements. GEF and MEF2A, isolated from nuclei of transfected cells, bound domain I and the MEF2 domain, respectively, which is consistent with activation through these regulatory elements. Finally, GEF and MEF2A coimmunoprecipitated in vivo, strongly supporting a mechanism of GLUT4 transcription activation that depends on this protein-protein interaction.