Overexpression of GLUT4 in mice causes up-regulation of UCP3 mRNA in skeletal muscle

Mitochondrial uncoupling protein 3 (UCP3) is expressed in skeletal muscles. We have hypothesized that increased glucose flux in skeletal muscles may lead to increased UCP3 expression. Male transgenic mice harboring insulin-responsive glucose transporter (GLUT4) minigenes with differing lengths of 5'-flanking sequence (-3237, -2000, -1000 and -442 bp) express different levels of GLUT4 protein in various skeletal muscles. Expression of the GLUT4 transgenes caused an increase in UCP3 mRNA that paralleled the increase of GLUT4 protein in gastrocnemius muscle. The effects of increased intracellular GLUT4 level on the expression of UCP1, UCP2 and UCP3 were compared in several tissues of male 4 month-old mice harboring the -1000 GLUT4 minigene transgene. In the -1000 GLUT4 transgenic mice, expression of GLUT4 mRNA and protein in skeletal muscles, brown adipose tissue (BAT), and white adipose tissue (WAT) was increased by 1.4 to 4.0-fold. Compared with non-transgenic littermates, the -1000 GLUT4 mice exhibited about 4- and 1.8-fold increases of UCP3 mRNA in skeletal muscle and WAT, respectively, and a 38% decrease of UCP1 mRNA in BAT. The transgenic mice had a 16% increase in oxygen consumption and a 14% decrease in blood glucose and a 68% increase in blood lactate, but no change in FFA or beta-OHB levels. T3 and leptin concentrations were decreased in transgenic mice. Expression of UCP1 in BAT of the -442 GLUT4 mice, which did not overexpress GLUT4 in this tissue, was not altered. These findings indicate that overexpression of GLUT4 up-regulates UCP3 expression in skeletal muscle and down-regulates UCP1 expression in BAT, possibly by increasing the rate of glucose uptake into these tissues.     

Tea catechins enhance the mRNA expression of uncoupling protein 1 in rat brown adipose tissue

The aim of the present study was to determine whether the antiobesity effects of tea catechins (TCs) are associated with the expression of uncoupling protein 1 (UCP1) in brown adipose tissue (BAT). Male Sprague–Dawley rats were fed a high-fat (HF; 35% fat) diet for 5 weeks, then divided into four groups and fed an HF, HF with 0.5% TC (HFTC), normal-fat (NF; 5% fat) or NF with 0.5% TC (NFTC) diet for 8 weeks. At the end of the experimental period, perirenal and epididymal white adipose tissues (WATs) and interscapular BAT were isolated. The NFTC group had significantly lower perirenal WAT weights than the NF group (NF: 12.7±0.53 g; NFTC: 10.2±0.43 g; P<.01), but the HF and HFTC groups did not differ significantly. TC intake had no effects on epididymal WAT weights. The NFTC and HFTC groups had significantly lower BAT weights than the NF and HF groups, respectively. The NFTC group had significantly higher UCP1 mRNA levels in BAT than the NF group (NF: 0.35±0.02; NFTC: 0.60±0.11; P<.05), but the HF and HFTC groups did not differ significantly. Thus, TC intake in the context of the NF diet reduced perirenal WAT weight and up-regulated UCP1 mRNA expression in BAT. These results suggest that the suppressive effect of TC on body fat accumulation is associated with UCP1 expression in BAT.     

Angiotensin II Receptor Blocker Ameliorates Stress-Induced Adipose Tissue Inflammation and Insulin Resistance

A strong causal link exists between psychological stress and insulin resistance as well with hypertension. Meanwhile, stress-related responses play critical roles in glucose metabolism in hypertensive patients. As clinical trials suggest that angiotensin-receptor blocker delays the onset of diabetes in hypertensive patients, we investigated the effects of irbesartan on stress-induced adipose tissue inflammation and insulin resistance. C57BL/6J mice were subjected to 2-week intermittent restraint stress and orally treated with vehicle, 3 and 10 mg/kg/day irbesartan. The plasma concentrations of lipid and proinflammatory cytokines [Monocyte Chemoattractant Protein-1 (MCP-1), tumor necrosis factor-α, and interleukin-6] were assessed with enzyme-linked immunosorbent assay. Monocyte/macrophage accumulation in inguinal white adipose tissue (WAT) was observed with CD11b-positive cell counts and mRNA expressions of CD68 and F4/80 using immunohistochemistry and RT-PCR methods respectively. The mRNA levels of angiotensinogen, proinflammatory cytokines shown above, and adiponectin in WAT were also assessed with RT-PCR method. Glucose metabolism was assessed by glucose tolerance tests (GTTs) and insulin tolerance tests, and mRNA expression of insulin receptor substrate-1 (IRS-1) and glucose transporter 4 (GLUT4) in WAT. Restraint stress increased monocyte accumulation, plasma free fatty acids, expression of angiotensinogen and proinflammatory cytokines including MCP-1, and reduced adiponectin. Irbesartan reduced stress-induced monocyte accumulation in WAT in a dose dependent manner. Irbesartan treatment also suppressed induction of adipose angiotensinogen and proinflammatory cytokines in WAT and blood, and reversed changes in adiponectin expression. Notably, irbesartan suppressed stress-induced reduction in adipose tissue weight and free fatty acid release, and improved insulin tolerance with restoration of IRS-1 and GLUT4 mRNA expressions in WAT. The results indicate that irbesartan improves stress-induced adipose tissue inflammation and insulin resistance. Our results suggests that irbesartan treatment exerts additive benefits for glucose metabolism in hypertensive patients with mental stress.     

Soybean and sunflower oil‐induced insulin resistance correlates with impaired GLUT4 protein expression and translocation specifically in white adipose tissue

Free fatty acids are known for playing a crucial role in the development of insulin resistance. High fat intake is known for impairing insulin sensitivity; however, the effect of vegetable‐oil injections have never been investigated. The present study investigated the effects of daily subcutaneous injections (100 µL) of soybean (SB) and sunflower (SF) oils, during 7 days. Both treated groups developed insulin resistance as assessed by insulin tolerance test. The mechanism underlying the SB‐ and SF‐induced insulin resistance was shown to involve GLUT4. In SB‐ and SF‐treated animals, the GLUT4 protein expression was reduced ～20% and 10 min after an acute in vivo stimulus with insulin, the plasma membrane GLUT4 content was ～60% lower in white adipose tissue (WAT). No effects were observed in skeletal muscle. Additionally, both oil treatments increased mainly the content of palmitic acid (～150%) in WAT, which can contribute to explain the GLUT4 regulations. Altogether, the present study collects evidence that those oil treatments might generate insulin resistance by targeting GLUT4 expression and translocation specifically in WAT. These alterations are likely to be caused due to the specific local increase in saturated fatty acids that occurred as a consequence of oil daily injections. Copyright © 2010 John Wiley & Sons, Ltd.     

Different metabolic responses of human brown adipose tissue to activation by cold and insulin

We investigated the metabolism of human brown adipose tissue (BAT) in healthy subjects by determining its cold-induced and insulin-stimulated glucose uptake and blood flow (perfusion) using positron emission tomography (PET) combined with computed tomography (CT). Second, we assessed gene expression in human BAT and white adipose tissue (WAT). Glucose uptake was induced 12-fold in BAT by cold, accompanied by doubling of perfusion. We found a positive association between whole-body energy expenditure and BAT perfusion. Insulin enhanced glucose uptake 5-fold in BAT independently of its perfusion, while the effect on WAT was weaker. The gene expression level of insulin-sensitive glucose transporter GLUT4 was also higher in BAT as compared to WAT. In conclusion, BAT appears to be differently activated by insulin and cold; in response to insulin, BAT displays high glucose uptake without increased perfusion, but when activated by cold, it dissipates energy in a perfusion-dependent manner.     

GLUT4 expression and subcellular localization in the intrauterine growth-restricted adult rat female offspring

Intrauterine growth restriction (IUGR) leads to obesity, glucose intolerance, and type 2 diabetes mellitus in the adult. To determine the mechanism(s) behind this "metabolic imprinting" phenomenon, we examined the effect of total calorie restriction during mid- to late gestation modified by postnatal ad libitum access to nutrients (CM/SP) or nutrient restriction (SM/SP) vs. postnatal nutrient restriction alone (SM/CP) on skeletal muscle and white adipose tissue (WAT) insulin-responsive glucose transporter isoform (GLUT4) expression and insulin-responsive translocation. A decline in skeletal muscle GLUT4 expression and protein concentrations was noted only in the SM/SP and SM/CP groups. In contrast, WAT demonstrated no change in GLUT4 expression and protein concentrations in all experimental groups. The altered in utero hormonal/metabolic milieu was associated with a compensatory adaptation that persisted in the adult and consisted of an increase in the skeletal muscle basal plasma membrane-associated GLUT4 concentrations. This perturbation led to no further exogenous insulin-induced GLUT4 translocation, thereby disabling the insulin responsiveness of the skeletal muscle but retaining it in WAT. These changes, which present at birth, collectively maximize basal glucose transport to the compromised skeletal muscle with a relative resistance to exogenous/postprandial insulin. Preservation of insulin responsiveness in WAT may serve as a sink that absorbs postprandial nutrients that can no longer efficiently access skeletal muscle. We speculate that, in utero, GLUT4 aberrations may predict type 2 diabetes mellitus, whereas postnatal nutrient intake may predict obesity, thereby explaining the heterogeneous phenotype of the IUGR adult offspring.     

Altered GLUT4 trafficking in adipocytes in the absence of the GTPase Arfrp1

The GTPase ADP-ribosylation factor related protein 1 (ARFRP1) controls the recruitment of proteins such as golgin-245 to the trans-Golgi. ARFRP1 is highly expressed in adipose tissues in which the insulin-sensitive glucose transporter GLUT4 is processed through the Golgi to a specialized endosomal compartment, the insulin-responsive storage compartment from which it is translocated to the plasma membrane in response to a stimulation of cells by insulin. In order to examine the role of ARFRP1 for GLUT4 targeting, subcellular distribution of GLUT4 was investigated in adipose tissue specific Arfrp1 knockout (Arfrp1^ad−/−) mice. Immunohistochemical and ultrastructural studies of brown adipocytes demonstrated an abnormal trans-Golgi in Arfrp1^ad−/− adipocytes. In addition, in Arfrp1^ad−/− adipocytes GLUT4 protein accumulated at the plasma membrane rather than being sequestered in an intracellular compartment. A similar missorting of GLUT4 was produced by siRNA-mediated knockdown of Arfrp1 in 3T3-L1 adipocytes which was associated with significantly elevated uptake of deoxyglucose under basal conditions. Thus, Arfrp1 appears to be involved in sorting of GLUT4.     

GLUT4 protein is differently modulated during development of obesity in monosodium glutamate-treated mice

The aim of the present study was to investigate the GLUT4 protein expression during the development of obesity in monosodium glutamate- (MSG) treated mice. Control (C) and neonatally MSG-treated 2-month-old (2-mo), 4-month-old (4-mo) and 7-month-old (7-mo) mice were analyzed. Anthropometric data, basal glycemia and insulinemia were measured; and the GLUT4 protein was assessed by Western blotting in white adipose tissue (WAT), skeletal muscle gastrocnemius (SM) and heart (H). Compared to age-matched C mice, the 2-mo and 4-mo MSG mice were already obese, but metabolically they showed increased or preserved whole-body insulin sensitivity, respectively. At these ages they showed unchanged total GLUT4 content in SM and H. However, in plasma membrane fraction from WAT, the MSG showed increased GLUT4 content at both 2- (by 60%) and 4-month (by 45%) of age. When the GLUT4 protein was expressed by unit of adipocyte surface area the protein amount was increased by 36 and 220% in 2-mo and 4-mo MSG mice, respectively. At 7 months of age, obesity was fully established in MSG mice, showing a strongly insulin resistant condition. Additionally, in the 7-mo MSG-mice the GLUT4 protein was reduced in SM (by 40%), H (by 28%), PM and M fractions of WAT (by approximately 70%), and PM expressed by unit of adipocyte surface area (by 92%). The data demonstrate that early, during the accelerated development of obesity in MSG-treated mice, the GLUT4 content was increased in WAT, and that may play a key role in the development of obesity. Later on, when obesity is fully established, the GLUT4 protein was reduced in SM, heart and WAT, and that may be involved in the insulin resistance present in this condition.     

Metallothionein gene expression and secretion in white adipose tissue

White adipose tissue (WAT) has been examined to determine whether the gene encoding metallothionein (MT), a low-molecular-weight stress response protein, is expressed in the tissue and whether MT may be a secretory product of adipocytes. The MT-1 gene was expressed in epididymal WAT, with MT-1 mRNA levels being similar in lean and obese (ob/ob) mice. MT-1 mRNA was found in each of the main adipose tissue sites (epididymal, perirenal, omental, subcutaneous), and there was no major difference between depots. Separation of adipocytes from the stromal-vascular fraction of WAT indicated that the MT gene (MT-1 and MT-2) was expressed in adipocytes themselves. Treatment of mice with zinc had no effect on MT-1 mRNA levels in WAT, despite strong induction of MT-1 expression in the liver. MT-1 gene expression in WAT was also unaltered by fasting or norepinephrine. However, administration of a beta(3)-adrenoceptor agonist, BRL-35153A, led to a significant increase in MT-1 mRNA. On differentiation of fibroblastic preadipocytes to adipocytes in primary culture, MT was detected in the medium, suggesting that the protein may be secreted from WAT. It is concluded that WAT may be a significant site of MT production; within adipocytes, MT could play an antioxidant role in protecting fatty acids from damage.