Effects of statins on the adipocyte maturation and expression of glucose transporter 4 (SLC2A4): implications in glycaemic control
Hyperlipidaemia often occurs in patients with type 2 diabetes mellitus. Though HMG-CoA reductase inhibitors (statins) are widely used for controlling hypercholesterolemia, atorvastatin has also been reported to have an adverse effect on glucose metabolism. Based on these findings, the aim of this study was to investigate the effects of statins on adipocytes, which play pivotal roles in glucose metabolism.
In 3T3-L1 cells, effects of statins on adipocyte maturation were determined morphologically. Protein and mRNA levels of SLC2A4 and adipocyte marker proteins were determined by immunoblotting and RT-PCR, respectively. Type 2 diabetic NSY mice were treated with atorvastatin for 15 weeks, followed by glucose and insulin tolerance tests and examination of SLC2A4 expression in white adipose tissue (WAT). Seventy-eight Japanese subjects with type 2 diabetes and hypercholesterolaemia were treated with atorvastatin (10 mg/day), and its effects on lipid and glycaemic profiles were measured 12 weeks after treatment initiation.
Treatment with atorvastatin inhibited adipocyte maturation, SLC2A4 and C/EBPα expressions and insulin action in 3T3-L1 cells. Atorvastatin also attenuated SLC2A4 and C/EBPα expressions in differentiated 3T3-L1 adipocytes. These effects were reversed by l-mevalonate or geranylgeranyl pyrophosphate. In NSY mice, atorvastatin accelerated glucose intolerance as a result of insulin resistance and decreased SLC2A4 expression in WAT. In addition to improving hyperlipidaemia, atorvastatin treatment significantly increased HbA1c but not fasting glucose levels in diabetic patients, and this effect was greater in the non-obese subgroup.
These results demonstrate that atorvastatin attenuates adipocyte maturation and SLC2A4 expression by inhibiting isoprenoid biosynthesis, and impairs glucose tolerance. These actions of atorvastatin could potentially affect the control of type 2 diabetes.
KeywordsAdipocyte C/EBPα GLUT4 HMG-CoA reductase inhibitors Insulin resistance Obesity SLC2A4 Statins
CCAAT/enhancer binding protein
fatty acid synthase
insulin receptor β-subunit
peroxisome proliferator-activated receptor
solute carrier family 2 (facilitated glucose transporter), member 4
CHD is the major cause of mortality in subjects with type 2 diabetes [1, 2], and one of the key factors contributing to cardiovascular disease in type 2 diabetic patients is the impairment of lipid metabolism . Several studies have shown that correction of dyslipidaemia significantly decreases the risk of CHD events [3, 4]. This may be achieved through use of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors (statins), which can control both hypercholesterolaemia and hypertriglyceridaemia. Several clinical reports have shown that these agents reduce CHD and total mortality in diabetic subjects [5, 6, 7].
The retrospective analysis of data from the West of Scotland Coronary Prevention Study (WOSCOPS) showed that pravastatin therapy resulted in a 30% reduction in the hazard of developing diabetes . On the other hand, it has been reported that atorvastatin increased the incidence of diabetes in the Anglo-Scandinavian Cardiac Outcomes Trial (ASCOT) , and that atorvastatin impaired glucose metabolism in some cases of type 2 diabetes [9, 10]. However, few studies have addressed the potential effects of statins upon glucose metabolism. Elucidation of the mechanisms underlying the pathological effects of statins on glucose metabolism is of importance, especially when deciding upon the most suitable of several statins for treatment of dyslipidaemia in type 2 diabetic patients. The adipocyte is one of the major targets for insulin action. During the course of insulin-stimulated glucose uptake in adipocytes, insulin activates the tyrosine kinase activity of the insulin receptor, which, in turn phosphorylates IRS-1, resulting in the recruitment of the insulin-sensitive solute carrier family 2 (facilitated glucose transporter), member 4 (SLC2A4, formerly known as GLUT4) to the plasma membrane and uptake of glucose . The decreased expression of SLC2A4 contributes to insulin resistance and type 2 diabetes . It was shown that TNFα, a factor that induces insulin resistance, decreased SLC2A4 expression in adipocytes , and that lovastatin administered at a super-pharmacological dose also decreased SLC2A4 expression . Adipocytes influence glucose metabolism and type 2 diabetes, at least partly, by releasing adipocytokines that either impair or improve insulin action [15, 16]. Whether adipocytes exert beneficial or deleterious effects on glucose metabolism depends on the state of adipocyte differentiation .
In this study we aimed to clarify the effects of atorvastatin on adipocyte differentiation and SLC2A4 expression in 3T3-L1 cells and on glucose metabolism and SLC2A4 expression in NSY mice. This mouse reportedly exhibits moderate levels of obesity, insulin resistance, and impaired insulin response to glucose, thus displaying the characteristics of type 2 diabetes in humans. Furthermore, Japanese subjects with type 2 diabetes and hypercholesterolaemia were treated with atorvastatin, and its effects on glycaemic profiles were examined.
Subjects, materials and methods
All standard culture reagents were obtained from Gibco BRL (Grand Island, NY, USA). Pravastatin, atorvastatin and simvastatin were gifts from Sankyo (Tokyo, Japan). Pitavastatin was a gift from Kowa (Tokyo, Japan). Geranylgeranyl pyrophosphate (GGPP) and dl-mevalonic acid lactone (dl-mevalonate) were purchased from Sigma Chemical (St Louis, MO, USA).
Cell culture, induction of adipocyte differentiation and treatment with statins
The 3T3-L1 cells were maintained as previously described . Differentiation of 3T3-L1 cells into adipocytes was induced by treatment of confluent monolayers for 2 days with 10 μg/ml insulin, 0.4 μg/ml dexamethasone and 0.5 mmol/l 3-isobutyl-1-methylxanthine (differentiation cocktail) in DMEM containing 10% foetal bovine serum (FBS), followed by subsequent culture to day 8 in most experiments. The day when this culture started was denoted as day 1. At day 3, the culture medium was changed to one containing 10% FBS and 10 μg/ml insulin. Insulin was removed on day 5, and the cells were fed every 2 days with DMEM containing 10% FBS.
To investigate the effects of HMG-CoA reductase inhibitors on adipocyte differentiation, the drug was added to the culture for the specific 2-day culture periods from day 1 to day 3 (days 1–3), days 3–5 or days 5–7. The cells on day 8 were used for most measurements. In the study to examine the effect of statins in the differentiated adipocytes, the cells were further cultured for 2 days (days 8–10) with or without atorvastatin at the indicated concentrations.
Cell culture, induction of myotube differentiation and treatment with statins
The C2C12 cells were cultured in DMEM with 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin at 37°C in an atmosphere of 5% CO2, 95% air. Differentiation of C2C12 cells into myotubes was induced by treatment of confluent monolayers with 10 μg/ml insulin, 5 μg/ml transferrin, 10 nmol/l selenite and 1 mg/ml BSA in culture for 3 days.
Treated cells were lysed in lysis buffer (100 mmol/l NaCl, 0.5% NP40, 1 mmol/l EDTA, 10 mmol/l Tris-HCl [pH 7.5], 0.5 U/ml aprotinin, 1 mmol/l PMSF). To extract nuclear proteins, cells were lysed in lysis buffer containing 1% SDS. For Akt phosphorylation, the cells were incubated with insulin (100 nmol/l) and lysed in lysis buffer containing 100 mmol/l sodium fluoride, 10 mmol/l sodium pyrophosphate, 2 mmol/l sodium orthovanadate. Cell lysates were subjected to SDS-PAGE through an 8 or 10% gel. SLC2A4 was detected with the SLC2A4 polyclonal antibody (a gift from Y. Oka, Tohoku University, Sendai, Japan). Hormone-sensitive lipase (HSL) was detected with the HSL polyclonal antibody . IRβ, IRS-1, peroxisome proliferator-activated receptor γ (PPARγ) and caveolin-1 proteins were detected with anti-IR, anti-IRS-1, anti-PPARγ and anti-caveolin-1 primary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA). Perilipin and CCAAT/enhancer binding protein α (C/EBPα) were detected with anti-perilipin and anti-C/EBPα primary antibodies (ABR—Affinity BioReagents, Golden, CO, USA). Fatty acid synthase (FAS) was detected with anti-FAS primary antibody (BD, San Jose, CA, USA). Phospho-Ser Akt was detected with the polyclonal antibody (CST, Beverly, MA, USA). Immunoreactive proteins were detected with HRP-conjugated secondary antibody and the ECL system (Amersham, Arlington Heights, IL, USA). For immunoblotting of Akt, membranes were reprobed and detected using anti-Akt polyclonal antibody.
Real-time RT-PCR analysis
Total RNA was isolated using TRIzol (Invitrogen, Tokyo, Japan), and first-strand cDNA synthesis was completed using the ReverTra Ace (Toyobo, Osaka, Japan). Using a QuantiTect SYBR Green PCR kit, real-time PCR was performed in an ABI-prism 7700 sequence detector (Applied Biosystems Japan, Tokyo, Japan). Using β-actin for internal control, data were analysed according to the 2−ΔΔCT method. Primers were as follows: β-actin, 5′-TTCCCCTCCATCGTGGGCCGC-3′ and 5′-GATGGCTACGTACATGGCTGG-3′; Slc2a4, 5′-CTTCTTTGAGATTGGCCCTGG-3′ and 5′-AGGTGAAGATGAA GAAGCCAAGC-3′; Cebpa, 5′-CCGGGAGAACTCTAACTC-3′ and 5′-GATGTAGGCGCTGATGT-3′.
Oil Red O staining and measurements of triglyceride content
At day 8, cells were fixed and stained with Oil Red O to morphologically assess adipocyte differentiation. The triglyceride content of lipid extracts was measured using an enzymatic assay kit (Triglyceride E-test; Wako, Osaka, Japan) and normalised to cell protein.
Measurement of 2-NBDG uptake
3T3-L1 cells were grown and differentiated with or without statins in 24-well culture plates. Before assaying, cells were preincubated with serum-free DMEM for 2 h. After washing with Krebs–Ringer phosphate buffer (KRB) containing 2.8 mmol/l glucose, cells were preincubated in 500 μl KRB for 30 min at 37°C. Cells were then stimulated with insulin (100 nmol/l) (Sigma) for 10 min in KRB at 37°C. Uptake was initiated by addition of a fluorescent derivative of 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose (2-NBDG) at 600 μmol/l in 2.8 mmol/l glucose containing KRB, as described previously . After 15 min, the reaction was terminated by quickly washing with ice-cold KRB. Cells were lysed by freeze–thaw. Fluorescence of 2-NBDG was measured using fluorescence spectrophotometer F-2500 (Hitachi, Tokyo, Japan) at a wavelength of 540 nm (excitation wavelength 485 nm).
Tests of glucose metabolism in type 2 diabetes NSY mice
Male NSY mice weighing 25–28 g were purchased from Japan SLC (Hamamatsu, Japan) . A single mouse was housed per cage, with a light–dark cycle of 12 h each (light from 07.00–19.00 h), and free access to food and water. From 5–20 weeks of age, NSY mice were given either a regular diet (CE-2; CLEA Japan, Tokyo, Japan) or one containing 0.05 g of atorvastatin per 100 g. All experimental procedures were carried out in accordance with the Japanese Physiological Society’s guidelines for animal care and approved by the institutional animal care and use committee. For glucose tolerance tests, 6-h-fasted NSY mice aged 20 weeks were injected i.p. with glucose (2.0 g/kg). For insulin tolerance tests, 6-h-fasted NSY mice aged 20 weeks were injected i.p. with rapid insulin (0.5 IU/kg). Glucose and insulin levels in blood samples obtained from the tail vein were determined by using Glucocard (Arkray, Kyoto, Japan) and ELISA (Shibayagi, Gumma, Japan), respectively.
Clinical research design
Baseline characteristics of the patients
Non-obese group (BMI<26 kg/m2)
Obese group (BMI>26 kg/m2)
Oral hypoglycaemic agents
All data are presented as means±SEM (n=number of observations). The statistical analysis of experimental data was carried out with the Student’s t-test. For the clinical research study, paired results obtained at baseline and 12 weeks after initiation of atorvastatin medication were compared using the Wilcoxon matched-pairs signed-ranks test. Differences were considered statistically significant when the p value was less than 0.05.
Effects of statins on differentiation of 3T3-L1 cells
Effects of atorvastatin on levels of insulin signalling-associated and/or adipocyte-associated proteins and glucose uptake in 3T3-L1 cells during the adipocyte differentiation period
Effects of atorvastatin on levels of insulin signalling-associated and/or adipocyte-associated proteins and glucose uptake in differentiated 3T3-L1 cells
Effects of atorvastatin on glucose metabolism and SLC2A4 expression in NSY mice
Patients’ glycaemic and lipid parameters before and after the follow-up period
Non-obese group (n=39) (BMI<26)
Obese group (n=39) (BMI>26)
Body weight (kg)
Total cholesterol (mmol/l)
HDL cholesterol (mmol/l)
Fasting plasma glucose (mmol/l)
This study demonstrates that treatment with atorvastatin at clinical doses inhibits adipocyte differentiation, decreases SLC2A4 expression in both differentiating and mature adipocytes, and impairs insulin sensitivity and post-challenge glucose tolerance in an animal model of type 2 diabetes. In type 2 diabetic patients, particularly in those without obesity, atorvastatin treatment was followed by an increase in HbA1c.
In this study, differentiation of 3T3-L1 cells into adipocytes was induced, and the stage of differentiation/maturation was judged by measurement of adipocyte markers and markers of insulin signalling; these included oil droplet formation, triglyceride accumulation, levels of SLC2A4, FAS, perilipin and caveolin-1, and insulin-induced glucose uptake and Akt phosphorylation. Atorvastatin administered at days 3–5 of culture markedly reduced all these parameters.
PPARγ and C/EBPα co-operate to induce the production of adipocyte-specific proteins, such as perilipin and SLC2A4, during the late stages of adipocyte differentiation, resulting in the accumulation of intracellular lipids and promotion of insulin-stimulated SLC2A4 translocation and glucose uptake . In this study, atorvastatin suppression of PPARγ and C/EBPα levels appeared at day 5 of culture, while suppression of SLC2A4 and caveolin-1 levels was observed at day 8 of culture. We therefore suggest that the atorvastatin-induced decrease in PPARγ and C/EBPα levels during the early stages of differentiation is at least partly responsible for the subsequent reduction in SLC2A4 and caveolin-1 levels at late stages of differentiation. In adipocytes, the insulin receptor is localised in caveolae, and insulin stimulation translocates SLC2A4 to caveolae, a process implicated in glucose uptake . Therefore, suppression of SLC2A4 and caveolin-1 expression by atorvastatin may be linked to the reduction in the insulin-stimulated glucose uptake.
These inhibitory effects of atorvastatin were counteracted by the co-administration of mevalonate or GGPP, indicating that atorvastatin inhibits adipocyte differentiation by inhibiting HMG-CoA reductase. These effects of atorvastatin were maximal when administered at days 3–5 of culture, in accordance with a previous report that the expression of cholesterol metabolic enzymes including HMG-CoA reductase is remarkably increased at day 3 of differentiation of 3T3-L1 cells . When administered after differentiation (on days 8–10), atorvastatin suppressed C/EBPα and SLC2A4 levels and insulin-induced glucose uptake. The inhibition of SLC2A4 expression in adipocytes by atorvastatin, both during and after differentiation, was reversed by GGPP. Inhibition of isoprenoid biosynthesis is associated with the reduced expression of SLC2A4 . C/EBPα directly binds to the Slc2a4 promoter to induce expression of the gene , maintaining insulin-stimulated SLC2A4 translocation . Thus, atorvastatin may reduce SLC2A4 expression primarily via inhibition of the synthesis of isoprenoid cholesterol precursors and downregulating C/EBPα production.
The effective concentrations of atorvastatin observed in this study are consistent with those reported in the plasma of patients treated with this drug . Simvastatin also suppressed adipocyte differentiation, but only at 1,000 ng/ml, which is 2–4 logarithmic orders higher than its plasma concentration in patients receiving this drug [29, 30]. Pravastatin had little effect on adipocyte differentiation. Therefore, the suppression of adipocyte differentiation observed in this study may not occur in patients treated with simvastatin or pravastatin. The lack of effect of pravastatin may be due to its hydrophilicity. The reason why atorvastatin is more potent than simvastatin in inhibiting the differentiation of adipocytes and SLC2A4 expression in these cells remains to be clarified. However, the simplest explanation is that adipocytes are more permeable to atorvastatin, giving this agent easier access to HMG-CoA reductase . We previously reported that simvastatin inhibited insulin secretion by interacting with rat islet beta cells in vitro . In the present study, atorvastatin had little influence on insulin secretion in vivo in NSY mice. These results suggest that beta cells have a higher affinity for simvastatin than for atorvastatin, although it should be noted that there were differences in experimental conditions between the two studies. Thus, the effects of statins could be produced through relatively selective interaction between the specific statin and the specific target tissue/cell.
In type 2 diabetic NSY mice, oral administration of atorvastatin impaired glucose tolerance, primarily as a result of induction of insulin resistance. Furthermore, atorvastatin reduced SLC2A4 levels in the adipocytes of NSY mice. Our finding is in accordance with the report that the adipose tissue-specific decrease in SLC2A4 expression causes insulin resistance not only in adipocytes but also in muscle and liver . Thus, the downregulation of SLC2A4 in adipocytes can lead to insulin resistance of the whole body and accelerate type 2 diabetes , which may occur in the atorvastatin-administered NSY mice.
In this study, treatment with atorvastatin was followed by an increase in HbA1c, but not fasting plasma glucose levels, in Japanese patients with type 2 diabetes, and this effect was more prominent in non-obese patients. These results suggest that atorvastatin treatment may impair postprandial glucose levels; however, these results are limited by the fact that there was no control group. This finding is consistent with a recent study on patients with hypercholesterolaemia, in which the HbA1c level was significantly increased in the atorvastatin-treated, but not the pravastatin-treated, group . Japanese non-obese type 2 diabetic patients are often characterised by a more prominent impairment in insulin secretion than insulin action . Therefore, we suggest a scenario in which atorvastatin inhibits adipocyte functions, thereby inducing and/or enhancing insulin resistance, and impairs post-prandial glucose tolerance; these effects could influence glycaemic control in diabetes, especially in non-obese patients, who are characteristically associated with impaired insulin secretion. In the obese subgroup of type 2 diabetic patients, the influence of atorvastatin on glycaemic control was less pronounced. It is widely accepted that elevation of the triglyceride-rich lipoproteins associated with obesity accelerates glucose intolerance, a process known as lipotoxicity . Several clinical studies have reported that the statins used in the current study all effectively lower plasma levels of both total and LDL cholesterol, and raise plasma HDL cholesterol levels, while atorvastatin is distinctly stronger in lowering the plasma triglyceride concentration . In obese type 2 diabetic patients with hypertriglyceridaemia, a favourably greater ability of atorvastatin to lower triglycerides could result in an effective correction of lipotoxicity, and could therefore help maintain glycaemic control. The anti-inflammatory action of statins could also act against diabetes . These beneficial effects of atorvastatin could counteract possible negative side effects associated with this drug.
Taken together, atorvastatin appears to influence multiple processes involved in glucose metabolism; these changes have both beneficial and disadvantageous effects. Their balance and net outcome may be determined by the metabolic conditions of individuals and, in turn, influence whether, overall, this drug corrects or worsens insulin resistance and, ultimately, type 2 diabetes. Since a large number of diabetic patients with diverse metabolic conditions are treated with statins over a long period, and adipocytes play a central role in regulating glucose and lipid metabolism, atorvastatin’s inhibitory effects on adipocytes and their possible impact on the glycaemic control should be kept in mind.
We thank T. Miyoshi and S. Usui for expert technical assistance. This study was supported by grants from a Grant-in-Aid for Scientific Research (to M. Natkata, T. Yada, S. Ishibashi) and a Grant-in-Aid for Scientific Research on Priority Areas (15081101) (to T. Yada) from the Japan Society for the Promotion of Science, a grant from the 21st Century Center of Excellence program (to T. Yada, S. Ishibashi), and an Insulin Research Award from Novo Nordisk Pharma (to T. Yada).
Duality of interest
This work was supported in part by a research grant from Sankyo.
- 5.Pyorala K, Pedersen TR, Kjekshus J, Faergeman O, Olsson AG, Thorgeirsson G (1997) Cholesterol lowering with simvastatin improves prognosis of diabetic patients with coronary heart disease: a subgroup analysis of the Scandinavian Simvastatin Survival Study (4S). Diabetes Care 20:614–620PubMedCrossRefGoogle Scholar
- 6.Goldberg RB, Mellies MJ, Sacks FM, et al (1998) Cardiovascular events and their reduction with pravastatin in diabetic and glucose-intolerant myocardial infarction survivors with average cholesterol levels: subgroup analyses in the Cholesterol and Recurrent Events (CARE) trial: the Care Investigators. Circulation 98:2513–2519PubMedGoogle Scholar
- 9.Sever PS, Dahlof B, Poulter NR et al (2003) Prevention of coronary and stroke events with atorvastatin in hypertensive patients who have average or lower-than-average cholesterol concentrations, in the Anglo-Scandinavian Cardiac Outcomes Trial-Lipid Lowering Arm (ASCOT-LLA): a multicentre randomised controlled trial. Lancet 361:1149–1158PubMedCrossRefGoogle Scholar
- 10.Diabetes Atorvastin Lipid Intervention (DALI) Study Group (2001) The effect of aggressive versus standard lipid lowering by atorvastatin on diabetic dyslipidemia: the DALI study: a double-blind, randomized, placebo-controlled trial in patients with type 2 diabetes and diabetic dyslipidemia. Diabetes Care 24:1335–1341CrossRefGoogle Scholar
- 12.Garvey WT, Maianu L, Huecksteadt TP, Birnbaum MJ, Molina MJ, Ciaraldi TP (1991) Pretranslational suppression of a glucose transporter protein causes insulin resistance in adipocytes from patients with non-insulin-dependent diabetes mellitus and obesity. J Clin Invest 87:1072–1081PubMedCrossRefGoogle Scholar
- 13.Stephens JM, Lee J, Pilch PF (1997) Tumor necrosis factor-α-induced insulin resistance in 3T3-L1 adipocytes is accompanied by a loss of insulin receptor substrate-1 and GLUT4 expression without a loss of insulin receptor-mediated signal transduction. J Biol Chem 272:971–976PubMedCrossRefGoogle Scholar
- 17.Kadowaki T, Hara K, Yamauchi T, Terauchi Y, Tobe K, Nagai R (2003) Molecular mechanism of insulin resistance and obesity. Exp Biol Med 228:1111–1117Google Scholar