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

Abstract

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.

FIG. 1.

Effects of etomoxir on CPT-1 activity. The 2- to 3-month-old male mice were given intraperitoneal injections of etomoxir (50 mg/kg body wt). A: After 18 h, mitochondria were isolated from hindquarter muscle (HQT), heart, and liver tissues, and CPT-1 activity was measured. B: Random fed blood glucose was measured from a tail vein blood sample. C: Wet weight of control and etomoxir-treated perigonadal white adipose tissue (WAT) and subscapular brown adipose tissue (BAT) from male mice was determined. D: Triacylgyceride was measured in HQT, heart (HRT), and liver after the Folch lipid extraction. All data are presented as mean and SE. Data were analyzed using a two-tailed Student's t test. *Significant difference between control (CON) and etomoxir-treated (ETO) (P < 0.05).

FIG. 2.

Effects of etomoxir on CAT and GLUT4 expression. The 2- to 3-month-old male transgenic mice were injected intraperitoneally with 50 mg/kg body wt of etomoxir. A: This depicts the schematic drawing of the −895-HG4-CAT construct used to construct this line of transgenic mice. B: After 18 h, total RNA was isolated from white adipose tissue (WAT), brown adipose tissue (BAT), hindquarter muscle (HQT), and heart (HRT) tissues. qrt-PCR was used to determine mRNA levels of CAT, GLUT4, and actin. CAT and GLUT4 expression was normalized to actin expression levels. Data were analyzed using a two-tailed Student's t test. *Significant difference between control (CON) and etomoxir-treated (ETO) (P < 0.05).

FIG. 3.

Effect of the loss of insulin signaling on CAT and GLUT4 transcription in cardiac tissue. A: Hearts from CIRKO-hGLUT4-CAT mice (Cre) and wild-type controls (CON) were removed, and total RNA was analyzed for GLUT4 mRNA and transgenic CAT mRNA using RNAse protection assay as described in research design and methods. B: Quantification of bands from ³²P-labeled probes to the 3′ untranslated region of CAT and GLUT4. All densitometry measurements were made with ImageJ (National Institutes of Health). The insulin receptor does not directly regulate the levels of CAT and GLUT4 mRNA.

FIG. 4.

GLUT4 LXRE is required for etomoxir-dependent downregulation of GLUT4 mRNA. A: The transgenic constructs used to construct the lines of transgenic mice are shown. B: The 2- to 3-month-old transgenic mice were injected intraperitoneally with 50 mg/kg body wt of etomoxir. After 18 h, total RNA was isolated from white adipose tissue (WAT) and brown adipose tissue (BAT) from each control and etomoxir-treated mice. qrt-PCR was used to determine mRNA levels of CAT, GLUT4, and actin. CAT and GLUT4 expression was normalized to actin expression levels. The ratio of CAT mRNA to GLUT4 mRNA was calculated for each sample. Data were analyzed using a two-tailed Student's t test. *Significant difference between control (CON) and etomoxir-treated (ETO) (P < 0.05).

FIG. 5.

Effects of etomoxir on GLUT4 mRNA expression are cell autonomous. 3T3–L1 adipocytes (days 6–8 post-differentiation) were treated with 25 nmol/l etomoxir. A: After 18 h, total RNA was isolated and qrt-PCR was used to determine mRNA levels of GLUT4 and actin. B: Cells were treated without or with 25 nmol/l etomoxir (ETO) as described above. During the final 4 h of incubation, 1 mmol/l palmitoyl-l-carnitine (PLC) in HEPES-buffered saline, pH 7.4, was added to etomoxir-treated cells. Total RNA was isolated and qrt-PCR was used to determine mRNA levels of GLUT4 and actin. Data were analyzed using a two-tailed Student's t test. *Significantly different from control (CON) (P < 0.05).

FIG. 6.

Role of the GLUT4 promoter LXRE in GLUT4 gene transcription. A: 3T3–L1 adipocytes (day 5 post-differentiation) were transfected with GLUT4 reporter/luciferase (firefly luciferase) reporter plasmids, GLUT4 enhancer factor (GEF) and MEF2 (+ indicates inclusion in transfection), and pRLTKluc (Renilla luciferase, for transfection efficiency). Immediately after transfection, the cells were treated without or with 25 nmol/l etomoxir (ETO). After 24 h, cells were harvested and luciferase assays performed in cell lysates. Data were analyzed using a Student's t test. Statistical significance between groups is indicated by lines. B: Chromatin immunoprecipitation using nuclear extracts from adipocytes (8 days post-differentiation) was performed using an anti–LXR-α antibody and nonimmune IgG. Quantification of this representative chromatin immunoprecipitation is presented in Fig. 7C. CON, control.

FIG. 7.

Treatment with LXR agonist recovers GLUT4 mRNA expression in etomoxir-treated cells. A: 3T3–L1 adipocytes (day 6–8 post-differentiation) were treated with 25 nmol/l etomoxir without or with 0.1 μmol/l TO901317. After 18 h, total RNA was isolated for quantification of GLUT4 and actin mRNA. B: Chromatin immunoprecipitation (ChIP) using nuclear extracts from adipocytes (8 days post-differentiation) was performed using an anti–LXR-α antibody and nonimmune IgG. Cells were treated without and with 25 nmol/l etomoxir (ETO) and/or 0.1 μmol/l TO901317 as described in A. C: Quantification of three independent ChIP experiments using qrt-PCR. IgG background is subtracted from the LXR antibody ChIPs. Data were analyzed using two-tailed Student's t test. Bars with different lowercase letters are significantly different from one another (P < 0.05). CON, control.