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Departments of Metabolic Diseases (T.Y., K.E., Y.O., S.Y., T.Y., N.S., T.K.) and Cardiovascular Medicine (R.N.), University of Tokyo Graduate School of Medicine, Tokyo 113-8655, Japan; CREST of Japan Science and Technology Corp. (K.E., Y.O., T.Y., M.N., T.K.), Saitama 332-0012, Japan; National Institute of Health and Nutrition (M.N., T.K.), Tokyo 162-8636, Japan; and Institute for Diabetes Care and Research, Asahi Life Foundation (M.N.), Tokyo 100-0005, Japan
Address all correspondence and requests for reprints to: Dr. Takashi Kadowaki, Department of Metabolic Diseases, University of Tokyo Graduate School of Medicine, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. E-mail: kadowaki-3im{at}h.u-tokyo.ac.jp.
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Introduction |
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Sterol regulatory element-binding proteins (SREBPs) are a family of three transcription factors that bind to sterol regulatory elements (SREs) on the promoter regions of genes involved in the lipid biosynthesis pathway and activate their transcription (20). SREBPs have in common the N-terminal basic helix-loop-helix leucine zipper domains and two transmembrane domains that direct them to the endoplasmic reticulum. SREBPs are converted to transcriptionally active truncated forms after proteolytic release of the N-terminal domains into the cytosol, which allows them to further enter the nucleus and activate gene transcription. There are three types of SREBP proteins: SREBP-1a, SREBP-1c, and SREBP-2. SREBP-1a and -1c, which are produced from a single gene through alternative use of promoters and first exons, preferentially activate genes involved in the biosynthesis of FFA and TG (21, 22, 23), whereas SREBP-2 is more potent at stimulating the biosynthesis of cholesterol than at stimulating that of FFA (24, 25). Transgenic mice overexpressing SREBP-1a and -1c in the liver indeed showed enlargement of the liver and increased TG content of the tissue, although SREBP-1a transgenic mice showed a stronger phenotype than SREBP-1c transgenic mice, because of the N-terminal acidic region specific to SREBP-1a conferring it with a stronger transactivating capability (26). Recently, an increased expression level of SREBP-1c was reported in the islets of ZDF rats (fa/fa), with ectopic overaccumulation of fat (27), suggesting that SREBP-1c may be a key molecule in inducing TG accumulation and ß-cell dysfunction.
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Materials and Methods |
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-32P]deoxy-CTP (3000 mCi/mmol) were purchased from PerkinElmer (Boston, MA). ExpressHyb hybridization solution and the adeno-X expression system were purchased from Clontech Laboratories (Palo Alto, CA). The bicinchoninic acid (BCA) protein assay kit was purchased from Pierce Chemical Co. (Rockford, IL), Carbosorbe E was purchased from Packard (Meriden, CT), and the Oligofectamine reagent was purchased from Invitrogen (Carlsbad, CA). The Picagene luciferase assay kit was purchased from Tokyo, Inc. (Tokyo, Japan). The Effectene transfection reagent and RNeasy were purchased from Qiagen (Tokyo, Japan). The L-type TG kit and triolein were purchased from Wako Biochemicals (Osaka, Japan). The TaqMan probe and primer sets for rat ATP citrate-lyase (ACL; NM_016987), ACC (NM_022193), acyl-CoA synthase (ACS; NM_130739), fatty acid synthase (FAS; NM_17332), UCP-2 (NM_019353), and 36B4 (NM_022402) were designed by and purchased from Applied Biosystems Japan (Tokyo, Japan). The RT system was purchased from Promega Corp. (Madison, WI). HEK293 cells were purchased from the Japan Health Science Foundation (Osaka, Japan).
Cell culture
INS-1 cells were cultured in RPMI 1640 medium (containing 11.1 mM glucose) supplemented with 10% heat-inactivated fetal calf serum, 10 mM HEPES, 2 mM L-glutamine, 1 mM sodium pyruvate, 50 µM ß- mercaptoethanol, 100 IU/ml penicillin, and 100 µg/ml streptomycin at 37 C in a humidified atmosphere (5% CO2 and 95% air). HEK293 cells were cultured in DMEM supplemented with 10% heat-inactivated fetal calf serum, 100 IU/ml penicillin, and 100 µg/ml streptomycin at 37 C in a humidified atmosphere (5% CO2 and 95% air).
Preparation of recombinant adenovirus
Recombinant adenovirus was generated using the adeno-X expression system according to the manufacturer’s instructions. Briefly, a constitutively active form of rat SREBP-1c cDNA encoding amino acids 1–403 was subcloned in the pShuttle plasmid vector and transferred directionally by restriction digestion with PI-SceI and I-CeuI into adeno-X viral DNA. Ad-SREBP-1c DNA was transfected into HEK293 cells using the Effectene transfection reagent, and recombinant viral particles were obtained from the cell lysates. Ad-LacZ that expressed Escherichia coli ß-galactosidase (LacZ) was prepared in the same way, to be used as the control adenovirus.
Immunoblot analysis
INS-1 cells were lysed in a lysis buffer [25 mM Tris-HCl (pH 7.4), 2 mM sodium orthovanadate, 10 mM sodium pyrophosphate, 10 mM sodium fluoride, 1 mM EDTA, 1 mM EGTA, 0.1 mM phenylmethylsulfonylfluoride, 1 µg/ml aprotinin, 1 µg/ml pepstatin A, 1 µg/ml leupeptin, and 1% Triton X-100], and the protein content was measured with a BCA protein assay kit (Pierce Chemical Co.). For the Western blot analysis, the samples were separated on sodium dodecyl sulfate-polyacrylamide gels, transferred to polyvinylidene fluoride membranes, and probed with the primary and secondary antibodies. Antibody against SREBP-1 (1:2000 dilution), UCP-2 (1:2000 dilution), phospho-AMPK (1:2000 dilution), phospho-ACC (1:1000 dilution), AMPK (1 µg/ml), or ACC (1 µg/ml) was used as the primary antibody. The secondary antibody against antirabbit IgG antibody conjugated to horseradish peroxidase was used at a 1:5000 dilution. Signal detections were performed using the enhanced chemiluminescence kit.
Luciferase reporter assay
Five copies of the SRE sequences (TCACCCACT) connected to the luciferase reporter gene were transiently introduced into INS-1 cells using the Effectene transfection reagent. The cells were harvested 48 h after transfection. Luciferase activity was measured using Picagene with a Lumat LB9507 luminometer (Berthold, Bad Wildbad, Germany).
Insulin secretion and content
After the indicated culture periods, for the insulin secretion assays, INS-1 cells were washed with Krebs-Ringer bicarbonate (KRB) buffer composed of 129 mM NaCl, 4.8 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 2.5 mM CaCl2, 5 mM NaHCO3, 0.2% BSA (fatty acid free), and 10 mM HEPES (pH 7.4), preincubated at 37 C for 30 min in KRB buffer containing 1 mM glucose, and incubated at 37 C for 60 min with the indicated concentrations of glucose. Insulin secreted into KRB buffer was measured by RIA using rat insulin as the standard. Cellular insulin was extracted with acid-ethanol (0.18 M HCl in 95% ethanol) overnight at 4 C, and its concentration was determined by RIA after appropriate dilutions.
Assay of adenine nucleotides
Assay of adenine nucleotides in INS-1 cells was performed as described previously (28). After preincubation at 37 C for 30 min in KRB buffer containing 1 mM glucose, INS-1 cells were incubated at 37 C for 30 min in KRB buffer containing 10 mM glucose. After aspiration of medium, the metabolism was snap-stopped by the addition of ice-cold 5% trichloroacetic acid. Then the samples were sonicated three times for 10 sec each time, and trichloroacetic acid was removed by extraction three times with diethyl ether. The samples were diluted with buffer A (20 mM HEPES and 3 mM MgCl2, pH 7.75), and the ATP content was measured using the luciferase-luciferin ATP kit. For determining the ADP content, the sum of the ATP content and the ADP content was determined. The conversion from ADP to ATP was performed in buffer A containing 2.3 U/ml pyruvate kinase and 1.5 mM phosphoenolpyruvate at room temperature for 15 min.
RNA preparation, Northern blot analyses, and quantitative real-time PCR
Total RNA was extracted from INS-1 cells with TRIzol (Life Technologies, Gaithersburg, MD). Northern blot analyses were performed using a standard protocol. Briefly, total RNA was separated on denaturing formaldehyde agarose gel (1%) and transferred to Hybond N+ nylon membranes. cDNA probes of mouse ACL, acetyl-coenzyme A (acetyl-CoA) carboxylase (ACC), FAS, ACS, UCP-2, and 36B4 were labeled by a random priming method using the Megaprime DNA labeling system and [-32P]deoxy-CTP (PerkinElmer). After overnight hybridization, the membranes were washed stringently, and signals were detected and quantified with the BAS 2000 system (Fuji Film, Tokyo, Japan). For quantitative real-time PCR, total RNA was extracted from the INS-1 cells with RNeasy (Qiagen), and first-strand cDNAs were synthesized with an RT system (Promega Corp.). The samples were subjected to quantitative real-time PCR using then TaqMan probe and primer sets for rat ACL, ACC, FAS, ACS, and UCP-2 (forward primer, 5'-TCCTGAAA GCCAACCTCATGA-3'; reverse primer, 5'-CAATGACGGTGGTGCAGAAG-3'; TaqMan probe, 5'-6-carboxy fluorescein-AGACGACCTCCCTTGCCACTTCACTTC-tetramethylrhodamine-3') and 36B4 (forward primer, 5'-TGCCTCACTCCATCATCAATG-3'; reverse primer, 5'-GCAGCCGCAAATGCA GAT-3'; TaqMan probe, 5'-6-carboxy fluorescein-CTTCCCACTGGCTGAAAAGGTCAAGGC-tetramethylrhodamine-3'). The ABI PRISM 7000 system was used for the reaction and detection (Applied Biosystems Japan).
TG content
The TG contents were determined as described previously (29). Briefly, cellular TG was extracted three times with chloroform and methanol (2:1). The extraction solvent was evaporated, and TG resuspended in isopropanol was measured with the L-type TG kit using triolein as the standard.
Fatty acid oxidation
To measure the fatty acid oxidation level, INS-1 cells were washed with PBS and preincubated at 37 C for 30 min in KRB buffer containing 1 mM carnitine in the presence of 1 mM glucose. The cells were then incubated for 2 h at 37 C in KRB buffer containing 1 mM carnitine in the presence of 1 mM or 10 mM glucose with 1.0 µCi of [1-14C]palmitic acid (50 mCi/mmol). At the end of the incubation period, an equal volume of 5 N HCl was added. The liberated CO2 was trapped with Carbosorb E (Packard). After overnight incubation at room temperature, the radioactivity of the trapped 14CO2 was measured with a liquid scintillation counter.
Introduction of small interfering RNA (siRNA) for UCP-2
The siRNA specific for rat UCP-2 (5'-CCUCAUGACAGACGACCUCdTdT-3' and 5'-GAGGUCGUCUGUCAUGAGGdTdT-3') was synthesized (Dharmacon Research, Lafayette, CO). The siRNA was targeted 624 nucleotides downstream of the start codon. The INS-1 cells were transfected with these siRNAs using the Oligofectamine reagent. The transfection efficiency was checked using, fluorescein-labeled, double-strand RNA (Dharmacon Research) and was determined to be 70–80%. The cells transfected with siRNA were assayed 48 h after the transfection.
Statistical analysis
All results were expressed as the mean ± SEM. For multiple comparisons, one-way ANOVA, followed by Tukey’s honestly significant difference test was used. For unpaired comparisons of two groups, a t test was used. P < 0.05 denoted significance.
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Results |
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). To confirm the transcriptional activity of the overexpressed SREBP-1c, a luciferase reporter assay was performed. Infection with Ad-SREBP-1c increased luciferase activity under the transcriptional control of five copies of SREs (Fig. 1B). Transcriptional activity was increased more than 30-fold at 10 MOI compared with that in controls infected with Ad-LacZ. The amount of cellular protein at 10 MOI after the 48 h of culture was checked. The cellular protein contents in the cells infected with the adenovirus were not different from those in control cells not infected with the adenovirus (Fig. 1C).
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). Quantification of the mRNA levels of these genes was performed by quantitative real-time PCR. The mRNA level for ACL, which converts citrate to acetyl-CoA and oxaloacetate, was increased by 3.8-fold (P < 0.05; Fig. 2B). The expression of ACC, which produces malonyl-CoA from acetyl-CoA, was increased by 1.9-fold (P < 0.05; Fig. 2C). Induction of FAS mRNA by SREBP-1c was the strongest among the lipogenic genes examined, and it was increased by 16.4-fold (P < 0.01) compared with the control value (Fig. 2D). The transcripts for ACS were also increased by 7.5-fold (P < 0.01; Fig. 2E). These mRNA levels were normalized to the 36B4 mRNA level.
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). Overexpression of LacZ did not affect the intracellular TG content at any MOI. We next studied insulin secretion from INS-1 cells overexpressing SREBP-1c. Insulin secretion in response to 10 mM glucose was decreased by 13% at 1 MOI compared with that in controls, and the secretion was progressively attenuated with increasing MOI. At 10 MOI, insulin secretion was decreased by 42% compared with that in controls (Fig. 3B). In contrast, basal insulin secretion in response to 1 mM glucose was increased by 64% at 10 MOI (Fig. 3C). Insulin content was not affected by adenovirus infection (Fig. 3D).
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). Quantification of the mRNA level of UCP-2 was performed by quantitative real-time PCR. The UCP-2 mRNA level was increased by 2.4-fold (P < 0.05) in INS-1 cells overexpressing SREBP-1c compared with that in control cells (Fig. 4B). The UCP-2 protein level was also increased by 38% (P < 0.01; Fig. 4D). The up-regulation of UCP-2 in SREBP-1c-overexpressing cells was predicted to be coupled with a decrease in the intracellular ATP/ADP ratio. To determine the intracellular ATP/ADP ratio, INS-1 cells were preincubated at 37 C for 30 min in KRB buffer containing 1 mM glucose and incubated at 37 C for 30 min in KRB buffer containing 10 mM glucose. The ATP/ADP ratio was decreased by 45% in SREBP-1c-overexpressing cells compared with that in control cells overexpressing LacZ (Fig. 4C).
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). The mRNA level of UCP-2 was quantified by quantitative real-time PCR. Overexpression of Ad-SREBP-1c was associated with a 2.1-fold increase in the UCP-2 mRNA expression level (P < 0.01 compared with that in controls infected with Ad-LacZ; Fig. 5B). The enhanced UCP-2 gene expression in association with SREBP-1c overexpression was suppressed 40% by siRNA treatment (P < 0.05 compared with that in the control cells overexpressing SREBP-1c treated with control siRNA). Under these conditions, the decreased intracellular ATP/ADP ratio observed in association with SREBP-1c overexpression was increased by 1.8-fold by siRNA treatment (P < 0.05 compared with that in control cells overexpressing SREBP-1c treated with control siRNA; Fig. 5C). Overexpression of SREBP-1c was associated with an approximately 53% decrease in 10 mM-glucose-stimulated insulin secretion (P < 0.01 compared with that in controls infected with Ad-LacZ; Fig. 6A). The impaired glucose-stimulated insulin secretion was increased 1.7-fold by siRNA treatment (P < 0.05 compared with that in control cells overexpressing SREBP-1c treated with control siRNA; Fig. 6A). Although overexpression of SREBP-1c increased insulin secretion in response to 1 mM glucose, siRNA treatment did not affect insulin secretion in response to 1 mM glucose (Fig. 6B). Under these conditions, accumulation of intracellular TG failed to change with siRNA treatment (Fig. 6C).
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). For insulin secretion, AICAR did not have any effect on insulin secretion in cells overexpressing LacZ. In contrast, impaired glucose-stimulated insulin secretion in cells overexpressing SREBP-1c was increased 1.5-fold by exposure to AICAR (Fig. 7B). The decreased TG content associated with AICAR treatment was considered to be due in part to increased ß-oxidation of FFA. Thus, oxidation of [1-14C]palmitic acid in cells overexpressing SREBP-1c was increased 1.7-fold in response to 1 mM glucose (Fig. 7C) and 2.6-fold fold in response to 10 mM glucose (Fig. 7D) by AICAR treatment. Enhancement of FFA oxidation by AICAR was also observed in the cells overexpressing LacZ, but it did not contribute to the reduction of TG content.
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-subunits of AMPK by 1.5-fold in both SREBP-1c- and LacZ-overexpressing cells compared with that in controls not exposed to AICAR (Fig. 8, A and C). The amount of AMPK protein was not changed by infection with Ad-SREBP-1c or addition of AICAR (Fig. 8, A and B). AICAR also increased the phosphorylation level of ACC in SREBP-1c- and LacZ-overexpressing cells by 1.7- and 1.6-fold, respectively, compared with that in controls not exposed to AICAR (Fig. 8, A and E). Although the amount of total ACC protein seemed to be slightly increased by infection with Ad-SREBP-1c, this was not significant and did not change with the addition of AICAR (Fig. 8, A and D).
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) and failed to affect the cellular ATP/ADP ratio in either cells overexpressing LacZ or those overexpressing SREBP-1c (Fig. 9B).
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Discussion |
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In this study we found that UCP-2 gene expression was enhanced in our lipotoxicity model. UCP-2 is ubiquitously expressed in mammalian tissues, including in the pancreatic islets (37), and has been hypothesized to be involved in dissipation of the mitochondrial proton gradient (38). It has been reported that the UCP-2 gene has the SRE sequence on its promoter region, suggesting that its expression may be regulated by direct interaction of SREBP-1c with the cis-acting element (31, 39, 40). Recently, adenovirus-mediated overexpression of UCP-2 in isolated islets or INS-1 cells was reported to suppress glucose-stimulated insulin secretion (41, 42, 43). Furthermore, glucose-stimulated insulin secretion was reported to be elevated in UCP-2-deficient mice (30, 44). These complementary results indicate that UCP-2 may be a negative regulator of ß-cell insulin secretion in response to the fuel secretagogue. However, contrary to these findings, Wang et al. (45) reported that adenovirus-mediated UCP-2 overexpression improved glucose-stimulated insulin secretion in islets from ZDF rats. In this case, as the overexpression of UCP-2 increased fatty acid oxidation (43, 45), ß-cell lipotoxicity in ZDF rats might be ameliorated by unloading lipid from ß-cells despite the decreased cellular ATP/ADP ratio in response to glucose. In our lipotoxicity model overexpressing SREBP-1c, up-regulation of UCP-2 was evident, and the cellular ATP/ADP ratio was significantly decreased. It has been known that the cellular ATP/ADP ratio is important in the regulation of insulin secretion due to its influence in inhibition of the KATP channel (46). To examine the causal relationship between UCP-2 up-regulation and decreased ATP/ADP ratio, we used a novel knockdown technology of target gene expression using a small, double-strand RNA (21–23 mer long) (47, 48, 49), called siRNA. Treatment with siRNA specific for UCP-2 of cells compromised by lipotoxicity suppressed the enhancement of UCP-2 expression, and concurrently the ATP/ADP ratio recovered without any change in cellular TG accumulation. As a result, the impaired glucose-stimulated insulin secretion recovered partially. The partial suppression of UCP-2 gene expression led to considerable recovery of the ATP/ADP ratio and glucose-stimulated insulin secretion. Thus, UCP-2 did play a key causal role in the impaired insulin secretory function in this model. At present, it remains to be ascertained whether the impaired glucose-stimulated insulin secretion can be fully corrected if UCP-2 expression is completely suppressed. The relationship between up-regulation of UCP-2 expression and lipotoxicity is still not clear. Nevertheless, up-regulation of UCP-2 has also been reported in the islets of obese animals (37) and in ß-cells treated with fatty acids (39, 40). Therefore, such up-regulation of UCP-2 expression may have an important role in lipotoxicity.
The siRNA treatment was unable to reduce the lipid content. To unload the excess lipid, AICAR, a well characterized agonist of AMPK was used. AMPK belongs to a family of serine-threonine kinases (50). AMPK is allosterically activated by high concentrations of AMP (51, 52). It has been reported that activation of AMPK causes FFA oxidation in primary hepatocytes (53) and skeletal muscle (34, 53, 54) and decreases lipogenesis in adipocytes (55). AICAR is an AMP analog that mimics the effect of AMP on AMPK, causing activation of AMPK (52, 56, 57, 58). AICAR has been reported to inhibit lipogenesis and increase fatty acid oxidation via AMPK activation in hepatocytes and muscle (33, 34, 53). In our ß-cell lipotoxicity model overexpressing SREBP-1c, exposure to AICAR was associated with increased oxidation of FFA, decreased TG content, and improved impaired insulin secretion in response to glucose. We observed that AICAR phosphorylated AMPK and ACC; on the other hand, it did not affect UCP-2 gene expression or the ATP/ADP ratio. These data suggest that disposal of the accumulated lipid by AICAR could release ß-cells from lipotoxicity. Although Li et al. (59) reported that the increased fatty acid oxidation by AICAR was associated with increased UCP-2 expression, UCP-2 expression by AICAR might be due to the influence of experimental conditions. Li et al. (59) also reported decreased fatty acid oxidation in response to high concentrations of glucose (27 mM). Under these conditions, AICAR failed to increase fatty acid oxidation. We also demonstrated that 10 mM glucose decreased fatty acid oxidation to a greater extent than 1 mM glucose. We cultured ß-cells in the presence of 11.1 mM glucose before the effect of AICAR was examined. Thus, experimental conditions under which fatty acid oxidation is attenuated may affect the influence of AICAR on UCP-2 expression. The relationship between TG content and UCP2 expression is not fully understood, although these are important factors in lipotoxicity, and additional studies are needed to clarify this relationship.
The precise effects of AICAR on insulin secretion remain controversial. Akkan and Malaisse (60) first described the effect of AICAR on insulin secretion. They reported that AICAR increased glucose-stimulated insulin secretion in isolated islets and perfused pancreases from rats. On the other hand, da Silva Xavier et al. (61) reported that AICAR inhibited glucose-stimulated insulin secretion. In yet another study, Zhan and Kim (62) reported no effect of AICAR on glucose-stimulated insulin secretion in a pancreatic ß-cell line. Interestingly, Salt et al. (63) reported that AICAR had both inhibitory and stimulatory effects on glucose-stimulated insulin secretion. Thus, acute exposure (10 min) to AICAR increased glucose-stimulated insulin secretion, but prolonged exposure inhibited glucose-stimulated insulin secretion in INS-1 cells. They also reported that AICAR enhanced insulin secretion in response to a low glucose concentration, but inhibited it in response to a high glucose concentration in rat islets. These reports suggest that the effects of AICAR on insulin secretion may be easily influenced by experimental conditions, such as the duration of incubation and the ambient glucose concentration.
Recently, Eto et al. (64) reported that a constitutively active form of the AMPK-1 subunit decreased glucose-stimulated insulin secretion despite reducing the TG content. These results seem to be incompatible with the results described here. However, there is a very important difference between the two experimental conditions, that is, in the extent of the accumulated intracellular lipid. It is known that lipid metabolism is important in the regulation of insulin secretion (11, 65). Depletion of lipid has been reported to be associated with decreased glucose-stimulated insulin secretion (66, 67). Under normal culture conditions, overexpression of AMPK seemed to cause depletion of the intracellular lipid and decreased glucose-stimulated insulin secretion (64). However, our lipotoxicity model overexpressing SREBP-1c showed accumulation of intracellular lipid. Under these conditions, activation of AMPK by AICAR normalized the accumulated lipid and improved glucose-stimulated insulin secretion, suggesting that there is a critical range of lipid accumulation for ß-cells to maintain their normal secretory function.
In summary, we have reported that ß-cell-specific overexpression of a constitutively active form of SREBP-1c up-regulated the expression of lipogenic genes and increased TG accumulation and glucose-stimulated insulin secretion. These results suggest that SREBP-1c may play a crucial role in the development of ß-cell lipotoxicity. Inhibition of the downstream pathways of SREBP-1c by down-regulating UCP-2 expression or facilitating disposal of the accumulated lipid by activating AMPK could be novel targets for protecting and rescuing ß-cells from the lipotoxicity seen in cases of type 2 diabetes.
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Acknowledgments |
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Footnotes |
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Abbreviations: ACC, Acetyl-coenzyme A carboxylase; ACL, ATP citrate-lyase; ACS, acyl-CoA synthase; AICAR, aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside; AMPK, AMP-activated protein kinase; BCA, bicinchoninic acid; CoA, coenzyme A; FAS, fatty acid synthase; FFA, free fatty acid; KRB, Krebs-Ringer bicarbonate; LacZ, ß-galactosidase; MOI, multiplicity of infection; siRNA, small interfering RNA; SRE, sterol regulatory element; SREBP, sterol regulatory element-binding protein; TG, triglyceride; UCP, uncoupling protein; ZDF, Zucker diabetic fatty.
Received November 25, 2003.
Accepted for publication March 25, 2004.
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References |
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) or Ad-SREBP-1c (
) at 1, 3, or 10 MOI were transiently transfected with a plasmid containing the luciferase reporter gene connected downstream of five copies of SRE sequences. After 24-h culture, the cells were lysed, and luciferase activities were measured. Data are shown as relative luciferase activities to those of control cells infected with Ad-LacZ. Values are the mean ± SEM of three experiments. **, P < 0.01. C, INS-1 cells were infected with or without adenovirus at MOI 10 and cultured for 48 h. The cells were lysed, and the protein contents were measured with a BCA protein assay kit (Pierce Chemical Co.). Values are the mean ± SEM of five experiments.
