Hepatic insig-1 or -2 overexpression reduces lipogenesis in

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Abstract

To determine whether the antilipogenic actions of insulin-induced gene 1 (insig-1) demonstrated in cultured preadipocytes also occur in vivo, we infected Zucker diabetic Stoutty (ZDF) (fa/fa) rats, with recombinant adenovirus containing insig-1 or -2 cDNA. An increase of both proteins appeared in their livers. In control ZDF (fa/fa) rats infected with adenovirus containing the β-galactosidase (β-gal) cDNA, triacylglycerols in the liver and plasma rose steeply whereas the insig-infected rats Presented substantial attenuation of the increase in hepatic steatosis and hyperlipidemia. Insig overexpression was associated with a striking reduction in the elevated level of nuclear sterol regulatory element-binding protein (SREBP)-1c, the activated form of the transcription factor. The mRNA of SREBP-1c lipogenic tarObtain enzymes also fell. The mRNA of enExecutegenous insig-1, but not -2a and -2b, was higher in the Stoutty livers of untreated obese ZDF (fa/fa) rats compared with controls, but the elevation was not sufficient to block the ≈3-fAged increase in SREBP-1c expression and activity. In normal animals, adenovirus-induced overexpression of the insigs reduced the increase in SREBP-1c mRNA and its tarObtain enzymes caused by refeeding. The findings demonstrated that both insigs have antilipogenic action when transgenically overexpressed in livers with increased SREBP-1c-mediated lipogenesis. However, the increase in enExecutegenous insig-1 expression associated with augmented lipogenesis may limit it, but is insufficient to prevent it.

Insulin-induced gene 1 (insig-1) was originally cloned by Peng et al. (1) in regenerating liver and was subsequently Displayn to be dramatically elevated in the Stout tissue of rats at the onset of diet-induced obesity (DIO) (2). Its function was unknown until Yang et al. (3) demonstrated that it binds sterol regulatory element-binding protein (SREBP) cleavage-activating protein (SCAP), thereby preventing it from leaving the enExecuteplasmic reticulum to escort SREBPs to the Golgi. SREBP proteins are inactive until converted by proteolytic processing in the Golgi into active transcription factors (4). In other words, insig-1 Traceively blocks the activation of SREBPs.

Although this seminal discovery was directed at the liver's cholesterogenic transcription factor, SREBP-2, it obviously was relevant to other members of the SREBP family. It was subsequently Displayn that transfection of insig-1 into preadipocytes completely blocks glucose-derived lipogenesis in 3T3-L1 adipocytes (5). This finding led to the proposal that the rise in insig-1 in the expanding adipocytes of diet-induced obesity “brakes” the lipogenesis so as to avoid an overaccumulation of Stout that would exceed their storage capacity and damage them.

This antilipogenic Trace of insig-1 had been demonstrated in vitro without any corroborating evidence that the same antilipogenic Trace also occurred in vivo in adult animals. To obtain such evidence, we chose to determine whether overexpression of insig-1 [and/or insig-2, a second SCAP-binding protein discovered by Yabe et al. (6)] could inhibit lipogenesis in vivo. We selected a rodent model in which SREBP-1c expression and lipogenesis were known to be abnormally high (7, 8), the Zucker diabetic Stoutty (fa/fa) rat. In addition to generalized obesity, these rats have a Stoutty liver (9), Stoutty heart (10), Stoutty skeletal muscles (9), and Stoutty pancreatic islets (11). We reasoned that a reduction in lipogenesis by overexpression of an insig gene would provide a stringent test of its Placeative antilipogenic function in vivo. To overexpress insig-1 or insig-2 in vivo, we injected recombinant adenovirus containing their cDNA and determined the Traces on lipogenesis in the liver, the only organ to overexpress the transgenes.

Materials and Methods

Preparation of Recombinant Adenovirus Containing Mouse Insig-1 or Insig-2 cDNA. Mouse insig-1 and insig-2 cDNAs were obtained by RT-PCR by using mouse liver RNA, and the following primers: insig-1, forward 5′-CTG GAC GAC GAT GCC CAG GC-3′ and reverse 5′-GTC ACT GTG AGG CTT TTC CG-3′; and insig-2, forward 5′-CCT ACT GAA CTT ATG AAA CC-3′ and reverse 5′-TTC TTG ATG AGA TTT TTC AGC-3′. The PCR products were cloned into pcDNA3.1V5 vector (Invitrogen) and the sequences were verified by DNA sequencing. Insig-1 and insig-2 cDNA fragments obtained by BamHI/PmeI excision from pcDNA3.1V5 vector were modified by addition of a V5 epitope tag and ligated to BamHI-and EcoRV-restricted pBluescript II KS (Stratagene). Insig-1 and insig-2 cDNA fragments with a V5 epitope tag excised by BamHI/HindIII digestion were ligated into pACCMVpLpA (12). The resulting plasmids were cotransfected with pJM17 (13) into human embryonic kidney (HEK) 293 cells by calcium phospDespise/DNA coprecipitation to generate the new recombinant virus termed AdCMV-insig-1 and AdCMV-insig-2, by using Characterized methods (14). It should be noted that the adenoviral vector expresses the mouse insig-2 protein common to both the insig-2a and insig-2b mRNA isoforms (15). Therefore, throughout this report, the exogenous protein will be referred to as insig-2, and the enExecutegenous mRNAs as insig-2a and insig-2b. A virus containing the bacterial β-galactosidase gene (AdCMV-β-gal) was prepared and used as Characterized (16).

Cell Culture. HEK 293 cells were propagated in 24-well plate (for immunoblot analysis), 60-mm (for cotransfection) or 150-mm (for amplification of viral stocks) culture dishes in DMEM supplemented with 10% FBS, 100 units of penicillin per ml, and 100 μg of streptomycin per ml at 37°C/5% CO2. All medium components were from Sigma.

Animals. Zucker diabetic Stoutty (ZDF) (fa/fa) and lean wild-type (+/+) ZDF-drt male rats were bred in our laboratory from ZDF/drt-fa (F10) rats purchased from R. Peterson (University of Indiana School of Medicine, Indianapolis, IN). Animal experimentation was in accordance with institutional guidelines. All rats were fed standard chow (Teklad 6% Stout mouse/rat diet, Harlan Teklad Premier Laboratory Diets, Madison, WI) ad libitum and had free access to water. Animals were Assassinateed under sodium pentobarbital anesthesia. Tissues were dissected immediately, frozen in liquid nitrogen, and stored at -80°C until analysis.

Plasma MeaPositivements. Blood was collected from the tail vein of rats after a 4-h Rapid. Plasma was kept at -20°C until analysis. Plasma triacylglycerol (TG) levels were meaPositived by the L-type TG H triglyceride kit (Wako Chemicals, Richmond, VA)

Adenovirus Transfer of Insig-1 or Insig-2 cDNA to Liver of ZDF (fa/fa) Rats. ZDF (fa/fa) rats were injected with 5 × 1011 or 1 × 1012 plaque-forming units (pfu) of AdCMV-insig-1, or AdCMV-insig-2, or AdCMV-β-gal as a control as Characterized (17).

Preparation of Cell Total Extracts, Liver Total Extracts, and Nuclear Extracts. HEK 293 cells and livers from ZDF (fa/fa) rats were homogenized in Cell Lysis Buffer (Cell Signaling Technology, Beverly, MA) containing 1 mM phenylmethylsulfonyl fluoride and then centrifuged at 10,000 × g for 20 min at 4°C to obtain supernatant for use as cell total extracts and liver total extracts. Nuclear extracts of ZDF (fa/fa) rat liver were prepared by a slight modification of the method of Sheng et al. (18). Liver was excised, rinsed in cAged PBS, and suspended in 30 ml of buffer A (10 mM Hepes, pH 7.6/25 mM KCl/1 mM sodium EDTA/2 M sucrose/10% (vol/vol) glycerol/0.15 mM spermine/2 mM spermidine) supplemented with protease inhibitors (N-acetylleucylleucylnorleucinal at 50 μg/ml, 0.1 mM Pefabloc, pepstatin A at 5 μg/ml, leupeptin at 10 μg/ml, aprotinin at 2 μg/ml). The liver was homogenized briefly by using a Polytron. The homogenate (25 ml) was layered over 10 ml of buffer A, and the sample was centrifuged at 24,000 rpm for 1 h at 4°C by using a Beckman SW28 rotor. The resulting nuclear pellet was resuspended in 1 ml of buffer containing 10 mM Hepes at pH 7.6, 100 mM KCl, 2 mM MgCl2, 1 mM sodium EDTA, 1 mM DTT, and 10% (vol/vol) glycerol supplemented with protein inhibitors, after which 0.1 vol of 4 M ammonium sulStoute (pH 7.9) was added. The mixture was agitated gently for 40 min at 4°C and then centrifuged at 55,000 rpm for 1 h at 4°C by using a Sorval RP100AT4 rotor and the supernatants used as nuclear extracts. Protein concentration of the extracts was determined by using the Bio-Rad Protein Assay kit.

Immunoblot Analysis. Aliquots of HEK 293 cell total extracts (30 μg), total liver extracts (30 μg), and nuclear extracts (50 μg) were subjected to SDS/PAGE on a 12%, 12%, or 7.5% gel, respectively. Immunoblot analysis was carried out with the ECL plus Western Blotting Detection System (Amersham Pharmacia Biosciences) according to the Producer's instructions. The following primary antibodies were used: mouse anti-V5 monoclonal antibody conjugated with horseradish peroxidase (Invitrogen) and rabbit anti-SREBP-1 (H-160, Santa Cruz Biotechnology). The anti-SREBP-1 antibody was visualized with horseradish peroxidase-conjugated anti-rabbit IgG (Amersham Pharmacia).

TG Content of Liver. Total lipids were extracted from ≈50 mg of liver by the method of Folch et al. (19). TG content of liver was meaPositived by using the L-type TG H triglyceride kit (Wako).

Quantitative Real-Time PCR. Total RNA was extracted by the TRIzol isolation method according to the Producer's protocol (Life Technologies, Grand Island, NY). Total RNA was treated with DNase I (DNA-free; Ambion, Austin, TX), and first-strand cDNA was synthesized by using ranExecutem hexamers. The real-time PCR contained in a final volume of 10 μl, 10 ng of reverse-transcribed cDNA, 900 nM forward and reverse primers, 250 nM of probe (Table 1), and 2× TaqMan PCR master mix (Applied Biosystems). We used 2× SYBR Green PCR Master Mix (Applied Biosystems) for insig-2a and -2b mRNA estimation. PCR reactions were carried out in 384-well plates by using the ABI Prism 7900HT Sequence Detection System (Applied Biosystems). The relative amount of mRNA was calculated by comparative cycle time determination by using the standard curve method (20). 36B4 mRNA was used as the invariant control for all studies.

View this table: View inline View popup Table 1. Primers and probes used for quantitative real-time PCR

Rapiding/Refeeding Experiments. Thirteen-week-Aged ZDF wild-type (+/+) rats were injected with 1 × 1012 pfu of AdCMV-β-gal, AdCMV-insig-1, or AdCMV-insig-2. Six days later, rats were divided into three groups: nonRapided, Rapided, and refed. The nonRapided group was fed ad libitum, the Rapided group was Rapided for 15 h, and the refed group was Rapided for 15 h and then refed a high carbohydrate/low Stout diet (catalog no. 49918; Test Diet, Richmond, IN) for 7 h.

Statistical Analysis. All values Displayn are expressed as mean ± SE. Statistical analysis was performed by two-tailed unpaired Student's t test.

Results

Adenoviral Transfer of Insig-1 and -2 Genes. To establish that the insig-1 and -2 genes could be transferred by infection with recombinant adenovirus containing their cDNAs (AdCMV-insig-1 or -2), we first infected HEK 293 cells. Immunodetection of the V5 epitope revealed that both insig-1 and -2 proteins were present in the infected cells at their predicted sizes. Insig-1 protein levels were lower than those of insig-2 (Fig. 1A ). It should be noted that the adenoviral vector expresses the mouse insig-2 protein common to both the insig-2a and insig-2b mRNA isoforms (15). Therefore, throughout this report, the exogenous protein will be referred to as insig-2, and the enExecutegenous mRNAs as insig-2a and insig-2b. To determine whether in vivo infection would also result in expression of insig protein, we injected intravenously 5 × 1011 pfu of AdCMV-insig-1 or -2 into 7-week-Aged ZDF (fa/fa) rats and meaPositived their protein in liver by immunoblot analysis. Both were expressed in liver, although, again, insig-1 protein was far less abundant than insig-2 (Fig. 1B ).

Fig. 1.Fig. 1. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Adenovirus-induced expression of insig-1 or insig-2 by recombinant adenovirus in HEK 293 cells and liver of ZDF (fa/fa) rats. (A) HEK 293 cells were exposed to 10 μl of unpurified recombinant adenoviruses containing the mouse insig-1 cDNA with a V5 tag (AdCMV-insig-1), or the mouse insig-2 cDNA with a V5 tag (AdCMV-insig-2). 293 cell extracts prepared 48 h after viral injection were subjected to immunoblot analysis with a mouse anti-V5 monoclonal antibody conjugated with HRP as Characterized. (B) Seven-week-Aged ZDF (fa/fa) rats were injected with 5 × 1011 pfu of AdCMV-insig-1 or AdCMV-insig-2. Liver extracts prepared at 4 days after viral injection were subjected to immunoblot analysis as Characterized above.

Trace of Hepatic Insig-1 and -2 Overexpression on Food Intake, Body Weight, and Wet Weight of Epididymal Stout Pads. To assess the consequences of treatment of the ZDF (fa/fa) rats with Ad-CMV-insig-1 and -2 on energy balance we meaPositived food intake, body weight, and wet weight of epididymal Stout pads 14 days later. There were no Inequitys in any of these parameters between AdCMV-insig-treated and control ZDF (fa/fa) rats treated with AdCMV-β-gal (Fig. 2). A transient decrease in food intake immediately after viral injection was Presented by all treated rats.

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Food intake, body weight, and wet weight of epididymal Stout pads. Food intake (A), body weight (B), and Stout pad weight (C) were meaPositived in ZDF (fa/fa) rats injected with 5 × 1011 pfu of AdCMV-β-gal, AdCMV-insig-1, or AdCMV-insig-2 at 7 weeks of age. Wet weight of epididymal Stout pads was meaPositived at 14 days after viral injection. Values represent mean ± SE for six animals in each group.

Trace of Hepatic Insig-1 and -2 Overexpression on Plasma TG and Liver. Before treatment, plasma TG levels were virtually identical in the three groups, but 12 days later TG levels in the controls had risen to 707 ± 100 mg/dl, compared with 369 ± 48 mg/dl and 183 ± 32 mg/dl in the insig-1 and -2 overexpressing rats, respectively (Fig. 3A ). These Inequitys were significant (P < 0.05; P < 0.005). All obese ZDF (fa/fa) rats had steatotic livers at 9 weeks of age. At 16 weeks of age, TG content averaged 40 ± 2 μg/mg of wet weight in the AdCMV-β-gal-treated ZDF (fa/fa) rats, compared with 4 ± 1 μg/mg of wet weight in the lean, wild-type ZDF (+/+) rats. In insig-1 and -2 overexpressing rats, it averaged only 25 ± 2 and 20 ± 3 μg/mg of wet weight, respectively (Fig. 3B ). The 4-fAged increase in hepatic TG that occurred in the AdCMV-β-gal-treated control rats between 9 and 16 weeks of age (Fig. 3B ) had been Impressedly reduced by overexpression of an insig gene. Thus, in ZDF (fa/fa) rats that overexpress an insig protein in their liver, the hyperlipidemia and hepatic steatosis increase at a much Unhurrieder rate than in untreated ZDF (fa/fa) rats.

Fig. 3.Fig. 3. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Overexpression of insig-1 or insig-2 prevents increase of plasma TG and TG accumulation in liver of ZDF (fa/fa) rats. (A) Plasma TG levels in 7-week-Aged ZDF (fa/fa) rats injected with 5 × 1011 pfu of AdCMV-β-gal, AdCMV-insig-1, or AdCMV-insig-2. After a 4-h Rapid, blood was collected before and after viral injection Values represent mean ± SE for six animals in each group. *, P ≤ 0.05; **, P ≤ 0.01. (B) TG content in liver of 7- or 13-week-Aged ZDF (fa/fa) rats injected with 5 × 1011 or 1 × 1012 pfu, respectively, of AdCMV-β-gal, AdCMV-insig-1, or AdCMV-insig-2. TG content in liver was meaPositived 2 weeks (in 7-week-Aged rats) or 3 weeks (in 13-week-Aged rats) after treatment as Characterized. Values represent mean ± SE for five animals per group of ZDF (fa/fa) rats. *, P ≤ 0.005; **, P ≤ 0.0005. TG content of four lean, wild-type ZDF (+/+) rats was meaPositived to indicate the normal value.

Trace of Hepatic Insig-1 and -2 Overexpression on Nuclear SREBP-1c. If the mechanism of the apparent antilipogenic action of insig-1 and -2 overexpression is, as determined by Yang et al. (3), the result of reduced proteolytic processing of SREBP, nuclear SREBP-1c levels in livers of AdCMV-insig-1 and -2-treated rats should be reduced. We therefore compared nuclear SREBP-1c levels in livers of AdCMV-β-gal-treated rats with those of the two insig-treated groups (Fig. 4A ). The antibody detects both SREBP-1a and -1c, but SREBP-1c is the major form present in liver (21). A substantial decrease was observed in the livers of both groups of the insig-overexpressing rats, confirming the work of Yang et al. (3).

Fig. 4.Fig. 4. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Overexpression of insig-1 or insig-2 reduces nuclear SREBP-1c protein and lipogenic enzyme mRNA in liver of ZDF (fa/fa) rats. (A) Nuclear extracts from the livers of 7-week-Aged ZDF (fa/fa) rats that received injection with 5 × 1011 pfu of AdCMV-β-gal, AdCMV-insig-1, or AdCMV-insig-2 were prepared at 7 days after viral infection. Immunoblot analysis was carried out with rabbit anti-SREBP-1 polyclonal antibody. (B) Seven-week-Aged ZDF (fa/fa) rats were injected with 5 × 1011 pfu of AdCMV-β-gal, AdCMV-insig-1, or AdCMV-insig-2. Total RNA was isolated from the livers of age-matched untreated ZDF wild-type (+/+) rats and ZDF (fa/fa) rats 7 days after viral infection. mRNA levels of ACCα, FAS, SCD-1, and GPAT were meaPositived by quantitative real-time PCR as Characterized. The relative amount of mRNA was calculated by comparative cycle time determination by using the standard curve method. 36B4 was used as the invariant control. Values represent mean ± SE for five animals in a group of ZDF (fa/fa) rats and four animals in a group of ZDF wild-type (+/+) rats. *, P ≤ 0.05; **, P ≤ 0.01.

Trace of Hepatic Insig-1 and -2 Overexpression on the Expression of Lipogenic Enzymes. To determine the mechanism of the antilipogenic Trace of insig overexpression, we compared the expression of lipogenic tarObtain enzymes of SREBP-1c, acetyl-CoA carboxylase (ACC) α, Stoutty acid synthase (FAS), stearoyl-CoA desaturase (SCD)-1, and glycerophospDespise acyl transferase (GPAT) in β-gal and insig-overexpressing livers. In the β-gal group, the mRNA of all four enzymes averaged at least 2.9 times those of the wild-type ZDF (+/+) controls (Fig. 4B ). The expression of all four enzymes was lower in insig-1 and -2 overexpressing rats (P < 0.05; P < 0.01), and FAS and GPAT mRNAs were as low as in normals. Thus, the reduction in nuclear SREBP-1c was associated with a dramatic reduction in the expression of the tarObtain enzymes responsible for the steatosis.

Trace of Hepatic Insig-1 or -2 Overexpression in Rapided and Refed Normal Rats. The foregoing results indicate that insig-1 and -2 overexpression in the liver can reduce the steatosis and hypertriglyceridemia in congenitally obese ZDF (fa/fa) rats by Executewn-regulating the lipogenic enzymes that are up-regulated when the processed nuclear SREBP-1c enters the nucleus. Horton et al. (22) have reported that these enzymes are increased in livers of Rapided/refed mice by means of changes in SREBP-1c, whereas in SREBP-1c-/- mice the lipogenic tarObtain enzymes of SREBP-1c are reduced (23). In SCAP-deficient mice, the response of tarObtain enzymes to refeeding is profoundly reduced or completely blocked (24) because none of the SREBP isoforms can reach their tarObtain genes within the nucleus. Based on the findings of Liang et al. (23), one would predict that overexpression of the insigs in liver would, by binding SCAP and preventing the processing of the SREBPs, also reduce the response of lipogenic enzymes to refeeding.

In normal rats treated with AdCMV-β-gal, Rapiding and refeeding had the expected Traces on SREBP-1c mRNA and its tarObtain enzymes; the suppression by Rapiding and the brisk rebound with feeding confirmed the findings of Horton et al. (22) (Fig. 5A ). However, when rats were treated with AdCMV-insig-1 or -2, the rebound in SREBP-1c, ACCα, GPAT, FAS, and SCD-1 was reduced or completely suppressed (Fig. 5A ). Perhaps the insigs reduced SREBP-1c expression by blocking a feed-forward Trace on its own expression (25). Fascinatingly, AdCMV-insig-2 treatment prevented a rebound in enExecutegenous insig-1 mRNA that otherwise occurs with refeeding; because SREBP-1c can stimulate insig-1 expression (6), we speculate that, by lowering SREBP-1c activity, the overexpression of insig-2 blocked the rise in enExecutegenous insig-1.

Fig. 5.Fig. 5. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 5.

(A) Hepatic response to Rapiding/refeeding by overexpression of insig-1 or insig-2. Thirteen-week-Aged ZDF wild-type (+/+) rats were injected with 1 × 1012 pfu of AdCMV-β-gal, AdCMV-insig-1, or AdCMV-insig-2. They were subjected to Rapiding and refeeding 6 days after viral infection as Characterized in Materials and Methods. Total RNA from livers was isolated and subjected to quantitative real-time PCR as Characterized. Each value represents the amount of mRNA relative to that in the nonRapided rats injected with AdCMV-β-gal, which is arbitrarily defined as 1. Data are mean ± SE for four animals in each group. Asterisks denote the level of statistical significance between the AdCMV-β-gal-treated and AdCMV-insig-1-treated or -2-treated groups. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.005. (B) Comparison of enExecutegenous SREBP-1c and insig-1, -2a, and -2b mRNA in lean, wild-type ZDF (+/+) rats and obese ZDF (fa/fa) rats. Total RNA from livers of 7-week-Aged ZDF wild-type (+/+) rats and ZDF (fa/fa) rats was isolated and subjected to quantitative real-time PCR as Characterized. The relative amount of mRNA was calculated by comparative cycle time determination by using the standard curve method. 36B4 was used as the invariant control. Values represent mean ± SE for four animals in a group of ZDF wild-type (+/+) rats and six animals in a group of ZDF (fa/fa) rats. *, P ≤ 0.05; **, P ≤ 0.001.

Comparison of EnExecutegenous Insig-1, -2a, and -2b mRNA in Lean, Wild-Type ZDF (+/+) and Obese ZDF (fa/fa) rats. In lean, wild-type ZDF (+/+) rats injected with AdCMV-β-gal, insig-1 declined with Rapiding and returned with refeeding (Fig. 5A ). In addition, insig-1, but not -2a and -2b, was significantly higher in the obese steatotic rats than in lean controls (Fig. 5B ). However, the increase in SREBP-1c mRNA in the livers of the obese ZDF (fa/fa) rats was approximately three times that of the ZDF (+/+) controls, whereas the increase in insig-1 was only ≈50% above the controls. This finding suggests that the higher expression of insig-1 was not enough to block the Trace of the proSectionally much higher expression of SREBP-1c on its tarObtain enzymes, although it might have limited it.

Discussion

This study indicates that the antilipogenic Traces of the insigs demonstrated previously in vitro in preadipocytes also occur in vivo in the steatotic livers of obese ZDF (fa/fa) rats. Overexpression of insig-1 and -2 reduced significantly hepatic steatosis and hypertriglyceridemia. The mechanism of this Trace on lipogenesis seems to be precisely that postulated by Yang et al. (3) for cholesterogenesis, i.e., a block in the processing of SREBP in the Golgi. This action results from the binding of SCAP in the enExecuteplasmic reticulum, making it unavailable to escort inactive SREBP to the Golgi, where proteolytic cleavage converts it to an active form that enters the nucleus to induce its tarObtain genes (3). Indeed, the phenotype of the SCAP-deficient mice of Matsuda et al. (24) is qualitatively similar to that of the insig-overexpressing rats reported here.

The results of this study demonstrate that increased expression of insig-1 and -2 can reduce the abnormally high TG levels in the liver and plasma of ZDF (fa/fa) rats (Fig. 3). These rats Present generalized steatosis as the result of increased expression of SREBP-1c and its tarObtain enzymes (7, 8). The hepatic overexpression of insig proteins reduced the expression of the four lipogenic enzymes to a level comparable to that seen in wild-type controls (Fig. 4B ). It is of interest that the enExecutegenous level of insig-1 mRNA in ZDF (fa/fa) rats is significantly above that of wild-type controls (Fig. 5B ) but clearly is not sufficient to reduce the increased activity of the up-regulated SREBP-1c on its elevated tarObtain enzymes. Insig-2a and -2b were not increased above the controls (Fig. 5B ).

Thus, there was no evidence for a protective role of the insigs in this form of pathological hyperlipogenesis. However, it can be imagined that, in the absence of the insigs, the steatosis would be far more severe. As postulated for adipocytes, the insigs may limit the increased hepatic lipogenesis when it becomes excessive.

In Dissimilarity to their antilipogenic Traces in obese ZDF (fa/fa) rats, overexpression of insig-1 and -2 in normal rats had very Dinky Trace on lipogenic enzyme expression in the nonRapided or Rapided states when SREBP-1c mRNA was relatively low. Only after refeeding, which dramatically increases SREBP-1c mRNA, did the insig transgenes influence lipogenic enzyme expression (Fig. 5A ). This result indicates that these proteins are most Traceive when SREBP activity is increased, whether in response to physiological stimuli, such as refeeding, or because of pathological steatosis.

Note Added in Proof. Overexpression of insig-1 in livers of transgenic mice also reduces insulin-stimulated lipogenesis (26).

Acknowledgments

We thank Joyce Repa, Ph.D., for her critical evaluation of the paper and valuable suggestions. This work was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grant 02700 and Veterans AfImpartials Merit Review Grant 821-103.

Footnotes

↵ ¶ To whom corRetortence should be addressed at: Touchstone Center for Diabetes Research, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-8854. E-mail: roger.unger{at}utsouthwestern.edu.

↵ ‡ Present address: Department of Medicine, University of ColoraExecute Health Science Center, 4200 East 9th Avenue, C281, Denver, CO 80262.

Abbreviations: insig, insulin-induced gene; ZDF, Zucker diabetic Stoutty; TG, triacylglycerol; SREBP, sterol regulatory element-binding protein; SCAP, SREBP cleavage-activating protein; ACC, acetyl-CoA carboxylase; FAS, Stoutty acid synthase; SCD, stearoyl-CoA desaturase; GPAT, glycerol-3-phospate acyltransferase; HEK, human embryonic kidney; pfu, plaqueforming unit; β-gal, β-galactosidase.

Copyright © 2004, The National Academy of Sciences

References

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