Treatment with Scd1 ASO decreases liver Scd1 expression and activity.

Following 2 injections of Scd1 ASO over 5 days (Figure ​(Figure1B),1B), liver Scd1 mRNA was decreased by 80%, as determined by quantitative real-time PCR (Q-PCR). Similarly, total SCD activity was decreased by approximately 50% in the liver of rats treated with Scd1 ASO compared with animals treated with scrambled ASO (SCR) (Figure ​(Figure1B).1B). The decrease in Scd1 activity was sufficient to alter the total pools of C18 acyl-CoAs, leading to a decrease in the C18 desaturation index, the ratio of C18:1 to C18:0 (oleyl-CoA/stearoyl-CoA) (Figure ​(Figure1B).1B). ASOs delivered i.p. were mainly taken up by the liver, but they also reached the peritoneal fat. Indeed, Scd1 mRNA levels were also decreased in epididymal fat pads. Food intake and body weight were not significantly different between the 2 treatment groups (Supplemental Table 1; supplemental material available online with this article; 10.1172/JCI26991DS1).

Effects of Scd1 deficiency on lipid metabolism.

Consistent with observations in chronic models of SCD1 deficiency (24, 27, 28), the levels of circulating triglycerides (TGs) were decreased by SCD1 ASO treatment (data not shown). However, the hepatic concentration of long-chain fatty acyl-CoAs (LCFA-CoAs) and of TGs was increased (Figure ​(Figure1C).1C). Consistent with these increases in tissue lipid levels, treatment with Scd1 ASO also activated the JNK pathway in the liver as manifested by increased JNK phosphorylation and activity (Figure ​(Figure1C). 1C).

Since SCD1 deficiency has been previously shown to be associated with decreases in the hepatic expression of lipogenic genes, we next examined the hepatic expression of fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC), and cholesterol hydroxylase 7α (CYP7A1) and the phosphorylation of AMP kinase (AMPK) and ACC at the completion of the insulin clamp procedure (Figure ​(Figure2,2, A and B) and under basal conditions (Figure ​(Figure2,2, C and D). At the completion of the clamp studies, the short-term downregulation of Scd1 expression had only marginal effects on these liver enzymes (Figure ​(Figure2B).2B). Under basal (overnight fast) conditions, treatment with Scd1 ASO moderately decreased the expression of FAS, ACC, and CYP7A1 (Figure ​(Figure2D).2D). These findings suggest that increased liver lipogenesis is not likely to account for the increase in liver TG and LCFA-CoA levels in rats treated with Scd1 ASO.

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Figure 2

Scd1 deficiency moderately decreases the expression of lipogenic genes in the liver.

(A and B) At the completion of insulin clamp studies, hepatic Scd1 deficiency in OF animals (black bars) did not significantly modify the levels of AMPK and FAS or the phosphorylation of AMPK and only modestly decreased ACC, p-ACC, and CYP7A1 in the liver when compared with SCR ASO OF (gray bars) and SC animals (white bars). (C and D) Under basal (overnight fast) conditions, hepatic Scd1 deficiency in OF animals (black bars) significantly decreased the expression of FAS, ACC, and CYP7A1 in the liver when compared with SCR ASO OF (gray bars) animals. *P < 0.05 versus SCR ASO OF.

Scd1 is required for diet-induced insulin resistance in rats.

In order to examine the impact of Scd1 deficiency on diet-induced insulin resistance, we next performed insulin clamp studies in OF rats (Figure ​(Figure3A).3A). Under conditions of physiological hyperinsulinemia, the rates of glucose infusion required to maintain euglycemia were markedly lower in OF rats compared with control rats fed a standard chow (SC) diet (7.9 ± 0.7 versus 15.4 ± 0.2 mg/kg/min; P < 0.01) when SCR was administered (Figure ​(Figure3B).3B). On the other hand, when Scd1 ASO was given, the rate of glucose infusion required to maintain euglycemia in OF rats was markedly increased up to the levels observed in control SC rats (15.2 ± 0.4 mg/kg/min; Figure ​Figure3B).3B). Scd1 ASO did not significantly alter the levels of glucoregulatory hormones (Supplemental Table 1) and the rate of glucose uptake (Figure ​(Figure3B).3B). Conversely, this intervention markedly suppressed liver glucose production (Figure ​(Figure3,3, B and D), and this effect entirely accounted for its effect on glucose metabolism. Glucose production represents the net contribution of gluconeogenesis and glycogenolysis (Figure ​(Figure3C).3C). However, a portion of glucose entering the liver via phosphorylation of glucose is also a substrate for dephosphorylation via glucose-6-phosphatase (Glc-6-Pase, encoded in rodents by the G6pc gene), creating a futile (glucose) cycle (Figure ​(Figure3C).3C). To delineate the mechanisms by which Scd1 inhibition modulates liver glucose homeostasis, we estimated the in vivo flux through Glc-6-Pase (Glc-6-Pase flux) and the relative contribution of glucose cycling, gluconeogenesis, and glycogenolysis to glucose output (Figure ​(Figure3,3, D and E). Treatment with Scd1 ASO markedly and similarly decreased the rates of glucose cycling and Glc-6-Pase flux (Figure ​(Figure3D)3D) in parallel to its effects on glucose production (Figure ​(Figure3D).3D). The decrease in glucose production was accounted for by a marked inhibition of gluconeogenesis as well as glycogenolysis (Figure ​(Figure3E3E and Supplemental Table 2).

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Figure 3

Scd1 deficiency negates the effects of OF on hepatic glucose fluxes.

(A) Protocol for the hyperinsulinemic-pancreatic clamp procedure. (B) Glucose infusion rate, glucose uptake, and inhibition of glucose production (expressed as percentage inhibition from basal) during the clamp period in SC (white bars), control OF (gray bars), and Scd1-deficient OF animals (black bars). (C) Schematic of the major pathways and enzymatic reactions contributing to glucose production in the liver. [¹⁴C]-PEP, ¹⁴C-phosphoenolpyruvate; ³H/¹⁴C-UDPG, ³H/¹⁴C-UDP glucose; GK, glucokinase. (D) Glucose production, Glc-6-Pase flux, and glucose cycling during the clamp period in control SC (white bars), control OF (gray bars), and liver Scd1–deficient OF animals (black bars). (E) Rate of hepatic gluconeogenesis and glycogenolysis in control SC (white bars), control OF (gray bars), and liver Scd1–deficient OF animals (black bars). *P < 0.05, versus SCR ASO OF; n = 5–6 per condition.

Liver-selective Scd1 deficiency normalizes hepatic insulin action in OF rats.

The i.p. delivery of Scd1 ASO led to a marked decrease in Scd1 expression not only in the liver but also in epididymal adipose tissue. In order to target Scd1 ASO to the liver, we delivered a 4-fold lower dose of ASO as a slow intraportal infusion rather than an i.p. bolus (Figure ​(Figure4A).4A). This protocol resulted in a significant decrease in liver expression of Scd1 in the absence of significant changes in Scd1 expression in epididymal fat (Figure ​(Figure4B).4B). Under conditions of physiological hyperinsulinemia, intraportal infusion of Scd1 ASO markedly improved insulin’s ability to restrain glucose production (Figure ​(Figure4B).4B). To more directly examine whether the decreased expression of SCD1 in adipose tissue is required for the effects of SCD1 ASO treatment on liver glucose fluxes, we compared subgroups of OF rats displaying similar decreases in liver Scd1 expression induced by either i.p. or intraportal delivery of ASO (Figure ​(Figure4,4, C–F). The i.p. administration of Scd1 ASO dramatically suppressed Scd1 expression in epididymal fat pads while the intraportal infusion of the same ASO had no detectable impact on adipose tissue Scd1 expression (Figure ​(Figure4,4, C and E). However, the 2 treatment regimens led to similar and dramatic improvements in hepatic insulin action (Figure ​(Figure4,4, D and F). Thus, the approximately 70% decrease in liver Scd1 expression is sufficient to negate the onset of hepatic insulin resistance in this model.

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Figure 4

Selective attenuation of liver Scd1 expression normalizes hepatic insulin action in OF rats.

(A) Protocol for the intraportal Scd1 ASO treatment (top panel). An indwelling catheter was placed into the portal vein, and 2 days later (day 0), the animals received a first intraportal injection of either SCR ASO or Scd1 ASO (25 mg/kg of body weight). On day 3, the rats were switched to a lard-enriched diet (HF) and received a second injection of ASOs. Lastly, on day 5, an insulin clamp procedure was performed (See Figure 2A). (B) Intraportal Scd1 ASO (black bar) decreased liver Scd1 mRNA when compared with SCR ASO (white bar; left panel). Selective attenuation of liver Scd1 (black bar) in OF rats improved hepatic insulin action when compared with SCR ASO (white bar) as shown by a significant decrease in glucose production (GP) (center panel) and a significant increase in insulin’s ability to inhibit GP (right panel). (C–F) To examine whether the decreased expression of SCD1 in adipose tissue is required for the effects of Scd1 ASO on GP, we compared subgroups of OF rats displaying similar decreases in liver SCD1 induced by either i.p. (C and D) or intraportal (E and F) ASO. (C) i.p. Scd1 ASO markedly suppressed Scd1 expression in liver and white fat. (E) Intraportal Scd1 ASO markedly decreased Scd1 expression in the liver but not in adipose tissue. (D and F) The 2 treatment regimens led to similar improvements in hepatic insulin action. *P < 0.05 versus SCR ASO OF; n = 5–6.

Scd1 is required for diet-induced insulin resistance in mice.

In order to examine whether Scd1 also plays a critical role in the development of diet-induced hepatic insulin resistance in HF-fed mice, we next performed insulin clamp studies in Scd1-deficient conscious mice (Figure ​(Figure5,5, A–C). Treatment with Scd1 ASO markedly decreased hepatic Scd1 mRNA and protein (Figure ​(Figure5B).5B). The enzymatic (desaturase) activity of Scd1 was markedly increased by HF feeding in mice and dramatically lowered by treatment with Scd1 ASO (Figure ​(Figure5D).5D). Under conditions of physiological hyperinsulinemia, the rates of glucose production were markedly increased in HF-fed mice compared with control mice fed an SC diet when the SCR was administered (Figure ​(Figure5D).5D). Importantly, in HF-fed mice treated with Scd1 ASO, the rate of glucose production was markedly suppressed to levels similar to those of control mice (Figure ​(Figure5D).5D). Of interest, Scd1 deficiency did not have an impact on the modest decrease in glucose uptake that was induced by HF feeding (Figure ​(Figure5D).5D). It should be noted that Scd1 ASO used in these experiments was directed against an entirely different region of Scd1 mRNA. Overall, our findings indicate that Scd1 deficiency exerts a protective effect against the development of hepatic insulin resistance in HF-fed rats and mice.

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Figure 5

Scd1 deficiency normalizes hepatic insulin action in HF-fed mice.

(A) Protocol for Scd1 ASO treatment in mice. Male C57BL/6J mice were allocated to a HF diet for 3 weeks. Five days before the completion of the protocol (day 0), the mice received an i.p. injection of either a SCR ASO or Scd1 ASO (100 mg/kg of body weight); on day 3, the animals received a second ASO injection, and vascular catheters were implanted; finally, on day 5, the mice were subjected to an insulin clamp procedure. (B) Scd1 ASO (black bars) markedly decreased the hepatic levels of Scd1 mRNA and protein compared with SCR ASO (grey bars) in HF diet–fed mice. (C) Protocol for the insulin clamp procedure in mice. (D) The enzymatic (desaturase) activity of Scd1 was markedly increased by HF feeding in mice (gray versus white bars) and dramatically lowered by treatment with Scd1 ASO (black bars). The rapid attenuation of liver Scd1 expression and activity prevented the development of hepatic insulin resistance induced by HF feeding in mice but did not modify insulin action on glucose uptake. *P < 0.05 versus SCR ASO; n = 6–10.

Scd1 deficiency enhances Akt phosphorylation and suppresses phosphoenolpyruvate carboxykinase and Glc-6-Pase expression in OF rats.

These metabolic data indicated that Scd1 ASO treatment enhanced insulin action to an extent that restored insulin sensitivity to normal levels in insulin-resistant animals. Enhanced insulin signaling could account for these effects. Thus, we next assessed the effect of short-term Scd1 deficiency on Akt phosphorylation and on the expression of Glc-6-Pase, encoded by G6pc, and phosphoenolpyruvate carboxykinase (PEPCK), encoded by Pck1, in the liver. Decreased expression of Scd1 led to a marked increase in the hepatic phosphorylation of Akt. Western blot analysis (Figure ​(Figure6A)6A) showed increased phosphorylation of Akt at both Ser473 (4-fold; Figure ​Figure6B)6B) and Thr308 (3-fold; Figure ​Figure6B)6B) in animals treated with Scd1 ASO as compared with control (scrambled ASO [SCR]) ASO–treated animals. Hepatic Akt phosphorylation in these OF Scd1-deficient animals was even higher than in animals that are not insulin resistant on a regular chow diet. We next investigated whether these changes in Akt activation may have led to transcriptional modulation of the glucoregulatory enzymes PEPCK and Glc-6-Pase. Q-PCR experiments showed that the expression of PEPCK was suppressed by almost 90% in animals deficient in Scd1 as compared with control animals (Figure ​(Figure6C).6C). Similar measurements showed an even more dramatic effect on liver Glc-6-Pase, the expression of which was virtually abolished (Figure ​(Figure6C)6C) in Scd1 ASO–treated animals. Thus, it is likely that the decreases in in vivo Glc-6-Pase flux and gluconeogenesis are at least partly due to decreased hepatic expression of these 2 critical glucoregulatory enzymes.

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Figure 6

Scd1 deficiency enhances hepatic insulin action in OF rats.

(A) Western blot analysis of Akt phosphorylation (Ser473) in the liver of SC control rats (SCR ASO SC) and in OF rats treated with either control (SCR ASO OF) or Scd1 ASO (Scd1 ASO OF). (B) OF (SCR ASO OF; gray bars) attenuated hepatic Akt phosphorylation at Ser473 and Thr308 as compared with SC diet (SCR ASO SC, white bars); Scd1 deficiency (Scd1 ASO OF, black bars) prevented the effect of OF and enhanced Akt phosphorylation 2- to 3-fold compared with animals on SC. (C) Liver expression of Pck1 and G6pc mRNA determined by Q-PCR. OF (gray bars) resulted in a marked increase in the hepatic expression of PEPCK and Glc-6-Pase when compared with an SC diet (white bars); ASO-mediated liver Scd1 deficiency (black bars) prevented the hepatic upregulation of these enzymes in response to OF and brought it down to levels below those of SC-fed animals. *P < 0.05 versus SCR ASO OF.

Animals and experimental design: mouse studies.

Adult male C57BL/6J mice (27–32 g) were anesthetized with xylazine and ketamine and catheterized through the right internal jugular vein. The venous catheter was used for infusion, and blood samples were collected from the tail vein. Each animal was monitored for food intake and weight gain after surgery to ensure complete recovery. Mice were randomized in 3 groups. During the 3 weeks preceding the clamp study, 1 group received an SC diet (Purina Mills Inc.) from which 59% of calories were provided by carbohydrate, 28% by protein, and 12% by fat (Figure ​(Figure5A).5A). The remaining 2 groups received HF chow (Purina Mills Inc.) from which 45% of calories were provided by carbohydrate, 22% by protein, and 33% by fat). Thus, we studied 3 groups of mice: group 1 consisted of SC-fed mice treated with control ASO; group 2 consisted of HF-fed mice treated with control ASO; group 3 consisted of HF-fed mice treated with Scd1 ASO. All mice received 2 i.p. injections (100 mg/kg) of either control ASO (groups 1 and 2) or Scd1 ASO (group 3) during the week preceding the clamp study.

In vivo glucose kinetics and pancreatic insulin clamp procedure in rats.

Pancreatic insulin clamp studies (Figure ​(Figure3A)3A) were performed as previously described to attain physiological hyperinsulinemia (10, 37). Rats were implanted with chronic catheters. At t = 0, a primed, continuous infusion of HPLC-purified [³H-3]-glucose (PerkinElmer; 40 mCi bolus, 0.4 mCi/min for duration of the study) was initiated and maintained throughout the 4 hours of the study. Samples for determination of ³H-glucose–specific activity were obtained at 10-minute intervals throughout infusions. At t = 2 hours, a pancreatic hyperinsulinemic-euglycemic clamp was initiated and maintained for the final 2 hours of the study. This procedure involved the infusion of somatostatin (3 μg/kg/min) and insulin (3 mU/kg/min) and a variable infusion of 25% glucose periodically adjusted to clamp the plasma glucose concentration at approximately 8 mM. At the end of the in vivo studies, rats were anesthetized (pentobarbital, 55 mg/kg body weight, i.v.), and tissue samples were freeze clamped in situ with aluminum tongs precooled in liquid nitrogen. All tissue samples were stored at –80°C for subsequent analysis.

In vivo glucose kinetics and pancreatic insulin clamp procedure in mice.

Euglycemic clamps (Figure ​(Figure5C)5C) were performed in conscious, unrestrained, catheterized mice as previously described (38). Food was removed for 5 hours before beginning of in vivo studies. The infusion studies lasted a total of 90 minutes. A solution of glucose (10% wt/vol) was infused at a variable rate as required to maintain euglycemia (8 mM). Mice received a constant infusion of HPLC-purified [³H-3]-glucose (0.1 μCi/min; PerkinElmer) and insulin (3.6 mU/min/kg body weight). Thereafter, plasma samples were collected to determine glucose levels (at t = 10, 20, 30, 40, 50, 60, 70, 80, and 90 minutes) as well as [³H-3]–glucose–specific activity (at t = 40, 50, 60, 70, 80, and 90 minutes). Additionally, in 5 mice from each experimental group, 10 minutes before the end of the in vivo studies, [U-¹⁴C]–lactate (5-μCi bolus, 0.4 μCi/min; PerkinElmer) was administered to determine the contribution of gluconeogenesis to the hepatic Glc-6-Pase pool. Steady state conditions for both plasma glucose concentration and specific activity were achieved by 40 minutes in these studies. At the end of the in vivo studies, mice were anesthetized (pentobarbital, 60 mg/kg, i.v.), the abdomen was quickly opened, and adipose tissue and liver were freeze clamped in situ with aluminum tongs that were cooled in liquid nitrogen. The time between the injection of anesthesia and the freeze clamping of tissue samples was less than 60 seconds. Tissue samples were stored at –80°C for further analysis.

Analytical procedures and calculations.

Plasma glucose was measured by the glucose oxidase method (Glucose Analyzer II, Beckman Coulter). Plasma insulin and leptin, adiponectin, and glucagon levels were determined by RIA (Linco Research Inc.). Plasma nonesterified fatty acid concentrations were determined using an enzymatic method by an automated kit according to the manufacturer’s specifications (Waco Pure Chemical Industries Ltd.). The radioactivity of [³H-3]-glucose in plasma was measured from supernatants of Ba(OH)₂ and from ZnSO₄ precipitates (Somogyi procedure) after each was evaporated to dryness for the removal of tritiated water. Measurements of hepatic ¹⁴C-phosphoenolpyruvate and ³H/¹⁴C-UDP glucose–specific activities were obtained from 2 sequential chromatographic separations, as previously described (39–42). Calculations were performed as described (43). In brief, the rates of ⁴C-phosphoenolpyruvate-gluconeogenesis, Glc-6-Pase flux, and glucose cycling (glucose cycling = Glc-6-Pase flux – glucose production) were calculated as previously described (43). Net glycogenolysis was calculated as the difference between glucose production and gluconeogenesis.

Scd1 activity.

Liver Scd1 activity assays were performed essentially as described (26). At the end of incubation, long-chain acyl-CoAs were extracted and fractionated by reverse-phase HPLC as previously reported (44). Chromatographically resolved stearoyl-CoA (C18:0) and oleyl-CoA (C18:1) peaks were collected and counted in a β-scintillation counter. Protein was assayed by the bicinchoninic acid method using a commercially available kit (Pierce Biotechnology) according to the manufacturer’s instructions.

Gene expression.

Scd1, Glc-6-Pase (G6pc), and PEPCK (Pck1) mRNAs were measured by Q-PCR as described (37). In brief, total RNA was extracted from either liver or fat tissue using the TRIzol reagent (Invitrogen Corp.) according to the manufacturer’s instructions. cDNA was then synthesized from 2 μg of total RNA using the Superscript III First Strand kit (Invitrogen Corp.). Samples of RNA in which the reverse transcriptase was omitted (no-RT controls) were also included. The cDNA and no-RT controls were diluted 1:20 with PCR grade water, and 2 μl from each sample was used for PCR in a total volume of 20 μl. The FastStart DNA Master SYBR Green I kit (Roche Applied Science) and a LightCycler 2 instrument (Roche Applied Science) were used in all experiments. The 18S mRNA expression was routinely measured for normalization purposes. The primer sequences used were as follows: 5′-CTACAAGCCTGGCCTCCTGC-3′ and 5′-GGACCCCAGGGAAACCAGGA-3′ for Scd1; 5′-GCGACCGTCCCCTTTGCATCTGTC-3′ and 5′-CCACCAAACACTCCCCCTCCTCC-3′ for G6pc; 5′-GCCTCCTCAGCTGCATAATGGTCT-3′ and 5′-GAATGCTTTCTCGAAGTCCTCTTCTG-3′ for Pck1; and 5′-AGGGTTCGATTCCGGAGAGG-3′ and 5′-CAACTTTAATATACGCTATTGG-3′ for ribosomal 18S. The mRNA copy number was calculated from standard curves obtained using cloned target templates (plasmid DNA) of known copy number for each gene of interest.

LCFA-CoA.

Liver LCFA-CoAs were extracted and measured as previously described (44).

TGs.

Hepatic TGs were extracted by the Folch method (45). Liver and plasma TG were measured using a commercially available colorimetric assay kit (Sigma-Aldrich) according to the manufacturer’s instructions.

Western blot analysis.

Liver tissue (50–100 mg) was homogenized in a detergent-based lysis buffer (M-PER; Pierce Biotechnology) supplemented with protease and phosphatase inhibitors. The extracts were incubated on ice for 20 minutes and then centrifuged at 15,000 g to remove tissue debris; the supernatants were saved and frozen at –80°C until analysis. Then, 50 μg of protein were heat denatured in SDS-PAGE sample buffer and resolved on denaturing (SDS) 10% polyacrylamide gels (SDS-PAGE). Fractionated proteins were electrophoretically transferred to nitrocellulose membranes by standard procedures (46). Membranes were immunoblotted with the appropriate primary antibodies; immune complexes were detected by ECL (Western Lightning, PerkinElmer) using horseradish peroxidase–conjugated secondary antibodies. Bands were imaged on x-ray film and digitized for quantification using ImageQuant software version 5.0 (GE Healthcare). β-tubulin, β-actin, or GAPDH was used to control for loading differences.

JNK activity assay.

Liver JNK activity assay was performed using a kit from Cell Signaling Technology per the manufacturer’s instructions. In brief, 200 μg of liver protein were used to pull down JNK using immobilized glutathione-S-transferase–tagged c-Jun. After extensive washing, the immobilized affinity complexes were incubated 30 minutes at 37°C in the absence or presence of 200 μM ATP. The reactions were stopped by the addition of Laemmli sample buffer and subsequent boiling. Phosphorylated c-Jun (p–c-Jun) was detected by Western blotting using p–c-Jun antibodies and ECL imaging.

Insulin signaling protocol.

To examine early insulin signaling events in the liver, another group of OF rats were treated with either control (SCR) or Scd1 ASO as described in Animals and experimental design: rat studies. These animals were not subjected to pancreatic clamps but were instead challenged with an i.p. bolus of either insulin (5 U/kg) or saline after overnight fasting. Five minutes later, the animals were sacrificed and the liver was collected and processed immediately for analysis. About 300–500 mg of liver was homogenized in 10 volumes of lysis buffer (50 mM HEPES, pH = 7.7, 100 mM sodium pyrophosphate, 100 mM sodium fluoride, 50 mM β-glycerophosphate, 10 mM sodium orthovanadate, 10 mM EDTA, 1% NP40, 2 mM PMSF, complete protease inhibitor cocktail; Roche Applied Science). The homogenates were incubated for 30 minutes on ice and then centrifuged at 3,800 g to remove tissue debris; the supernatants were used for the subsequent procedures. Protein concentration was measured by the bicinchoninic acid method described above, and protein concentration in all samples was adjusted to 20 mg/ml. We incubated 16 mg of total protein from each lysate with 10 μg of either anti-IRS1 antibody (Upstate USA Inc.) or 4G10 phosphotyrosine antibody (Upstate USA Inc.) at 4°C for 2 hours. Next, 20 μl of protein-A sepharose (for IRS1) or protein-G sepharose (for phosphotyrosine) beads (Amersham Biosciences) was added and incubated at 4°C for 1 hour. The beads were then washed 5 times with 1 ml of lysis buffer and boiled with 60 μl of Laemmli buffer. The immunoprecipitates were size fractionated by SDS-PAGE (10 μl of sample was applied in each lane), transferred to nitrocellulose as described above, and subjected to Western blot analysis.

Antibodies.

Rabbit polyclonal or monoclonal antibodies to ACC, phospho-ACC (Ser79), Akt, p-Akt (Ser473 and Thr308), AMPK-α, p–AMPK-α (Thr172), JNK, and p-SAPK/JNK (Thr183/Tyr185) were from Cell Signaling Technology; mouse monoclonal β-tubulin antibodies were obtained from Covance Research Products; FAS antibodies were from BD Biosciences — Pharmingen. The following were obtained from Santa Cruz Biotechnology Inc.: CYP7A1 rabbit polyclonal antibodies, insulin receptor β subunit (IRβ) antibodies, and mouse Scd1 goat polyclonal antibodies. IRS1 antibodies were from Upstate USA Inc., and p85α subunit of PI3K antibodies were from Cell Signaling Technology. Rabbit polyclonal antibodies to PTP1B were obtained from Abcam, and GAPDH mouse monoclonal antibodies were from Research Diagnostics Inc.

We wish to thank Bing Liu, Clive Baveghems, Stanislaw Gaweda, and Yuhua Wang for expert technical assistance. H. Ono is the recipient of a fellowship from the Sankyo Foundation of Life Science. This work was supported by grants from the NIH (DK 48321, DK 45024, AG 21654, and DK 20541) to L. Rossetti.