Mechanism of Hepatic Insulin Resistance in Non-alcoholic Fatty Liver Disease

Short term high fat feeding in rats results specifically in hepatic fat accumulation and provides a model of non-alcoholic fatty liver disease in which to study the mechanism of hepatic insulin resistance. Short term fat feeding (FF) caused a ∼3-fold increase in liver triglyceride and total fatty acyl-CoA content without any significant increase in visceral or skeletal muscle fat content. Suppression of endogenous glucose production (EGP) by insulin was diminished in the FF group, despite normal basal EGP and insulin-stimulated peripheral glucose disposal. Hepatic insulin resistance could be attributed to impaired insulin-stimulated IRS-1 and IRS-2 tyrosine phosphorylation. These changes were associated with activation of PKC-ϵ and JNK1. Ultimately, hepatic fat accumulation decreased insulin activation of glycogen synthase and increased gluconeogenesis. Treatment of the FF group with low dose 2,4-dinitrophenol to increase energy expenditure abrogated the development of fatty liver, hepatic insulin resistance, activation of PKC-ϵ and JNK1, and defects in insulin signaling. In conclusion, these data support the hypothesis hepatic steatosis leads to hepatic insulin resistance by stimulating gluconeogenesis and activating PKC-ϵ and JNK1, which may interfere with tyrosine phosphorylation of IRS-1 and IRS-2 and impair the ability of insulin to activate glycogen synthase. Short term high fat feeding in rats results specifically in hepatic fat accumulation and provides a model of non-alcoholic fatty liver disease in which to study the mechanism of hepatic insulin resistance. Short term fat feeding (FF) caused a ∼3-fold increase in liver triglyceride and total fatty acyl-CoA content without any significant increase in visceral or skeletal muscle fat content. Suppression of endogenous glucose production (EGP) by insulin was diminished in the FF group, despite normal basal EGP and insulin-stimulated peripheral glucose disposal. Hepatic insulin resistance could be attributed to impaired insulin-stimulated IRS-1 and IRS-2 tyrosine phosphorylation. These changes were associated with activation of PKC-ϵ and JNK1. Ultimately, hepatic fat accumulation decreased insulin activation of glycogen synthase and increased gluconeogenesis. Treatment of the FF group with low dose 2,4-dinitrophenol to increase energy expenditure abrogated the development of fatty liver, hepatic insulin resistance, activation of PKC-ϵ and JNK1, and defects in insulin signaling. In conclusion, these data support the hypothesis hepatic steatosis leads to hepatic insulin resistance by stimulating gluconeogenesis and activating PKC-ϵ and JNK1, which may interfere with tyrosine phosphorylation of IRS-1 and IRS-2 and impair the ability of insulin to activate glycogen synthase. In recent years, there has been an increasing appreciation for the significance of non-alcoholic fatty liver disease (NAFLD). 1The abbreviations used are: NAFLD, non-alcoholic fatty liver disease; IR, insulin resistance; IRS, insulin receptor substrate; PKC, protein kinase C; JNK, Jun N-terminal kinase; ANOVA, analysis of variance; 2,4-DNP, 2,4-dinitrophenol; FA, fatty acid; GS, glycogen synthase; EGP, endogenous glucose production; PI, phosphatidylinositol. Although the true prevalence is unknown, estimates of the prevalence of NAFLD in the general population range from 5 to 20% and up to 75% of patients with obesity and diabetes mellitus (1Sanyal A.J. Gastroenterology. 2002; 123: 1705-1725Google Scholar, 2McCullough A.J. J. Clin. Gastroenterol. 2002; 34: 255-262Google Scholar, 3Angulo P. N. Engl. J. Med. 2002; 346: 1221-1231Google Scholar). While it is accepted that hepatic fat accumulation is linked to insulin resistance, the exact mechanism is unclear (4Marchesini G. M B. Forlani G. Melchionda N. Am. J. Med. 1999; 107: 450-455Google Scholar). Some investigators have postulated that with insulin resistance, the combination of elevated plasma concentrations of glucose and fatty acids promote hepatic fatty acid synthesis and impair β-oxidation leading to hepatic steatosis (4Marchesini G. M B. Forlani G. Melchionda N. Am. J. Med. 1999; 107: 450-455Google Scholar, 5Sanyal A. Campbell-Sargent C. Clore J. Gastroenterology. 2001; 120: 1183-1192Google Scholar). In contrast, others have proposed that hepatic fat accumulation and hepatic insulin resistance can occur without the development of peripheral insulin resistance (6Kim J.K. Fillmore J.J. Chen Y. Yu C. Moore I.K. Pypaert M. Lutz E.P. Kako Y. Velez-Carrasco W. Goldberg I.J. Breslow J.L. Shulman G.I. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7522-7527Google Scholar, 7Kraegen E.W. Clark P.W. Jenkins A.B. Daley E.A. Chisholm D.J. Storlien L.H. Diabetes. 1991; 40: 1397-1403Google Scholar). However, the mechanism by which hepatic fat accumulation might lead to hepatic insulin resistance has not been resolved. Determining the steps between hepatic fat accumulation and hepatic insulin resistance requires models in which hepatic fat accumulation occurs without peripheral fat accumulation. In a study examining the time course of hepatic and peripheral insulin resistance, Kraegen et al. (7Kraegen E.W. Clark P.W. Jenkins A.B. Daley E.A. Chisholm D.J. Storlien L.H. Diabetes. 1991; 40: 1397-1403Google Scholar) reported that rats fed a high fat diet for 3 days developed hepatic insulin resistance prior to the development of peripheral insulin resistance (7Kraegen E.W. Clark P.W. Jenkins A.B. Daley E.A. Chisholm D.J. Storlien L.H. Diabetes. 1991; 40: 1397-1403Google Scholar). We reasoned that feeding rats for a short duration would therefore provide an excellent model of NAFLD in which we could study the effect of hepatic fat accumulation on hepatic insulin responsiveness without the confounding effects of peripheral insulin resistance. In the current study, rats were subjected to a 3 day high fat diet to simulate NAFLD. Glucose metabolism and insulin response were then determined with a hyperinsulinemic-euglycemic clamp. A low dose of the mitochondrial uncoupler, 2,4-dinitrophenol, was used to increase energy expenditure and prevent hepatic fat accumulation. In this way, it was possible to determine if the hepatic insulin resistance specifically depended on hepatic fat accumulation. In addition, the model was used to determine the impact of hepatic fat accumulation on the insulin signaling pathway, glycogen synthase (GS) activation, and possible mediators of fat-induced hepatic insulin resistance. Animals and Diets—Normal, adult male Sprague-Dawley rats (300–350 g) were obtained from Charles River Labs (Wilmington, MA). The rats were placed on a 12-h day/night cycle and provided ad libitum access to food and water, except when specified by experimental protocol. They were housed individually and had their food consumption and weights measured daily. Rats received either regular rodent chow (60% CHO/10% fat/30% protein) or a high fat diet (26% CHO/59% fat/15% protein). Safflower oil was the major constituent of the high fat diet (Dyets Inc., Bethlehem, PA). Animals were fasted for 12 h prior to any study. The Yale Animal Care and Use Committee approved all protocols. Hyperinsulinemic-Euglycemic Clamps—Five days prior to the clamp, indwelling catheters were implanted into the right jugular vein extending to the right atrium, and the right carotid artery extending to the aortic arch. The catheters were externalized through a subcutaneous channel at the back of the neck, sealed with a polyvinylpyrrolidine/heparin solution, and closed. Animals were allowed 2 days to recover from surgery before starting on the diet. After 3 days of either a control or high fat diet, the animals were fasted for 12 h prior to the clamp. A primed (25 mg/kg)/continuous (0.25 mg/kg/min) infusion of [U-13C]glucose (>99%, Cambridge Isotope Laboratories, Andover, MA) was started at 0 min. From 90 to 120 min of the basal period, plasma samples are obtained every 10 min to determine the plasma enrichment of glucoseM+6. After the basal period, the animals receive a primed (150 milliunits/kg/continuous (4 milliunits/kg/min) infusion of insulin and a variable infusion of unlabeled 20% glucose to maintain euglycemia (∼100 mg/dl). Plasma samples were taken every 10 min to determine the steady state enrichment of glucoseM+6 from 90 to 120 min of the hyperinsulinemic-euglycemic clamp,. At the end of the clamp, the tissues were harvested in situ with aluminum tongs precooled in liquid nitrogen and stored at –80 C. Plasma samples were deproteinized with 5 volumes of 100% methanol, dried, and derivatized with 1:1 acetic anhydride/pyridine to produce the pentacetate derivative of glucose. The atom percent enrichment of glucoseM+6 was then measured by GC/MS analysis using a Hewlett-Packard 5890 gas chromatograph interfaced to a Hewlett-Packard 5971A mass selective detector operating the chemical ionization mode (8Hundal R.S. Krssak M. Dufour S. Laurent D. Lebon V. Chandramouli V. Inzucchi S.E. Schumann W.C. Petersen K.F. Landau B.R. Shulman G.I. Diabetes. 2000; 49: 2063-2069Google Scholar). GlucoseM+6 enrichment was determined from the ratio of m/z 337:331. Glucose incorporation into glycogen under hyperinsulinemic-euglycemic conditions was done by omitting the basal infusion to avoid contaminating the glycogen pool with [U-13C]glucose and by using 20% glucose that was 20% enriched with [U-13C]glucose. This higher level of plasma enrichment insured satisfactory detection of [U-13C]glucose incorporation into glycogen. The glycogen was extracted from liver homogenates and completely digested with amylogluccosidase. The resulting glucose concentration was measured by the glucose oxidase method (Glucose Analyzer II, Beckman Instruments, Fullerton, CA). GlucoseM+6 enrichment was then analyzed by GCMS as described above. 2-Deoxyglucose Uptake in Vitro—Measurement of 2-deoxyglucose uptake in isolated soleus strips was done as described previously (9Hansen P.A. Gulve E.A. Marshall B.A. Gao J. Pessin J.E. Holloszy J.O. Mueckler M. J. Biol. Chem. 1995; 270: 1679-1684Google Scholar). After an overnight fast, rats were anesthetized and had soleus muscles dissected out. The soleus muscles were split to yield ∼30 mg strips, which were held under resting tension between metal clips. The muscle strips were allowed 40 min of recover in Krebs-Henseleit bicarbonate buffer (KHB) supplemented with 2 mm pyruvate, 0.1% bovine serum albumin at 30 °C under 95% O2, 5% CO2. They were then preincubated for 20 min in either KHB or KHB+1 milliunits/ml insulin. Following preincubation, they underwent incubation in media identical to the preincubation with the addition of [14C]mannitol and 2-[3H]deoxyglucose. 2-Deoxyglucose Uptake in Vivo—Measurement of tissue 2-deoxyglucose was performed as described previously (10Griffin M.E. Marcucci M.J. Cline G.W. Bell K. Barucci N. Lee D. Goodyear L.J. Kraegen E.W. White M.F. Shulman G.I. Diabetes. 1999; 48: 1270-1274Google Scholar). Briefly, overnight fasted rats were subjected to a hyperinsulinemic-euglycemic clamp experiment as described in the preceding section. After 100 min, 20 mCi of 2-[14C]deoxyglucose was given as a single i.v. bolus. Plasma was collected at 100.5, 101, 102, 103, 105, 107.5, 110, 120, 130, and 140 min to determine 14C activity and plasma glucose concentration. Epididymal white adipose tissue, soleus, and gastrocnemius were clamped in situ with tongs precooled in liquid nitrogen and stored at –80 C until use. 2-Deoxyglucose uptake was calculated based on the intracellular 2-deoxyglucose content and the plasma 2-deoxyglucose area under the curve. Tissue Lipid Content—Lipid was extracted from frozen, ground tissues by homogenization in 10× volume of 2:1 chloroform: methanol followed by shaking at room temperature for 3–4 h. The organic and aqueous phases were removed by adding 1 volume of 1 m H2SO4 and centrifugation at 4000 rpm for 15 min. The organic phase was completely dried and resuspended in 1 ml of chloroform. A small aliquot (10–30 μl) was removed and dried again. The triglyceride concentration in this aliquot was determined using the Infinity triglyceride kit (Sigma). The measurement of the tissue fatty acyl-CoA concentrations was done as described previously (11Yu C. Chen Y. Cline G.W. Zhang D. Zong H. Wang Y. Bergeron R. Kim J.K. Cushman S.W. Cooney G.J. Atcheson B. White M.F. Kraegen E.W. Shulman G.I. J. Biol. Chem. 2002; 277: 50230-50236Google Scholar). Insulin Signaling—A separate group of rats were used to assess the impact of hepatic fat accumulation on the insulin signaling pathway. These rats were treated exactly as above and underwent a 20-min hyperinsulinemic-euglycemic clamp without a basal infusion. Tissues were harvested in situ immediately at the end of the clamp. Liver samples harvested in situ in fasting conditions (basal) at the end of the 20-min clamp (insulin-stimulated) were used to assess IR, IRS-1, and IRS-2 tyrosine phosphorylation, (11Yu C. Chen Y. Cline G.W. Zhang D. Zong H. Wang Y. Bergeron R. Kim J.K. Cushman S.W. Cooney G.J. Atcheson B. White M.F. Kraegen E.W. Shulman G.I. J. Biol. Chem. 2002; 277: 50230-50236Google Scholar) IRS-1 and IRS-2-associated PI 3-kinase activity (12Folli F. Saad M.J. Backer J.M. Kahn C.R. J. Biol. Chem. 1992; 267: 22171-22177Google Scholar), Akt2 activity (13Alessi D.R. Caudwell F.B. Andjelkovic M. Hemmings B.A. Cohen P. FEBS Lett. 1996; 399: 333-338Google Scholar), and GSK3 activity (14Cross D.A. Alessi D.R. Vandenheede J.R. McDowell H.E. Hundal H.S. Cohen P. Biochem. J. 1994; 303: 21-26Google Scholar). Primary antibodies used for these experiments were rabbit polyclonal IgG obtained from Upstate (Charlottesville, VA). For assessment of tyrosine phosphorylation, after the membrane was blotted with anti-phosphotyrosine antibody, it was stripped and reblotted with the same antibody used for immunoprecipitation to assess any differences in total protein (i.e. IR, IRS1, or IRS2) present. Glycogen synthase activity was also determined in basal and insulin-stimulated tissue using previously described methods (15Nuttall F.Q. Gannon M.C. Anal. Biochem. 1989; 178: 311-319Google Scholar). PKC Membrane Translocation and Activity—PKC membrane translocation was performed as described previously (16Qu X. Seale J.P. Donnelly R. J. Endocrinol. 1999; 162: 207-214Google Scholar). Briefly, 50 μg of crude membrane and cytosol protein extracts were resolved by SDS-PAGE using 8% gel and electroblotted onto polyvinylidene difluoride membrane (DuPont, Boston, MA) using a semidry-transfer cell (Bio-Rad). The membrane was then blocked for 2 h at room temperature in phosphate-buffered saline-Tween (PBS-T:10 mmol/liter NaH2PO4, 80 mmol/liter Na2HPO4, 0.145 mol/liter NaCl, and 0.1% Tween-20, pH 7.4) containing 5% (w/v) nonfat dried milk, washed twice, and then incubated overnight with rabbit anti-peptide antibody against PKC-α, -β1, -β2, -ϵ, -δ, -η,-ζ, -λ (Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1:100 in rinsing solution. After further washings, membranes were incubated with horseradish peroxidase-conjugated IgG fraction of goat anti-rabbit IgG (Bio-Rad) diluted 1: 5000 in PBS-T for 2 h. PKC translocation was expressed as the ratio of arbitrary units of membrane bands over cytosol bands. In addition, PKC-q levels and activity were measured from whole cell lystaes using two different PKC-q antibodies (from Santa Cruz Biotechnology, as above, and from BD Transduction Laboratories, San Diego, CA). JNK1 Immunoprecipitation and Activity—For JNK1 immunoprecipitation, 100 mg of liver tissues were lysed with Triton X-100 lysis buffer (50 mm Hepes, 150 mm NaCl, 1 mm EDTA, 2 mm Na3VO4, 20 mm Na4P2O7, 100 mm NaF, 1% Triton X-100, 2 mm phenylmethylsulfonyl fluoride, 20 μg/ml aprotinin, 1 μg/ml pepstatin, and leupeptin, pH 7.4). 8 mg of cell lysate was precleared with protein A/G-Sepharose for 1 h at 4 °C with gentle rocking. Either polyclonal anti-IRS-1 or polyclonal anti-JNK1 antibodies were added and incubated at 4 °C overnight with gentle rocking. Immunocomplexes were collected by incubation with protein A/G, washed three times with 1 ml of ice-cold lysis buffer, resuspended in Laemmli sample buffer, and separated using 8% SDS-PAGE. For association of JNK1 and IRS2, 20 μg of rabbit polyclonal IRS2 was linked to gel matrix using the Seize Primary kit (Pierce). This was then incubated with 2 mg of precleared cell lysate at 4 C overnight. After washing three times with TBS, the proteins were eluted in three fractions with elution buffer. The first fraction contained the majority of the IRS2 and was used for subsequent analysis. Proteins were electrophoretically transferred to Immobilon-P membranes (Millipore, Billerica, MA) and immunoblotted with the appropriate antibody followed by detection using ECL chemiluminescence (Amersham Biosciences). JNK1 activity assay was measured using the SAPK/JNK assay kit (Upstate, Charlottesville, VA). After autoradiograph was performed to detect 32P-labeled c-Jun-GST, the membrane was blotted with anti-JNK antibody to determine the efficiency of the immunoprecipitation. The 32P-c-Jun-GST signal was then normalized to the amount of JNK present in the immunoprecipitates. mRNA Analyses—Liver and muscle were harvested in situ using tongs pre-cooled in liquid nitrogen. Tissue was stored at –80 C until use. The mRNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA). Transcripts were analyzed by Northern blot using 32P-labeled cDNA probes for pyruvate carboxylase, phosphoenolpyruvate carboxykinase, fructose-1,6-bisphosphatase, and glucose-6-phosphatase and normalized to β-actin. Images obtained on a phosphorimager screen were analyzed using the Storm system with ImageQuant software (Amersham Biosciences) Calculations—Rates of whole body glucose uptake and basal glucose turnover were determined as the ratio of the [U-13C]glucose infusion rate (mg per kg per minute) to the atom percent enrichment of glucoseM+6 (%) during steady state of the basal and clamped periods. Endogenous glucose production (EGP) during the clamp procedure was determined by subtracting the glucose infusion rate from whole body glucose uptake. Statistics—All values are represented as the mean ± S.E. A two-way Student's t test was performed to determine difference between the control and treated group. Significance was accepted at p < 0.05. For multiple comparisons between groups, ANOVA was performed followed by Bonferroni's t test. Baseline Characteristics—The average caloric intake and weight gain were similar in between control and high fat-fed animals (Table I). Plasma glucose concentrations were not different between the two groups. While there was a trend for increased peripheral insulin, this did not reach statistical significance (16 ± 4 versus 31 ± 6 microunits/ml, p = 0.08, n = 10 per group). There were no differences in portal insulin (46 ± 16 versus 63 ± 16 microunits/ml) or portal glucagon concentration (81 ± 8 versus 79 ± 13 pg/ml). Plasma leptin concentration in the FF group increased in a similar fashion to what has previously been reported (1.2 ± 0.14 versus 2.2 ± 0.2 ng/ml, p = 0.002) (17Wang J. Obici S. Morgan K. Barzilai N. Feng Z. Rossetti L. Diabetes. 2001; 50: 2786-2791Google Scholar). Adiponectin concentrations were not significantly different between the two groups (2658 ± 283 versus 3302 ± 279 ng/ml, p = 0.12).Table ICharacteristics of rats fed 3 days of a control or high fat dietControlFat-fedDNP-treatedWeight (g)332 ± 3329 ± 4321 ± 6Weight gain (g)23 ± 122 ± 215 ± 2ap = 0.008 versus Control.,bp = 0.019 versus Fat-fed by Bonferroni's t test.Caloric consumption (kcal/kg-d)354 ± 11345 ± 9.6321 ± 10Glucose (mg/dl)120 ± 3114 ± 2112 ± 3a p = 0.008 versus Control.b p = 0.019 versus Fat-fed by Bonferroni's t test. Open table in a new tab Plasma fatty acid concentration was measured at several points throughout the day (Table II). Immediately after food withdrawal, both peripheral and portal FA concentration were elevated in the fat-fed group. Thereafter, a 4 h, 8 h, and after an overnight fast, plasma fatty acid concentration in the peripheral blood was identical between the two groups. Surprisingly, the portal FA concentration after an overnight fast was nearly 50% lower in the fat-fed group compared with the control group. In addition, mesenteric weight, an indicator of visceral fat stores, was unchanged (2.80 ± 0.32 versus 2.96 ± 0.20, p = 0.49).Table IIFatty acid concentration in during fasting and hyperinsulinemic-euglycemic clampTime after food removalControlFat-fedpPeripheral blood0 h0.15 ± 0.030.47 ± 0.080.014 h0.22 ± 0.030.26 ± 0.030.318 h0.21 ± 0.030.26 ± 0.010.14Overnight0.74 ± 0.070.69 ± 0.070.57Portal blood0 h0.21 ± 0.040.48 ± 0.050.0064h0.31 ± 0.030.30 ± 0.050.938h0.25 ± 0.030.32 ± 0.030.13Overnight1.20 ± 0.030.61 ± 0.110.02 Open table in a new tab As shown in Fig. 1a, hepatic triglyceride content was increased in the fat-fed rats after 3 days of high fat feeding (4.9 ± 0.8 versus 16.2 ± 2.5 mg/g liver, p = 0.004). In contrast, there was no change in muscle triglyceride content (1.5 ± 0.2 versus 1.8 ± 0.2 mg/g muscle). Total fatty acyl-CoA concentrations were measured by LC/MS/MS (Fig. 1b). Fat feeding results in a 3-fold elevation in hepatic total fatty acyl-CoA (58.7 ± 4.5 versus 163.2 ± 23.2 nmol/g, p = 0.004) in the liver without a significant change in the muscle (12.4 ± 0.9 versus 20.8 ± 4.4, p = 0.11). Analysis of the species of fatty acyl-CoA revealed the major species in the tissues reflected the major dietary species (18:2 fatty acid or linoleic acid). DNP Therapy for Fat-fed Animals—2,4-Dinitrophenol, has been used to promote fat oxidation by increasing energy expenditure through mitochondrial uncoupling (18Harper J.A. Dickinson K. Brand M.D. Obes. Rev. 2001; 2: 255-265Google Scholar). We used this agent as a pharmacological tool to prevent hepatic fat accumulation in fat-fed rats and examined whether or not this would prevent the development of hepatic insulin resistance. Separate groups of rats were subjected to either 3 days of fat feeding with 0.3 mg/g 2,4-dinitrophenol (16 mg/kg/day). Previous studies have shown that at doses below 20 mg/kg/day, no adverse affects have been observed (19Koizumi M. Yamamoto Y. Ito Y. Takano M. Enami T. Kamata E. Hasegawa R. J. Toxicol. Sci. 2001; 26: 299-311Google Scholar). Compared with the control and fat-fed group, total weight gain over the 3 days was reduced by 30% in the DNP-treated group. There was no difference in intrahepatic ATP content, as assessed by 31P magnetic resonance spectroscopy. Analysis of liver fatty acyl-CoA content showed that DNP treatment in fat-fed rats prevented an increase in hepatic fat content (Fig. 2a). Plasma FA concentrations from both peripheral and portal samples were not different from the fat-fed animals (0.76 ± 0.08 and 0.78 ± 0.08 mm for peripheral and portal samples, respectively). Assessment of Peripheral Insulin Action—In order to assess the effects of 2,4-DNP on peripheral glucose metabolism 2-deoxyglucose uptake was performed in isolated soleus muscle strips and in vivo during hyperinsulinemic-euglycemic clamp conditions. There was no difference in basal 2-deoxyglucose uptake between any groups. Insulin increased the uptake of 2-deoxyglucose over the basal state similarly in soleus strips for all groups (fold increase over basal: 1.47 ± 0.44 versus 1.62 ± 0.30 versus 2.01 ± 0.56, for control, fat-fed, and DNP, respectively. ANOVA p = 0.56). There was no significant difference in 2-deoxyglucose uptake in the gastrocnemius muscle between control and fat-fed rats during the hyperinsulinemic-euglycemic clamp (71.7 ± 11 versus 48.0 ± 11 nmol/g/min, p = 0.17). DNP treatment did increase 2-deoxyglucose uptake as compared with the fat-fed rats but not the control rats (106.3 ± 11.9 nmol/g/min, p = 0.005 versus fat fed, p = 0.06 versus cont). Neither fat feeding nor DNP treatment altered epididymal adipose tissue 2-deoxyglucose uptake during hyperinsulinemic-euglycemic clamps (10.3 ± 1.8 versus 17.0 ± 1.7 versus 12.0 ± 2.5 nmol/g/min for control, fat-fed, and DNP, respectively, ANOVA p = 0.10). In addition, during the hyperinsulinemic-euglycemic clamp, FA levels were suppressed to an equal degree in all three groups (0.35 ± 0.12 versus 0.36 ± 0.02 versus 0.29 ± 0.04 mm for control, fat-fed, and DNP-treated respectively). Hyperinsulinemic-Euglycemic Clamp—During the hyperinsulinemic-euglycemic clamp, insulin-stimulated peripheral glucose metabolism was similar between the control and fat-fed groups (23.2 ± 1.3 versus 25.4 ± 1.3 mg/kg/min, p = 0.27, Fig. 2b). Basal endogenous glucose production was similar in the control and fat-fed group (4.7 ± 0.4 versus 5.4 ± 0.4 mg/kg/min, p = 0.31). In contrast, insulin suppression of endogenous glucose production was impaired in the fat-fed group compared with the control group (74 ± 18% versus 8 ± 3%, p < 0.001) (Fig. 2c). DNP treatment did not affect either insulin-stimulated whole body glucose metabolism (22.6 ± 1.2 mg/kg/min) or basal endogenous glucose production (4.8 ± 0.3 mg/kg/day) (Fig. 2, b and c). However, the ability of insulin to suppress endogenous glucose production was improved in the DNP animals (8 ± 3% versus 39 ± 12%, p = 0.016, Fig. 2c). Furthermore, a positive linear correlation between liver triglyceride content and clamped EGP was observed (r2 = 0.52, Fig. 2d). Effect of Fat Feeding on Insulin Signaling Pathway—To determine the mechanism of fat-induced hepatic insulin resistance, the insulin signaling cascade was dissected into its key components. Activation of IR, IRS-1, and IRS-2 was determined by measuring the degree of tyrosine phosphorylation. The activity of the downstream kinases, IRS-2-associated PI 3-kinase, AKT2, and GSK3 were measured directly by immunoprecipitating the appropriate kinase and quantifying the phosphorylation on the target substrate. The results are reported as insulin-stimulated values compared with unstimulated values. As shown in Fig. 3a the increase in IR tyrosine phosphorylation was equal in both groups. However, the increase in both IRS-1 and IRS-2 tyrosine phosphorylation was diminished in the fat-fed group. (Fig. 3, b and c). This block in IRS tyrosine phosphorylation was reflected in diminished activation of IRS-associated PI 3-kinase. Both IRS-1- and IRS-2-associated PI 3-kinase activity increased with insulin in the control animals but was unchanged in the fat-fed animals (Fig. 3, d and e). This defect in insulin-stimulated IRS-1 and IRS-2 PI 3-kinase activity was prevented by DNP treatment in the fat-fed animals. This proximal block in the signaling cascade was propagated in the downstream signaling kinases. AKT2 activity increased 3.2 ± 0.15-fold with insulin stimulation in the control rats versus 1.0 ± 0.06-fold in the fat-fed rats (Fig. 4a). Again, this block in insulin-stimulated AKT2 activation was prevented with DNP treatment (fold increase 5.4 ± 1.1). Glycogen synthase kinase 3 (GSK-3) tonically phosphorylates and inactivates glycogen synthase. When it is phosphorylated by AKT2 it is inactivated. Thus the net effect would be to allow dephosphorylation and activation of glycogen synthase. GSK-3 activity was decreased to a greater extent in the control animals than in the fat-fed animals (Fig. 4b). DNP treatment in fat-fed animals maintained the ability of insulin to deactivate GSK-3.Fig. 4Alterations in downstream signaling kinases associated with hepatic fat accumulation.a, fold change in AKT2 activity. b, percent change in GSK3 activity. Values represent mean ± S.E. of 4–5 animals. *, p < 0.05 versus control; #, p < 0.01 versus control; and ‡, p < 0.005 versus fat-fed by Bonferroni's t test.View Large Image Figure ViewerDownload (PPT) Effect of Fat Feeding on Glycogen Synthase and Glycogen Synthesis—The activity of GS in liver homogenates was measured in the basal and insulin-stimulated state. As shown in Fig. 5a, the ability of insulin to increase GS activity was diminished in the fat-fed animals. Insulin increased total GS activity ∼4.7 ± 0.5-fold in the control animals, but only by 2.4 ± 0.2-fold in the fat-fed animals (p = 0.002). Glycogen synthesis was also assessed in vivo during a hyperinsulinemic-euglycemic clamp by comparing the enrichment of [U-13C]glucose in plasma versus glycogen. As shown in Fig. 5b, the percent of glycogen synthesized via the direct pathway was 28% in the control compared with 11% in the fat-fed group (p = 0.04) consistent with increased gluconeogenesis in the fat-fed animals. Effect of Fat Feeding on Protein Kinase C Activity—Activation the novel PKC (PKC-θ,-δ, and -βII) has been implicated in the pathogenesis of peripheral insulin resistance in rodents and humans (10Griffin M.E. Marcucci M.J. Cline G.W. Bell K. Barucci N. Lee D. Goodyear L.J. Kraegen E.W. White M.F. Shulman G.I. Diabetes. 1999; 48: 1270-1274Google Scholar, 20Itani S.I. Ruderman N.B. Schmieder F. Boden G. Diabetes. 2002; 51: 2005-2011Google Scholar). We assessed the activities of the major hepatic isoforms of PKC (α, β, δ, ϵ, and ζ) to determine if a similar activation occurred in the liver. Measuring the relative abundance of the particular PKC isoform in the membrane and cytosol fractions reflected PKC activation. An increase in the membrane to cytosol fraction was taken as an indication of PKC activation. As shown in Fig. 6, a and b, PKC-ϵ was most activated as a result of hepatic fat accumulation. The results of the membrane translocation assay were confirmed using a direct assay of PKC-ϵ activity (Fig. 6c). DNP treatment in the fat-fed animals prevented PKC-ϵ membrane translocation and prevented the increase in PKC-ϵ activity. This also suggests that PKC-ϵ activation may be linked to hepatic fat accumulation. As PK