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

Abstract

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 They were then for min in either or in to the with the of and 2-Deoxyglucose Uptake in of 2-deoxyglucose was as described previously N. D. Kraegen E.W. Shulman G.I. Diabetes. 1999; Scholar). overnight fasted rats were subjected to a hyperinsulinemic-euglycemic as described in the After of was as a Plasma was at and min to determine and plasma glucose and were in situ with tongs precooled in liquid nitrogen and stored at –80 2-Deoxyglucose uptake was on the 2-deoxyglucose content and the plasma 2-deoxyglucose under the was extracted from tissues by in of by at for The and were by of and at for min. The was completely and in of A was and The triglyceride concentration in this was determined using the triglyceride The of the fatty acyl-CoA concentrations was done as described previously C. Chen Y. D. Y. J.K. B. Kraegen E.W. Shulman G.I. J. Biol. Chem. 2002; Scholar). group of rats were used to the impact of hepatic fat accumulation on the insulin signaling These rats were as and a hyperinsulinemic-euglycemic without a basal were harvested in situ at the end of the clamp. samples harvested in situ in conditions at the end of the were used to IR, and IRS-2 tyrosine C. Chen Y. D. Y. J.K. B. Kraegen E.W. Shulman G.I. J. Biol. Chem. 2002; Scholar) IRS-1 and J. Biol. Chem. M. B.A. P. and P. J. Scholar). used for these were obtained from of tyrosine the was with it was and with the used for to any in total protein IR, or synthase was determined in basal and insulin-stimulated using previously described Scholar). and was as described previously J. 1999; Scholar). of and protein were by using and MA) using a The was then for 2 h at in and 0.1% and then overnight with in After were with of in for 2 was as the ratio of of In addition, and were measured from using as and from Laboratories, CA). and mg of liver tissues were with buffer mm mm mm 2 mm mm mm 2 mm and mg of was with protein for h at °C with or were and at °C overnight with were by with protein with of in and using of and of was linked to using the This was then with 2 mg of at After with the were in with The the of the and was used for were to MA) and with the by detection using was measured using the After was to the was with to determine the of the The was then to the of in the and muscle were harvested in situ using tongs in liquid was stored at –80 The was extracted using CA). were analyzed by using for and and to obtained on a were analyzed using the with of glucose uptake and basal glucose were determined as the ratio of the [U-13C]glucose infusion to the atom percent enrichment of glucoseM+6 steady state of the basal and glucose production (EGP) the was determined by the glucose infusion from glucose are as the S.E. A was to determine between the control and was accepted at between was by and were in between control and high animals Plasma glucose concentrations were not between the While there was a for increased peripheral this not significance 10 were in insulin or concentration Plasma concentration in the FF group increased in a to has previously been reported J. S. N. Diabetes. 2001; Scholar). concentrations were not between the of rats fed 3 days of a control or high fat by consumption by in a Plasma fatty acid concentration was measured at the day food peripheral and concentration were elevated in the a and an overnight fast, plasma fatty acid concentration in the peripheral was between the the concentration an overnight was in the group with the control In addition, an of visceral fat was acid concentration in and hyperinsulinemic-euglycemic food in a in hepatic triglyceride content was increased in the rats 3 days of high fat feeding liver, In contrast, there was in muscle triglyceride content fatty acyl-CoA concentrations were measured by feeding results in a in hepatic total fatty acyl-CoA in the liver without a significant in the muscle of the of fatty acyl-CoA the major in the tissues the major fatty acid or for has been used to promote fat by increasing energy expenditure through mitochondrial 2001; Scholar). We used this as a to prevent hepatic fat accumulation in rats and or not this would prevent the development of hepatic insulin resistance. of rats were subjected to either 3 days of fat feeding with 2,4-dinitrophenol have that at have been M. Y. Y. M. J. Sci. 2001; Scholar). with the control and group, total the 3 days was by in the was in as by of liver fatty acyl-CoA content that in rats an increase in hepatic fat content Plasma concentrations from peripheral and samples were not from the animals and mm for peripheral and of to the effects of on peripheral glucose metabolism 2-deoxyglucose uptake was in isolated soleus muscle strips and in hyperinsulinemic-euglycemic was in basal 2-deoxyglucose uptake between any increased the uptake of 2-deoxyglucose the basal state in soleus strips for all increase for and was significant in 2-deoxyglucose uptake in the muscle between control and rats the hyperinsulinemic-euglycemic increase 2-deoxyglucose uptake as with the rats not the control rats fat fat feeding 2-deoxyglucose uptake hyperinsulinemic-euglycemic for and In addition, the hyperinsulinemic-euglycemic clamp, were to an in all mm for and Hyperinsulinemic-Euglycemic the hyperinsulinemic-euglycemic clamp, insulin-stimulated peripheral glucose metabolism was between the control and endogenous glucose production was in the control and group In contrast, insulin of endogenous glucose production was impaired in the group with the control group not either insulin-stimulated glucose metabolism mg/kg/min) or basal endogenous glucose production and However, the ability of insulin to endogenous glucose production was in the animals a between liver triglyceride content and EGP was of on determine the mechanism of fat-induced hepatic insulin resistance, the insulin signaling was dissected into of IR, and IRS-2 was determined by the of tyrosine phosphorylation. The of the and were measured by the kinase and the phosphorylation on the The results are reported as insulin-stimulated with in the increase in tyrosine phosphorylation was in However, the increase in IRS-1 and IRS-2 tyrosine phosphorylation was diminished in the and This in tyrosine phosphorylation was in diminished activation of and increased with insulin in the control animals was in the animals and This in insulin-stimulated IRS-1 and IRS-2 was by in the This in the signaling was in the signaling increased with insulin in the control rats in the rats this in insulin-stimulated activation was with increase synthase kinase 3 and glycogen synthase. it is by it is the effect would be to and activation of glycogen synthase. was decreased to a in the control animals in the animals in animals the ability of insulin to in signaling associated with hepatic fat in percent in S.E. of and by of on and of in liver homogenates was measured in the basal and insulin-stimulated in the ability of insulin to increase was diminished in the increased total in the control by in the animals synthesis was in a hyperinsulinemic-euglycemic by the enrichment of [U-13C]glucose in plasma glycogen. in the percent of glycogen the was in the control with in the group with increased gluconeogenesis in the of on the and has been in the of peripheral insulin resistance in and N. D. Kraegen E.W. Shulman G.I. Diabetes. 1999; Scholar, G. Diabetes. 2002; Scholar). We the of the major hepatic of and to determine if a activation in the the of the in the and increase in the to was taken as an of in a and PKC-ϵ was as a of hepatic fat accumulation. The results of the were using a of PKC-ϵ in the animals PKC-ϵ and the increase in PKC-ϵ This that PKC-ϵ activation may be linked to hepatic fat accumulation. has been previously to be in the development of hepatic insulin resistance, was measured as A. Am. J. 2002; Scholar). In to the activation of there was in in control or animals as has been in the of fat muscle insulin resistance, were in with the in of were we could not any significant in These results the not a major in the development of hepatic insulin resistance. of on the N-terminal kinase has been to a in the of fat-induced insulin resistance J. G. M. 2002; Scholar). The of in was a in fat feeding in a increase in This activation was by is a which has phosphorylation on IRS-1 and In a was to to IRS-1 and in control and rats and has previously been as a for phosphorylation. in phosphorylation was between control and rats Although an between NAFLD and hepatic insulin resistance is a between hepatic fat accumulation and hepatic insulin resistance has not been In this we provide to support the between hepatic fat accumulation and hepatic insulin resistance. we a between hepatic fat accumulation and hepatic insulin resistance. we that hepatic fat accumulation the development of hepatic insulin resistance. we a between fat accumulation and insulin resistance. we provide to a mechanism hepatic fat accumulation can lead to hepatic insulin resistance. The of the current model is the increase in hepatic fat content without significant of peripheral fat content. days of high fat feeding hepatic and fatty acyl-CoA content. The in fat between the liver and diet, an of fatty that the of the liver fat is the diet. This of was in the days of high fat feeding not plasma glucose concentration or the basal of glucose was in that peripheral insulin was In contrast, short term high fat feeding in hepatic insulin in the control group EGP was by by in the this model allowed to the impact of hepatic fat accumulation on hepatic insulin without the confounding of peripheral fat accumulation. The between fatty acids and hepatic insulin has been previously using infusion of to fatty acids C. V. M. Am. J. Scholar, Diabetes. 1995; Scholar, G. A. Am. J. Scholar). These that the defects in hepatic glucose metabolism are a of insulin resistance in the visceral In the study, fat feeding elevated plasma fatty acids in the At and an overnight fast, the concentration was in the control and an overnight fast, concentration was in the animals the control insulin was by fat feeding as by 2-deoxyglucose uptake and of plasma the hyperinsulinemic-euglycemic clamp. was by fat that visceral fat were in this hepatic insulin resistance without any insulin resistance. hepatic fat accumulation is for the development of hepatic insulin resistance, then fat accumulation in rats prevent hepatic insulin resistance. This was by using a dose of the mitochondrial the mitochondrial and the of the as We that low of would increase energy expenditure and prevent hepatic fat accumulation in rats subjected to the high fat diet. The concentration of to the high fat diet in an dose of on a study of in M. Y. Y. M. J. Sci. 2001; Scholar). Although there was significant in food there was a in of However, the of had effect on had on peripheral insulin of rats the accumulation of fat and fatty acid the in rats the hepatic insulin as by the ability to EGP the hyperinsulinemic-euglycemic clamp. there was a between hepatic fat content and hepatic insulin as by EGP the hyperinsulinemic-euglycemic clamp. These results that hepatic fat accumulation in rats on a high fat diet hepatic insulin resistance. We the insulin signaling to determine the mechanism hepatic fat accumulation hepatic insulin Although there was effect of fat feeding on insulin receptor tyrosine insulin-stimulated IRS-1 and IRS-2 tyrosine phosphorylation was in the insulin activation of and of was impaired in the in animals the development of this in the insulin signaling and insulin-stimulated activation and hepatic fat accumulation to be specifically linked to the development of this impaired insulin signaling in Ultimately, the in the signaling insulin activation of glycogen synthase with a insulin-stimulated increase in in the control group, increased by in the synthesis was in using to determine the of the and of glycogen The percent of glycogen the was in the control group in the group that hepatic steatosis is associated with increased gluconeogenesis. The mechanism for this increase is of the and was not by fat feeding not The increase could be to either activation or of of these or increased through this by the of S. G. Shulman G. Am. J. 1999; Scholar). these data that hepatic fat accumulation is to increase EGP that it hepatic insulin resistance. This can be attributed in to decreased insulin-stimulated tyrosine phosphorylation of IRS-1 and which in the ability of insulin to activate glycogen synthase and the ability of the liver to glucose as glycogen. In addition, hepatic steatosis was associated with increased gluconeogenesis. This may maintain EGP under that higher insulin concentrations are to gluconeogenesis with J.L. J.E. Diabetes. Scholar). have activation of the in the of skeletal muscle insulin resistance in N. D. Kraegen E.W. Shulman G.I. Diabetes. 1999; Scholar, C. Chen Y. D. Y. J.K. B. Kraegen E.W. Shulman G.I. J. Biol. Chem. 2002; Scholar) and and G. Diabetes. 2002; Scholar). in of all the a PKC, to be the In the that activation of PKC-ϵ that that activation is specifically linked to hepatic et al. A. Am. J. 2002; Scholar) have as a possible for fat-induced hepatic insulin resistance A. Am. J. 2002; Scholar). to a rats to plasma and peripheral and hepatic insulin resistance, as with the increase in level and the development of peripheral insulin resistance this model from the in this study and In the current study, there was increased of increased of from on the data we that hepatic PKC-ϵ is by accumulation of an fatty acid and may in the of hepatic insulin resistance. This may be to the in the of peripheral insulin resistance. possible of PKC-ϵ is the N-terminal kinase a of the protein in and that is by in response to and D. A. V. C. Biol. 2001; Scholar, N. W. C. J. Biol. Chem. 1999; Scholar). was to a in the of fat-induced insulin resistance, caused by phosphorylation of IRS-1 J. G. M. 2002; Scholar, J. J. Biol. Chem. Scholar). in liver was increased in the was to and in control and this and the increased with fat we were to an increase in phosphorylation. Although have increased hepatic phosphorylation J. G. M. 2002; Scholar, G. S. J. the models used are the in that are models of insulin resistance the and the phosphorylation of may be a in the of insulin resistance that is not in the described hepatic fat accumulation to be associated with increased the for this kinase PKC-ϵ and activation were by that have a between hepatic fat accumulation and hepatic insulin resistance. to be to determine the exact of of in the of fat hepatic insulin resistance. In conclusion, the data in this support a for hepatic fat accumulation in the of hepatic insulin resistance. days of high fat feeding specifically hepatic fat accumulation and hepatic insulin resistance in the of significant peripheral fat accumulation or peripheral insulin resistance. These changes were not associated with any in visceral fat mass or vein fatty acid hepatic insulin resistance may from activation of PKC-ϵ JNK1, which may then lead to impaired IRS-1 and IRS-2 tyrosine phosphorylation. This in the insulin signaling then the ability of insulin to activate glycogen synthase. In addition, fat accumulation the of gluconeogenesis to total mitochondrial with low dose in animals hepatic fat accumulation and activation of PKC-ϵ and JNK1. This in the insulin signaling and the development of hepatic insulin resistance. These support the between hepatic fat accumulation and hepatic insulin resistance. We and Chen for with the We for the