Modulation of the JNK Pathway in Liver Affects Insulin Resistance Status

The c-Jun N-terminal kinase (JNK) pathway is known to be activated under diabetic conditions and to possibly be involved in the progression of insulin resistance. In this study, we examined the effects of modulation of the JNK pathway in liver on insulin resistance and glucose tolerance. Overexpression of dominant-negative type JNK in the liver of obese diabetic mice dramatically improved insulin resistance and markedly decreased blood glucose levels. Conversely, expression of wild type JNK in the liver of normal mice decreased insulin sensitivity. The phosphorylation state of crucial molecules for insulin signaling was altered upon modification of the JNK pathway. Furthermore, suppression of the JNK pathway resulted in a dramatic decrease in the expression levels of the key gluconeogenic enzymes, and endogenous hepatic glucose production was also greatly reduced. Similar effects were observed in high fat, high sucrose diet-induced diabetic mice. Taken together, these findings suggest that suppression of the JNK pathway in liver exerts greatly beneficial effects on insulin resistance status and glucose tolerance in both genetic and dietary models of diabetes. The c-Jun N-terminal kinase (JNK) pathway is known to be activated under diabetic conditions and to possibly be involved in the progression of insulin resistance. In this study, we examined the effects of modulation of the JNK pathway in liver on insulin resistance and glucose tolerance. Overexpression of dominant-negative type JNK in the liver of obese diabetic mice dramatically improved insulin resistance and markedly decreased blood glucose levels. Conversely, expression of wild type JNK in the liver of normal mice decreased insulin sensitivity. The phosphorylation state of crucial molecules for insulin signaling was altered upon modification of the JNK pathway. Furthermore, suppression of the JNK pathway resulted in a dramatic decrease in the expression levels of the key gluconeogenic enzymes, and endogenous hepatic glucose production was also greatly reduced. Similar effects were observed in high fat, high sucrose diet-induced diabetic mice. Taken together, these findings suggest that suppression of the JNK pathway in liver exerts greatly beneficial effects on insulin resistance status and glucose tolerance in both genetic and dietary models of diabetes. The hallmark of type 2 diabetes is insulin resistance and pancreatic β-cell dysfunction (1Shulman G.I. J. Clin. Investig. 2000; 106: 171-176Crossref PubMed Scopus (2215) Google Scholar, 2Saltiel A.R. Kahn C.R. Nature. 2001; 414: 799-806Crossref PubMed Scopus (4066) Google Scholar). Normal β-cells can compensate for insulin resistance by increasing insulin secretion, but insufficient compensation leads to the onset of glucose intolerance. Once hyperglycemia becomes apparent, insulin sensitivity is further reduced, and β-cell function progressively deteriorates (3Weir G.C. Laybutt D.R. Kaneto H. Bonner-Weir S. Sharma A. Diabetes. 2001; 50: S154-S159Crossref PubMed Google Scholar). Various studies have demonstrated that hyperglycemia is the direct cause of these phenomena, collectively called “glucose toxicity” (4Robertson R.P. Zhang H.-J. Pyzdrowski K.L. Walseth T.F. J. Clin. Investig. 1992; 90: 320-325Crossref PubMed Scopus (197) Google Scholar, 5Sharma A. Olson L.K. Robertson R.P. Stein R. Mol. Endocrinol. 1995; 9: 1127-1134Crossref PubMed Google Scholar, 6Tokuyama Y. Sturis J. DePaoli A.M. Takeda J. Stoffel M. Tang J. Sun X. Polonsky K.S. Bell G.I. Diabetes. 1995; 44: 1447-1457Crossref PubMed Google Scholar, 7Zangen D.H. Bonner-Weir S. Lee C.H. Latimer J.B. Miller C.P. Habener J.F. Weir G.C. Diabetes. 1997; 46: 258-264Crossref PubMed Scopus (127) Google Scholar, 8Jonas J.-C. Sharma A. Hasenkamp W. Iikova H. Patane G. Laybutt R. Bonner-Weir S. Weir G.C. J. Biol. Chem. 1999; 274: 14112-14121Abstract Full Text Full Text PDF PubMed Scopus (485) Google Scholar). It has been reported that the activity of c-Jun N-terminal kinase (JNK) 1The abbreviations used are: JNK, c-Jun N-terminal kinase; WT, wild type; DN, dominant-negative type; Ad, adenovirus; GFP, green fluorescent protein; PFU, plaque-forming unit; GIR, glucose infusion rate; HGP, hepatic glucose production; IRS-1, insulin receptor substrate-1; PEPCK, phosphoenolpyruvate carboxykinase; FFA, free fatty acid; TNF-α, tumor necrosis factor-α; IPGTT, intraperitoneal glucose tolerance test; MOPS, 4-morpholinepropanesulfonic acid. (9Hibi M. Lin A. Smeal T. Minden A. Karin M. Genes Dev. 1993; 7: 2135-2148Crossref PubMed Scopus (1724) Google Scholar, 10Derijard B. Hibi M. Wu I.-H. Barrett T. Su B. Deng T. Karin M. Davis R.J. Cell. 1994; 76: 1025-1037Abstract Full Text PDF PubMed Scopus (2994) Google Scholar, 11Kyriakis J.M. Avruch J. J. Biol. Chem. 1996; 271: 24313-24316Abstract Full Text Full Text PDF PubMed Scopus (1028) Google Scholar, 12Davis R. Cell. 2000; 103: 239-252Abstract Full Text Full Text PDF PubMed Scopus (3701) Google Scholar, 13Chang L. Karin M. Nature. 2001; 410: 37-40Crossref PubMed Scopus (4472) Google Scholar), which is known to be activated by various stress signals such as cytokines or oxidative stress, is abnormally elevated in various tissues under diabetic conditions (14Hirosumi J. Tuncman G. Chang L. Gorgun C.Z. Uysal K.T. Maeda K. Karin M. Hotamisligil G.S. Nature. 2002; 420: 333-336Crossref PubMed Scopus (2707) Google Scholar, 15Liu W. Schoenkerman A. Lowe Jr., W.L. Am. J. Physiol. 2000; 279: E782-E790Crossref PubMed Google Scholar, 16Purves T. Middlemas A. Agthong S. Jude E.B. Boulton A.J. Fernyhough P. Tomlinson D.R. FASEB J. 2001; 15: 2508-2514Crossref PubMed Scopus (229) Google Scholar) and that activation of the JNK pathway interferes with insulin action (14Hirosumi J. Tuncman G. Chang L. Gorgun C.Z. Uysal K.T. Maeda K. Karin M. Hotamisligil G.S. Nature. 2002; 420: 333-336Crossref PubMed Scopus (2707) Google Scholar, 17Aguirre V. Uchiuda T. Yenush L. Davis R. White M.F. J. Biol. Chem. 2000; 275: 9047-9054Abstract Full Text Full Text PDF PubMed Scopus (1199) Google Scholar). Indeed, it was shown that insulin resistance is substantially decreased in mice homozygous for a targeted mutation in the JNK gene (14Hirosumi J. Tuncman G. Chang L. Gorgun C.Z. Uysal K.T. Maeda K. Karin M. Hotamisligil G.S. Nature. 2002; 420: 333-336Crossref PubMed Scopus (2707) Google Scholar). Furthermore, we reported recently that JNK activation is involved in the reduction of insulin gene expression by oxidative stress and that suppression of the JNK pathway can protect β-cells from glucose toxicity (18Kaneto H. Xu G. Fujii N. Kim S. Bonner-Weir S. Weir G.C. J. Biol. Chem. 2002; 277: 30010-30018Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar). Thus, it is likely that JNK is a crucial mediator of the progression of insulin resistance as well as β-cell dysfunction found in type 2 diabetes. Here we report that suppression of the JNK pathway in the liver improves insulin resistance in the whole body and markedly ameliorates glucose intolerance in diabetic mice. Preparation of Recombinant Adenoviruses—Recombinant adenoviruses expressing wild type (WT) and dominant-negative type (DN) JNK were prepared using the AdEasy system (kindly provided by Dr. Bert Vogelstein, the Johns Hopkins Oncology Center) (33He T.-C. Zhou S. DaCosta L.T. Yu J. Kinzler K.W. Vogelstein B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2509-2514Crossref PubMed Scopus (3276) Google Scholar). In brief, the encoding region of WT- and DN-JNK (K55R) was cloned into a shuttle vector pAdTrack-CMV, and to allow for homologous recombination, the linearized plasmid containing WT- or DN-JNK and the adenoviral backbone plasmid, pAdEasy-1, were introduced into electrocompetent Escherichia coli BJ5183 cells. The linearized plasmids were transfected into the adenovirus packaging cell line 293 using LipofectAMINE (Invitrogen), and the adenovirus titers were increased up to 1 × 108 plaque-forming units (PFU)/ml in the 293 cells. Control adenovirus expressing green fluorescent protein (Ad-GFP) was prepared in the same manner. Adenovirus titers were further increased up to 1 × 1010 PFU/ml using the Adeno-X™ virus purification kit (Clontech). Virus titers were estimated using the Adeno-X™ titer kit (Clontech). Animals and Administration of Recombinant Adenoviruses—Male C57BL6 and C57BL/KsJ-db/db mice were purchased from Japan SLC (Hamamatsu, Japan). Mice (8 weeks old) were injected with Ad-WT-JNK, Ad-DN-JNK, or Ad-GFP (1 × 1010 PFU/ml for Ad-WT-JNK and 2 × 109 PFU/ml for Ad-DN-JNK) from the cervical vein. After adenovirus injection, blood glucose levels were measured regularly with a portable glucose meter (Glu-test Sensor, Sanwa, Japan) after tail snipping. For measurement of serum insulin levels, blood samples of mice after a 6-h fast were collected into heparinized capillary tubes, and serum insulin levels were determined with the Insulin-EIA test kit (Glazyme). Glucose Tolerance Tests—After a 6-h fast, mice were injected intraperitoneally with glucose (2.0 g/kg of body weight). Blood samples were taken at various time points (0–120 min), and blood glucose levels and serum insulin levels were determined as described above. Insulin Tolerance Tests—After a 6-h fast, mice were injected intraperitoneally with insulin (2.0 units/kg for C57BL/KsJ-db/db mice). Blood samples were taken at various time points (0–90 min), and blood glucose levels were measured as described above. Euglycemic Hyperinsulinemic Clamp—Fourteen days before the clamp study, Ad-WT-JNK (1 × 1010 PFU/ml) or Ad-DN-JNK (2 × 109 PFU/ml) was injected from the left jugular vein. Three days before the clamp study, a silicon catheter (Phicon Tube, Fuji-Systems, Tokyo, Japan) was inserted into the right jugular vein under general anesthesia with sodium pentobarbital. The catheter, which is required for infusion in the clamp study, was exteriorized at the back of the neck through a subcutaneous tunnel and filled with heparinized saline (200 units/ml). Clamp studies were performed on mice under conscious and unstressed conditions after a 6-h fast. A euglycemic hyperinsulinemic clamp with a tracer dilution method was applied to determine peripheral glucose uptake and endogenous glucose production. Experiments consisted of a 120-min euglycemic hyperinsulinemic clamp period (15 pmol of regular human insulin/kg/min for C57BL6 mice and 27 pmol/kg/min for C57BL/KsJ-db/db mice during the 120-min clamp period). During this period, blood glucose levels were monitored every 5 min, and the rate of 50% glucose containing 10% 6,6-2H2glucose infusion into the jugular vein was adjusted to maintain blood glucose concentrations at 120 ± 10 mg/dl. Measurement of Endogenous Hepatic Glucose Production (HGP) by Stable Isotope-labeled Glucose Enrichment—To estimate HGP, stable isotope-labeled glucose enrichment was determined. Blood samples were taken at 90, 105, and 120 min, and 20 μl of each plasma sample were deproteinized with 60 μl of 99.5% ethanol. The supernatant was evaporated, and the residue was derivatized by the following procedure. First, 7.5 μl of N-methyl-bis(trifluoroacetamide) (MBTFA, Pierce) and 7.5 μl of pyridine were added to the residue, and the mixture was heated for 1 h at 60 °C. The reaction product (1 μl) containing trifluoroacetylated glucose was then analyzed by gas chromatography and mass spectrometry (model TSQ-700, Finningan-MAT, San Jose, CA) with a silicon SE-30 capillary column (30 m × 0.25 mm inner diameter, Gasukuro Kogyo, Tokyo, Japan). The trifluoroacetyl derivative of glucose was separated from the other compounds by gas chromatography and was analyzed by electron impact mass spectrometry at 70 eV. The fragment ion peaks of unlabeled glucose and 6,6-2H2glucose were measured at a mass/electrical charge (m/e) of 319 and 321, respectively. Western Blot Analysis—Whole cell extracts obtained from liver were fractionated by 10% SDS-PAGE and transferred to a reinforced cellulose nitrate membrane (Optitran BA-S85, Schleicher 420: 333-336Crossref PubMed Scopus (2707) Google Scholar, 17Aguirre V. Uchiuda T. Yenush L. Davis R. White M.F. J. Biol. Chem. 2000; 275: 9047-9054Abstract Full Text Full Text PDF PubMed Scopus (1199) Google Scholar). the involved in the of insulin the JNK we phosphorylation in the liver of C57BL6 mice. shown in phosphorylation was increased in mice with mice. also found a decrease in phosphorylation in mice with mice. Furthermore, a decrease in phosphorylation was observed in C57BL6 mice with mice. we examined the involved in the reduction of insulin resistance by DN-JNK in the shown in phosphorylation was markedly decreased in mice with mice. also found an in phosphorylation in mice with mice. of phosphorylation was observed in C57BL/KsJ-db/db mice with mice. an in phosphorylation be with the of insulin resistance by JNK we examined the expression levels of the key gluconeogenic enzymes, and both of which are known to be by insulin shown in expression levels of both enzymes, and PEPCK, were increased by Ad-WT-JNK in C57BL6 mice. In expression levels of both were markedly decreased by Ad-DN-JNK in C57BL/KsJ-db/db mice. that suppression of the JNK pathway insulin which leads to a decrease in and of glucose tolerance. of the JNK in the Insulin and Glucose it is well known that a high and high sucrose diet insulin we fed C57BL6 mice a high fat, high sucrose diet and examined suppression of the JNK pathway has on high fat, high sucrose diet-induced insulin resistance. was in body and between the two groups shown in blood glucose weeks after the in the mice. weeks after the high fat, high sucrose blood glucose and insulin concentrations were in mice with mice blood glucose of ± ± insulin of ± ± further this we performed the shown in blood glucose levels in mice were with of mice. that suppression of the JNK pathway in the liver improves insulin resistance and glucose tolerance by a high fat, high sucrose Taken together, that suppression of the JNK pathway in the liver improves insulin resistance and ameliorates glucose tolerance in two diabetic models with insulin C57BL/KsJ-db/db diabetic mice and high fat, high sucrose diet-induced diabetic mice In this study, we that activation of the JNK pathway in the liver using adenovirus insulin resistance and in suppression of the JNK pathway using DN-JNK adenovirus insulin resistance and markedly improves glucose tolerance in two diabetic models with insulin resistance. suggest that the JNK pathway be a for diabetes In it is well known that various insulin tissues such as and as well as the liver are involved in the progression of insulin in insulin resistance of the whole body in mice was markedly improved by DN-JNK in the was in glucose suggest that JNK activation in the liver a in insulin resistance and glucose tolerance in the whole The JNK pathway is known to be activated by such as oxidative stress, free fatty and tumor necrosis of which are known to be increased under diabetic diabetic T. Nature. 2000; PubMed Scopus Google Scholar) are in various tissues P. K. S. B. J. T. 1996; PubMed Scopus Google Scholar, J. T. S. K. T. H. H. P. V. A. H. 1997; PubMed Scopus Google Scholar, Y. S. K. H. T. H. H. Y. Y. Diabetes. 1999; PubMed Scopus Google Scholar, T. S. T. Y. M. Nature. 2000; PubMed Scopus Google Scholar) and are involved in the of insulin resistance A. R. R. H. N. Diabetes. 1998; PubMed Scopus Google Scholar, 2002; PubMed Scopus Google Scholar, Diabetes. PubMed Scopus Google Scholar) as well as the progression of β-cell (18Kaneto H. Xu G. Fujii N. Kim S. Bonner-Weir S. Weir G.C. J. Biol. Chem. 2002; 277: 30010-30018Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar, T. Y. H. Kaneto H. M. Y. Y. T. R. Y. J. Clin. Investig. 1997; PubMed Scopus Google Scholar, P. L. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar, H. Xu G. K. Bonner-Weir S. Sharma A. Weir G.C. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, K. K. T. M. K. Y. J. T. K. M. PubMed Scopus Google Scholar, H. Y. J. T. Y. Y. T. Y. Y. M. Diabetes. 1999; PubMed Scopus Google Scholar, Y. Robertson R.P. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar). and are also likely to be involved in the of insulin levels of and are increased under obese diabetic conditions with insulin which leads to a further in insulin resistance. Thus, examined in study, we that of insulin resistance by the suppression of the JNK pathway was at in the effects of such as oxidative stress, and Here we report the dramatic of insulin resistance by suppression of the JNK the of the JNK pathway in insulin resistance. the of the kinase pathway in insulin resistance has been reported M. N. Lee J. L. Karin M. S.E. 2001; PubMed Scopus Google Scholar, Kim Y. Lee J. M. Karin M. P. S.E. G.I. J. Clin. Investig. 2001; PubMed Scopus Google which the improves insulin resistance in and blood glucose levels in type 2 diabetic suggest that of the JNK pathway for the liver are for insulin resistance in diabetic A.M. Davis R.J. PubMed Scopus Google Scholar). In activation of the JNK pathway as under diabetic conditions insulin and in suppression of the JNK pathway insulin resistance and markedly improves glucose tolerance in diabetic Dr. Bert Vogelstein Hopkins Oncology Center) for the AdEasy also for and for also Dr. for on the