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. 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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% [6,6-2H2]glucose 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 [6,6-2H2]glucose 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 & Schuell). After blocking, the membranes were incubated at 4 °C overnight in TBS buffer (50 mm Tris-HCl, 150 mm NaCl) containing a 1:1000 dilution of rabbit anti-IRS-1, anti-IRS-1-Ser(P)-307 antibody (Upstate Biotechnology), anti-Akt, or anti-Akt-Ser(P)-473 antibody (Cell Signaling, Beverly, MA) and then incubated for 1 h at room temperature in TBS containing a 1:1000 dilution of anti-rabbit IgG antibody coupled to horseradish peroxidase (Bio-Rad). Immunoreactive bands were visualized by incubation with LumiGLO (Cell Signaling) and exposed to light-sensitive film. Northern Blot Analysis—Ten μg of total RNA isolated from freeze-clamped liver tissues were electrophoresed on 1.0% formaldehyde-denatured agarose gel in 1× MOPS running buffer and then transferred overnight to a Hybond-N+ membrane (Amersham Biosciences). The phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (Glc-6-Pase) cDNA probes were labeled with [α-32P]dCTP using the Rediprime labeling system kit (Amersham Biosciences). After overnight hybridization with a 32P-labeled probe at 42 °C, the membranes were washed in 2× saline/sodium phosphate/EDTA (SSPE), 0.1% SDS at 42 °C. The probed membranes were exposed to an imaging plate, BAS-MS 2040 (Fujifilm, Tokyo, Japan), and the hybridization intensity was quantified using a BAS2500 system (Fujifilm). Diet Study—Six-week-old male mice (C57BL6) were divided at random into two groups. One group was fed a high fat, high sucrose diet (AIN93G, Oriental Yeast, Suita, Japan), and the other was fed standard laboratory mouse chow (Clea Japan, Tokyo, Japan) for 4 weeks. Statistical Analysis—Results are expressed as mean ± S.E. Differences between groups were examined for statistical significance using the Student's t test or analysis of variance (ANOVA) with the Fisher's test. Activation of the JNK in the Insulin in C57BL6 the effects of JNK in the liver on insulin sensitivity and glucose tolerance in we prepared adenovirus and adenovirus (Ad-GFP) and each adenovirus to C57BL6 mice from the cervical vein. It is that this Ad-WT-JNK expressed as well as shown in a in JNK expression was observed in the but in other tissues such as and The left of a liver after to by the in the liver were with the was in body and between the two groups and blood glucose concentrations in mice were also with in mice to the of in the liver on glucose we performed the intraperitoneal glucose tolerance test shown in was in glucose tolerance between and mice. serum insulin concentrations in mice were in mice at the blood glucose levels that JNK insulin resistance. this we performed the euglycemic hyperinsulinemic clamp test. shown in the left of in the clamp study, the glucose infusion rate in C57BL6 mice was that in mice ± ± that activation of the JNK pathway in the liver insulin sensitivity in C57BL6 mice. Furthermore, we endogenous in mice using tracer shown in the right of in mice was that in mice ± ± In was in glucose rate between the two which glucose in the peripheral tissues that of JNK insulin sensitivity by increasing in mice. the other JNK is well known to be involved in the and of and in the liver after that the in insulin resistance by is of the of in the of the JNK in the Insulin and Glucose Tolerance in C57BL/KsJ-db/db the of suppression of the JNK pathway in the liver on insulin resistance and glucose intolerance in diabetic we prepared adenovirus and adenovirus (Ad-GFP) and each adenovirus the cervical vein to C57BL/KsJ-db/db obese diabetic mice. was in body and between the two groups shown in C57BL/KsJ-db/db mice were with Ad-DN-JNK, blood glucose levels were markedly from ± 42 to ± (2 weeks after adenovirus such was observed in mice. blood glucose concentrations a 6-h were also in mice with was in serum insulin concentrations between these two groups the of suppression of JNK activity in the liver on insulin we performed the intraperitoneal insulin tolerance test shown in the to insulin was in C57BL/KsJ-db/db mice with mice. this we performed the euglycemic hyperinsulinemic clamp test. shown in in the clamp study, the in mice was that in mice ± ± that suppression of the JNK pathway in the liver insulin resistance and ameliorates glucose tolerance in C57BL/KsJ-db/db mice. Furthermore, hepatic glucose production was in mice with mice ± ± In was in the glucose rate between these two groups that reduction of insulin resistance and of glucose tolerance by DN-JNK are a of suppression of hepatic glucose production. we examined Ad-DN-JNK exerts on glucose tolerance and insulin sensitivity in C57BL6 mice. In to C57BL/KsJ-db/db was in blood glucose levels between and C57BL6 after a 6-h fast, blood glucose levels in and C57BL6 mice were ± and ± respectively. was in the between and C57BL6 mice. to the on insulin we measured serum insulin levels. After a 6-h fast, serum insulin levels in and C57BL6 mice were the same at ± and ± respectively. Furthermore, to the of Ad-DN-JNK on insulin sensitivity in we performed the euglycemic hyperinsulinemic glucose clamp test in C57BL6 mice. In to C57BL/KsJ-db/db was in the between and C57BL6 mice ± ± are with the that Ad-DN-JNK the JNK which is activated under diabetic conditions and leads to a decrease of insulin resistance and of glucose tolerance. of Insulin in the the JNK with the of Insulin and of has been reported that phosphorylation of insulin receptor phosphorylation of IRS-1, to an in insulin resistance (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). 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. 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