Insulin Is a Stronger Inducer of Insulin Resistance than Hyperglycemia in Mice with Type 1 Diabetes Mellitus (T1DM)
2009; Elsevier BV; Volume: 284; Issue: 40 Linguagem: Inglês
10.1074/jbc.m109.016675
ISSN1083-351X
AutoresHui-Yu Liu, Sophia Y. Cao, Hong Tao, Jianmin Han, Zhenqi Liu, Wenhong Cao,
Tópico(s)Natural Antidiabetic Agents Studies
ResumoSubjects with type 1 diabetes mellitus (T1DM) eventually develop insulin resistance and other features of T2DM such as cardiovascular disorders. The exact mechanism has been not been completely understood. In this study, we tested the hypothesis that excessive or inappropriate exposure to insulin is a primary mediator of insulin resistance in T1DM. We found that continuous exposure of mice with non-obese diabetes to insulin detemir, which is similar to some current conventional treatment of human T1DM, induced severe insulin resistance, whereas untreated hyperglycemia for the same amount of time (2 weeks) did not cause obvious insulin resistance. Insulin resistance was accompanied by decreased mitochondrial production as evaluated by mitochondrial DNA and levels of transcripts and proteins of mitochondrion-associated genes, increased ectopic fat accumulation in liver and skeletal muscle (gastrocnemius) evaluated by measurements of triglyceride content, and elevated oxidative stress detected by the GSH/GSSG ratio. Prolonged exposure of cultured hepatocytes to insulin induced significant insulin resistance, whereas the same length of exposure to a high level of glucose (33 mm) did not cause obvious insulin resistance. Furthermore, our results showed that prolonged exposure to insulin caused oxidative stress, and blockade of mitochondrion-derived oxidative stress by overexpression of manganese-superoxide dismutase prevented insulin resistance induced by the prolonged exposure to insulin. Together, our results show that excessive exposure to insulin is a primary inducer of insulin resistance in T1DM in mice. Subjects with type 1 diabetes mellitus (T1DM) eventually develop insulin resistance and other features of T2DM such as cardiovascular disorders. The exact mechanism has been not been completely understood. In this study, we tested the hypothesis that excessive or inappropriate exposure to insulin is a primary mediator of insulin resistance in T1DM. We found that continuous exposure of mice with non-obese diabetes to insulin detemir, which is similar to some current conventional treatment of human T1DM, induced severe insulin resistance, whereas untreated hyperglycemia for the same amount of time (2 weeks) did not cause obvious insulin resistance. Insulin resistance was accompanied by decreased mitochondrial production as evaluated by mitochondrial DNA and levels of transcripts and proteins of mitochondrion-associated genes, increased ectopic fat accumulation in liver and skeletal muscle (gastrocnemius) evaluated by measurements of triglyceride content, and elevated oxidative stress detected by the GSH/GSSG ratio. Prolonged exposure of cultured hepatocytes to insulin induced significant insulin resistance, whereas the same length of exposure to a high level of glucose (33 mm) did not cause obvious insulin resistance. Furthermore, our results showed that prolonged exposure to insulin caused oxidative stress, and blockade of mitochondrion-derived oxidative stress by overexpression of manganese-superoxide dismutase prevented insulin resistance induced by the prolonged exposure to insulin. Together, our results show that excessive exposure to insulin is a primary inducer of insulin resistance in T1DM in mice. 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Antibodies against total and phospho-Akt at Ser473, IRS-1(pSer636/639) and PI 3-kinases (p55pTyr199) were from Cell Signaling Technology (Danvers, MA). Antibodies against TFAM were from Santa Cruz Biotechnology (catalog No. CS-23588). Antibodies against Mn-SOD and total/phospho-IRS-1 at Tyr416 were from Abcam. Insulin detemir was from Novo Nordisk. GSH/GSSG-412™ assay kit was from Bioxytech® (Foster City, CA). Blood glucose concentrations were measured by using a Breese® 2 glucose meter (Bayer HealthCare). The protein assay kit was from Bio-Rad. Other materials were all obtained commercially and were of analytical quality. Animals were housed under the usual day (12 h daylight) and night (12 h darkness) circadian rhythm and fed ad libitum. NOD/ShiLtj mice were purchased from The Jackson Laboratory (Bar Harbor, ME). When fasting blood glucose level reached ∼300 mg/dl, NOD mice were treated with either detemir or the vehicle solution (saline, 100 μl) via subcutaneous injections once every 12 h for 2 weeks. Euglycemia was reached and maintained for at least 2 days in each animal. Detemir doses varied among animals to achieve euglycemia. Application of detemir was similar to the so called current conventional treatment of human T1DM (48DCCTAm J. Cardiol. 1995; 75: 894-903Abstract Full Text PDF PubMed Scopus (582) Google Scholar). All animal studies were approved by the Institutional Animal Care and Use Committee of The Hamner Institutes for Health Sciences and fully complied with the guidelines from the National Institutes of Health. Mouse primary hepatocytes were isolated from C57BL/6 mice that were fed with the regular chow diet and cultured in Williams' medium E supplemented with 10% fetal bovine serum as described previously (49Cao W. Collins Q.F. Becker T.C. Robidoux J. Lupo Jr., E.G. Xiong Y. 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Data were confirmed by measuring the ratio of cytochrome b to glucagon genomic DNA of the same sample. ITT was performed after an overnight fast or 14 h after the last dose of detemir. Initial blood glucose levels were determined followed by intraperitoneal injection of human insulin (catalog No. I9278, Sigma) (0.75 unit/kg). Blood glucose levels were measured via tail vein blood at 15, 30, 60, 90, and 120 min after the injection. Levels of GSH and GSSG in cell or tissue lysates were determined with a kit from OXIS International, Inc. (Foster City, CA) and normalized to protein levels. The GSH/GSSG ratio was calculated according to the instructions provided by the manufacturer. Liver and muscle lysates of NOD mice were prepared in ice-cold Nonidet P-40 lysis buffer in the presence of 1 mm sodium orthovanadate (Na3VO4) and protease inhibitors. PI 3-kinase-α was purified with the anti-PI 3-kinase-α antibody (catalog No. 06-195, Millipore, Billerica, MA). The activity of PI 3-kinase in the purified PI 3-kinase-α was then evaluated by measuring the amount of PI(3,4,5)P3 converted from PI(4,5)P2 (substrate) with a PI 3-kinase enzyme-linked immunosorbent assay kit from Echelon Biosciences Inc. (Salt City, UT). Immunoblotting was performed as described previously (49Cao W. Collins Q.F. Becker T.C. Robidoux J. Lupo Jr., E.G. Xiong Y. Daniel K.W. Floering L. Collins S. J. Biol. Chem. 2005; 280: 42731-42737Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 50Collins Q.F. Xiong Y. Lupo Jr., E.G. Liu H.Y. Cao W. J. Biol. Chem. 2006; 281: 24336-24344Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 51Collins Q.F. Liu H.Y. Pi J. Liu Z. Quon M.J. Cao W. J. Biol. Chem. 2007; 282: 30143-30149Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar, 52Xiong Y. Collins Q.F. An J. Lupo Jr., E. Liu H.Y. Liu D. Robidoux J. Liu Z. Cao W. J. Biol. Chem. 2007; 282: 4975-4982Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 53Liu H.Y. Collins Q.F. Xiong Y. Moukdar F. Lupo Jr., E.G. Liu Z. Cao W. J. Biol. Chem. 2007; 282: 14205-14212Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 54Liu H.Y. Collins Q.F. Moukdar F. Zhuo D. Han J. Hong T. Collins S. Cao W. J. Biol. Chem. 2008; 283: 12056-12063Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 55Liu H.Y. Wen G.B. Han J. Hong T. Zhuo D. Liu Z. Cao W. J. Biol. Chem. 2008; 283: 30642-30649Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). In brief, cells were lysed in Nonidet P-40 lysis buffer (1% Nonidet P-40, 150 mm NaCl, 10% glycerol, 2 mm EDTA, 20 mm Tris (pH 8.0), 1 mm dithiothreitol, 1 mm sodium orthovanadate, 1 mm phenylmethylsulfonyl fluoride, 2 μg/ml leupeptin, and 10 μg/ml aprotinin. Cell lysates (15 μg/lane) were resolved in 4–20% Tris/glycine gels (Invitrogen) and transferred to nitrocellulose membranes (Bio-Rad). Target proteins were detected by immunoblotting with primary antibodies as indicated and alkaline phosphatase-conjugated secondary antisera. The fluorescent bands were visualized with a Typhoon 9410 variable mode imager from GE Healthcare and then quantified by densitometry using ImageQuant 5.2 software (GE Healthcare). Total RNAs were extracted from cells or tissues with an RNeasy mini kit (Qiagen) and reverse-transcribed into cDNAs, which were quantified by TaqMan® real-time PCR with specific probes and primers from Applied Biosciences and normalized to levels of β-actin. Data are presented as mean ± S.E. Data were compared by Student's t test using GraphPad Prism version 5.0 for Windows (San Diego, CA). Differences at values of p < 0.05 were considered significant. Both hyperglycemia and treatment with insulin have been considered as inducers of insulin resistance in subjects with T1DM (9Reaven G.M. Diabetes. 1988; 37: 1595-1607Crossref PubMed Google Scholar, 57DeFronzo R.A. Simonson D. Ferrannini E. Diabetologia. 1982; 23: 313-319Crossref PubMed Scopus (489) Google Scholar, 58Rossetti L. Giaccari A. DeFronzo R.A. Diabetes Care. 1990; 13: 610-630Crossref PubMed Scopus (895) Google Scholar). To determine which of these two inducers is a stronger inducer of insulin resistance, a group of NOD diabetic mice with hyperglycemia (over 300 mg/dl) was treated with saline for 2 weeks, and another group of diabetic NOD mice was treated with two doses of the long acting insulin reagent detemir, with one dose in the early morning and another in the evening. Given that detemir is long acting, insulin was basically present in the blood at all times. Doses of detemir were gradually increased to achieve euglycemia. Detemir treatment started when fasting blood glucose levels reached ∼300 mg/dl. The application of detemir in this study was similar to the current conventional treatment of human T1DM (48DCCTAm J. Cardiol. 1995; 75: 894-903Abstract Full Text PDF PubMed Scopus (582) Google Scholar). NOD sibling mice of the same ages, with no diabetes, were used as negative controls. To achieve euglycemia, the dose of detemir was gradually increased to as high as 20 units/kg of body weight (Fig. 1A). As shown in Fig. 1B, blood glucose levels increased gradually and reached as high as 600 mg/dl at the end of the 2-week period in diabetic NOD mice treated with saline. Blood glucose levels in diabetic NOD mice treated with detemir decreased gradually and reached euglycemia during the last 2 days of the 2-week treatment when the dosage of detemir reached ∼20 units/kg of body weight (Fig. 1B). To determine the levels of insulin sensitivity in different groups of mice, ITT was performed. As shown in Fig. 1, C and D, the NOD diabetic mice treated with detemir for 2 weeks showed no response to acute challenge with the regular fast acting insulin, whereas the NOD diabetic mice with hyperglycemia for 2 weeks without detemir treatment were equally as responsive to insulin challenge as the NOD mice without diabetes. Body weights for all groups were not significantly different (Fig. 1E). Intake levels of food and water in diabetic NOD mice tended to increase in comparison with the NOD mice with no diabetes (p = 0.06 and 0.08, respectively) but were similar between the NOD diabetic mice treated with either saline or detemir (Fig. 1, F and G). The changes in intake of food and water and body weight were not as dramatic as usually thought. There are several probable explanations. First, glucose reabsorption via the Na(+)-glucose transporter-2 (SGLT-2) in the kidney is increased when insulin is deficient (59Freitas H.S. Anhê G.F. Melo K.F. Okamoto M.M. Oliveira-Souza M. Bordin S. Machado U.F. Endocrinology. 2008; 149: 717-724Crossref PubMed Scopus (154) Google Scholar), leading to milder loss of glucose and water as predicted. Second, the NOD mice used in this study were based on A/J mice, which are resistant to body weight change even under the high fat diet (60Surwit R.S. Kuhn C.M. Cochrane C. McCubbin J.A. Feinglos M.N. Diabetes. 1988; 37: 1163-1167Crossref PubMed Scopus (0) Google Scholar). Third, treatment with insulin was not sufficient until the last 2 days (Fig. 1, A and B). Thus, changes in the average body weight were not expected to be very significant. Taken together, these results show that: (a) continuous exposure to insulin for 2 weeks is a strong inducer of insulin resistance, whereas hyperglycemia for 2 weeks does not cause obvious insulin resistance in mice; and (b) no matter how strong the insulin resistance, blood glucose level can be brought to a normal level as long as sufficient amount of insulin is provided. To determine levels of basal insulin signaling in different groups of animals, phosphorylation levels of p55 subunit of PI 3-kinase and Akt were measured in liver and gastrocnemius. Phosphorylation of both the p55 PI 3-kinase subunit and Akt was elevated by treatment with detemir in liver (Fig. 2, A and B). PI 3-kinase activity was also increased by detemir (Fig. 2C). Similarly, phosphorylation of both the p55 PI 3-kinase subunit and Akt and PI 3-kinase activity were stimulated by detemir in gastrocnemius. (Fig. 2, D and E). These results show that the basal insulin signaling is increased in diabetic mice treated with detemir (insulin). To examine the mechanism by which treatment with detemir caused insulin resistance, we examined levels of IRS-1 phosphorylation at serines 636 and 639. As shown in Fig. 3, IRS-1 phosphorylation at serines 636 and 639 was increased in both liver and gastrocnemius of detemir-treated mice. These results imply that prolonged exposure to insulin induces insulin resistance, likely through IRS-1 serine phosphorylation. Note that these results do not suggest that appropriate application of the right type of insulin reagents for the appropriate amount of time via a physiological route cause insulin resistance. To further examine the mechanism by which insulin induces insulin resistance, mtDNA and transcripts of some mitochondrion-related genes were quantified. As shown in Fig. 4A, levels of mtDNA tended to increase in both liver and gastrocnemius of NOD diabetic mice without detemir treatment (p > 0.05) but were decreased in liver (p < 0.05) and tended to decrease in gastrocnemius (p > 0.05) of NOD diabetic mice that were treated with detemir. Transcript levels of several mitochondrion-related genes including ATP synthase, estrogen receptor-related receptor α (ERRα), NADH dehydrogenase (Ndufv1), and mitochondrial transcription factor A (Tfam) were significantly decreased by continuous exposure to detemir in comparison with the NOD diabetic mice without insulin treatment in liver but not in gastrocnemius (Table 1). The levels of many other mitochondrion-related gene transcripts were also decreased by insulin treatment without reaching statistical significance in liver. Protein levels of the key mitochondrial transcription factor TFAM was increased in the liver of diabetic NOD mice, but the increase was reversed by treatment with detemir (Fig. 4B). TFAM protein levels were also increased in the gastrocnemius of diabetic NOD mice, and this increase tended to be reversed by treatment with detemir although without reaching statistical significance (Fig. 4C). Together, these results suggest that mitochondrial production is increased in the absence of insulin in NOD diabetic mice but is decreased by treatment with continuous exposure to insulin (detemir).TABLE 1Transcript levels of mitochondrion-associated genes in liver and gastrocnemius of NOD miceDetemir vs. vehiclep value-foldLiverATP5a10.6731 ± 0.09590.0214*COXIV0.9764 ± 0.12400.8953Cycs0.7874 ± 0.11960.3671ERRα0.5537 ± 0.13260.0213*Ndufv10.5886 ± 0.10500.0425*NRF11.2520 ± 0.34760.4798PGC-1α0.9553 ± 0.12130.7796PGC-1β1.0270 ± 0.32460.9405COXI1.214 ± 0.263400.4810Sdhc0.8064 ± 0.08780.1019Tfam0.7486 ± 0.07980.0407*GastrocnemiusATP5a11.044 ± 0.23320.8807COXIV0.925 ± 0.26570.8383Cycs1.013 ± 0.15160.9564ERRα1.141 ± 0.29550.7422Ndufv10.819 ± 0.24710.5893NRF11.296 ± 0.06120.3826PGC-1α1.024 ± 0.23140.9252PGC-1β0.931 ± 0.12780.6296COXI1.035 ± 0.18640.5770Sdhc1.106 ± 0.35240.8181Tfam0.586 ± 0.41840.4878 Open table in a new tab As ectopic fat accumulation is a necessary component of insulin resistance (23Kim J.Y. van de Wall E. Laplante M. Azzara A. Trujillo M.E. Hofmann S.M. Schraw T. Durand J.L. Li H. Li G. Jelicks L.A. Mehler M.F. Hui D.Y. Deshaies Y. Shulman G.I. Schwartz G.J. Scherer P.E. J. Clin. Investig. 2007; 117: 2621-2637Crossref PubMed Scopus (1018) Google Scholar, 24Wan
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