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Effect of Rosiglitazone on the Differential Expression of Diabetes-associated Proteins in Pancreatic Islets of C57Bl/6 lep/lep Mice

2002; Elsevier BV; Volume: 1; Issue: 7 Linguagem: Inglês

10.1074/mcp.m200033-mcp200

1535-9484

Jean‐Charles Sanchez, Véronique Converset, Anna L. Nolan, G. M. Schmid, Steven Wang, Manfred Heller, Matthew V. Sennitt, Denis F. Hochstrasser, Michael A. Cawthorne,

Adipokines, Inflammation, and Metabolic Diseases

The insulin sensitizer drug, rosiglitazone, has been shown to have a protective effect on pancreatic islet cell structure and function in animal models of type 2 diabetes. The identification of new molecular targets associated both with islet cell dysfunction and protection is a crucial research goal. In the present study, a proteomics approach has been used to identify such targets. Obese C57Bl/6J lep/lep mice and lean littermates were given the insulin sensitizer drug BRL49653, rosiglitazone. It normalized the impaired glucose tolerance in lep/lep mice but had no significant effect on glucose tolerance in the lean mice. Pancreatic islet polypeptides were arrayed by a two-dimensional gel electrophoresis system that separated more than 2500 individual spots. Three overexpressed and six underexpressed proteins were significant (p < 0.05) between lep/lep and lean mice, and four were modulated significantly (p < 0.05) by the rosiglitazone treatment of the obese mice. The identity of these differentially expressed proteins was made using mass spectrometric analysis and provided evidence that differential expression of actin-binding proteins may be an important aspect of defective islet function. Rosiglitazone increased carboxypeptidase B expression in both lep/lep and normal mice suggesting that this might be an independent effect of rosiglitazone that contributes to improved insulin processing. The insulin sensitizer drug, rosiglitazone, has been shown to have a protective effect on pancreatic islet cell structure and function in animal models of type 2 diabetes. The identification of new molecular targets associated both with islet cell dysfunction and protection is a crucial research goal. In the present study, a proteomics approach has been used to identify such targets. Obese C57Bl/6J lep/lep mice and lean littermates were given the insulin sensitizer drug BRL49653, rosiglitazone. It normalized the impaired glucose tolerance in lep/lep mice but had no significant effect on glucose tolerance in the lean mice. Pancreatic islet polypeptides were arrayed by a two-dimensional gel electrophoresis system that separated more than 2500 individual spots. Three overexpressed and six underexpressed proteins were significant (p < 0.05) between lep/lep and lean mice, and four were modulated significantly (p < 0.05) by the rosiglitazone treatment of the obese mice. The identity of these differentially expressed proteins was made using mass spectrometric analysis and provided evidence that differential expression of actin-binding proteins may be an important aspect of defective islet function. Rosiglitazone increased carboxypeptidase B expression in both lep/lep and normal mice suggesting that this might be an independent effect of rosiglitazone that contributes to improved insulin processing. Type 2 diabetes is a complex metabolic disease involving defects in both insulin secretion and insulin action and is influenced by a range of genetic and environmental factors (1.Zimmet P. Alberti K.G. Shaw J. Global and societal implications of the diabetes epidemic.Nature. 2001; 414: 782-787Google Scholar). As in other polygenic diseases, including cancers and cardiovascular diseases, advances in molecular genetic approaches have made it possible to start to identify susceptibility genes of type 2 diabetes, but so far the search for candidate genes has been largely negative (2.Elbein S.C. The genetics of human non insulin-dependent (type 2) diabetes mellitus.J. Nutr. 1997; 127: 1891S-1896SGoogle Scholar) probably as a result of redundancy and compensatory mechanisms (3.Tautz D. Redundancies, development and the flow of information.Bioessays. 1992; 14: 263-266Google Scholar). Linear genetic strategies are thus not sufficient to predict specific phenotypes, and more global approaches that combine genomic and proteomic investigation are required.Whether insulin resistance or insulin secretion defects are primary in the development of type 2 diabetes has been a highly debated topic, and there is no consensus. However, it is clear that overt type 2 diabetes only occurs when the insulin output from the pancreatic islet fails to match the insulin requirement as a result of the insulin resistance (4.Gerich J.E. Is reduced first-phase insulin release the earliest detectable abnormality in individuals destined to develop type 2 diabetes?.Diabetes. 2002; 51: 117-121Google Scholar).Current therapies targeted to the pancreatic islet are agents such as sulfonylureas that increase meal-induced insulin secretion. However, it is clear that although these agents can be effective in the short term they can also accelerate the progression of the diabetic state (5.Scheen A.J. Lefebvre P.J. Antihyperglycaemic agents. Drug interactions of clinical importance.Drug Saf. 1995; 12: 32-45Google Scholar). There is a growing opinion that new agents are required that increase the pancreatic islet cell mass and/or enhance the production of insulin by the islet cell (6.Bernard-Kargar C. Ktorza A. Endocrine pancreas plasticity under physiological and pathological conditions.Diabetes. 2001; 50: 30-35Google Scholar). Recently, insulin sensitizer drugs, such as BRL49653 (rosiglitazone), have become available. These agents increase insulin sensitivity of adipose tissue, skeletal muscle, and liver (7.Ciaraldi T. Henry R.R. Thiazolidinediones and their effects on glucose transporters.Eur. J. Endocrinol. 1997; 137: 610-612Google Scholar) and reduce the insulin requirement. BRL49653 has also been shown to prevent or alleviate islet hyperplasia in Zucker fa/fa rats (8.Buckingham R.E. Al-Barazanji K.A. Toseland C.D. Slaughter M. Connor S.C. West A. Bond B. Turner N.C. Clapham J.C. Peroxisome proliferator-activated receptor-gamma agonist, rosiglitazone, protects against nephropathy and pancreatic islet abnormalities in Zucker fatty rats.Diabetes. 1998; 47: 1326-1334Google Scholar), and human studies provide evidence of islet cell protection (9.Jones N.P. Charbonnel B. Lannqvist F. Owen S. Patwardhan R. Rosiglitazone reduces plasma insulin and its precursor while decreasing glycaemia in type 2 diabetes.Diabetologia. 1999; 42 (Abstr. 22): 859Google Scholar). Animal studies have also shown that islet cell mass might affect significantly the susceptibility to develop diabetes. Thus, C57Bl/6 mice have twice the islet cell mass of C57Bl/Ks mice (10.Swenne I. Andersson A. Effect of genetic background on the capacity for islet cell replication in mice.Diabetologia. 1984; 27: 464-467Google Scholar), and the latter mice are significantly more susceptible to diabetes induced by chemical damage (11.Korsgren O. Jansson L. Sandler S. Andersson A. Hyperglycemia-induced B cell toxicity. The fate of pancreatic islets transplanted into diabetic mice is dependent on their genetic background.J. Clin. Invest. 1990; 86: 2161-2168Google Scholar) or as a result of the acquisition of the obesity or obesity receptor gene mutations lep or lepR, previously known as ob and db (12.Kaku K. Province M. Permutt M.A. Genetic analysis of obesity-induced diabetes associated with a limited capacity to synthesize insulin in C57BL/KS mice: evidence for polygenic control.Diabetologia. 1989; 32: 636-643Google Scholar).In the present study, we have used two separate phenotypic paradigms to undertake a systematic identification of proteins associated with anatomical and functional abnormalities of pancreatic islets. First we have used C56Bl/6J lep/lep mice and lean littermates that have been treated with the insulin sensitizer BRL49653. Because this agent normalizes glucose tolerance and insulin sensitivity in the lep/lep mice but has little effect in the littermates (7.Ciaraldi T. Henry R.R. Thiazolidinediones and their effects on glucose transporters.Eur. J. Endocrinol. 1997; 137: 610-612Google Scholar), it follows that islet proteins that are expressed differentially between lep/lep and lean C57Bl/6J mice and whose expression is modulated by BRL49653 only in the lep/lep mice are disease-associated proteins. Second, we have examined the differential expression of islet proteins in C57Bl/6 and C57Bl/Ks mice to identify proteins potentially associated with low islet cell mass.EXPERIMENTAL PROCEDURESReagents and Apparatus—All reagents and apparatus used have been described in detail elsewhere (13.Hochstrasser D.F. Frutiger S. Paquet N. Bairoch A. Ravier F. Pasquali C. Sanchez J.C. Tissot J.D. Bjellqvist B. Vargas R. Appel R.D. Human liver protein map: a reference database established by microsequencing and gel comparison.Electrophoresis. 1992; 13: 992-1001Google Scholar). Mice were obtained from Jackson Laboratories, Bar Harbor, ME.Mice Treatment—All mice used in this study were 8-week-old females, and they were housed on a 12-h light cycle (light turned on at 08:00) at 21 °C and given food (rat and mouse standard diet; B+K Universal, Hull, United Kingdom) and water ad libitum. Each group of mice had its food consumption measured daily. Lean (n = 4) and obese (n = 4) control C57Bl/6J lep/lep mice were given powdered diet. Lean (n = 4) and obese (n = 4) treated C57Bl/6J lep/lep mice were given BRL49653, rosiglitazone (7.Ciaraldi T. Henry R.R. Thiazolidinediones and their effects on glucose transporters.Eur. J. Endocrinol. 1997; 137: 610-612Google Scholar) (Dextra Laboratories, Readings Berks, United Kingdom) by dietary admixture at the rate of 10 mg/kg diet for 7 days. After an overnight fast, mice were given glucose (3.0 g/kg body weight, per os). Blood from the tail (20 μl) was taken every 30 min (−30 min to +180 min after glucose was given). Blood glucose concentrations were measured as described previously (14.Ibrahimi A. Teboul L. Gaillard D. Amri E.Z. Ailhaud G. Young P. Cawthorne M.A. Grimaldi P.A. Evidence for a common mechanism of action for fatty acids and thiazolidinedione antidiabetic agents on gene expression in preadipose cells.Mol. Pharmacol. 1994; 46: 1070-1076Google Scholar). All animal studies were approved by the local ethical review committee and were carried out in accordance with United Kingdom government regulations and NIH guidelines on the care and welfare of laboratory animals.Pancreatic Islet Preparations and Solubilization—Mice were anesthetized with 50% hyponovel and 50% hyponorm and then killed with carbon dioxide gas. The pancreas was removed, and pancreatic islets were isolated by collagenase digestion at 37 °C using a physiological saline solution supplemented with 1 mm CaCl2, 4 mm glucose and equilibrated with CO2:O2 (5%:95%), pH 7.4 (15.Pallett A.L. Morton N.M. Cawthorne M.A. Emilsson V. Leptin inhibits insulin secretion and reduces insulin mRNA levels in rat isolated pancreatic islets.Biochem. Biophys. Res. Commun. 1997; 238: 267-270Google Scholar). Islets were handpicked using a binocular microscope, washed, microdissected to remove residual acinar material, and finally snap-frozen in liquid nitrogen and stored at −80 °C until analysis. 200 (analytical gels) or 1,000 (preparative gels) islets were mixed with 60 μl of a solution containing urea (8 m), CHAPS 1The abbreviations used are: CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; 2-DE, two-dimensional gel electrophoresis; IPG, immobilized pH gradient. (4% w/v), Tris (40 mm), dithiothreitol (65 mm), SDS (0.05% w/v), and a trace of bromphenol blue. The whole final diluted sample was loaded in a cup at the cathodic end of the IPG gels.Two-dimensional Gel Electrophoresis—A commercial sigmoidal IPG (18 cm nonlinear from Amersham Biosciences) going from pH 3.5 to 10.0 was used for first-dimensional separation (16.Bjellqvist B. Pasquali C. Ravier F. Sanchez J.-C. Hochstrasser D.F. A nonlinear wide-range immobilized pH gradient for two-dimensional electrophoresis and its definition in a relevant pH scale.Electrophoresis. 1993; 14: 1357-1365Google Scholar, 17.Gorg A. Postel W. Gunther S. Weser J. Strahler J.R. Hanash S.M. Somerlot L. Kuick R. Approach to stationary two-dimensional pattern: influence of focusing time and immobiline/carrier ampholytes concentrations.Electrophoresis. 1988; 9: 37-46Google Scholar). After equilibration, the IPG gel strips were transferred for the second dimension onto vertical gradient slab gels (9–16% T, 2.6% C) and run with the Laemmli-SDS-discontinuous system (18.Hochstrasser D.F. Harrington M.G. Hochstrasser A.C. Miller M.J. Merril C.R. Methods for increasing the resolution of two-dimensional protein electrophoresis.Anal. Biochem. 1988; 173: 424-435Google Scholar, 19.Hochstrasser D.F. Merril C.R. "Catalysts" for polyacrylamide gel polymerization and detection of proteins by silver staining.Appl. Theor. Electrophor. 1988; 1: 35-40Google Scholar, 20.Hochstrasser D.F. Patchornik A. Merril C.R. Development of polyacrylamide gels that improve the separation of proteins and their detection by silver staining.Anal. Biochem. 1988; 173: 412-423Google Scholar). Protein detection was achieved using a sensitive ammoniacal silver stain (16.Bjellqvist B. Pasquali C. Ravier F. Sanchez J.-C. Hochstrasser D.F. A nonlinear wide-range immobilized pH gradient for two-dimensional electrophoresis and its definition in a relevant pH scale.Electrophoresis. 1993; 14: 1357-1365Google Scholar, 21.Oakley B.R. Kirsch D.R. Morris N.R. A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels.Anal. Biochem. 1980; 105: 361-363Google Scholar) or Coomassie Brilliant Blue R-250 (0.1% w/v) and methanol (50% v/v) for 30 min. Destaining was done in a solution containing methanol (40% v/v) and acetic acid (10% v/v).Image Acquisition and Data Analysis—Duplicate gels, which had a reproducibility of ±45% for the volume of any of the 2528 detected spots, were run. Gels were scanned using a laser densitometer (Amersham Biosciences). The 2-DE image computer analysis was carried out using the MELANIE 3 software package (GeneBio, Geneva, Switzerland) (22.Appel R.D. Palagi P.M. Walther D. Vargas J.R. Sanchez J.C. Ravier F. Pasquali C. Hochstrasser D.F. Melanie II–a third-generation software package for analysis of two-dimensional electrophoresis images: I. Features and user interface.Electrophoresis. 1997; 18: 2724-2734Google Scholar). Spots were detected and quantified automatically. The optical density, the area, and the volume were computed and related directly to protein concentration. The relative optical density and relative volume were also calculated to correct for differences in gel staining. Differential analysis (>200%) and Student's t test (p < 0.05) using the relative volume of each spot (>0.04%) allowed the detection of significantly over- and underexpressed polypeptides with a minimum ratio of two.Protein Identification by Mass Spectrometry—The identity of the differentially expressed proteins was made using mass spectrometric analysis including peptide mass fingerprinting analysis and then peptide fragmentation for their validation. Coomassie Blue 2-DE spots were destained with 100 μl of 30% acetonitrile in 50 mm ammonium bicarbonate at 37 °C for 45 min. The supernatant was discarded, and the gel spots were dried in a SpeedVac for 30 min. The gel spots were rehydrated with 25 μl of a solution containing 0.2 μg of porcine trypsin and 50 mm ammonium bicarbonate for 2 h at 35 °C. Then the gel spots and supernatant were dried in a SpeedVac for 30 min, rehydrated with 20 μl of H2O for 30 min at 35 °C, and dried again for 30 min. 5 μl of a solution containing 50% acetonitrile and 0.1% trifluoroacetic acid was added to the spots and sonicated for 10 min.Protein Identification by Peptide Mass Fingerprinting—1 μl of the supernatant was loaded in each well of a 96-well matrix-assisted laser desorption ionization target plate. The samples were air-dried. Then 2 μl of a solution containing 4 mg/ml of α-cyano-4-hydroxycinnamic acid, 50% acetonitrile, and 0.1% trifluoroacetic acid was added on each well and air-dried. The peptide mixtures were analyzed by a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (Applied Biosciences Voyager Elite MALDI-TOF-MS) with a nitrogen laser (337 nm) and operated in reflectron-delayed extraction mode. Protein identification was carried out using SmartIdent (www.expasy.ch/sprot/SmartIdent.html). It is a tool that allows the identification of proteins using pI, molecular weight, and peptide mass fingerprinting data. Experimentally measured, user-specified peptide masses were compared with the theoretical peptides calculated for all proteins in the SWISS-PROT/TREMBL databases.Protein Identification by Peptide Fragmentation—4 μl of the supernatant was desalted by a liquid reversed-phase chromatography system with C18 (75-μm diameter and 10-cm-long) microcolumns. Peptides were applied by nanoflow (in-house nanospray) sample introduction to a tandem mass spectrometer that consists of two quadrupoles and an orthogonal time of flight tube (Q-TOF-MS) from MicroMass (Manchester, United Kingdom). Fragment ion spectra were interpreted with the SEQUEST database search (ThermoFinnigan, San Jose, CA) and compared with theoretical ones from different databases such as SWISS-PROT, TREMBL, and/or expressed sequence tag databases.RESULTSOral Glucose Tolerance Test—The obese C56Bl/6J lep/lep strain shows hyperphagia, insulin resistance, glucose intolerance, and fasting hyperglycemia (23.Duhault J. Boulanger M. Espinal J. Marquie G. Petkov P. du Boistesselin R. Latent autoimmune diabetes mellitus in adult humans with non-insulin-dependent diabetes: is Psammomys obesus a suitable animal model?.Acta Diabetol. 1995; 32: 92-94Google Scholar) relative to its lean littermates. To check the efficacy of rosiglitazone on blood glucose concentration, an oral glucose tolerance test was performed on the four groups of mice (i.e. lean control, obese (lep/lep) control, lean treated, and obese (lep/lep) treated). Fig. 1 shows that the area under the blood glucose concentration curve of the obese control group is much greater than the lean control. Treatment of the obese group with rosiglitazone normalized the glucose tolerance, whereas treatment of the lean group had no further effect. These results validated that rosiglitazone improves the glycemia of the lep/lep group and does not provoke hypoglycemia in the lean littermates.2-DE Gel Separation and Identification of Proteins—Mouse islets were arrayed on the 2-DE gel system, and proteins were identified by mass spectrometric analysis. From the 2528 detected spots corresponding approximately to 1500 proteins and representing only 10% of the whole islet proteome, 63 have been identified that correspond to 44 protein entries. The data have been established in the mouse SWISS-2DPAGE database (24.Sanchez J.-Ch. Chiappe D. Converset V. Hoogland C. Binz P.-A. Paesano S. Appel R.D. Wang S. Sennitt M. Nolan A. Cawthorne M.A. Hochstrasser D.F. The mouse SWISS-2DPAGE database: a tool for proteomics study of diabetes and obesity.Proteomics. 2001; 1: 136-163Google Scholar). The database can be found on the ExPASy server (www.expasy.ch/cgi-bin/map2/def?ISLETS_MOUSE). Fig. 2 shows the 2-DE gel electrophoresis image of mouse islets of Langerhans with differentially expressed protein locations marked.Fig. 2Silver-stained mouse islet of Langerhans 2-DE image. 200 islets were loaded on an IPG gel (3.5–10 nonlinear IPG, 18 cm). The second dimension was a vertical gradient slab gel (9–16% T). The gene names mark the location of the corresponding differentially expressed proteins. TM1, tropomyosin isoform 1; DHPR, dihydropteridine reductase; DHPRf, dihydropteridine reductase fragment; GSTP1, glutathione S-transferase P1; BGR, β-granin; S100A9, calgranulin B; FABP4, adipocyte fatty acid-binding protein; PFN1, profilin; PFN1f, profilin fragment; CPB, carboxypeptidase B.View Large Image Figure ViewerDownload (PPT)Changes of Protein Expression between lep/lep Mouse Islets and Lean Littermates—Image analysis allowed the detection and identification (Fig. 3) of six underexpressed (4-fold reduction in tropomyosin isoform 1, 10-fold reduction of dihydropteridine reductase and dihydropteridine reductase fragment, 4-fold reduction in glutathione S-transferase P1, 4-fold reduction of β-granin, and 3-fold reduction of calgranulin B) and three overexpressed polypeptides (3-fold increase in adipocyte fatty acid-binding protein and 4-fold increase of profilin and profilin fragment) in obese controls relative to lean mice (p < 0.05).Fig. 3Bar charts representing the relative spot volume detected by a differential expression analysis. Six underexpressed (tropomyosin isoform 1, dihydropteridine reductase, dihydropteridine reductase fragment, glutathione S-transferase P1, β-granin, and calgranulin B) and three overexpressed polypeptides (adipocyte fatty acid-binding protein, profilin, and profilin fragment) were significant (p < 0.05) between obese control (n = 4) and lean control mice (n = 4).View Large Image Figure ViewerDownload (PPT)Changes of Protein Expression Induced by Treatment with BRL49653—Of the nine polypeptides differentially expressed between lep/lep mouse islets and lean littermates, four were modulated significantly (p < 0.05) after treatment of the lep/lep mice, but not in the lean littermates, with BRL49653. These (tropomyosin isoform 1, adipocytes fatty acid-binding protein, profilin, and profilin fragment) had their expression level altered toward the level in the lean littermates (Fig. 4). In addition, BRL49653 increased carboxypeptidase B precursor protein expression in both lep/lep and normal mice (Fig. 5).Fig. 4Bar charts representing the relative spot volume detected by a differential expression analysis. Four differentially expressed proteins (tropomyosin isoform 1, adipocyte fatty acid-binding protein, profilin, and profilin fragment) were modulated in obese models (n = 4) to the level in lean control (n = 4) after rosiglitazone treatment (p < 0.05).View Large Image Figure ViewerDownload (PPT)Fig. 5Bar chart representing the relative spot volume detected by a differential expression analysis. Overexpression of carboxypeptidase B precursor protein in obese and lean models (n = 4) after rosiglitazone treatment relative to obese and lean controls (n = 4) (p < 0.05).View Large Image Figure ViewerDownload (PPT)Differential Polypeptide Expression between C57Bl/6 and C57Bl/Ks Mice—The yield of islets from lean C57Bl/Ks mice was on average only 50% of the yield from C57Bl/6 mice, and the Ks islets were smaller, in agreement with the studies of Swenne and Andersson (10.Swenne I. Andersson A. Effect of genetic background on the capacity for islet cell replication in mice.Diabetologia. 1984; 27: 464-467Google Scholar). Of the 31 polypeptides found to be differentially expressed between these two genotypes (data not shown), two of the proteins (tropomyosin and profilin) had similar levels of expression in the C57Bl/Ks mice to that in the C57Bl/6 lep/lep mice (Fig. 6).Fig 6Bar charts representing the relative spot volume detected by a differential expression analysis. Expression of tropomyosin and profilin in islets from lean (n = 4), obese (n = 4), and obese treated C57Bl/6J (n = 4), as well as from lean C57Bl/Ks (n = 4), is shown. Expression in both obese mice and lean C57Bl/Ks mice was significantly different from expression in C57Bl/6J mice (p < 0.05).View Large Image Figure ViewerDownload (PPT)DISCUSSIONThe obese C57Bl/6J lep/lep mouse strain shows obesity, hyperphagia, insulin resistance, glucose intolerance, and mild fasting hyperglycemia (23.Duhault J. Boulanger M. Espinal J. Marquie G. Petkov P. du Boistesselin R. Latent autoimmune diabetes mellitus in adult humans with non-insulin-dependent diabetes: is Psammomys obesus a suitable animal model?.Acta Diabetol. 1995; 32: 92-94Google Scholar). Hyperplasia and hypertrophy of β-cell mass are also present (25.Tomita T. Doull V. Pollock H.G. Krizsan D. Pancreatic islets of obese hyperglycemic mice (ob/ob).Pancreas. 1992; 7: 367-375Google Scholar). The obese phenotype results from a nonsense mutation in codon 105 of the ob gene located in chromosome 6 (26.Zhang Y. Proenca R. Maffei M. Barone M. Leopold L. Friedman J.M. Positional cloning of the mouse obese gene and its human homologue.Nature. 1994; 372: 425-432Google Scholar). The ob gene is expressed mainly in white adipose tissue, and its mutation leads to a failure to secrete the gene product into the circulation. The gene product, leptin ("leptos" in Greek means thin), acts on the hypothalamus to regulate appetite (27.Stephens T.W. Basinski M. Bristow P.K. Bue-Valleskey J.M. Burgett S.G. Craft L. Hale J. Hoffmann J. Hsiung H.M. Kriauciunas A. The role of neuropeptide Y in the antiobesity action of the obese gene product.Nature. 1995; 377: 530-532Google Scholar). However, leptin receptors (product of the db gene) are also present in a variety of tissues (28.Caro J.F. Sinha M.K. Kolaczynski J.W. Zhang P.L. Considine R.V. Leptin: the tale of an obesity gene.Diabetes. 1996; 45: 1455-1462Google Scholar) including pancreatic islet cells, and leptin affects insulin secretion (29.Wang M.Y. Koyama K. Shimabukuro M. Newgard C.B. Unger R.H. OB-Rb gene transfer to leptin-resistant islets reverses diabetogenic phenotype.Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 714-718Google Scholar). The hyperplasia and hypertrophy of the β-cell mass in C57Bl/6J lep/lep mice is not solely due to the lep mutation, because when the mutation is expressed on the C57Bl/Ks background, islet cells become damaged, and the mice develop frank diabetes (12.Kaku K. Province M. Permutt M.A. Genetic analysis of obesity-induced diabetes associated with a limited capacity to synthesize insulin in C57BL/KS mice: evidence for polygenic control.Diabetologia. 1989; 32: 636-643Google Scholar). Other studies have shown fundamental differences in islet cell mass between wild-type C57Bl/6 and C57Bl/Ks mice (10.Swenne I. Andersson A. Effect of genetic background on the capacity for islet cell replication in mice.Diabetologia. 1984; 27: 464-467Google Scholar).Recently it has been shown that, in addition to its primary effect on insulin sensitivity, rosiglitazone increases the insulin content of the pancreas in diabetic rats, as well as the number and total mass of islet cells (8.Buckingham R.E. Al-Barazanji K.A. Toseland C.D. Slaughter M. Connor S.C. West A. Bond B. Turner N.C. Clapham J.C. Peroxisome proliferator-activated receptor-gamma agonist, rosiglitazone, protects against nephropathy and pancreatic islet abnormalities in Zucker fatty rats.Diabetes. 1998; 47: 1326-1334Google Scholar, 9.Jones N.P. Charbonnel B. Lannqvist F. Owen S. Patwardhan R. Rosiglitazone reduces plasma insulin and its precursor while decreasing glycaemia in type 2 diabetes.Diabetologia. 1999; 42 (Abstr. 22): 859Google Scholar). Whether this is a direct effect of rosiglitazone through activation of a peroxisome proliferator-activated receptor γ-mediated pathway in islets or an indirect result of normalizing the glycemia is not known, but the molecular changes that are associated with this islet cell remodeling are potentially important to the long term efficacy of rosiglitazone. In the current study, nine significant (p < 0.05) differentially expressed polypeptides were detected between lep/lep and control mice, and four of these were modulated by rosiglitazone treatment only in lep/lep mice.Of the differentially expressed proteins not modulated by rosiglitazone, dihydropteridine reductase (and a fragment) were reduced 10-fold in islets from lep/lep mice. Dihydropteridine reductase converts dihydrobiopteridin (BH2) to tetrahydrobiopteridin (BH4) in a NADH-mediated reaction. BH4 is an essential molecule in the synthesis of serotonin and catecholaminergic neurotransmitters. As demonstrated by Masiello et al. (30.Masiello P. Balestreri E. Bacciola D. Bergamini E. Influence of experimental diabetes on brain levels of monoamine neurotransmitters and their precursor amino acids during tryptophan loading.Acta Diabetol. Lat. 1987; 24: 43-50Google Scholar), in the brain of streptozotocin-induced diabetic rats, its deficiency potentially results from reduced neurotransmitter synthesis in islets. Glutathione S-transferase was reduced 4-fold in lep/lep mice, as it was also in white adipose tissue. 2J.-C. Sanchez, V. Converset, A. Nolan, G. Schmid, S. Wang, M. Heller, M. V. Sennitt, D. F. Hochstrasser, and M. A. Cawthorne, unpublished results. Glutathione S-transferase has also been found to be reduced in liver of lep/lep mice, and streptozotocin-induced diabetic rats (31.Barnett C.R. Abbott R.A. Bailey C.J. Flatt P.R. Ioannides C. Cytochrome P-450-dependent mixed-function oxidase and glutathione S-transferase activities in spontaneous obesity-diabetes.Biochem. Pharmacol. 1992; 43: 1868-1871Google Scholar), and in the placenta of overt diabetic patients (32.Glover D.D. McRobie D.J. Tracy T.S. Effects of gestational and overt diabetes on placental cytochromes P450 and glutathione S-transferase.Prim. Care Update Ob. Gyns. 1998; 5: 189Google Scholar). Reactive intermediates generated through metabolism are detoxicated by several systems. One mechanism is the conjugation of biologically active electrophiles with endogenous tripeptide glutathiones leading to the final formation and excretion of mercapturic acid. The reaction is catalyzed by glutathione S-transferase (33.Habig W.H. Pabst M.J. Jakoby W.B. Glutathione S-transferases. The first enzymatic step in mercapturic acid formation.J. Biol. Chem. 1974; 249: 7130-7139Google Scholar).