The molecular pathways leading to islet fibrosis in diabetes are unknown. Therefore, we studied gene expression in islets of 4-month-old Goto-Kakizaki (GK) and Wistar control rats. Of 71 genes found to be overexpressed in GK islets, 24% belong to extracellular matrix (ECM)/cell adhesion and 34% to inflammatory/immune response families. Based on gene data, we selected several antibodies to study fibrosis development during progression of hyperglycemia by immunohistochemistry. One-month-old GK and Wistar islets appeared to be similar. Two-month-old GK islets were strongly heterogenous in terms of ECM accumulation compared with Wistar islets. GK islet vascularization, labeled by von Willebrand factor, was altered after 1 month of mild hyperglycemia. Numerous macrophages (major histocompatibility complex class II+ and CD68+) and granulocytes were found in/around GK islets. These data demonstrate that marked inflammatory reaction accompanies GK islet fibrosis and suggest that islet alterations in this nonobese model of type 2 diabetes develop in a way reminiscent of microangiopathy.

Islet fibrosis has been observed in humans and also in various spontaneous rodent models of type 2 diabetes, with or without obesity. In 4-month-old diabetic nonobese Goto-Kakizaki (GK) rats, Kakizaki et al. (1) first described the presence of large islets where endocrine cells were disrupted by fibrotic tissue (“starfish-like islets”), which later was confirmed by other groups (2,3). In nondiabetic Zucker fatty rats, occasional fibrotic and irregular islets are seen by 12 weeks of age (4), while these fibrotic islets are more frequent in younger (5- to 7-week-old) Zucker diabetic fatty (ZDF) rats. By 12 weeks of age, many ZDF rat islets are markedly abnormal with fibrosis and irregular projections in the exocrine tissue (4). The Otsuka Long-Evans Tokushima fatty (OLETF) rats also show disorganized fibrotic islets with vessel anomalies (5). A more recently established inbred strain, the spontaneously diabetic Torii (SDT) rat, develops hyperglycemia without obesity after 20 weeks of age (6). The primary changes of SDT islets consist of microvascular events at 8–10 weeks. At that time, SDT rats have lower plasma insulin and lower β-cell mass. These vascular alterations are followed by inflammation and progressive fibrosis and eventually islet atrophy. Islet architectural changes are also observed in type 2 diabetic mice, in association with presence of inflammatory cells and some extracellular matrix (ECM) deposition (7).

In humans, amyloid deposition leads to morphological islet alterations close to those observed in spontaneous rat type 2 diabetic models (8). Amyloid can be observed as perivascular deposits adjacent to capillaries surrounding the islet and also adjacent to capillaries penetrating the islet core. The most extensive islet amyloid deposits are associated with decreased β-cell mass (8).

Fibrosis is usually a consequence of an inflammatory reaction. Taking into account the increasing role of inflammation in type 2 diabetes pathogenesis (9,10), we investigated in greater depth islet alterations occurring in GK rats, a type 2 diabetes model that we have previously and extensively studied from fetal life onwards (2,11,12). Before birth, GK rats present decreased β-cell mass and low plasma insulin as compared with Wistar control rats. Then, they develop mild hyperglycemia and insulin resistance around weaning (3–4 weeks of age). Here, using a complementary approach that associated gene expression analysis (Affymetrix microarrays), quantitative RT-PCR, and immunohistochemical studies of pancreata as a function of hyperglycemia duration, we demonstrate that an inflammatory reaction is associated with islet fibrosis in 4-month-old diabetic GK rats according to a process reminiscent of microangiopathy. These alterations worsened with hyperglycemia duration and might contribute to enhanced GK β-cell impairment.

For gene analysis and immunohistochemistry, experimentation was conducted on fed 15- to 16-week-old and 4-, 8-, and 16-week-old male GK rats and age- and sex-matched nondiabetic Wistar rats from our local colonies, respectively. The characteristics of the nonobese GK rat model of type 2 diabetes maintained in our colony at University Paris 7 have been described previously (12). Glycemia was determined with a glucose analyzer (Beckman). At 1 (unweaned animals), 2, and 4 months of age, basal morning blood glucose levels were: 6.6 ± 0.2 and 10.8 ± 0.5 mmol/l (n = 5–8, P < 0.001), 7.3 ± 0.3 and 10.7 ± 0.8 mmol/l (n = 6–7, P < 0.01), and 6.5 ± 0.3 and 11.9 ± 0.8 mmol/l (n = 5–7, P < 0.002), for Wistar and GK rats, respectively (unpaired Student’s t test). Animal experimentation was performed in accordance with accepted standards of animal care as established in the French National Center for Scientific Research guidelines.

Islet isolation.

Rats were injected intraperitoneally with pentobarbital sodium (1 ml/kg body wt; Ceva Santé Animale, Libourne, France). For glycemia determination, blood samples were collected after decapitation and immediately centrifuged at 4°C. Pancreata were digested with collagenase according to standard procedures. Subsequently, islets were purified using a continuous Histopaque (Sigma, St. Louis, MO) gradient. Purified islet fractions were collected, and all islets (i.e., islets with normal architecture and islets more or less affected by fibrosis) were hand picked up under a stereomicroscope and lysate. The homogenized lysate was conserved at −80°C until RNA extraction. Six to 16 different islet isolation were done for Wistar and GK rats (n = 20 Wistar and n = 50 GK) with islet recovery per rat of 307 ± 32 and 72 ± 14, respectively.

RNA preparation and differential gene expression by cDNA analysis.

RNA was extracted from Wistar and GK islets using the RNeasy total RNA Isolation Kit (Qiagen, Hilder, Germany). Using the Superscript Choice System (Invitrogen, Groningen, Netherlands), 7 μg RNA was used to synthesize double-stranded cDNA. In vitro transcription was carried out on 6 μl cDNA using Bioarray High Yield RNA transcript-labeling reagents (Enzo Diagnostics, New York, NY). Reactions yielded 50–70 μg biotin-labeled cRNA, which was purified on RNeasy affinity columns (Qiagen) and fragmented at 94°C for 35 min in fragmentation buffer as previously described (13). Then, 15–20 μg fragmented cRNA was hybridized to the Affymetrix RG-U34A oligonucleotide microarrays representative of 8,799 rat genes. Arrays were scanned, and the data obtained were analyzed using Microarray suite 5.0, Affymetrix Data Mining tool 2.0, and Genespring 6 (Agilent Technologies, Palo Alto, CA). The microarray experiments were performed in collaboration with the Genomics Platform of the NCCR Frontiers, Geneva. Gene expression was determined relative to the housekeeping genes glyceraldehyde-3-phosphate dehydrogenase and the ribosomal gene L3. Genes were considered up- or downregulated if the averaged fold change was at least two in duplicate experiments. Genes were assigned to functional groups by database searches on PubMed and Affymetrix websites.

Quantitative RT-PCR.

RNA was isolated using RNeasy Mini kit (Qiagen). cDNA was synthesized with Superscript II (Invitrogen, Basel, Switzerland), using 1 μg of total RNA in a 20-μl reaction volume. The dsDNA-specific dye SYBR Green I (Eurogentech, Bruxels, Belgium) and fluorescein (Biorad, Hercules, CA) were incorporated into the PCR buffer (qPCR core kit, Eurogentech) to allow for quantitative detection of the PCR product (for primers see Table 1). The results were analyzed using the iCycler iQ System (Biorad). The housekeeping genes glyceraldehyde-3-phosphate dehydrogenase and ribosomal protein (L3) were used as internal controls.

Immunohistochemistry.

For insulin labeling, pancreata from Wistar and GK rats were excised, fixed in aqueous Bouin’s solution, and embedded in paraplast, according to standard procedures. Histological sections of pancreas (7-μm thick) were prepared and mounted on glass microscope slides (Superfrost Plus, Kindler O, Freiburg, Germany). Slides were incubated overnight at 4°C in a humidified chamber with guinea pig anti-porcine insulin. For insulin detection, a peroxidase-conjugated rabbit anti–guinea pig IgG was used (for antibodies see Table 2). After washing, peptide immunoreactivity was localized with 3,3′-diaminobenzidine-tetra-hydrochloride using a peroxidase substrate kit (DAB; Vector Laboratories, Burlingame, CA). Tissue sections were counterstained with hematoxylin and mounted under glass coverslips.

For ECM protein and immune cell labeling, pancreata were removed, embedded in optimal cutting temperature (Tissue-Tek, Miles, Elkart, IN), and frozen in n-hexane on dry ice–chilled alcohol. Tissues were stored at −80°C until immunohistochemistry was performed. For 10 min, 6-μm thick cryostat sections were fixed in acetone. After washing with PBS containing 0.05% Tween-20 (Merck, Paris, France) (PBS/Tween), slides were incubated with primary antibodies for 30 min at room temperature in a moist chamber. Subsequently, slides were washed twice with PBS/Tween and incubated with appropriate peroxidase-coupled secondary antibodies in the absence or presence of 1% normal rat serum for 30 min at room temperature (for antibodies see Table 2). Following further PBS/Tween washing, slides were incubated with 3-amino-9-ethylcarbazole (Sigma, Saint-Quentin-Fallavier, France) as the substrate in 50 mmol/l sodium acetate and 0.02% H2O2 and washed in water after 3 min. Finally, slides were counterstained for 3 min in Harri’s hematoxylin (Merck), dehydrated in serially graded ethanol baths, and mounted. For each series of pancreas sections, one slide was stained only with the second antibody as a control for endogenous peroxidase activity and nonspecific antibody binding.

Large disorganized islets are present in 4-month-old diabetic GK pancreas.

At the age of 4 months, total β-cell mass has been shown to be decreased by 60% in parallel to pancreatic insulin stores in GK rats as compared with Wistar rats (2). Two populations of islets were observed in GK rats: small islets with heavily stained β-cells and normal architecture (Fig. 1A) and large islets with spots of heterogeneously insulin-stained cells intermingled with fibrosis (Fig. 1B). This latter type of islet strongly differed from normal islets of age-matched Wistar control rats, where β-cells covered almost homogeneously the islet area, with only thin connective tissue spaces (Fig. 1C).

Gene expression analysis highlights the presence of an inflammatory/immune reaction in GK islets.

We used Affymetrix microarrays to detect gene expression changes in islets of 4-month-old GK rats as compared with those of the nondiabetic Wistar controls. In GK islets, 71 genes were overexpressed and 19 genes were underexpressed as compared with Wistar control islets (Table 3). The analysis of the 71 overexpressed sequences according to known cellular function has led us to identify seven clusters: 23.9% of overexpressed genes are implicated in ECM/cell adhesion; 16.9% in inflammation; 16.9% in immune response; 2.8% in oxidative stress; 9.9% in metabolism; 5.6% in growth control, survival, and differentiation; and 23.9% are not classified (Table 3). ECM/cell adhesion, inflammation, immune response, and oxidative stress are the four principal families of potential interest in the context of our investigation. To validate our Affymetrix data, we selected a few genes: collagen I, collagen III, decorin, fibronectin, membrane-type 1 matrix metalloproteinase (also called MMP-14), and tissue inhibitor of metalloproteinase-1 for ECM/cell adhesion; annexin 1 (or lipocortin 1), lipocalin 2 (or neutrophil gelatinase-associated lipocalin), lipopolysaccharide-induced tumor necrosis factor α, and osteopontin for inflammation; CD53, CD74, and major histocompatibility complex (MHC) II for immune response; and thioredoxine-interacting protein and glutathione peroxidase for oxidative stress. The overexpression of all of these genes has been successfully confirmed by RT-PCR.

Fibrotic islet alterations result from mild and short-duration hyperglycemia.

We selected antibodies against three proteins that belong to the ECM/cell adhesion family and had genes we found to be overexpressed in GK islets: collagen I and III and fibronectin. These antibodies were used for a immunohistochemical analysis of development of islet fibrosis as a function of duration of diabetes in GK rats.

At 1 month of age, islets of unweaned GK rats showed no sign of fibrosis as compared with control Wistar islets (14). However, at 2 months of age (i.e., after 1 month of chronic mild hyperglycemia), marked differences were observed (Fig. 1). In Wistar islets, fine labeling for collagen I and III and fibronectin, which are all known to be produced by vascular endothelial and/or smooth muscle cells (15,16) was present with intra- and peri-islet localization, therefore indicative of the presence of vessels (Fig. 1D–F). In GK islets, however, a peri- and intraislet thickening was observed in many medium-sized islets, and increased labeling of all the ECM components examined was observed: e.g., collagen I (Fig. 1G versus D, GK versus Wistar, respectively), collagen III (Fig. 1H versus E), and fibronectin (Fig. 1I versus F). In 4-month-old control Wistar rat islets, the labeling pattern for the various ECM molecules was similar to that described in 2-month-old Wistar rat islets (Fig. 1J–L versus DF at 4 versus 2 months of age for collagen I and III and fibronectin, respectively). However, in 4-month-old GK rats, more precisely after 3 months of chronic hyperglycemia, when microarray analysis was performed, the largest islets showed massive fibrosis, as illustrated for collagen I (Fig. 1M), collagen III (Fig. 1N), and fibronectin (Fig. 1O). Nevertheless, some small and nonfibrotic islets were still present (Fig. 1M), which is in agreement with insulin labeling of GK islets (Fig. 1A).

Alteration of vascularization time correlates with hyperglycemia in GK islets.

As described above, as early as 2 months of age (i.e., 1 month after diabetes onset), immunohistochemistry for the various ECM molecules suggested that vascularization might be the starting point of fibrosis formation in GK islets. Therefore, we compared the expression of a specific endothelial cell marker, von Willebrand factor (vWF), which is known to be increased in the blood of type 2 diabetic patients (10), on serial sections of 2-month-old Wistar and GK pancreata. Representative stainings for vWF and fibronectin of control Wistar islets are shown in Fig s. 2A and B, respectively. As expected, Wistar islets are characterized by fine labeling of endothelial cells for both molecules. In age-matched GK pancreata, however, islet vascularization differed markedly from that of Wistar islets, as assessed by vWF labeling (Fig. 2C, E, and G versus A). Moreover, in a given GK rat pancreas, islets were extremely heterogenous in terms of vascularization; indeed, vessels appeared to be more or less hypertrophied (Fig. 2C and E) or even greatly disorganized (Fig. 2G). As demonstrated in Fig. 2C and D, for vWF and fibronectin, respectively, expression of both molecules is partially colocalized in midly hypertrophied vessels. Finally, while sized-matched GK islets showed different degrees of vascularization alteration (Fig. 2C, E, and G), the extent of fibronectin labeling in corresponding serial sections greatly varied (Fig. 2D, F, and H).

Macrophage and granulocyte infiltration time correlates with hyperglycemia in GK islets.

Our microarray data revealed that 16.9% of the genes that were found to be overexpressed in GK islets coded for molecules involved in immune response. Using quantitative RT-PCR, we confirmed the overexpression of three of these genes, namely CD53, CD74, and MHC class II (Table 3). Since MHC class II is a well-known macrophage marker (17), we investigated the presence of macrophages in the pancreata of both groups at the different ages. We used several antibodies that were available in rats: 1) anti-MHC class II; 2) ED1, which recognizes a 110-kD single-chain glycoprotein (human equivalent CD68), which is expressed predominantly on the lysosomal membrane of myeloid cells and is found on the majority of tissue macrophages and weakly on peripheral granulocytes (18); 3) ED2, which reacts with a membrane antigen (human equivalent CD163) on resident rat macrophages (18); and 4) ED3 (CD169), which recognizes a receptor for syalyated glycoconjugates and characterized tissue macrophage subpopulations involved in autoimmune disease (18). No difference was observed in 1-month-old Wistar and GK islets concerning MHC class II and CD68 islet labeling (data not shown). However, at 2 months of age (i.e., after 1 month of chronic mild hyperglycemia), labeling shows constantly more macrophages in/around GK islets than Wistar islets (Fig. 3B versus A for MHC class II, and Fig. 3D versus C for CD68). A similar observation has been done for both markers in 4-month-old animals of the two groups (14). However, at 4 months of age, macrophage GK islet infiltration appeared less pronounced than at 2 months. Using the ED2 macrophage antibody, no difference was observed between the two strains regardless of age, and macrophages recognized by the ED3 antibody were never detected (data not shown).

CD53 cell-surface antigen is a 43-kDa glycoprotein that is expressed by all myeloid and peripheral lymphoid B- and T-cells and also a small subset of thymocytes (19). In 1-month-old animals, CD53+ cells were found to be located near the ducts, particularly in GK rats (data not shown). At 2 months of age, a more- or less-marked CD53+ cell islet infiltration was observed in/around GK islets, while these cells were scarce in age-matched Wistar islets (Fig. 3F versus E). A similar observation has been done in 4-month-old GK pancreata, in which more CD53+ cells were reproducibly found at the islet-ductal pole (data not shown). For a better characterization of this cell population, we used several antibodies against granulocytes (clone HIS48), mature T-cells (anti-CD6), or B-cells (anti-CD45R). At 2 months of age in both strains, T- and B- cells were exceptionally observed in (or close to) islets (data not shown). While granulocytes were scarce in 2-month-old Wistar pancreata (Fig. 3G), they were much more numerous in/around GK islets (Fig. 3H), thus confirming the myeloid phenotype of CD53+ cells.

To investigate the mechanisms leading to fibrosis in GK islets, we performed a gene expression analysis using Affymetrix microarrays on islets of 4-month-old GK and age-matched control Wistar rats. Notably, around 60% of the genes that were overexpressed in 4-month-old GK rat islets (i.e., after 3 months of mild hyperglycemia) belong to ECM/cell adhesion (23.9%), inflammation (16.9%), immune response (16.9%), and oxidative stress (2.8%). For fifteen genes of interest, the relative expression levels were assessed by RT-PCR and found to be comparable to the microarray values.

While our gene analysis confirms the presence of fibrosis, more pertinently it highlights the presence of an inflammatory/immune reaction in islets of a nonobese animal model of spontaneous type 2 diabetes. But previously, no signs of inflammation had been described in the islets or acinar parenchyma of 3- to 3.5-month-old GK rats, while 28% of starfish islets were present (3). However, fibrosis is usually linked to inflammation and a common feature of type 2 diabetes in several animal models and also in humans (amyloid deposition) (48,20). Therefore, local islet inflammation might be a general phenomenom in type 2 diabetes, in addition to the presently well-acknowledged peripheral and adipose tissue inflammation (10).

However, at a given time, as shown here at 4 months of age (Fig. 1M–O), small (recently formed and not yet affected) and older, larger, more- or less-affected islets coexist. We also showed that islet fibrosis progresses with the duration of hyperglycemia. Since hyperglycemia is known to stimulate the secretion of fibronectin and collagen I and III by endothelial cells and/or vascular smooth muscle cells (15,16), we analyzed islet vascularization in Wistar and GK rats at various ages using vWF, a specific marker of endothelial cells (10). At 2 months of age, GK islet vascularization was heterogenous; it could be similar to that of Wistar islets, or more developed (in a way that is closely associated to fibronectin deposition) or greatly disorganized. These data suggest that ECM deposition progresses from intra- and peri-islet vessels, as it is known to happen in microangiopathy (21). Abnormalities of islet vascularization have been described in two other spontaneous models of type 2 diabetes, namely OLETF and SDT rats, but these alterations were not detected in a colony of GK rats that differ from ours (5,6,22). In humans, amyloid deposition exists as “perivascular deposits adjacent to capillaries surrounding the islet or those penetrating the islet core with limited accumulation or extensive deposits within the islet” (8).

Altered vascularization and abnormal blood flow have been described in spontaneous animal models of type 2 diabetes, particularly GK and OLETF rats (11,2326). Increased islet blood flow, which is present in young diabetic GK rats, is reverted to decreased flow when animals reach the age of 1 year (23). In OLETF rats, increased islet blood flow is also observed during the pre-diabetic phase (26), and, as the rats advance in age, the fine capillaries that form the intraislet network are extremely sparse in the lean diabetic group as compared with the age-matched obese OLETF group or control Long-Evans Tokushima Otsuka (LETO) group (25). In GK rats, early increased capillary blood flow might be one of the factors that will damage the islet endothelium, induce thickening of the capillary walls, reduce islet blood flow, as described in diabetic retina and kidneys, and finally contribute to decline of islet function (24). Therefore, we looked for genes that are overexpressed in GK islets and belong to the families depicted above but are also expressed normally in endothelial and vascular smooth muscle cells and involved in angiogenesis and atherosclerosis. Decorin, galectin 3 (or lectin galactose binding, soluble 3) (Table 3), tissue inhibitor of metalloproteinase-1, and membrane-type 1 matrix metalloproteinase are known to be involved in angiogenesis and/or atherosclerosis (2730). In addition expression of osteopontin or galectin 3 is increased in human diabetic arteries and/or under high glucose concentration in rat aortic diabetic arteries and/or also under high glucose concentration in rat aortic smooth muscle (27,31,32).

Endothelial dysfunction is the hallmark of diabetes complications regardless of its type (33,34). Indeed, hyperglycemia is known to activate intra- and peri-islet endothelial cells, leading to proinflammatory cytokine production and adhesion molecule expression that facilitate the recruitment, adhesion, and migration of leukocytes (3538). In GK kidney and aorta, endothelial modifications have been described where they are associated with monocyte/macrophage infiltration and increased macrophage-induced angiogenesis, respectively (39,40). Our immunohistochemical data reveal the presence of more MHC class II+ and CD68+ macrophages in and/or around GK islets than in those of Wistar at 2 and 4 months of age. More CD68+ macrophages are also present in human type 2 diabetes islets (J. Ehses, A. Perren, M. Donath, personal communication). In addition, we found overexpression of the gene for CD74 (macrophage inhibitory factor receptor): CD74 coexists with CD68 on macrophages (41,42). Finally, ED3+ macrophages and mature T- and B-cells are absent in the early phase of the disease, arguing against a precocious autoimmune reaction in this type 2 diabetic model.

Activated macrophages produce various ECM-related molecules, chemokines (and their receptors), inflammatory cytokines, and growth factors (43,44). The secretion of some inflammatory factors such as interleukin-1 and -6, tumor necrosis factor α, transforming growth factor β1, and macrophage chemoattractant protein by monocytes/macrophages is stimulated by hyperglycemia (45,46). From our gene expression analysis (Table 3), overexpression of some other genes might be attributed to effect on, or production by, islet macrophages: this is the case for immediate early serum-responsive JE gene that corresponded to macrophage chemoattractant protein-1 gene and genes coding for proinflammatory factors, such as lipopolysaccharide-induced tumor necrosis factor α (47) and galectin 3 (48), or anti-inflammatory factors, such as apolipoprotein E (49) and lipocortin 1 (annexin 1) (50) and also transferrin, a circulating negative acute-phase protein that is downregulated in inflammatory conditions such as diabetes (51) and might be of macrophage origin (43).

An impressive infiltration of cells positive for CD53 (another gene that was found to be overexpressed in GK islets) is present in diabetic GK pancreata. These CD53+ cells correspond to granulocytes and not to peripheral lymphocytes. Notably, macrophage (and endothelium)-produced galectin 3 is known to facilitate binding of neutrophils to the endothelium (48). Activated granulocytes are potential producers of toxic oxygen metabolites, lytic and toxic proteases, nitric oxide, and inflammatory cytokines (52). These data are also in line with the gene overexpression for lipocaline 2 (neutrophil gelatinase–associated lipocalin) (Table 3), which is produced mainly by granulocytes. Lipocalin 2 is considered a sign of leukocyte activation in various diseases, particularly hypertension and acute cerebral ischemic attack (53). In addition, lipocortin 1 (or annexin 1), as mentioned above for macrophages, is also produced by granulocytes (54). Finally, granulocytes are associated with capillary closure in spontaneously diabetic monkey retinas (55), and granulocytes might play a role in atherosclerosis (56).

In conclusion, our data demonstrate, for the first time, that an inflammatory reaction takes place at the islet level in a type 2 diabetic animal model, resembling microangiopathy with subsequent fibrosis leading to loss of islet architecture and probably responsible for increased β-cell impairment. In various type 2 diabetic animal models, treatments that preserve islet architecture, thiazolinediones (rosiglitazone), ACE inhibitor (ramipril), and protease inhibitor (camostat) improve β-cell function (5,20,57,58). Also, glitazones ameliorate endothelial dysfunction in patients with diabetes and lower inflammatory markers and reactive oxygen species in serum (59). Therefore, our data contributing to a better understanding of the pathogenesis of the disease might lead to design β-cell–sensitizing molecules with improved anti-inflammatory and anti-atherosclerotic effects.

FIG. 1.

Islet fibrosis progresses rapidly in mild hyperglycemic GK rats. Insulin labeling demonstrates the concomitant presence of small normal and large fibrotic islets (A and B, respectively) in 4-month-old GK pancreas as compared with age-matched control Wistar pancreas (C) (×500). As early as 1 month of chronic mild hyperglycemia (i.e., at 2 months of age), many GK islets show, compared with Wistar islets, peri- and intraislet thickening of vascularization, as demonstrated by labeling for collagen I (G versus D), collagen III (H versus E), or fibronectin (I versus F) (×500). At 4 months of age, fibrosis is extensive in large GK islets, as shown by collagen I (M versus J for GK and Wistar, respectively), collagen III (N versus K), and fibronectin (O versus L) labelings (×250). Of note, small intact islets coexist with large fibrotic islets, as shown in M (collagen I labeling), confirming the insulin data (A and B). d, duct; i, islet.

FIG. 1.

Islet fibrosis progresses rapidly in mild hyperglycemic GK rats. Insulin labeling demonstrates the concomitant presence of small normal and large fibrotic islets (A and B, respectively) in 4-month-old GK pancreas as compared with age-matched control Wistar pancreas (C) (×500). As early as 1 month of chronic mild hyperglycemia (i.e., at 2 months of age), many GK islets show, compared with Wistar islets, peri- and intraislet thickening of vascularization, as demonstrated by labeling for collagen I (G versus D), collagen III (H versus E), or fibronectin (I versus F) (×500). At 4 months of age, fibrosis is extensive in large GK islets, as shown by collagen I (M versus J for GK and Wistar, respectively), collagen III (N versus K), and fibronectin (O versus L) labelings (×250). Of note, small intact islets coexist with large fibrotic islets, as shown in M (collagen I labeling), confirming the insulin data (A and B). d, duct; i, islet.

FIG. 2.

Islet vascularization is altered in 2-month-old GK rats. Labelings for vWF (left) and fibronectin (right), two factors known to be produced by endothelial cells, show the normal organization of islet vascularization in 2-month-old Wistar rats (A and B for vWF and fibronectin, respectively). C, E, and G: Islet vascularization differs markedly in age-matched GK rats and appears to be either hypertrophied (C and E) or atrophied (G). At least in the first steps of the lesion formation, fibronectin and vWF labelings progress similarly (C versus D, for vWF and fibronectin, respectively), before invading the islet and desorganizing vascularization (G versus H) (×500).

FIG. 2.

Islet vascularization is altered in 2-month-old GK rats. Labelings for vWF (left) and fibronectin (right), two factors known to be produced by endothelial cells, show the normal organization of islet vascularization in 2-month-old Wistar rats (A and B for vWF and fibronectin, respectively). C, E, and G: Islet vascularization differs markedly in age-matched GK rats and appears to be either hypertrophied (C and E) or atrophied (G). At least in the first steps of the lesion formation, fibronectin and vWF labelings progress similarly (C versus D, for vWF and fibronectin, respectively), before invading the islet and desorganizing vascularization (G versus H) (×500).

FIG. 3.

Inflammatory cells rapidly infiltrate the islets of mildy hyperglycemic GK rats. Compared with 2-month-old Wistar rats, numerous macrophages are present in/around GK islets, as shown by MHC class II (B versus A; ×250) and CD68 labelings (D versus C; ×500). Other myeloid cells are also more abundant in GK islets, as demonstrated by CD53 (F versus E; ×500) and, particularly, granulocyte labeling (H versus G; ×500). The concomitant presence of macrophages and granulocytes together with the quasiabsence of T- and B-cells and ED3 macrophages that are involved in autoimmune reaction (data not shown) suggests a pure inflammatory process in this spontaneous model of type 2 diabetes, at least at this age.

FIG. 3.

Inflammatory cells rapidly infiltrate the islets of mildy hyperglycemic GK rats. Compared with 2-month-old Wistar rats, numerous macrophages are present in/around GK islets, as shown by MHC class II (B versus A; ×250) and CD68 labelings (D versus C; ×500). Other myeloid cells are also more abundant in GK islets, as demonstrated by CD53 (F versus E; ×500) and, particularly, granulocyte labeling (H versus G; ×500). The concomitant presence of macrophages and granulocytes together with the quasiabsence of T- and B-cells and ED3 macrophages that are involved in autoimmune reaction (data not shown) suggests a pure inflammatory process in this spontaneous model of type 2 diabetes, at least at this age.

TABLE 1

Primer oligonucleotide sequences

Sequences
Annexin 1/lipocortin 1 Forward: 5′ CCT GGA GGA GGT TGT TTT GG 3′ 
Annexin 1 Reverse: 5′ CAG CAC GGA GTT CAT CTG CAT 3′ 
CD53 Forward: 5′ GCG TGG TTT CAC TCC AAT TTC 3′ 
CD53 Reverse: 5′ GGA CAT CCC CAG CAC CTG TA 3′ 
CD74 Forward: 5′ CCA GGA CCA CGT GAT GCA 3′ 
CD74 Reverse: 5′ CCC CTT CAG CTG TGG GTA GTT 3′ 
Collagen type I Forward: 5′ CCC AAC CCC CAA AAA CG 3′ 
Collagen type I Reverse: 5′ CTG CGT CTG GTG ATA CAT ATT CTT CT 3′ 
Collagen type III Forward: 5′ AGC TGG CCT TCC TCA GAC TTC 3′ 
Collagen type III Reverse: 5′ GCT GTT TTT GCA GTG GTA TGT AAT G 3′ 
Decorin Forward: 5′ GCG GCA ACC CAC TGA AAA 3′ 
Decorin Reverse: 5′ TGT ATC CGA GAC CCT TCA TTC C 3′ 
Fibronectin Forward: 5′ CCT ACG GAT GAC TCA TGC TTT G 3′ 
Fibronectin Reverse: 5′ CAG ATA ACC GCT CCC ATT CC 3′ 
Glutathione peroxidase Forward: 5′ TGC TCA TTG AGA ATG TCG CG 3′ 
Glutathione peroxidase Reverse: 5′ TGT AGT CCC GGG TCG TGG T 3′ 
Lipocaline 2 Forward: 5′ TCC GAT GAA CTG AAG GAG CG 3′ 
Lipocaline 2 Reverse: 5′ GAG GCC CAG AGA CTT GGC A 3′ 
Lipopolysaccharide-induced tumor necrosis factor α Forward: 5′ TGG CTC TCC TGT GGC AGT CT 3′ 
Lipopolysaccharide-induced tumor necrosis factor α Reverse: 5′ CGC AAA ACG GAA TGA AGC A 3′ 
MHC class II Forward: 5′ CCC TCC AGC GGT CAA TGT 3′ 
MHC class II Reverse: 5′ TGA CAC GCC TTT GGT GAC A 3′ 
Membrane type 1-matrix metalloproteinase Forward: 5′ GCC ATC ATG GCT CCC TTT TA 3′ 
Membrane type 1-matrix metalloproteinase Reverse: 5′ GCG GCG ATC GTC ATC AG 3′ 
Osteopontin Forward: 5′ CCT TCA CTG CCA GCA CAC AA 3′ 
Osteopontin Reverse: 5′ CCG TCA GGG ACA TCG ACT GT 3′ 
Tissue inhibitor of metalloproteinase-1 Forward: 5′ CCG CAG CGA GGA GTT TCT C 3′ 
Tissue inhibitor of metalloproteinase-1 Reverse: 5′ GGC AGT GAT GTG CAA ATT TCC 3′ 
Vdup1 (or thioredoxin-interacting protein) Forward: 5′ GCA AGA GTC TCC GAG TGC AGA 3′ 
Vdup1 Reverse: 5′ TGT TGC AGC CCA GAA TGG A 3′ 
Sequences
Annexin 1/lipocortin 1 Forward: 5′ CCT GGA GGA GGT TGT TTT GG 3′ 
Annexin 1 Reverse: 5′ CAG CAC GGA GTT CAT CTG CAT 3′ 
CD53 Forward: 5′ GCG TGG TTT CAC TCC AAT TTC 3′ 
CD53 Reverse: 5′ GGA CAT CCC CAG CAC CTG TA 3′ 
CD74 Forward: 5′ CCA GGA CCA CGT GAT GCA 3′ 
CD74 Reverse: 5′ CCC CTT CAG CTG TGG GTA GTT 3′ 
Collagen type I Forward: 5′ CCC AAC CCC CAA AAA CG 3′ 
Collagen type I Reverse: 5′ CTG CGT CTG GTG ATA CAT ATT CTT CT 3′ 
Collagen type III Forward: 5′ AGC TGG CCT TCC TCA GAC TTC 3′ 
Collagen type III Reverse: 5′ GCT GTT TTT GCA GTG GTA TGT AAT G 3′ 
Decorin Forward: 5′ GCG GCA ACC CAC TGA AAA 3′ 
Decorin Reverse: 5′ TGT ATC CGA GAC CCT TCA TTC C 3′ 
Fibronectin Forward: 5′ CCT ACG GAT GAC TCA TGC TTT G 3′ 
Fibronectin Reverse: 5′ CAG ATA ACC GCT CCC ATT CC 3′ 
Glutathione peroxidase Forward: 5′ TGC TCA TTG AGA ATG TCG CG 3′ 
Glutathione peroxidase Reverse: 5′ TGT AGT CCC GGG TCG TGG T 3′ 
Lipocaline 2 Forward: 5′ TCC GAT GAA CTG AAG GAG CG 3′ 
Lipocaline 2 Reverse: 5′ GAG GCC CAG AGA CTT GGC A 3′ 
Lipopolysaccharide-induced tumor necrosis factor α Forward: 5′ TGG CTC TCC TGT GGC AGT CT 3′ 
Lipopolysaccharide-induced tumor necrosis factor α Reverse: 5′ CGC AAA ACG GAA TGA AGC A 3′ 
MHC class II Forward: 5′ CCC TCC AGC GGT CAA TGT 3′ 
MHC class II Reverse: 5′ TGA CAC GCC TTT GGT GAC A 3′ 
Membrane type 1-matrix metalloproteinase Forward: 5′ GCC ATC ATG GCT CCC TTT TA 3′ 
Membrane type 1-matrix metalloproteinase Reverse: 5′ GCG GCG ATC GTC ATC AG 3′ 
Osteopontin Forward: 5′ CCT TCA CTG CCA GCA CAC AA 3′ 
Osteopontin Reverse: 5′ CCG TCA GGG ACA TCG ACT GT 3′ 
Tissue inhibitor of metalloproteinase-1 Forward: 5′ CCG CAG CGA GGA GTT TCT C 3′ 
Tissue inhibitor of metalloproteinase-1 Reverse: 5′ GGC AGT GAT GTG CAA ATT TCC 3′ 
Vdup1 (or thioredoxin-interacting protein) Forward: 5′ GCA AGA GTC TCC GAG TGC AGA 3′ 
Vdup1 Reverse: 5′ TGT TGC AGC CCA GAA TGG A 3′ 
TABLE 2

List of antibodies

AntibodiesSpecificitySupplierDilution
Primary antibodies    
    Collagen I Rabbit anti-rat Novotec 1:40 
    Collagen IIII Rabbit anti-rat Novotec 1:50 
    Fibronectin Rabbit anti-rat Novotec 1:40 
    Insulin Guinea pig anti-porcine ICN Pharmaceuticals 1:1000 
    vWF Rabbit anti-human DakoCytomation 1:100 
    MHC class II Mouse anti-mouse/rat I-A Serotec 1:300 
    ED1 Mouse anti-rat CD68 Serotec 1:100 
    ED2 Mouse anti-rat CD153 BMA Biomedicals 1:500 
    ED3 Mouse anti-rat CD169 BMA Biomedicals 1:500 
    CD53 Mouse anti-rat Serotec 1:30 
    Granulocytes Mouse anti-rat Serotec 1:30 
    T-cell (CD6) Mouse anti-rat CD6 Serotec 1:30 
    B-cell (CD45R) Mouse anti-rat CD45R Serotec 1:30 
Secondary antibodies    
    PO399 Swine anti-rabbit IgG: HRP Dako 1:100 
    M3007 Goat anti-mouse IgG: HRP Caltag 1:100 + 1% rat serum 
    Star77 Goat anti-mouse IgG: HRP (rat absorbed) Serotec 1:50 
    Star86P Goat anti-mouse IgM: HRP Serotec 1:100 + 1% rat serum 
    PO141 Rabbit anti–guinea pig IgG:HRP Dako 1:50 
AntibodiesSpecificitySupplierDilution
Primary antibodies    
    Collagen I Rabbit anti-rat Novotec 1:40 
    Collagen IIII Rabbit anti-rat Novotec 1:50 
    Fibronectin Rabbit anti-rat Novotec 1:40 
    Insulin Guinea pig anti-porcine ICN Pharmaceuticals 1:1000 
    vWF Rabbit anti-human DakoCytomation 1:100 
    MHC class II Mouse anti-mouse/rat I-A Serotec 1:300 
    ED1 Mouse anti-rat CD68 Serotec 1:100 
    ED2 Mouse anti-rat CD153 BMA Biomedicals 1:500 
    ED3 Mouse anti-rat CD169 BMA Biomedicals 1:500 
    CD53 Mouse anti-rat Serotec 1:30 
    Granulocytes Mouse anti-rat Serotec 1:30 
    T-cell (CD6) Mouse anti-rat CD6 Serotec 1:30 
    B-cell (CD45R) Mouse anti-rat CD45R Serotec 1:30 
Secondary antibodies    
    PO399 Swine anti-rabbit IgG: HRP Dako 1:100 
    M3007 Goat anti-mouse IgG: HRP Caltag 1:100 + 1% rat serum 
    Star77 Goat anti-mouse IgG: HRP (rat absorbed) Serotec 1:50 
    Star86P Goat anti-mouse IgM: HRP Serotec 1:100 + 1% rat serum 
    PO141 Rabbit anti–guinea pig IgG:HRP Dako 1:50 

HRP, horseradish peroxidase.

TABLE 3

List of genes modified in GK versus Wistar islets (Affymetrix analysis)

Gene overexpression
Gene ID numberGene nameGene symbolFold change
AffymetrixRT-PCR
ECM/Cell adhesion     
    AI169327 Tissue inhibitor of metalloproteinase 1 TIMP1 30.6 5.6 
    X70369 Collagen, type 3, α1 COL3A1 17.3 10 
    X05834 Fibronectin 1 FN1 10.3 11 
    AI231472 Collagen, type 1, α1 COL1A1 8.5 27 
    X62952 Vimentin VIM 6.9  
    X81449 Keratin 19 KRT19 6.4  
    S77494 Lysyl oxidase LOX 5.4  
    AA892506 Coronin, actin-binding protein 1A CORO1A 5.1  
    X59859 Decorin DCN 4.1 2.2 
    X89963 Thrombospondin 4 THBS4 4.0  
    X83537 Matrix metalloproteinase 14, membrane inserted MMP14 3.9 6.2 
    M83107 Transgelin TAGLN 3.6  
    AF016503 Procollagen C proteinase enhancer protein PCOLCE 3.0  
    AA892333 α Tubulin TUBA6 2.8  
    U75928 Secreted protein acidic and rich in cystein (osteonectin) SPARC 2.5  
    AF083269 Actin-related protein complex 1 ARPC1B 2.3  
    X54617 Regulatory light-chain A gene for myosin regulatory light chain RCL-A 2.2  
Inflammation     
    AA946503 Lipocalin 2 LCN2 72.8 22 
    M98049 Pancreatitis-associated protein 1 PAP1 45.5  
    M14656 Osteopontin SPP1 14.9 17 
    L20869 Pancreatitis-associated protein 3 PAPIII0 9.0  
    AI171962 Annexin 1 (p35) (lipocortin 1) ANXA1 8.7 
    J02962 Lectin, galactose binding, soluble 3 (galectin 3) LGALS3 6.2  
    X17053 Immediate early serum responsive JE gene (macrophage chemoattractant protein-1) JE 5.0  
    D88250 Complement component 1, s subcomponent C1S 3.2  
    U53184 Lipopolysaccharide-induced tumor necrosis factor α LITAF 2.4 
    AI136891 Zn finger protein 36, C3H type-like 1 ZFP3611 2.3  
    L03201 Cathepsin S CTSS 2.2  
    X61381 Interferon inducible protein IFITM3 2.2  
Immune response     
    M12822 Ig germline κ-chain  17.4  
    AA801174 Ig rearanged μ-chain C region  8.7  
    AI234828 IgA heavy chain  4.3  
    X13044 CD74 antigen CD74 3.7 
    M57276 CD53 antigen CD53 3.2 10 
    X68782 Ig heavy-chain VDJ region CH1-CH2  3.1  
    U39609 Anti–nerve growth factor 30 antibody light chain  3.0  
    AA850138 Ig active λ2-like chain  2.8  
    M15562 Major histocompatibility complex class II RT1u D α chain  2.8 
    S81289 IgM κ-chain variable region  2.6  
    X14254 Major histocompatibility complex class II associated invariant chain  2.5  
    U75411 Anti-idiotype IgM light chain  2.5  
Oxidative stress     
    AI237654 Upregulated by 1,25-dihydroxyvitamin D-3 (thioredoxin-interacting protein) VDUP1 5.4 5.6 
    X07365 Gluthatione peroxidase 1 GPX1 2.7 2.2 
Metabolism     
    X15679 Preprotrypsinogen IV  17.9  
    X02291 Aldolase B ALDOB 11.9  
    X59012 Trypsin V a-form  9.0  
    D28560 Ectonucleotide pyrophosphatase/phosphodiesterase ENPP2 3.7  
    AA858673 Pancreatic secretory trypsin inhibitor type II PSTI-II 3.6  
    M54926 Lactate deshydrogenase A LDHA 2.2  
    D00753 Serine protease inhibitor SPIN2C 2.0  
Not classified     
Growth control, survival, and differentiation     
    E01983 Reg REG 12.4  
    D23676 Regenerating islet derived 3α REG3A 11.1  
    L13039 Calpactin I heavy chain ANXA2 3.3  
    U10071 Cocaine- and amphetamine-regulated transcript CART 3.3  
    D26307 Jun-D gene JUND 13.1  
    U32681 Deleted in malignant brain tumors 1 DMBT1 11.6  
    AF014503 Nuclear protein 1 NUPR1 8.2  
    AB012234 Nuclear factor I/X NFIX 7.8  
    AA875531 Cyclin-dependent kinase 110 CDK110 7.6  
    D38380 Transferrin TF 6.7  
    J03627 S-100 related protein S100a10 3.5  
    M58716 Secretory granule membrane glycoprotein GP2 3.2  
    AI137583 Inhibitor DNA binding 2 ID2 3.1  
    S87544 Polyprotein 1-microglobulin/bikunin AMBP 2.9  
    M27440 Apolipoprotein B APOB 2.8  
    AA899854 Topoisomerase (DNA) 2 α TOP2A 2.7  
    AA849769 Follistatin-related protein FSTL1 2.7  
    U95178 Disabled homolog 2, mitogen-responsive phophoprotein DAB2 2.3  
    X06916 S100 calcium binding protein A4 S100a4 2.3  
    AF020757 Purinergic receptor P2X, ligand-gated ion channel, 2 P2RX2 2.3  
    S76779 Apolipoprotein E APOE 2.0  
ESTs 12 sequences     
8 Repetitive sequences     
Gene overexpression
Gene ID numberGene nameGene symbolFold change
AffymetrixRT-PCR
ECM/Cell adhesion     
    AI169327 Tissue inhibitor of metalloproteinase 1 TIMP1 30.6 5.6 
    X70369 Collagen, type 3, α1 COL3A1 17.3 10 
    X05834 Fibronectin 1 FN1 10.3 11 
    AI231472 Collagen, type 1, α1 COL1A1 8.5 27 
    X62952 Vimentin VIM 6.9  
    X81449 Keratin 19 KRT19 6.4  
    S77494 Lysyl oxidase LOX 5.4  
    AA892506 Coronin, actin-binding protein 1A CORO1A 5.1  
    X59859 Decorin DCN 4.1 2.2 
    X89963 Thrombospondin 4 THBS4 4.0  
    X83537 Matrix metalloproteinase 14, membrane inserted MMP14 3.9 6.2 
    M83107 Transgelin TAGLN 3.6  
    AF016503 Procollagen C proteinase enhancer protein PCOLCE 3.0  
    AA892333 α Tubulin TUBA6 2.8  
    U75928 Secreted protein acidic and rich in cystein (osteonectin) SPARC 2.5  
    AF083269 Actin-related protein complex 1 ARPC1B 2.3  
    X54617 Regulatory light-chain A gene for myosin regulatory light chain RCL-A 2.2  
Inflammation     
    AA946503 Lipocalin 2 LCN2 72.8 22 
    M98049 Pancreatitis-associated protein 1 PAP1 45.5  
    M14656 Osteopontin SPP1 14.9 17 
    L20869 Pancreatitis-associated protein 3 PAPIII0 9.0  
    AI171962 Annexin 1 (p35) (lipocortin 1) ANXA1 8.7 
    J02962 Lectin, galactose binding, soluble 3 (galectin 3) LGALS3 6.2  
    X17053 Immediate early serum responsive JE gene (macrophage chemoattractant protein-1) JE 5.0  
    D88250 Complement component 1, s subcomponent C1S 3.2  
    U53184 Lipopolysaccharide-induced tumor necrosis factor α LITAF 2.4 
    AI136891 Zn finger protein 36, C3H type-like 1 ZFP3611 2.3  
    L03201 Cathepsin S CTSS 2.2  
    X61381 Interferon inducible protein IFITM3 2.2  
Immune response     
    M12822 Ig germline κ-chain  17.4  
    AA801174 Ig rearanged μ-chain C region  8.7  
    AI234828 IgA heavy chain  4.3  
    X13044 CD74 antigen CD74 3.7 
    M57276 CD53 antigen CD53 3.2 10 
    X68782 Ig heavy-chain VDJ region CH1-CH2  3.1  
    U39609 Anti–nerve growth factor 30 antibody light chain  3.0  
    AA850138 Ig active λ2-like chain  2.8  
    M15562 Major histocompatibility complex class II RT1u D α chain  2.8 
    S81289 IgM κ-chain variable region  2.6  
    X14254 Major histocompatibility complex class II associated invariant chain  2.5  
    U75411 Anti-idiotype IgM light chain  2.5  
Oxidative stress     
    AI237654 Upregulated by 1,25-dihydroxyvitamin D-3 (thioredoxin-interacting protein) VDUP1 5.4 5.6 
    X07365 Gluthatione peroxidase 1 GPX1 2.7 2.2 
Metabolism     
    X15679 Preprotrypsinogen IV  17.9  
    X02291 Aldolase B ALDOB 11.9  
    X59012 Trypsin V a-form  9.0  
    D28560 Ectonucleotide pyrophosphatase/phosphodiesterase ENPP2 3.7  
    AA858673 Pancreatic secretory trypsin inhibitor type II PSTI-II 3.6  
    M54926 Lactate deshydrogenase A LDHA 2.2  
    D00753 Serine protease inhibitor SPIN2C 2.0  
Not classified     
Growth control, survival, and differentiation     
    E01983 Reg REG 12.4  
    D23676 Regenerating islet derived 3α REG3A 11.1  
    L13039 Calpactin I heavy chain ANXA2 3.3  
    U10071 Cocaine- and amphetamine-regulated transcript CART 3.3  
    D26307 Jun-D gene JUND 13.1  
    U32681 Deleted in malignant brain tumors 1 DMBT1 11.6  
    AF014503 Nuclear protein 1 NUPR1 8.2  
    AB012234 Nuclear factor I/X NFIX 7.8  
    AA875531 Cyclin-dependent kinase 110 CDK110 7.6  
    D38380 Transferrin TF 6.7  
    J03627 S-100 related protein S100a10 3.5  
    M58716 Secretory granule membrane glycoprotein GP2 3.2  
    AI137583 Inhibitor DNA binding 2 ID2 3.1  
    S87544 Polyprotein 1-microglobulin/bikunin AMBP 2.9  
    M27440 Apolipoprotein B APOB 2.8  
    AA899854 Topoisomerase (DNA) 2 α TOP2A 2.7  
    AA849769 Follistatin-related protein FSTL1 2.7  
    U95178 Disabled homolog 2, mitogen-responsive phophoprotein DAB2 2.3  
    X06916 S100 calcium binding protein A4 S100a4 2.3  
    AF020757 Purinergic receptor P2X, ligand-gated ion channel, 2 P2RX2 2.3  
    S76779 Apolipoprotein E APOE 2.0  
ESTs 12 sequences     
8 Repetitive sequences     
Gene underexpression
Gene ID numberGene nameGene symbolFold change
AffymetrixRT-PCR
    AA848563 Heat shock protein 70-1 HSPA1A 16.7  
    U48246 Protein kinase C binding protein NELL1 NELL1 16.1  
    M72422 Glutamate decarboxylase 2 GAD2 4.9  
    X70871 Cyclin G1 CCNG1 3.9  
    M13588 Pancreatic polypeptide PPY 3.7  
    M15880 Neuropeptide Y NPY 2.7  
    J05592 Protein phosphatase 1, regulatory (inhibitor) subunit 1A PPP1R1A 2.7  
    U20796 Nuclear receptor subfamily 1, group D, member 2 NRLD2 2.6  
    U08290 Neuronatin NNAT 2.5  
    M85214 Trk precursor NTRK1 2.3  
    E12625 sterol-C4-methyl oxidase like SC4MOL 2.3  
    M10094 RT1 class Ib gene RT1AW2 2.3  
    D85183 Protein tyrosine phosphatase PTPNS1 2.2  
    AA852004 Glutamine synthetase GLUL 2.2  
    L27075 ATP-citrate lyase ACLY 2.2  
    J03481 Quinoid dihydropteridine reductase QDPR 2.1  
    S75952 Glucagon-like peptide 1 receptor GLP1R 2.1  
    U17254 Immediate early gene transcription factor nerve growth factor-1B NR4A1 2.1  
    M23601 Monoamine oxidase B MAOB 2.1  
ESTs 3 sequences     
Gene underexpression
Gene ID numberGene nameGene symbolFold change
AffymetrixRT-PCR
    AA848563 Heat shock protein 70-1 HSPA1A 16.7  
    U48246 Protein kinase C binding protein NELL1 NELL1 16.1  
    M72422 Glutamate decarboxylase 2 GAD2 4.9  
    X70871 Cyclin G1 CCNG1 3.9  
    M13588 Pancreatic polypeptide PPY 3.7  
    M15880 Neuropeptide Y NPY 2.7  
    J05592 Protein phosphatase 1, regulatory (inhibitor) subunit 1A PPP1R1A 2.7  
    U20796 Nuclear receptor subfamily 1, group D, member 2 NRLD2 2.6  
    U08290 Neuronatin NNAT 2.5  
    M85214 Trk precursor NTRK1 2.3  
    E12625 sterol-C4-methyl oxidase like SC4MOL 2.3  
    M10094 RT1 class Ib gene RT1AW2 2.3  
    D85183 Protein tyrosine phosphatase PTPNS1 2.2  
    AA852004 Glutamine synthetase GLUL 2.2  
    L27075 ATP-citrate lyase ACLY 2.2  
    J03481 Quinoid dihydropteridine reductase QDPR 2.1  
    S75952 Glucagon-like peptide 1 receptor GLP1R 2.1  
    U17254 Immediate early gene transcription factor nerve growth factor-1B NR4A1 2.1  
    M23601 Monoamine oxidase B MAOB 2.1  
ESTs 3 sequences     

F.H.-D., S.C., and J.-C.I. contributed equally to this work.

DOI: 10.2337/db05-1526

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

This work was supported in parts by grants from the National Center for Scientific Research and the Swiss National Science Foundation. S.C. was a recipient of doctoral fellowships from the Ministère de l’Education Nationale, de l’Enseignement et de la Recherche, and from the Fondation pour la recherche médicale. S.C. also thanks Naturalia et Biologia Association for a travel grant.

Parts of this work were presented at the 18th International Diabetes Federation Congress, Paris, France, 24–29 August 2003 and at the 41st Annual Meeting of the European Association of the Study of Diabetes, Athens, Greece, 12–15 September 2005.

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