Impairment of extracellular sugar detection in GLUT2-expressing tissues in transgenic mice.

<p>Effect of a glucose-rich diet on gene expression in liver (A) and adipose tissue (C). Transgenic (Tg) and wild-type (WT) mice were fasted for 48 h and refed for 15 h before liver and epididymal fat pad biopsies. Levels of mRNA were analyzed by real time PCR. Values are presented as means±S.E.M. (n = 3 to 5 mice/group). Statistical differences between refed and fasted mice are indicated by *P<0.05, **P<0.01, and ns non significant. B: GLUT2 protein levels in total membrane preparations from the liver of mice fed a glucose-rich diet for five days. D: Blood glucose concentrations during an insulin tolerance test in wild-type and transgenic mice. Values are presented as means±S.E.M (n = 8 to 10 mice/group).</p

Urinary excretion in mice after impairment of extracellular sugar detection. Metabolic and electrolyte levels in 24-h urine samples from wild-type (WT) or transgenic mice fed a standard or a glucose-rich diet. Values are presented as means±S.E.M. (n = 6 per group). Statistical differences between wild-type and transgenic mice are indicated as *P<0.05 ns non significant, nd indicates not determined.

<p>Urinary excretion in mice after impairment of extracellular sugar detection. Metabolic and electrolyte levels in 24-h urine samples from wild-type (WT) or transgenic mice fed a standard or a glucose-rich diet. Values are presented as means±S.E.M. (n = 6 per group). Statistical differences between wild-type and transgenic mice are indicated as *P<0.05 ns non significant, nd indicates not determined.</p

Kidney function in mice after impairment of extracellular sugar detection.

<p>A: Effect of a glucose-rich diet on gene expression in the kidney of transgenic (Tg) and wild-type (WT) mice fasted for 48 h and refed for 15 h. Levels of mRNA were analyzed by real-time PCR. Values are presented as means±S.E.M. (n = 3 to 4 mice/group). Statistical differences between refed and fasted mice are indicated as *P<0.05, **P<0.01 and ns non significant. B: GLUT2 protein levels in total membrane preparations of kidney from mice fed with a glucose-rich diet for five days. C: Structure, size and weight of kidneys from wild-type and transgenic mice shown by ultrasonic image (transverse cross section). D: Urine and blood glucose concentrations during an oral glucose tolerance test in fasted wild-type and transgenic mice (n = 3 mice per group). Statistical differences between transgenic and wild-type mice are indicated as **P<0.01 (two-way ANOVA) for the areas under the curves. E: Levels of SGLT mRNA were analyzed by real-time PCR. Values are presented as means±S.E.M. (n = 3 to 4 mice/group). Statistical differences between refed and fasted mice are indicated as **P<0.01 and ns non significant.</p

Generation of GLUT2-loop transgenic mice.

<p>A: Quantification of the transgene copy number in genomic DNA from independent lines of mice (Tg G, P, B and W) to the reference gene Apolipoprotein A1 (ApoA1). B: RT-PCR analysis of transgene and L19 control mRNA levels in various tissues. C: Immunoprecipitation and immunoblot analysis showing the presence of GLUT2 loop in liver homogenate from transgenic mice.</p

Pancreatic function in mice after impairment of extracellular sugar detection.

<p>A: Effect of a glucose-rich diet on gene expression in the pancreas of wild-type (WT) and transgenic (Tg) mice fasted for 48h and refed for 15 h. Levels of mRNA were analyzed by real-time PCR. Values are presented as means±S.E.M. (n = 3 to 4 mice/group). Statistical differences between refed and fasted mice are indicated as **P<0.01 and ns non significant. B: GLUT2 protein levels in total membrane preparations of pancreas from mice fed a glucose-rich diet for five days. C: Left panel: Blood glucose concentrations during an oral glucose tolerance test in wild-type and transgenic mice fasted for 24 h (n = 17 for wild-type mice, n = 2 to 5 for transgenic mice). Statistical differences between transgenic and wild-type mice are indicated as ***P<0.001, *P<0.05 and ns non significant (two-way ANOVA) for the areas under the curves. Right panel: Blood glucose concentrations in wild-type and transgenic mice in the fasted state or 6 h after being refed with a glucose-rich diet. Values are presented as means±S.E.M. (n = 4 to 8 mice/group). D: Upper panel: Plasma insulin concentrations during an oral glucose tolerance test in fasted wild-type and transgenic mice (n = 10 to 13 mice/group). Statistical differences between transgenic and wild-type mice are indicated as ***P<0.001 (two-way ANOVA) for the areas under the curves. Lower panel: Pancreatic insulin content in mice 6h after being refed a standard diet. Values are presented as means±S.E.M. (n = 5 mice/group). Statistical differences between transgenic and wild-type mice are indicated as *P<0.05. E: Upper panel : Representative immunostaining with antibody against insulin of pancreatic sections from wild-type and transgenic mice. Arrows indicate small islets. The bar corresponds to 100 µm. Lower panel: Histomorphometric comparisons of islet number, size and ß-cell mass. Proportion of small islets (<25 µm) to total number of islets is statistically different indicated as *P<0.03 between transgenic and wild-type mice.</p

<i>In vitro</i> cytokine/chemokine release by small and large islets from 3-month-old Wistar (□) and GK (▪) rats.

<p>The islets were selected as a function of their size by handpicking under a stereomicroscope. (A) Representative photomicrographs of islets cultured on ECM matrix in the presence of 11 mM glucose. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090045#pone.0090045.s006" target="_blank">Figure S6</a> is indicative of the groups of small and large islets which were compared between Wistar and GK rats. Note the disturbed shape and/or the ring of small cells (possibly, acinar and/or myeloid) around both types of isolated GK islets. (B, C, D, E): IL-6, CCL2, CCL3 and CXCL1 islet release, respectively. Wistar and GK islets were pooled separately and plated in triplicate. In a given experiment, cytokine/chemokine levels were normalized to total islet protein content after 48 h culture; in (B–E), data are presented as mean values ± SEM, n = 3 different islet isolations for each group of rats; in (F) data are presented for large islets, as mean values ± SEM (n = 3) of the ratio of GK <i>versus</i> control Wistar values in each experiment. *p<0.05 and **p<0.01 <i>vs</i> corresponding Wistar islets, as analyzed by Student’s <i>t</i> test for unpaired data. CCL2, CC-chemokine ligand-2 or monocyte chemoattractant protein-1 (MCP-1); CCL3, CC-chemokine ligand-3 or macrophage inflammatory protein-1α (MIP-1α); CXCL1, CXC-chemokine ligand-1 or chemokine GRO1/KC (murine IL-8 equivalent); IL-6, interleukin-6.</p

Circulating cytokines/chemokines in Wistar and GK rats as a function of age.

<p>All data were determined under fed conditions. Values (pg/ml) are mean ± SEM for the number (<i>n</i>) of animals. Statistical analysis used the Student <i>t</i>-test for unpaired data. No significant difference in circulating cytokine levels was observed between 3- and 4-month-old Wistar and GK rats. CCL2, CC-chemokine ligand-2 or monocyte chemoattractant protein-1 (MCP-1); CCL3, CC-chemokine ligand-3 or macrophage inflammatory protein-1α (MIP-1α); CXCL1, CXC-chemokine ligand-1 or chemokine GRO1/KC (murine IL-8 equivalent); IL-6, Interleukin-6.</p

Regenerating gene-1(<i>Reg1</i>) and <i>Reg3β</i> gene expression in the pancreas of 4-month-old Wistar (□) and GK (▪) rats.

<p>Values are means ± SEM of three distinct experiments measured by quantitative RT-PCR for: A) islet-depleted exocrine-to-endocrine (isolated islets) tissue ratio; B) isolated islets: mRNA levels were expressed as the fold change in GK <i>vs</i> corresponding Wistar group. Glyceraldehyde-3-phosphate dehydrogenase was used as housekeeping gene. Three different islet isolations for each group of rats, * <i>p</i><0.05 and ** <i>p</i><0.01 <i>vs</i> Wistar rats, as analyzed by Student’s <i>t</i> test for unpaired data.</p

Immunohistochemistry for CD68 (A) and MHC class II (B) in 2-month-old GK rat pancreas sections.

<p>Analysis of serial pancreas sections for 3 GK rats. Correlation coefficient (r = 0.573, <i>p</i><0.025) between CD68⁺ area and total islet area (C). Correlation coefficient (r = 0.950, <i>p</i> = 0.003) between MHC class II⁺ area and total islet area (D). Correlations were assessed with non-parametric Spearman’s rank correlation test. MHC, major histocompatibity complex.</p

Representative pancreas immunochemistry in 4-month-old male Wistar (A–G) and GK (H–N) rats.

<p>Serial staining (brown) for: (A, H) insulin (β-cell marker), (B, I) glucagon+somatostatin+pancreatic polypeptide cocktail (non-β cell marker), (C, J) α-amylase (acinar cell marker), and (D, K, and F–G, M–N) REG-1. For REG-1 labeling, we used the monoclonal anti-rat REG-1 antibody from Hiroshi Okamoto (Japan). REG-1 negative controls are shown in (E, L). In (F–G) and (M–N), the border of islets is defined by the red dashed line. In Fig. 2N, “d” means “duct”.</p