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. 2012 Jul;61(7):1719-25.
doi: 10.2337/db11-1531. Epub 2012 Apr 20.

Overexpression of monocarboxylate transporter-1 (SLC16A1) in mouse pancreatic β-cells leads to relative hyperinsulinism during exercise

Timothy J Pullen  1 , Lykke Sylow, Gao Sun, Andrew P Halestrap, Erik A Richter, Guy A Rutter
Affiliations

Overexpression of monocarboxylate transporter-1 (SLC16A1) in mouse pancreatic β-cells leads to relative hyperinsulinism during exercise

Timothy J Pullen et al. Diabetes. 2012 Jul.

Abstract

Exercise-induced hyperinsulinism (EIHI) is an autosomal dominant disorder characterized by inappropriate insulin secretion in response to vigorous physical exercise or pyruvate injection. Activating mutations in the monocarboxylate transporter-1 (MCT1, SLC16A1) promoter have been linked to EIHI. Expression of this pyruvate transporter is specifically repressed (disallowed) in pancreatic β-cells, despite nearly universal expression across other tissues. It has been impossible to determine, however, whether EIHI mutations cause MCT1 expression in patient β-cells. The hypothesis that MCT1 expression in β-cells is sufficient to cause EIHI by allowing entry of pyruvate and triggering insulin secretion thus remains unproven. Therefore, we generated a transgenic mouse capable of doxycycline-induced, β-cell-specific overexpression of MCT1 to test this model directly. MCT1 expression caused isolated islets to secrete insulin in response to pyruvate, without affecting glucose-stimulated insulin secretion. In vivo, transgene induction lowered fasting blood glucose, mimicking EIHI. Pyruvate challenge stimulated increased plasma insulin and smaller excursions in blood glucose in transgenic mice. Finally, in response to exercise, transgene induction prevented the normal inhibition of insulin secretion. Forced overexpression of MCT1 in β-cells thus replicates the key features of EIHI and highlights the importance of this transporter's absence from these cells for the normal control of insulin secretion.

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Figures

FIG. 1.
FIG. 1.
A: Transgenic mouse strain for β-cell–specific, inducible expression of MCT1. MCT1 transgenic mice bearing Mct1 and firefly luciferase (Luc) cDNA under the control of a bidirectional tetracycline-regulated promoter (TRE) (22) were crossed with RIP7-rtTA mice in which the reverse tetracycline transactivator (rtTA) is expressed under control of the rat insulin promoter (RIP). In DT offspring, treatment with doxycycline induces expression of luciferase and MCT1 selectively in β-cells. B: Regulation of transgene expression is shown in islets isolated from DT mice. Islets from a single mouse were divided and cultured in the presence (+Dox) or absence (−Dox) of 5 μg/mL doxycycline for 48 h. Six size-matched islets were lysed, and luciferase activity was measured. Data are presented as mean ± SEM (n = 3). C: Immunofluorescence of pancreatic slices showing islets from ST control and DT mice, both treated with doxycycline, visualized with anti-insulin antibodies. Increased MCT1 staining is visible in DT mice. Scale bar = 50 μm. D: Quantification of immunofluorescence showing intensity of α-MCT1 signal within insulin-positive regions as mean ± SEM. E: Mct1 mRNA quantified by quantitative RT-PCR in tissues prepared from DT mice treated with (+Dox) or without (−Dox) doxycycline (n = 3). *P < 0.05 by Student t test; **P < 0.01 by Student t test. Sk., skeletal. (A high-quality digital representation of this figure is available in the online issue.)
FIG. 2.
FIG. 2.
Abnormal stimulation of insulin secretion by pyruvate from islets in vitro. Islets isolated from a DT mouse were isolated, divided, and cultured for 48 h in the presence (+Dox) or absence (−Dox) of 5 μg/mL doxycycline. Batches of six size-matched islets were incubated in Krebs buffer with 3 mmol/L glucose for 60 min and then transferred to various concentrations of glucose, pyruvate, or lactate in Krebs buffer for 30 min. Secreted insulin is presented as a percentage of total insulin content of islets. Data are presented as mean ± SEM (n = 3–6). Data for each induction state were analyzed by ANOVA with Dunnett multiple comparison test to compare each condition with 3 mmol/L glucose *P < 0.05; **P < 0.01.
FIG. 3.
FIG. 3.
Effects of Mct1 overexpression in β-cells on blood glucose homeostasis in vivo. A: Blood glucose after overnight fast was not significantly different between ST and DT mice without doxycycline treatment. After treatment with 1 g/L doxycycline in the drinking water for 5 days, fasting blood glucose in DT mice was significantly lower than in ST controls (n = 13 for both groups). B: Fasting plasma insulin levels were not significantly different between ST and DT mice (n = 14 for both groups). C and D: Pyruvate tolerance test. ST and DT mice were tested both before (−Dox) and after (+Dox) induction with doxycycline. Pyruvate (0.5 g/kg) was injected intraperitoneally into mice after an overnight fast, and blood glucose was measured at the time points indicated. Two-way ANOVA with Bonferroni post test was performed to detect differences between genotypes for each time point (ST n = 6, DT n = 12). E: Pyruvate-stimulated insulin secretion. ST and DT mice were tested after induction with doxycycline. Pyruvate (2 g/kg) was injected intraperitoneally into mice after an overnight fast, and blood was collected at time points indicated. Plasma insulin was measured by enzyme-linked immunosorbent assay (n = 5 for both groups). F and G: Plasma pyruvate (F) and lactate (G) levels both before and 15 min after injection with 2 g/kg pyruvate (n = 3 for both groups). H and I: Glucose tolerance test. ST and DT mice were induced with doxycycline as previously described, fasted overnight, then injected intraperitoneally with 1 g/kg glucose. Blood glucose was measured at the time points indicated. Because of sex-specific differences in glucose tolerance, data for females (H) and males (I) are displayed separately (ST females n = 6, DT females n = 8; ST males n = 8, DT males n = 12). No significant differences between genotypes were detected by two-way ANOVA with Bonferroni post hoc test. All data are presented as mean ± SEM. *P < 0.05; **P < 0.001.
FIG. 4.
FIG. 4.
DT mice display abnormal insulin secretion during treadmill exercise. Male mice were subjected to treadmill exercise at a 5% incline (see research design and methods). A and B: The maximum running speed (A) and blood glucose after a 4-h fast (B) of each genotype before (−Dox) and after (+Dox) doxycycline treatment. C–G: After fasting, mice were run at 80% of maximum running speed for 20 min. Blood glucose levels (C) were measured during exercise. Plasma insulin (D), lactate (F), and pyruvate (G) were measured before and immediately after exercise. HOMA2-B% values (E) were calculated from the glucose and insulin levels. Data are presented as mean ± SEM (ST n = 10, DT n = 8). *P < 0.05 within genotypes; **P < 0.001 within genotypes; #P < 0.05 between genotypes.
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