Effects of high-fat diet feeding on Znt8-null mice: differences between β-cell and global knockout of Znt8

Abstract

Genomewide association studies have linked a polymorphism in the zinc transporter 8 (Znt8) gene to higher risk of developing type 2 diabetes. Znt8 is highly expressed in pancreatic β-cells where it is involved in the regulation of zinc transport into granules. However, Znt8 is also expressed in other tissues including α-cells, where its function is as yet unknown. Previous work demonstrated that mice lacking Znt8 globally were more susceptible to diet-induced obesity (Lemaire et al., Proc Natl Acad Sci USA 106: 14872-14877, 2009; Nicolson et al., Diabetes 58: 2070-2083, 2009). Therefore, the main goal of this study was to examine the physiological impact of β-cell-specific Znt8 deficiency in mice during high-fat high-calorie (HFHC) diet feeding. For these studies, we used β-cell-specific Znt8 knockout (Ins2Cre:Znt8loxP/loxP) and whole body Znt8 knockout (Cre-:Znt8(-/-)) mice placed on a HFHC diet for 16 wk. Ins2Cre:Znt8loxP/loxP mice on HFHC diet had similar body weights throughout the study but displayed impaired insulin biosynthesis and secretion and were glucose intolerant compared with littermate control Ins2Cre mice. In contrast, Cre-:Znt8(-/-) mice became remarkably obese, hyperglycemic, hyperinsulinemic, insulin resistant, and glucose intolerant compared with littermate control Cre- mice. These data show that β-cell Znt8 alone does not considerably aggravate weight gain and glucose intolerance during metabolic stress imposed by an HFHC diet. However, global loss of Znt8 is involved in exacerbating diet-induced obesity and resulting insulin resistance, and this may be due to the loss of Znt8 activity in a tissue other than the β-cell. Thus, our data suggest that Znt8 contributes to the risk of developing type 2 diabetes through β-cell- and non-β-cell-specific effects.

Fig. 1.

Acute zinc transporter 8 (Znt8) gene deletion in Pdx1Cre:ERZnt8LoxP/LoxP mice. A: tamoxifen (125 mg/kg, 3 injections every other day) was used to trigger deletion of Znt8 through the CreER system driven by Pdx1 promoter. Tamoxifen activates CreER, promoting translocation of CreER into the nucleus. Then, Cre induces recombination between LoxP sites flanking exon 1 of SLC30A8, leading to excision of the start codon and inhibiting the expression of Znt8. B: genotyping strategy used to identify Pdx1Cre:ERLoxP/LoxP mice. C: qPCR analysis of the expression of Znt8 in isolated islets of corn oil (CO) and tamoxifen-injected mice (***P < 0.001, n = 3). D: immunohistochemistry experiments were performed on dispersed islet cells obtained from CO and tamoxifen-injected mice labeled for insulin and Znt8. E: % of cells labeled were calculated and summarized (n = 3). Scale bar, 25 μm. F and G: granule morphological analysis of β-cells in CO- and tamoxifen-injected Pdx1CreERLoxP/LoxP mice (n = 3). F: representative electron micrographs (scale bar, 500 nm). G: quantification of different types of β-cell granules (n = 3); *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 2.

Weekly body weight and area under the body weight curve in Cre-:Znt8^−/− (A and B, n = 11) Ins2Cre:Znt8loxP/loxP (C and D, n = 15), Pdx1CreER:Znt8loxP/loxP (E and F, n = 9) and their respective control mice fed a high-fat high-calorie (HFHC) diet. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 3.

Fasting blood glucose (A, E, I), plasma insulin (B, F, J), HOMA-IR index (C, G, K), and plasma glucagon (D, H, L) in Cre-:Znt8^−/− (A–D, n = 11), Ins2Cre:Znt8loxP/loxP (E–H, n = 11), and Pdx1CreER:Znt8loxP/loxP (I–L, n = 9) and their respective control mice fed an HFHC diet for 12 wk and following a 16-h fast. *P < 0.05, **P < 0.01.

Fig. 4.

Oral glucose tolerance test in 6-h-fasted Cre-:Znt8^−/− (A, n = 5) Ins2Cre:Znt8loxP/loxP (E, n = 5), and their respective control mice after 15 wk on an HFHC diet. Area under the glucose curve for the entire test was calculated (B and F). Insulin tolerance tests in 4-h-fasted Cre-:Znt8^−/− mice after 14 wk on HFHC diet (C, n = 9). D: area under the glucose curve for the entire test was calculated. *P < 0.05, **P < 0.01.

Fig. 5.

Islet morphological analysis in Cre-:Znt8^−/− (A–D, n = 3), Ins2Cre:Znt8loxP/loxP (E–H, n = 3), and their respective control mice fed an HFHC diet for 16 wk. Histological sections were used to calculate islet number per pancreatic slice area (B and F) and islet size (D and H). β-Cell area (A and E) and α-cell area (C and G) calculated from images of insulin and glucagon staining. *P < 0.05, **P < 0.01; scale bar, 100 μm.

Fig. 6.

Intracellular zinc content in primary pancreatic islet cells obtained from Cre-:Znt8^−/− (A–C, n = 3), Ins2Cre:Znt8loxP/loxP (F–H, n = 3), and their respective control mice after 16 wk on HFHC diet. Representative bright field images of primary pancreatic cells (A and F) loaded with zinquin (B and G). Intracellular zinc content was estimated by measuring zinquin fluorescence and is represented on bar graphs (C and H, n = 3, ***P < 0.001; scale bar, 10 μm). Granule morphological analysis in Cre-:Znt8^−/− (D and E, n = 3), Ins2Cre:Znt8loxP/loxP (I and J, n = 3), and their respective control mice after 16 wk on HFHC diet. Representative electron micrographs (D and I) and quantification of different types of β-cell granules (E and J, n = 3, ***P < 0.001; scale bar, 500 nm).

Fig. 7.

Fed plasma insulin, proinsulin, and proinsulin-to-insulin ratio in Cre-:Znt8^−/− (A–C, n = 5), Ins2Cre:Znt8loxP/loxP (n = 10, D–F) and their respective controls fed HFHC diet for 16 wk. *P < 0.05, **P < 0.01.

Fig. 8.

Glucose-stimulated insulin secretion from Cre-:Znt8^−/− (A, n = 6), Ins2Cre:Znt8loxP/loxP (D, n = 6), and their respective control islets measured at 0, 11.1, and 20 mmol/l glucose. Total insulin content of isolated islets from Cre-:Znt8^−/− (B, n = 10), Ins2Cre:Znt8loxP/loxP (E, n = 7), and controls. Glucagon secretion from Cre-:Znt8^−/− (C, n = 6), Ins2Cre:Znt8loxP/loxP (F, n = 6), and control islets measured at 0, 11.1, and 20 mmol/l glucose. Islets were collected after 16 wk of HFHC diet. *P < 0.05.

Fig. 9.

Znt8 and other Znt transcript expression in islets of Ins2Cre mice on chow or HFHC fed for 16 wk (A, n = 3). Znt8 transcript expression in Ins2Cre mouse islets (B, n = 3) and in human islets (C, n = 3) after 48-h incubation in culture medium only (control) or supplemented with oleate (0.4 mM) or palmitate (0.4 mM). Znt8 transcript expression in hypothalamus, adrenal gland, inguinal fat, and skeletal muscle (Sk. Muscle) in Cre- mice on chow or HFHC diet fed for 16 wk (D, n = 4–6). *P < 0.05.