Mice Deficient in Proglucagon-Derived Peptides Exhibit Glucose Intolerance on a High-Fat Diet but Are Resistant to Obesity

<div><p>Homozygous glucagon-GFP knock-in mice (<i>Gcg</i>^{<i>gfp/gfp</i>}) lack proglucagon derived-peptides including glucagon and GLP-1, and are normoglycemic. We have previously shown that <i>Gcg</i>^{<i>gfp/gfp</i>} show improved glucose tolerance with enhanced insulin secretion. Here, we studied glucose and energy metabolism in <i>Gcg</i>^{<i>gfp/gfp</i>} mice fed a high-fat diet (HFD). Male <i>Gcg</i>^{<i>gfp/gfp</i>} and <i>Gcg</i>^<i>gfp/+</i> mice were fed either a normal chow diet (NCD) or an HFD for 15–20 weeks. Regardless of the genotype, mice on an HFD showed glucose intolerance, and <i>Gcg</i>^{<i>gfp/gfp</i>} mice on HFD exhibited impaired insulin secretion whereas <i>Gcg</i>^<i>gfp/+</i> mice on HFD exhibited increased insulin secretion. A compensatory increase in β-cell mass was observed in <i>Gcg</i>^<i>gfp/+</i>mice on HFD, but not in <i>Gcg</i>^{<i>gfp/gfp</i>} mice on the same diet. Weight gain was significantly lower in <i>Gcg</i>^{<i>gfp/gfp</i>} mice than in <i>Gcg</i>^<i>gfp/+</i>mice. Oxygen consumption was enhanced in <i>Gcg</i>^{<i>gfp/gfp</i>} mice compared to <i>Gcg</i>^<i>gfp/+</i> mice on an HFD. HFD feeding significantly increased uncoupling protein 1 mRNA expression in brown adipose and inguinal white adipose tissues of <i>Gcg</i>^{<i>gfp/gfp</i>} mice, but not of <i>Gcg</i>^<i>gfp/+</i>mice. Treatment with the glucagon-like peptide-1 receptor agonist liraglutide (200 mg/kg) improved glucose tolerance in <i>Gcg</i>^{<i>gfp/gfp</i>} mice and insulin content in <i>Gcg</i>^{<i>gfp/gfp</i>} and <i>Gcg</i>^<i>gfp/+</i> mice was similar after liraglutide treatment. Our findings demonstrate that <i>Gcg</i>^{<i>gfp/gfp</i>} mice develop diabetes upon HFD-feeding in the absence of proglucagon-derived peptides, although they are resistant to diet-induced obesity.</p></div

Changes in body weight and quantification of energy expenditure. Indirect calorimetry was analyzed by using CLAMS.

<p>(A) Body weight changes during HFD feeding. (B) Body weight gain at 15 weeks of HFD-feeding. (C) Oxygen consumption (<i>VO</i>_<i>2</i>). (D) Carbon dioxide output (<i>VCO</i>_<i>2</i>). (E) Respiratory exchange rates (RER). (F) Energy intake. (G) Physical activity. White bars, mice fed NCD; black bars, mice fed HFD (n = 6–8). *p < 0.05; **p < 0.01; ***p < 0.001. Data are presented as means ± SEM.</p

Effect of liraglutide on glucose metabolism. Liraglutide (200 mg/kg) was subcutaneously administered once a day for last 4 weeks of HFD-feeding.

<p>(A) body weights during liraglutide treatment (n = 6–7). Open circles, liraglutide-treated <i>Gcg</i>^<i>gfp/+</i> mice on HFD; closed circles, liraglutide-treated <i>Gcg</i>^{<i>gfp/gfp</i>} mice on HFD. **p < 0.01 vs <i>Gcg</i>^<i>gfp/+</i> mice. ^#p < 0.05; ^##p < 0.01 vs. before liraglutide treatment. (B) Blood glucose levels during IPGTT (n = 6–7). Open circles, liraglutide-treated <i>Gcg</i>^<i>gfp/+</i> mice on HFD; closed circles, liraglutide-treated <i>Gcg</i>^{<i>gfp/gfp</i>} mice on HFD. *p < 0.05; ***p < 0.001 vs. <i>Gcg</i>^<i>gfp/+</i> mice. (C) Plasma insulin levels 0 min (white bars) and 15 min (black bars) after i.p. glucose loading (n = 6–7). (D) Insulin contents in pancreata. White bars, liraglutide-treated <i>Gcg</i>^<i>gfp/+</i> mice on HFD; black bars, liraglutide-treated <i>Gcg</i>^{<i>gfp/gfp</i>} mice on HFD (n = 5–7). Data are presented as means ± SEM.</p

<i>Gcg</i>^{<i>gfp/gfp</i>} mice fed HFD fail to show β-cell expansion and increased insulin contents.

<p>(A) β-cell area is shown as insulin-positive area relative to total pancreas area by morphometric analysis (n = 5–6). (B) Insulin contents in pancreata (n = 6–9). (C) Proliferation of pancreatic β-cells. Percentage of Ki-67-positive β-cells is shown as the number of Ki-67-positive cells relative to insulin-positive islet cells. White bars, mice fed NCD; black bars, mice fed HFD (n = 3–4). **p < 0.01. (D) mRNA Expression of <i>Irs2</i>, <i>Pdx1</i>, and <i>cyclin D2</i> in islets. White bars, mice fed NCD; black bars, mice fed HFD (n = 4–8). *p < 0.05; **p < 0.01. Data are presented as means ± SEM.</p

Analysis of adipose tissue with regard to energy metabolism.

<p>(A) Intrascapular BAT weights after NCD- or HFD-feeding (n = 6–8). (B) Representative H&E staining of BAT. (C) Triglyceride contents in BAT (n = 4). (D) mRNA expression of <i>Ucp1</i>, <i>Ppargc1a</i>, and <i>Dio2</i> in BAT (n = 5–8). (E) mRNA expression of <i>Ucp1</i>, <i>Ppargc1a</i>, <i>Dio2</i>, <i>F4/80</i>, <i>Mcp1</i>, <i>Tnfa</i>, <i>Tbx1</i>, <i>Cd137</i>, and <i>Cidea</i> in inguinal WAT. White bars, mice fed on a NCD; black bars, mice fed on an HF diet (n = 4–5). *p < 0.05; ***p < 0.001. Data are presented as means ± SEM.</p

<i>Gcg</i>^{<i>gfp/gfp</i>} mice fed HFD exhibit glucose intolerance and impaired insulin secretion.

<p>(A) Insulin tolerance test. Open circles, <i>Gcg</i>^<i>gfp/+</i> mice; closed circles, <i>Gcg</i>^{<i>gfp/gfp</i>} mice (n = 5–7). *p < 0.05; **p < 0.01. (B) Blood glucose levels during IPGTT. Open circles, <i>Gcg</i>^<i>gfp/+</i> mice; closed circles, <i>Gcg</i>^{<i>gfp/gfp</i>} mice (n = 4–6). *p < 0.05; **p < 0.01; ***p < 0.001. (C) Plasma insulin levels at 0 min (white bars) and 15 min (black bars) after i.p. glucose loading (n = 4–6). **p < 0.01; ***p < 0.001. (D) Glucose-induced insulin secretion from isolated islets. Isolated islets were stimulated by 2.8 mmol/L glucose (white bars) or 16.7 mmol/L glucose (black bars) for 30 min. Insulin secretion is expressed as the ratio of insulin released into the medium relative to insulin content (n = 6–12). ***p < 0.001. Data are presented as means ± SEM.</p

Long-Term Pancreatic Beta Cell Exposure to High Levels of Glucose but Not Palmitate Induces DNA Methylation within the Insulin Gene Promoter and Represses Transcriptional Activity

<div><p>Recent studies have implicated epigenetics in the pathophysiology of diabetes. Furthermore, DNA methylation, which irreversibly deactivates gene transcription, of the insulin promoter, particularly the cAMP response element, is increased in diabetes patients. However, the underlying mechanism remains unclear. We aimed to investigate insulin promoter DNA methylation in an over-nutrition state. INS-1 cells, the rat pancreatic beta cell line, were cultured under normal-culture-glucose (11.2 mmol/l) or experimental-high-glucose (22.4 mmol/l) conditions for 14 days, with or without 0.4 mmol/l palmitate. DNA methylation of the rat insulin 1 gene (<i>Ins1</i>) promoter was investigated using bisulfite sequencing and pyrosequencing analysis. Experimental-high-glucose conditions significantly suppressed insulin mRNA and increased DNA methylation at all five CpG sites within the <i>Ins1</i> promoter, including the cAMP response element, in a time-dependent and glucose concentration-dependent manner. DNA methylation under experimental-high-glucose conditions was unique to the <i>Ins1</i> promoter; however, palmitate did not affect DNA methylation. Artificial methylation of <i>Ins1</i> promoter significantly suppressed promoter-driven luciferase activity, and a DNA methylation inhibitor significantly improved insulin mRNA suppression by experimental-high-glucose conditions. Experimental-high-glucose conditions significantly increased DNA methyltransferase activity and decreased ten-eleven-translocation methylcytosine dioxygenase activity. Oxidative stress and endoplasmic reticulum stress did not affect DNA methylation of the <i>Ins1</i> promoter. High glucose but not palmitate increased ectopic triacylglycerol accumulation parallel to DNA methylation. Metformin upregulated insulin gene expression and suppressed DNA methylation and ectopic triacylglycerol accumulation. Finally, DNA methylation of the <i>Ins1</i> promoter increased in isolated islets from Zucker diabetic fatty rats. This study helps to clarify the effect of an over-nutrition state on DNA methylation of the <i>Ins1</i> promoter in pancreatic beta cells. It provides new insights into the irreversible pathophysiology of diabetes.</p></div

DNA methylation of <i>Ins1</i> promoter in pancreatic islets from male Zucker diabetic fatty (ZDF) rats.

<p>(A) DNA methylation of the <i>Ins1</i> promoter was examined by pyrosequencing analysis in the pancreatic islets isolated from 12-week-old ZDF rats. (B) The alpha/beta cell ratio was calculated in islets isolated from heterozygous and homozygous ZDF rats. (C) Isolated pancreases were immunostained for insulin (green), glucagon (red), and DAPI (blue) in heterozygous and homozygous ZDF rats. Scale bars indicate 100 μm. Results are mean ± SEM. A: <i>n</i> = 4 rats. B: <i>n</i> = 90 islets from 3 rats per group. Asterisks indicate statistically significant differences (*<i>p</i> < 0.05, **<i>p</i> < 0.01).</p

Insulin mRNA levels and DNA methylation of the <i>Ins1</i> promoter in high-glucose conditions.

<p>(A–D) INS-1 cells were cultured for 14 days. (E and F) under normal-culture-glucose (11.2 mmol/l; white bar) or experimental-high-glucose (22.4 mmol/l; black bar) conditions. (G and H) INS-1 cells cultured in 11.2 mmol/l glucose conditions with palmitate for 14 days. Insulin mRNA levels (A, C, E, and G) were examined by real-time PCR analysis. DNA methylation of the <i>Ins1</i> promoter (B, D, F, and H) was examined by pyrosequencing analysis. (I) INS-1 cells were cultured for 14 days under the indicated conditions. Following this, GSIS was performed with low glucose (2.8 mmol/l; white bar) or high glucose (16.7 mmol/l; black bar) for 30 min. All results are mean ± SEM (<i>n</i> ≥ 4). Asterisks indicate statistically significant differences (*<i>p</i> < 0.05, **<i>p</i> < 0.01).</p

Oxidative stress and endoplasmic reticulum (ER) stress did not induce DNA methylation of <i>Ins1</i> promoter.

<p>INS-1 cells were cultured for 14 days under the following conditions: (A and B) with H₂O₂ in 11.2 mmol/l glucose; (C and D) with N-acetyl-cysteine (NAC) in 22.4 mmol/l glucose; (E and F) with thapsigargin in 11.2 mmol/l glucose; and (G and H) with tauroursodeoxycholic acid (TUDCA) in 22.4 mmol/l glucose. Insulin mRNA levels (A, C, E, and G) were examined by real-time PCR. DNA methylation of the <i>Ins1</i> promoter (B, D, F, and H) was examined by pyrosequencing analysis. All results are means ± SEM (<i>n</i> ≥ 4). Asterisks indicate statistically significant differences (*<i>p</i> < 0.05, **<i>p</i> < 0.01).</p