Moderate Hypoxia Induces β-Cell Dysfunction with HIF-1–Independent Gene Expression Changes

<div><p>Pancreatic β-cell failure is central to the development and progression of type 2 diabetes. We recently demonstrated that β-cells become hypoxic under high glucose conditions due to increased oxygen consumption and that the pancreatic islets of diabetic mice but not those of control mice are moderately hypoxic. However, the impact of moderate hypoxia on β-cell number and function is unknown. In the present study, moderate hypoxia induced a hypoxic response in MIN6 cells, as evidenced by increased levels of HIF-1α protein and target genes. Under these conditions, a selective downregulation of <i>Mafa</i>, <i>Pdx1</i>, <i>Slc2a2</i>, <i>Ndufa5</i>, <i>Kcnj11</i>, <i>Ins1</i>, <i>Wfs1</i>, <i>Foxa2</i>, and <i>Neurod1</i>, which play important roles in β-cells, was also observed in both MIN6 cells and isolated pancreatic islets. Consistent with the altered expression of these genes, abnormal insulin secretion was detected in hypoxic MIN6 cells. Most of the hypoxia-induced gene downregulation in MIN6 cells was not affected by the suppression of HIF-1α, suggesting a HIF-1–independent mechanism. Moderate hypoxia also induced apoptosis in MIN6 cells. These results suggest that hypoxia is a novel stressor of β-cells and that hypoxic stress may play a role in the deterioration of β-cell function.</p></div

The role of apoptosis in hypoxia-induced gene downregulation in MIN6 cells.

<p>(A) MIN6 cells were incubated at 5% O2 for 10 h and the expression levels of <i>Ddit3</i> mRNA were examined by qPCR (n = 4). (B, C) Annexin V-positive cell (B) and PI-positive cell death (C) ratios were evaluated by flow cytometric analysis (n = 6) after MIN6 cells were cultured at 5% O2 for 10 h. (D) Expression levels of <i>Pdx1</i>, <i>Neurod1</i>, <i>Wfs1</i>, and <i>Slc2a2</i> were examined by qPCR (n = 4) in the same conditions as in (A). (E) Ctrl MIN6 cells (gray bars) and CHOP knockdown MIN6 cells (black bars) were incubated either in normoxia (20% O₂) or in hypoxia (5% O₂) for 40 h and the expression levels of <i>Ddit3</i> mRNA were examined by qPCR (n = 3). (F) Ctrl MIN6 cells (gray bars) (n = 5) and CHOP knockdown MIN6 cells (black bars) (n = 5) were incubated either in normoxia (20% O₂) or in hypoxia (5% O₂). The PI-positive cell death ratio was evaluated by flow cytometric analysis. (G) Ctrl MIN6 cells (gray bars) and CHOP knockdown MIN6 cells (black bars) were incubated in the same conditions as in (E). Downregulation of mRNA levels (Δ mRNA levels) by 5% O₂. Δ mRNA levels indicate (1-expression levels at 5% O₂/expression levels at 20% O₂) ×100 (%). In qPCR analysis, the mRNA value of each gene was normalized to that of <i>Tbp</i>. The data are shown as the means ± S.E. (error bars) of values from each group. *, p<0.05; **, p<0.01; ***, p<0.001.</p

The effect of moderate hypoxia on β-cell gene expression.

<p>(A, B) Gene expression analysis by qPCR of β-cell transcription factors (A) and components of the insulin secretion pathway (B) was performed using MIN6 cells incubated in normoxia (20% O₂, gray bars; n = 4) or in hypoxia (5% O₂, black bars; n = 4) for 30 h. (C) Gene expression analysis by qPCR was performed when mouse isolated islets were cultured in normoxia (20% O₂, gray bars; n = 3) or hypoxia (5% O₂, black bars; n = 3) for 16 h. (D) MIN6 cells were incubated in normoxia (20% O₂, gray bars; n = 4) or in hypoxia (5% O₂, black bars; n = 4) for 16 h. The mRNA value of each gene was normalized to that of <i>Tbp</i>. The means ± S.E. (error bars) of values from each group are shown. *, p<0.05; **, p<0.01; ***, p<0.001.</p

The effect of moderate hypoxia in MIN6 cells.

<p>(A) Gene expression analysis by qPCR of known HIF-1 target genes was performed using hypoxic MIN6 cells (n = 4). (B) Lactate concentration in media was measured after MIN6 cells were cultured in hypoxia (n = 4). (C) Gene expression analysis by qPCR of genes encoding mitochondrial respiratory chain complex components (n = 4). (D) Mitochondrial respiratory chain complex I activity under hypoxia was assessed (n = 7). (E) Cellular ATP content under hypoxic conditions was evaluated (n = 4). In all experiments, MIN6 cells were cultured in normoxia (20% O₂, gray bars) or in moderate hypoxia (5% O₂, black bars) for 30 h. Each value of mRNA was normalized to that of <i>TATA-binding protein (Tbp)</i>. The means ± S.E. (error bars) of values from each group are shown. *, p<0.05; **, p<0.01; ***, p<0.001. Lactate levels and cellular ATP content were standardized by cell number.</p

Altered insulin secretion by MIN6 cells under hypoxia.

<p>(A) Cellular insulin content was examined after MIN6 cells were incubated in normoxia (20% O₂, gray bars) or hypoxia (5% O₂, black bars) for 30 h. Insulin content was standardized by cell number. (B) Glucose-stimulated insulin secretion was examined when MIN6 cells that had been cultured at 20% O₂ or 5% O₂ for 40 h were stimulated with low glucose (2.2 mM glucose, gray bars) or high glucose (22 mM glucose, black bars) for 1 h (n = 4). Secreted insulin was normalized to cellular protein levels. Data are shown as the means ± S.E. (error bars) of values from each group. **, p<0.01; ***, p<0.001. N.S., not significant.</p

The role of HIF-1 in hypoxia-induced gene downregulation and insulin secretion defects in MIN6 cells.

<p>(A) Control (Ctrl) MIN6 cells (gray bars) and HIF-1α knockdown MIN6 cells (black bars) were cultured in normoxia (20% O₂) and the relative expression levels of β-cell genes were evaluated by qPCR analysis. Each value of mRNA was normalized to that of <i>Actb</i>. (B) Downregulation of mRNA levels (Δ mRNA levels) by 5% O₂. Δ mRNA levels indicate (1-expression levels at 5% O₂/expression levels at 20% O₂) ×100 (%). (C) Ctrl MIN6 cells (n = 11) and HIF-1α knockdown MIN6 cells (n = 9) that had been cultured in normoxic (20% O₂, gray bars) or hypoxic (5% O_2, black bars) conditions for 40 h were stimulated with 2.2 mM glucose or 22 mM glucose for 1 h. Secreted insulin was normalized to cellular protein levels. (D) The fold change in glucose-stimulated insulin secretion (insulin level at 22 mM glucose divided by that at 2.2 mM glucose) is indicated (n = 9). Data are shown as the means ± S.E. (error bars) of the values from each group. *, p<0.05; **, p<0.01; ***, p<0.001.</p