Thioredoxin-interacting protein: a critical link between glucose toxicity and beta-cell apoptosis

Abstract

Objective: In diabetes, glucose toxicity affects different organ systems, including pancreatic islets where it leads to beta-cell apoptosis, but the mechanisms are not fully understood. Recently, we identified thioredoxin-interacting protein (TXNIP) as a proapoptotic beta-cell factor that is induced by glucose, raising the possibility that TXNIP may play a role in beta-cell glucose toxicity.

Research design and methods: To assess the effects of glucose on TXNIP expression and apoptosis and define the role of TXNIP, we used INS-1 beta-cells; primary mouse islets; obese, diabetic BTBR.ob mice; and a unique mouse model of TXNIP deficiency (HcB-19) that harbors a natural nonsense mutation in the TXNIP gene.

Results: Incubation of INS-1 cells at 25 mmol/l glucose for 24 h led to an 18-fold increase in TXNIP protein, as assessed by immunoblotting. This was accompanied by increased apoptosis, as demonstrated by a 12-fold induction of cleaved caspase-3. Overexpression of TXNIP revealed that TXNIP induces the intrinsic mitochondrial pathway of apoptosis. Islets of diabetic BTBR.ob mice also demonstrated increased TXNIP and apoptosis as did isolated wild-type islets incubated at high glucose. In contrast, TXNIP-deficient HcB-19 islets were protected against glucose-induced apoptosis as measured by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling and caspase-3, indicating that TXNIP is a required causal link between glucose toxicity and beta-cell death.

Conclusions: These findings shed new light onto the molecular mechanisms of beta-cell glucose toxicity and apoptosis, demonstrate that TXNIP induction plays a critical role in this vicious cycle, and suggest that inhibition of TXNIP may represent a novel approach to reduce glucotoxic beta-cell loss.

Figures

FIG. 1

Glucose effects on TXNIP protein levels and apoptosis in INS-1 β-cells. INS-1 cells were incubated at low (5 mmol/l) or high (25 mmol/l) glucose for 24 h and analyzed by immunoblotting for the expression of TXNIP and cleaved caspase-3. Bars represent mean fold change in protein levels corrected for β-actin ± SE (n = 3 independent experiments).

FIG. 2

Expression of TXNIP and cleaved caspase-3 in islets of diabetic mice. Body weight (A) and blood glucose (B) in diabetic BTBR.ob and normoglycemic BTBR.lean mice are shown for comparison. Islets of 8-week-old BTBR.ob and control BTBR.lean mice were isolated and analyzed by immunoblotting for TXNIP protein levels (C) and cleaved caspase-3 (D). Three independent experiments were performed, and bars represent mean fold change ± SE in protein levels corrected for β-actin.

FIG. 3

Effects of high glucose exposure on caspase-3 activation in TXNIP-deficient HcB-19 and C3H control islets. Isolated primary islets of control C3H and TXNIP-deficient HcB-19 mice were incubated at low (5 mmol/l) or high (25 mmol/l) glucose for 24 h and assessed for TXNIP expression and apoptosis. A: Representative immunoblot. B: Quantification of TXNIP protein levels in C3H and HcB-19 islets. (As expected, no TXNIP protein was detected in HcB-19 islets.) *P < 0.005. C: Quantification of cleaved caspase-3 in C3H and HcB-19 islets. Bars represent mean fold change ± SE in protein levels corrected for β-actin (n = 3 independent experiments). *P < 0.05 high vs. low glucose. , 5 mmol/l glucose; ■, 25 mmol/l glucose.

FIG. 4

Protection against glucose toxicity–induced β-cell apoptosis in TXNIP-deficient HcB-19 islets. Isolated primary islets of control C3H mice (A and B) and TXNIP-deficient HcB-19 mice (C and D) were again incubated at low (5 mmol/l) or high (25 mmol/l) glucose for 24 h and then analyzed by TUNEL. Representative pictures (×40) are shown, and white arrows point at bright appearing TUNEL-positive nuclei. E: For quantification >500 nuclei and at least 10 different islets were analyzed per group and condition and the percentage of TUNEL-positive β-cells per islet was calculated. Bars represent means ± SE. *P < 0.001 high vs. low glucose. , 5 mmol/l glucose; ■, 25 mmol/l glucose.

FIG. 5

Glucose toxicity in C57BL/6 and human islets. Islets of wild-type C57BL/6 mice (A) or isolated human islets (B) were incubated at low (5 mmol/l) or high (25 mmol/l) glucose for 24 h and then analyzed by TUNEL. At least seven different islets were analyzed per group and condition and >1,000 mouse or human β-cell nuclei were evaluated. Bars represent means ± SE.

FIG. 6

TXNIP effects on mitochondrial damage and ER stress. A: Immunoblot of cytochrome C in mitochondrial (mito) and cytosolic (cyto) cell fractions obtained from INS-1 β-cells overexpressing TXNIP (INS-TXNIP) and control cells (INS-LacZ). A total of 25 μg of protein were loaded per lane, and β-actin is shown as a loading control. One representative of seven independent experiments is shown. B: Quantification of cytochrome C in the different cell fractions. Bars represent mean fold change ± SE of seven independent experiments corrected for β-actin. Expression of BiP (C), ChOP (D), and TXNIP (E) as measured by quantitative real-time RT-PCR in INS-TXNIP and INS-LacZ cells. Bars represent means ± SE of three independent experiments corrected for 18S.