Uncoupling Protein 2: A Possible Link Between Fatty Acid Excess and Impaired Glucose-Induced Insulin Secretion?
Article Figures & Tables
Figures
- FIG. 1.
Effects of a long-term exposure of INS-1 cells to oleate on insulin secretion in response to glucose, tolbutamide, and KCl. Cells were cultured in RPMI medium containing 10% FCS with 0.5% BSA for 72 h at 5 mmol/l glucose, in the absence or presence of 0.4 mmol/l oleate. They were then incubated for 30 min in KRBH buffer at 2.8 mmol/l glucose (G 2.8), 12 mmol/l glucose (G 12), 2.8 mmol/l glucose + 100 μmol/l tolbutamide (TOLB), or 30 mmol/l KCl (KC1). The insulin contents in cells cultured in the absence or presence of oleate were 231 ± 43 and 190 ± 8 ng/μg DNA, respectively (P < 0.05 by paired t test). Data are means ± SE of 3–5 experiments. **P < 0.02.
- FIG. 2.
Effect of a long-term exposure to oleate on glucose-induced changes in plasma membrane potential. INS-1 cells were cultured in RPMI medium containing 10% FCS with 0.5% BSA for 72 h at 5 mmol/l glucose, in the absence or presence of 0.4 mmol/l oleate. Cells were detached and incubated in KRBH buffer containing 2.8 mmol/l glucose and bisoxonol (100 μmol/l), and fluorescence was used to measure plasma membrane potential. At the indicated time, glucose was added at a final concentration of 12.8 mmol/l and then at 30 mmol/l KCl. Data are from one of four representative experiments.
- FIG. 3.
Effect of a long-term exposure to oleate on the mitochondrial membrane potential (ΔΨm) of INS-1 cells. Cells were cultured in RPMI medium containing 10% FCS with 0.5% BSA for 72 h at 5 mmol/l glucose, in the absence or presence of 0.4 mmol/l oleate, then detached and incubated 30 min in KRBH buffer containing 0.1% BSA and 2.8 mmol/l glucose with the mitochondrial potential sensitive dye TMRE, and analyzed by flow cytometry. CCCP (75 μmol/l) was added to uncouple the cells. A, ΔΨm in the absence of CCCP; B, ΔΨm in the presence of CCCP; Δ, A – B. Data are means of four experiments. *P < 0.05.
- FIG. 4.
Effect of a long-term exposure to oleate on glucose-induced changes in mitochondrial membrane potential (Δψm). INS-1 cells were cultured at 5 mmol/l glucose, in the absence or presence of 0.4 mmol/l oleate, then detached and incubated in KRBH buffer containing 0.1% BSA and 2.8 mmol/l glucose with the mitochondrial membrane potential sensitive dye rhodamine 123. At the indicated times, glucose was added at a final concentration of 12.8 mmol/l. At the end of each trace, CCCP (75 μmol/l) was added to uncouple the cells. Data are from one of four representative experiments.
- FIG. 5.
Distribution and expression of UCP2 in rat tissues and INS-1 cells. A: Comparison of UCP2 mRNA expression in rat tissues and INS-1 cells. UCP2 and 18S mRNA were detected on Northern blots containing 8 μg total RNA per lane and hybridized with 32P-labeled rat UCP2 or 18 S probes. L, liver; S, spleen; K, kidney; WAT, white adipose tissue. B: Comparison of UCP2 protein expression in INS-1 cells, rat pancreatic islets, and purified β-cells. Western blotting was performed with 30 μg protein from INS-1 cells, pancreatic islets, and β-cells using a polyclonal antibody to the COOH-terminal domain of UCP2. C: UCP2 is found in the same cellular fraction as the mitochondrial enzyme cytochrome oxidase. H, homogenate; S, postnuclear supernatant; mit, mitochondrial fraction; PM, crude plasma membrane fraction. Western blotting was performed with 30 μg protein from homogenate and subcellular fractions of INS-1 cells using a polyclonal antibody to the NH2-terminal domain of UCP2.
- FIG. 6.
Effects of oleate, palmitate, bromopalmitate, glucose, and forskolin on UCP2 mRNA expression. INS-1 cells were cultured for 18 h with 0.5% BSA, fatty acid bound to BSA (0.4 mmol/l), 25 mmol/l glucose, or 50 μmol/l forskolin. UCP2 transcript was measured by Northern blotting hybridization on 8 μg total RNA. Cont, control; OL, oleate; Palm, palmitate; BrP, 2-bromopalmitate; G25, 25 mmol/l glucose; Forsk, forskolin. The inset shows the signal of a representative experiment. Data are means ± SE of four to five separate experiments. *P < 0.05.
- FIG. 7.
Effect of oleate on UCP2 protein expression. INS-1 cells were cultured for 72 h with normal medium containing 0.5% BSA of BSA-oleate (0.4 mmol/l), and UCP2 was measured by Western blotting using 30 μg cell homogenate. Data are means of triplicate determinations. *P < 0.05.
Tables
- TABLE 1
Effects of a long-term or short-term exposure of INS-1 cells to oleate on the ATP content and ATP-to-ADP ratio at low and high glucose
Culture conditions ATP (nmole/μg DNA) ATP-to-ADP ratio Glucose (mmol/l) 2.8 12 2.8 12 Long-term BSA 72 h 0.420 ± 0.048 0.480 ± 0.043* 4.9 ± 0.1 8.3 ± 0.9† Oleate 0.4 mmol/l 72 h 0.500 ± 0.048 0.510 ± 0.056 6.4 ± 0.2 6.7 ± 0.5 Short-term BSA 15 min 0.296 ± 0.033 0.355 ± 0.029‡ ND ND Oleate 0.4 mmol/l 15 min 0.216 ± 0.021 0.329 ± 0.029§ ND ND Data are means ± SE of four independent experiments.
- *
* P ≤ 0.02 vs. 2.8 mmol/l glucose;
- †
† P ≤ 0.05 vs. 2.8 mmol/l glucose;
- ‡
‡ P < 0.02 vs. 2.8 mmol/l glucose by paired analysis; and
- §
§ P < 0.01 vs. 2.8 mmol/l glucose + oleate by paired analysis. ND, not determined.
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