Transport of Ca2+ and Ca2+-Dependent Permeability Transition in Rat Liver Mitochondria under the Streptozotocin-Induced Type I Diabetes

Abstract

Although diabetes mellitus is known to be a disease associated with mitochondrial dysfunction, not everything is clear about mitochondrial Ca²⁺ transport and Ca²⁺-induced permeability transition in diabetic cells. The objective of this work was to study the operation of MCU and Ca²⁺-dependent mitochondrial permeabilization in the liver cells of Sprague-Dawley rats under the streptozotocin-induced type I diabetes. It was shown that two weeks after the induction of diabetes, the rate of Ca²⁺ uptake by the mitochondria of diabetic animals increased ~1.4-fold. The expression of MCU and MICU1 subunits did not change, yet the quantity of dominant-negative MCUb channel subunits was almost twice as lower. The organelles also became more resistant to the induction of CsA-sensitive MPT pore and less resistant to the induction of CsA-insensitive palmitate/Ca²⁺-induced pore. The mitochondria of diabetic liver cells also showed changes in the lipid matrix of their membranes. The content of fatty acids in the membranes grew, and microviscosity of the lipid bilayer (assessed with laurdan) increased. At the same time, lipid peroxidation (assessed by the production of malonic dialdehyde) was stimulated. The paper discusses the consequences of the diabetes-related changes in mitochondria in the context of cell physiology.

Keywords: Ca2+ uniporter; MPT pore; calcium; diabetes mellitus; lipid pore; mitochondria; palmitic acid.

Figure 1

Diabetes leads to increased rates of Ca²⁺ uptake by rat liver mitochondria. (A) The dynamics of Ca²⁺ uptake (25 μM) by liver mitochondria of control (trace 1) and STZ-treated (trace 2) rats. (B) Rates of Ca²⁺ uptake by liver mitochondria of control and STZ-treated rats. The incubation medium contained 150 mM sucrose, 50 mM KCl, 2 mM KH₂PO₄, 5 mM succinic acid, 5 µM EGTA, 1 µM rotenone, and 10 mM Hepes-KOH, pH 7.4. The concentration of mitochondrial protein in the cuvette was 1 mg/mL. (C) The Ca²⁺/O ratio of liver mitochondria of control and STZ-treated rats. The incubation medium contained 120 mM KCl, 5 mM NaH₂PO₄, 5 mM succinic acid, 5 µM EGTA, 1 µM rotenone, 1 µM cyclosporin A (CsA), and 10 mM HEPES-KOH, pH 7.4. Mitochondrial oxygen consumption was stimulated by the addition of 200 μM CaCl₂. The concentration of mitochondrial protein in the cuvette was ~0.5 mg/mL. Values are given as means ± SEM (n = 5). * p < 0.05 compared to controls.

Figure 1

Diabetes leads to increased rates of Ca²⁺ uptake by rat liver mitochondria. (A) The dynamics of Ca²⁺ uptake (25 μM) by liver mitochondria of control (trace 1) and STZ-treated (trace 2) rats. (B) Rates of Ca²⁺ uptake by liver mitochondria of control and STZ-treated rats. The incubation medium contained 150 mM sucrose, 50 mM KCl, 2 mM KH₂PO₄, 5 mM succinic acid, 5 µM EGTA, 1 µM rotenone, and 10 mM Hepes-KOH, pH 7.4. The concentration of mitochondrial protein in the cuvette was 1 mg/mL. (C) The Ca²⁺/O ratio of liver mitochondria of control and STZ-treated rats. The incubation medium contained 120 mM KCl, 5 mM NaH₂PO₄, 5 mM succinic acid, 5 µM EGTA, 1 µM rotenone, 1 µM cyclosporin A (CsA), and 10 mM HEPES-KOH, pH 7.4. Mitochondrial oxygen consumption was stimulated by the addition of 200 μM CaCl₂. The concentration of mitochondrial protein in the cuvette was ~0.5 mg/mL. Values are given as means ± SEM (n = 5). * p < 0.05 compared to controls.

Figure 2

Contents and ratios of Ca²⁺ uniporter proteins in the liver mitochondria of control (CTR) and diabetic (DM) rats. (A) Western blot analysis of members of the Ca²⁺ uniporter protein family (MCU, MCUb, and MICU1). (B) Summarized data of densitometric band analysis of these proteins. In these experiments, VDAC1 was used as a marker. Values are given as means ± SEM (n = 4). * p < 0.05 compared to controls.

Figure 3

Diabetes increases the resistance of rat liver mitochondria to the opening of mitochondrial permeability transition (MPT) pore. (A) Changes in the external [Ca²⁺] upon successive addition of small Ca²⁺ doses (25 μM) to the suspension of liver mitochondria of control (trace 1) and STZ-treated (trace 2) rats. (B) Ca²⁺ capacity of liver mitochondrial of control and diabetic animals in the absence and presence 1 μM CsA. The values are given as means ± SEM (n = 5). The medium composition was as indicated in Figure 1A. (C) The amplitude of Ca²⁺-induced swelling of liver mitochondria of control (curve 1) and diabetic (curve 2) rats versus Ca²⁺ concentration. The incubation medium contained 210 mM mannitol, 70 mM sucrose, 5 mM succinate, 10 μM EGTA, 1 μM rotenone, and 10 mM Hepes/KOH buffer, pH 7.4. The concentration of mitochondrial protein was 0.4 mg/mL. The values are given as means ± SEM (n = 4). * p < 0.05 compared to controls; ** p < 0.01 compared to controls.

Figure 4

The levels of MPT-related proteins in the liver mitochondria of CTR and DM rats. (A) Western blot analysis of MPT-related proteins: CypD, ANT1, ANT2, and ATP5A. (B) Summarized data on the levels of these proteins in mitochondria. VDAC1 was used as a marker. Values are given as means ± SEM (n = 4). * p < 0.05 compared to controls.

Figure 5

Effect of diabetes mellitus on the opening of palmitate/Ca²⁺-induced lipid pores in rat liver mitochondria. (A) Absorbance changes in the suspension of liver mitochondria of control (trace 1) and STZ-treated (trace 2) rats in response to the addition of 20 μM palmitic acid (Pal) and 30 μM Ca²⁺. (B) The rate of CsA-insensitive palmitate/Ca²⁺-induced swelling of liver mitochondria in control (curve 1) and diabetic (curve 2) rats versus palmitic acid concentration. The incubation medium contained 210 mM mannitol, 70 mM sucrose, 5 mM succinate, 10 μM EGTA, 1 μM rotenone, 1 μM CsA, and 10 mM Hepes/KOH buffer, pH 7.4. Additions: 30 μM Ca²⁺. The concentration of mitochondrial protein was 0.4 mg/mL. Values are given as means ± SEM (n = 5). * p < 0.05 compared to controls.

Figure 6

Fluidity of liver mitochondrial membranes of control and diabetic rats. The composition of the incubation medium was as indicated in Figure 5. Values are given means ± SEM (n = 5). *** p < 0.001 compared to controls.

Figure 7

Diabetes stimulates lipid peroxidation in rat liver mitochondria. Lipid peroxidation was assessed by the level of thiobarbituric acid-reactive substances (TBARS) (MDA and other minor aldehyde species) in the liver mitochondria of control (CTR) and STZ-treated (DM) rats. Values are given as means ± SEM (n = 4). * p < 0.05 compared to controls.