Glucagon regulation of oxidative phosphorylation requires an increase in matrix adenine nucleotide content through Ca2+ activation of the mitochondrial ATP-Mg/Pi carrier SCaMC-3

Abstract

It has been known for a long time that mitochondria isolated from hepatocytes treated with glucagon or Ca(2+)-mobilizing agents such as phenylephrine show an increase in their adenine nucleotide (AdN) content, respiratory activity, and calcium retention capacity (CRC). Here, we have studied the role of SCaMC-3/slc25a23, the mitochondrial ATP-Mg/Pi carrier present in adult mouse liver, in the control of mitochondrial AdN levels and respiration in response to Ca(2+) signals as a candidate target of glucagon actions. With the use of SCaMC-3 knock-out (KO) mice, we have found that the carrier is responsible for the accumulation of AdNs in liver mitochondria in a strictly Ca(2+)-dependent way with an S0.5 for Ca(2+) activation of 3.3 ± 0.9 μm. Accumulation of matrix AdNs allows a SCaMC-3-dependent increase in CRC. In addition, SCaMC-3-dependent accumulation of AdNs is required to acquire a fully active state 3 respiration in AdN-depleted liver mitochondria, although further accumulation of AdNs is not followed by increases in respiration. Moreover, glucagon addition to isolated hepatocytes increases oligomycin-sensitive oxygen consumption and maximal respiratory rates in cells derived from wild type, but not SCaMC-3-KO mice and glucagon administration in vivo results in an increase in AdN content, state 3 respiration and CRC in liver mitochondria in wild type but not in SCaMC-3-KO mice. These results show that SCaMC-3 is required for the increase in oxidative phosphorylation observed in liver mitochondria in response to glucagon and Ca(2+)-mobilizing agents, possibly by allowing a Ca(2+)-dependent accumulation of mitochondrial AdNs and matrix Ca(2+), events permissive for other glucagon actions.

FIGURE 1.

SCaMC-3 is the main functional Ca²⁺-dependent ATP-Mg/P_i carrier in adult liver. A, expression levels of the main SCaMC paralogs were analyzed by Western blot in isolated mitochondria using different tissues from wild type (WT) and SCaMC-3-KO (KO) mice. β-ATPase was used as loading control. B, Western blot analysis of liver extracts from embryos (E14–E17), postnatal (P0–P4), and 3-month-old mice (3 mo) from wild type and SCaMC-3-KO mice. Antibodies against SCaMC-1 and SCaMC-3, as well as Hsp60 as loading control, were used. Absence of SCaMC-3 did not induce up-regulation of other ATP-Mg/P_i paralogs. C, respiratory rates were measured in isolated liver mitochondria from wild type and SCaMC-3-KO mice using substrates for complex I (glu + mal) or complex II (succinate) in the presence (state 3, st. 3) or absence (state 4, st. 4) of 0.5 mm ADP. Respiratory control ratios (state 3/state 4; RCR) using substrates for complex I or complex II are also shown. Results are expressed as mean ± S.E. of 8 (complex I) and 22 (complex II) independent experiments. D, SCaMC-3 transports ATP-Mg in a Ca²⁺-dependent way. Left panel shows ATP uptake in isolated liver mitochondria from wild type and SCaMC-3-KO mice in the presence or absence of Ca²⁺. Mitochondria were incubated at 30 °C in the presence of 4 mm ATP, 5 mm Mg²⁺, and 200 nm RR, with 20 μm Ca²⁺ or 1 mm EGTA in the medium, and mitochondrial ATP levels were determined. Results are expressed as mean ± S.E. of 3 independent experiments. In the right panel, kinetics of Ca²⁺ activation of ATP uptake in SCaMC-3 wild type liver mitochondria are shown. Data obtained were fitted by nonlinear regression to the following equation: V = V₀ + [((V_max − V₀) × [Ca²⁺]ⁿ)/(S_0.5ⁿ + [Ca²⁺]ⁿ)] (where V is transport activity at each [Ca²⁺], V₀ is the basal transport rate at 0 [Ca²⁺] (i.e. below the Calcium-Green detection limit), V_max is the maximal activity, n is the Hill index, and S_0.5 is the Ca²⁺ concentration that generates half-maximal transport activity) using Sigma Plot version 9. Pooled data from 4 independent experiments are shown.

FIGURE 2.

SCaMC-3 mediates the increase in Ca²⁺ retention capacity in liver mitochondria. Mitochondrial Ca²⁺ uptake was monitored using the fluorescent indicator Calcium Green-5N in the absence of AdNs in the medium (A) or in the presence of 0.2 mm ADP (B) or 1 mm ATP-Mg (C). Arrows indicate additions of 10 (A) or 20 (B and C) nmol of CaCl₂. Quantification of total Ca²⁺ retained by mitochondria in each case is shown on the right side of each panel. Results are expressed as mean ± S.E. of 3–5 independent experiments (*, p < 0.05; unpaired, two-tailed Student's t test).

FIGURE 3.

Addition of ADP to AdN-depleted mitochondria stimulates respiration through SCaMC-3. Representative electrode traces and respiratory rates of AdN-depleted liver mitochondria from wild type (WT) and SCaMC-3-KO mice respiring on succinate and stimulated with 2 mm ADP in the presence of Ca²⁺ (A) or in the presence of 1 mm EGTA and 1 mm EDTA (B). State 3 respiratory rate and respiratory control ratio (RCR) are only stimulated in wild type liver mitochondria in the presence of extramitochondrial Ca²⁺. Results are expressed as mean ± S.E. of 3–5 independent experiments. *, p < 0.05; **, p < 0.01; WT versus KO two-tailed, unpaired Student's t test; #, p < 0.05; ##, p < 0.01; state 3 versus uncoupled two-tailed, paired Student's t test. Scale bars: 10 nmol at O (vertical) and 1 min (horizontal).

FIGURE 4.

Respiration in mitochondria loaded with AdNs. A, wild type (WT) and SCaMC-3-KO (KO) mitochondria were depleted from AdNs and incubated at 30 °C in the presence of 200 nm RR, 2 mm ATP, and 5 mm MgCl₂ for different lengths of time before monitoring state 4, state 3, and uncoupled respiratory rates in the presence of 2 μm rotenone plus 5 mm succinate. Respiratory control ratios (RCR) are also shown. The maximal increase of state 3 respiratory rate is observed in wild type cells after 2 min of incubation. Results are expressed as mean ± S.E. of 5 independent experiments (*, p < 0.05; **, p < 0.01; two-tailed, unpaired Student's t test). B, nondepleted wild type and SCaMC-3-KO mitochondria were incubated at 30 °C in the presence of 200 nm RR, 10 mm ATP, 10 mm MgCl₂, and 20 μm CaCl₂ at different times before monitoring state 4, state 3, and uncoupled respiratory rates as in A. To correct for residual AdNs in the medium, state 4 was that obtained after the addition of oligomycin. In parallel, mitochondrial levels of ATP + ADP were also determined (bottom panel). Results are expressed as mean ± S.E. of 3–5 independent experiments (*, p < 0.05, two-tailed, unpaired Student's t test).

FIGURE 5.

Effects of glucagon and phenylephrine on cytosolic Ca²⁺ signals and respiratory parameters in primary hepatocytes from wild type and SCaMC-3-KO animals. A, representative traces corresponding to cytoplasmic Ca²⁺ concentration in primary wild type hepatocytes in response to 0.1 μm glucagon and 100 μm phenylephrine addition. Light gray traces correspond to individual cells, whereas the average is represented by a black trace. In both genotypes phenylephrine caused an immediate Ca²⁺ peak, whereas the effect of glucagon was detectable 2 min after its addition. The Ca²⁺ signals evoked by both agonists were identical in SCaMC-3-KO cells. B, a representative experiment of OCR in primary hepatocytes from wild type and SCaMC-3-KO and the response to phenylephrine is shown. OCR is expressed as the rate at each point with respect to the basal rate at the time of addition of the agonist. Where indicated, 100 μm phenylephrine, 6 μm oligomycin, 1 μm FCCP, and 1 μm rotenone plus 1 μm antimycin A (rot + AntA) were injected. C, OCR responses to phenylephrine (100 μm) and glucagon (0.1 μm) and the corresponding vehicle in hepatocytes from WT and SCaMC-3 KO mice. Data correspond to representative experiments. D, respiratory parameters after treatment with 0.1 μm glucagon or 100 μm phenylephrine with respect to vehicle in wild type and SCaMC-3-KO hepatocytes. Nonmitochondrial respiration (the lowest value remaining after rotenone plus antimycin A addition) was subtracted from all measurements after confirming that at longer times respiration was not further decreased. Respiratory parameters were calculated as the average of three consecutive measurements after each addition (agonist, oligomycin, and FCCP). The stimulations of glucagon and phenylephrine and maximal respiratory capacity were calculated as the percentage of respiration after agonist or FCCP addition with respect to basal respiration. Coupled respiration is the percentage of respiration in the presence of agonist sensitive to oligomycin inhibition. Proton leak was calculated as the difference between oligomycin-sensitive and nonmitochondrial respiration. Results are expressed as mean ± S.E. of 3 independent experiments with four replicates each (*, p < 0.05; **, p < 0.01; unpaired, two-tailed Student's t test, WT versus KO; #, p < 0.01 ##, p < 0.001 two-tailed Student's t test, WT versus vehicle).

FIGURE 6.

SCaMC-3 mediates glucagon-stimulated mitochondrial uptake of AdNs and increase in CRC and respiratory activity in vivo. Following in vivo administration of glucagon (2 mg/kg) or vehicle, liver mitochondria from wild type and SCaMC-3-KO mice were rapidly isolated and different mitochondrial parameters were evaluated. A, matrix AdN content, measured by HPLC. Left panel shows total AdNs, whereas the right panel shows the levels of the different AdNs. Results are expressed as mean ± S.E. of 3–5 independent experiments (*, p < 0.05; WT versus KO, two-tailed, unpaired Student's t test; #, p < 0.05; WT vehicle versus WT glucagon, one-tailed, unpaired Student's t test). B, Ca²⁺ retention in isolated mitochondria. Left panel shows a representative experiment, with arrows corresponding to 10 nmol of CaCl₂ additions. In the right panel, CRC is shown. Results are expressed as mean ± S.E. of 3 independent experiments (*, p < 0.05; WT versus KO, two-tailed, unpaired Student's t test). C, state 4 (st. 4), state 3 (st. 3), and uncoupled (unc) respiratory rates using different substrates. Results are expressed as mean ± S.E. of 6 independent experiments (*, p < 0.05; vehicle versus glucagon, two-tailed paired Student's t test; **, p < 0.01; vehicle versus glucagon, two-tailed paired Student's t test).