Role of Ca2+ ions in the regulation of intramitochondrial metabolism in rat epididymal adipose tissue. Evidence against a role for Ca2+ in the activation of pyruvate dehydrogenase by insulin

Abstract

1. The sensitivity of rat epididymal-adipose-tissue pyruvate dehydrogenase phosphate phosphatase, NAD+-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase to Ca2 + ions was studied both in mitochondrial extracts and within intact coupled mitochondria. It is concluded that all three enzymes may be activated by increases in the intramitochondrial concentration of Ca2 + and that the distribution of Ca2 + across the mitochondrial inner membrane is determined, as in rat heart mitochondria, by the relative activities of a uniporter (which transports Ca2 + into mitochondria and is inhibited by Mg2 + and Ruthenium Red) and an antiporter (which allows Ca2 + to leave mitochondria in exchange for Na+ and is inhibited by diltiazem). 2. Previous studies with incubated fat-cell mitochondria have indicated that the increases in the amount of active non-phosphorylated pyruvate dehydrogenase in rat epididymal tissue exposed to insulin are the result of activation of pyruvate dehydrogenase phosphate phosphatase. In the present studies, no changes in the activity of the phosphatase were found in extracts of mitochondria, and thus it seemed likely that insulin altered the intramitochondrial concentration of some effector of the phosphatase. 3. Incubation of rat epididymal adipose tissue with medium containing a high-concentration of CaCl2 (5 mM) was found to increase the active form of pyruvate dehydrogenase to much the same extent as insulin. However, the increases caused by high [Ca2 + ] in the medium were blocked by Ruthenium Red, whereas those caused by insulin were not. Moreover, whereas the increases resulting from both treatments persisted during the preparation of mitochondria and their subsequent incubation in the absence of Na+, only the increases caused by treatment of the tissue with insulin persisted when the mitochondria were incubated in the presence of Na+ under conditions where the mitochondria are largely depleted of Ca2 +. 4. It is concluded that insulin does not act by increasing the intramitochondrial concentration of Ca2 +. This conclusion was supported by finding no increases in the activities of the other two Ca2+-responsive intramitochondrial enzymes (NAD+-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase) in mitochondria prepared from insulin-treated tissue compared with controls.

Regulation of ATP production by mitochondrial Ca2+

“…PDHP1c and the L2 domain of the E2 subunit lack Ca 2+ binding motifs so the binding site for the cation is likely to be formed jointly by residues of both interacting proteins [30]. Ca 2+ activates the phosphatase in heart mitochondrial extracts with K 0.5 of ∼1 μM [31] and extramitochondrial Ca 2+ stimulates phosphatase activity over the physiological range (0.1–1.0 μM), in rat heart [32] liver [33] and adipose tissue [34] mitochondria.…”

Inhibition of Mitochondrial Na+-Ca2+ Exchanger Increases Mitochondrial Metabolism and Potentiates Glucose-Stimulated Insulin Secretion in Rat Pancreatic Islets

“…Activation of pyruvate dehydrogenase, isocitrate dehydrogenase, and ␣-ketoglutarate dehydrogenase by calcium has been described in several tissues, including heart, liver, and some endocrine tissues (42)(43)(44)(45), and occurs at [Ca 2ϩ ] m in the approximate range of 0.1-2.0 mol/l (46 -48). The peak [Ca 2ϩ ] m achieved in the present study in glucose-and glucose plus CGP37157-treated cells was within this range, consistent with our conclusion that the elevated [Ca 2ϩ ] m was responsible for the enhanced Krebs cycle flux and oxidative ATP production that we observed.…”

Evidence that adrenaline activates key oxidative enzymes in rat liver by increasing intramitochondrial [Ca2+]

“…When intact rat heart [4,5], skeletal muscle [7,8] or adipose tissue [9] fig.1) it was found to be also necessary to add some pyruvate (1 mM) to inhibit PDH, kinase (see 1331) otherwise PDH, values were very low and the effects of Ca2' were small and difficult to observe. This is analogous to the situation with heart mitochondria from starved or diabetic rats [33] and presumably means that in the absence of pyruvate the kinase activity is far greater than that of the phosphatase f33].…”

“…In the absence of Na+ and Mg2+ the activation curves are shifted to the left ( fig. 1) as with heart [4] or adipose tissue [9] but the shift is less, presumably because in liver mitochondria Na+-dependent egress of Ca2+ is slower, and Na+-independent egress faster, compared to these others [25].…”

“…)), NAD+-isocitrate dehydrogenase [2] and oxoglutarate dehydrogenase (OGDH) [3]. Studies on rat heart [4-61, skeletal muscle [7,8] and adipose tissue [9] and pig lymphocytes [lo] have demonstrated that these enzymes can be activated within mitochondria (incubated with physiological concentrations of Mg2+ and Na+) by increases in extramitochondrial [Ca"] within the expected physiological range [l 11. This led to the proposal [12,13] that hormones and other agents * Present address: Department of Biochemistry, The University of Leeds, Leeds LS2 9JT, England which increase cytoplasmic [Ca2'] [l l] may thereby increase intramitochondrial [Ca'+] and so activate these enzymes.…”