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Regulation of glycine catabolism in rat liver mitochondria.

ABSTRACT: 1. The catabolism of glycine was studied in isolated rat liver mitochondria by measuring release of 14CO2 from [1-14C]-glycine. Incubation of mitochondria in a medium containing 0.5 microM free Ca2+ resulted in an 8-fold increase in the rate of degradation of glycine. Intraperitoneal injection of glucagon (33 or 100 micrograms/100 g body wt.) 25 min before killing of rats also resulted in a 3-fold or 10-fold (depending on dosage) increase in the rate of catabolism of glycine. 2. Both the stimulation by free Ca2+ and that by injection of glucagon in vivo were dependent on phosphate in the incubation medium. This requirement for phosphate was specific, as replacement of phosphate by other permeant anions such as thiocyanate and acetate did not permit the stimulation. The phosphate-dependent stimulation of glycine catabolism by Ca2+ was also evident when mitochondria were incubated in the absence of K+. 3. Mitochondria isolated from rats previously injected with glucagon showed elevated rates of degradation of glycine even in the presence of rotenone, provided that regeneration of NAD+ was affected by providing acetoacetate. 4. Hypo-osmolarity of the medium markedly stimulated the rate of degradation of glycine by mitochondria. Although hypo-osmolarity-induced stimulation of glycine degradation was accompanied by parallel changes in mitochondrial matrix volume, no measurable changes in matrix volume were observed in mitochondria stimulated either by free Ca2+ (0.5 microM) or by injection of glucagon in vivo. Furthermore, Ca2+ stimulated glycine decarboxylation in mitochondria exposed to either hyper-osmolar (410 mosmol) or hypo-osmolar (210 mosmol) conditions. Although hyper-osmolarity decreased and hypo-osmolarity increased matrix volume, stimulation of glycine degradation by Ca2+ was not associated with any further changes in matrix volume. 5. These data demonstrate that the regulation of hepatic glycine oxidation by glucagon and by free Ca2+ is largely independent of changes in mitochondrial matrix volume.

SUBMITTER: Jois M

PROVIDER: S-EPMC1131053 | biostudies-other | 1992 Apr

REPOSITORIES: biostudies-other

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Project description:The mechanism whereby rat liver mitochondria regulate the extramitochondrial concentration of free Ca(2+) was investigated. At 30 degrees C and pH7.0, mitochondria can maintain a steady-state pCa(2+) (0) (the negative logarithm of the free extramitochondrial Ca(2+) concentration) of 6.1 (0.8mum). This represents a true steady state, as slight displacements in pCa(2+) (0) away from 6.1 result in net Ca(2+) uptake or efflux in order to restore pCa(2+) (0) to its original value. In the absence of added permeant weak acid, the steady-state pCa(2+) (0) is virtually independent of the Ca(2+) accumulated in the matrix until 60nmol of Ca(2+)/mg of protein has been taken up. The steady-state pCa(2+) (0) is also independent of the membrane potential, as long as the latter parameter is above a critical value. When the membrane potential is below this value, pCa(2+) (0) is variable and appears to be governed by thermodynamic equilibration of Ca(2+) across a Ca(2+) uniport. Permeant weak acids increase, and N-ethylmaleimide decreases, the capacity of mitochondria to buffer pCa(2+) (0) in the region of 6 (1mum-free Ca(2+)) while accumulating Ca(2+). Permeant acids delay the build-up of the transmembrane pH gradient as Ca(2+) is accumulated, and consequently delay the fall in membrane potential to values insufficient to maintain a pCa(2+) (0) of 6. The steady-state pCa(2+) (0) is affected by temperature, incubation pH and Mg(2+). The activity of the Ca(2+) uniport, rather than that of the respiratory chain, is rate-limiting when pCa(2+) (0) is greater than 5.3 (free Ca(2+) less than 5mum). When the Ca(2+) electrochemical gradient is in excess, the activity of the uniport decreases by 2-fold for every 0.12 increase in pCa(2+) (0) (fall in free Ca(2+)). At pCa(2+) (0) 6.1, the activity of the Ca(2+) uniport is kinetically limited to 5nmol of Ca(2+)/min per mg of protein, even when the Ca(2+) electrochemical gradient is large. A steady-state cycling of Ca(2+) through independent influx and efflux pathways provides a model which is kinetically and thermodynamically consistent with the present observations, and which predicts an extremely precise regulation of pCa(2+) (0) by liver mitochondria in vivo.

Project description:The FDA has approved 31 small-molecule kinase inhibitors (KIs) for human use as of November 2016, with six having black box warnings for hepatotoxicity (BBW-H) in product labeling. The precise mechanisms and risk factors for KI-induced hepatotoxicity are poorly understood. Here, the 31 KIs were tested in isolated rat liver mitochondria, an in vitro system recently proposed to be a useful tool to predict drug-induced hepatotoxicity in humans. The KIs were incubated with mitochondria or submitochondrial particles at concentrations ranging from therapeutic maximal blood concentrations (Cmax) levels to 100-fold Cmax levels. Ten endpoints were measured, including oxygen consumption rate, inner membrane potential, cytochrome c release, swelling, reactive oxygen species, and individual respiratory chain complex (I-V) activities. Of the 31 KIs examined only three including sorafenib, regorafenib and pazopanib, all of which are hepatotoxic, caused significant mitochondrial toxicity at concentrations equal to the Cmax, indicating that mitochondrial toxicity likely contributes to the pathogenesis of hepatotoxicity associated with these KIs. At concentrations equal to 100-fold Cmax, 18 KIs were found to be toxic to mitochondria, and among six KIs with BBW-H, mitochondrial injury was induced by regorafenib, lapatinib, idelalisib, and pazopanib, but not ponatinib, or sunitinib. Mitochondrial liability at 100-fold Cmax had a positive predictive power (PPV) of 72% and negative predictive power (NPV) of 33% in predicting human KI hepatotoxicity as defined by product labeling, with the sensitivity and specificity being 62% and 44%, respectively. Similar predictive power was obtained using the criterion of Cmax ≥1.1 µM or daily dose ≥100 mg. Mitochondrial liability at 1-2.5-fold Cmax showed a 100% PPV and specificity, though the NPV and sensitivity were 32% and 14%, respectively. These data provide novel mechanistic insights into KI hepatotoxicity and indicate that mitochondrial toxicity at therapeutic levels can help identify hepatotoxic KIs.

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Regulation of glycine catabolism in rat liver mitochondria.

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