Cyclophilin D and the mitochondrial permeability transition in kidney proximal tubules after hypoxic and ischemic injury

Mitochondrial matrix cyclophilin D (CyPD) is known to promote development of the mitochondrial permeability transition (MPT). Kidney proximal tubule cells are especially prone to deleterious effects of mitochondrial damage because of their dependence on oxidative mitochondrial metabolism for ATP production. To clarify the role of CyPD and the MPT in proximal tubule injury during ischemia-reperfusion (I/R) and hypoxia-reoxygenation (H/R), we assessed freshly isolated tubules and in vivo injury in wild-type (WT) and Ppif(-/-) CyPD-null mice. Isolated mouse tubules developed a sustained, nonesterified fatty acid-mediated energetic deficit after H/R in vitro that could be substantially reversed by delipidated albumin and supplemental citric acid cycle substrates but was not modified by the absence of CyPD. Susceptibility of WT and Ppif(-/-) tubules to the MPT was increased by H/R but was less in normoxic and H/R Ppif(-/-) than WT tubules. Correction of the energetic deficit that developed during H/R strongly increased resistance to the MPT. Ppif(-/-) mice were resistant to I/R injury in vivo spanning a wide range of severity. The data clarify involvement of the MPT in oxygen deprivation-induced tubule cell injury by showing that the MPT does not contribute to the initial bioenergetic deficit produced by H/R but the deficit predisposes to subsequent development of the MPT, which contributes pathogenically to kidney I/R injury in vivo.     

Calcium compartmentation in isolated renal tubules in suspension

Substantial increases of total cell Ca2+ have been observed in suspensions of isolated rabbit proximal tubules subjected to hypoxic injury or treated with exogenous ATP followed by apparent recovery with reoxygenation of the hypoxic tubules or continued incubation of ATP-treated tubules. Ca2+ compartmentation studies using digitonin and metabolic inhibitors were done to clarify the basis for these changes. Digitonin, 40-90 micrograms/mg tubule protein, rapidly permeabilized the tubule cells and did not impair mitochondrial Ca2+ sequestration. Most of the increases of tubule cell Ca2+ produced by hypoxia and ATP were accounted for by pools which could be rapidly removed by exposure of tubules to EGTA and the uncoupler carbonyl cyanide m-chlorophenyl hydrazone without concomitant use of digitonin, suggesting that the changes of Ca2+ predominantly reflect sequestration by mitochondria in severely damaged cells or mitochondria already released to the medium from them. The time course of uptake followed by spontaneous release of mitochondrial Ca2+ from tubule cells deliberately permeabilized with digitonin, then incubated for prolonged periods, indicated that the decreases of tubule cell Ca2+ during reoxygenation of hypoxic suspensions and prolonged incubation of ATP-treated tubules were likely to be attributable to loss of Ca2+ from free mitochondria and those in damaged cells rather than to extrusion by intact cells.     

Effects of hypoxia and fatty acids on the distribution of metabolites in rat heart

The effects of exogenous fatty acids and hypoxia on cardiac energy metabolism were studied by measuring mitochondrial and cytosolic adenine nucleotides as well as CoA and carnitine esters using a tissue fractionation technique in non-aqueous solvents. During normoxia, the administration of 0.5 mM palmitate caused a considerable increase in acyl-CoA and acylcarnitine, particularly in mitochondria. High-energy phosphates, however, were only slightly altered. A 90 min low-flow hypoxia caused a dramatic increase in mitochondrial acyl esters. The mitochondrial ATP content decreased significantly, while the cytosolic concentration was only slightly diminished, suggesting an inhibition of mitochondrial adenine nucleotide translocation by long-chain acyl-CoA. Addition of palmitate during hypoxia amplified hypoxic damage and reduced adenine nucleotides in both compartments considerably, while fatty acid metabolites were only slightly affected. In presence of an inhibitor of fatty acid oxidation (BM 42.304), the fatty-acid-induced acceleration of cardiac injury was prevented. Since BM 42.304 decreased mitochondrial acylcarnitine and increased the cytosolic concentration significantly, BM 42.304 was presumed to inhibit mitochondrial acylcarnitine translocase. However, a causal relationship between lipid metabolites and ischemic damage seemed unlikely.     

Regulation of the Ca-independent phospholipase A2 in liver mitochondria by changes in the energetic state

The effect of electron transport chain redox status on activity of the mitochondrial Ca²⁺-independent phospholipase A₂ (iPLA₂) has been examined. When oxidizing NAD-linked substrates, the enzyme is not active unless deenergization occurs. Uncoupler, rotenone, antimycin A, and cyanide are equally effective at upregulating the enzyme, while oligomycin is ineffective. Thenoyltrifluoroacetone causes deenergization and activates the enzyme, but only if succinate is the respiratory substrate. These findings show that the mitochondrial iPLA₂ responds to the energetic state overall, rather than to the redox status of individual electron transport chain complexes. With NAD-linked substrates, and using rotenone to deenergize, iPLA₂ activation can be reversed by adding succinate to reestablish a membrane potential. For this purpose, ascorbate plus N,N,N′N′-tetramethyl-phenylenediamine can be used instead of succinate and is equally effective. With succinate as substrate, the membrane potential can be reduced in a graded and stable fashion by adding increasing concentrations of malonate, which is a competitive inhibitor of succinate utilization. A partial and stable activation of the iPLA₂ accompanies partial deenergization. These findings suggest that in addition to the several functions that have been proposed, the mitochondrial iPLA₂ may help to coordinate local capillary blood flow with changing energy demands.     

Partial purification and product characterization of fatty acid ethyl ester synthases in rabbit myocardium

Homogenates of rabbit ventricular myocardium synthesize fatty acid ethyl esters using as substrates nonesterified fatty acid and ethanol in the absence of coenzyme A and ATP. This catalytic activity resides in two soluble cytosolic enzymes accounting for 19 and 81% of total fatty acid ethyl ester synthetic capability. These enzymes have been separated and partially purified by anion exchange chromatography. Gas chromatographic/mass spectrometric analyses of the catalytic products formed by these enzymes from nonesterified fatty acid and ethanol confirm their identity as ethyl esters of fatty acids. Kinetic studies indicate apparent Km values for ethanol of 0.65 M and 0.75 M for the minor and major activities, respectively. These data confirm the presence of a myocardial pathway for nonoxidative ethanol metabolism and for a metabolism of fatty acids independent of coenzyme A.     

Organic hydroperoxides at high concentrations cause energization and activation of ATP synthesis in mitochondria

The addition of high concentrations of cumene or tert-butyl hydroperoxide to previously deenergized mitochondria results in the energization of these mitochondria and activation of ATP synthesis. The energization effect was observed in the presence of 0.5-0.7 mM cumene hydroperoxide or 2.0-2.5 mM tert-butyl hydroperoxide. This energization of mitochondria and activation of oxidative phosphorylation by organic hydroperoxides required the presence of ADP in the mitochondrial matrix and does not depend upon the method of deenergization of the mitochondria.     

The role of short chain fatty acid substrates in aerobic and glycolytic metabolism in primary cultures of renal proximal tubule cells

Summary This study examined the role of odd and even short-chain fatty acid substrates on aerobic and glycolytic metabolism in well-aerated primary cultures of rabbit renal proximal tubule cells (RPTC). Increasing oxygen delivery to primary cultures of RPTC by shaking the dishes (SHAKE) reduced total lactate levels and lactate dehydrogenase (LDH) activity and reduced net glucose consumption compared to RPTC cultured under standard conditions (STILL). The addition of butyrate, valerate, heptanoate, or octanoate to SHAKE RPTC produced variable effects on glycolytic metabolism. Although butyrate and heptanoate further reduced total lactate levels and net glucose consumption during short-term culture (<24 h), no fatty acid tested further reduced total lactate levels, net glucose consumption, or LDH activity during long-term culture (7 days). During the first 12 h of culture, maintenance of aerobic metabolism in SHAKE RPTC was dependent on medium supplementation with fatty acid substrates (2 mM). However, by 24 h, SHAKE RPTC did not require fatty acid substrates to maintain levels of aerobic metabolism equivalent to freshly isolated proximal tubules and greater than STILL RPTC. This suggests that SHAKE RPTC undergo adaptive changes between 12 and 24 h of culture, which give RPTC the ability to utilize other substrates for mitochondrial oxidation, therefore allowing greater expression of mitochondrial oxidative potential in SHAKE RPTC than in STILL RPTC.     

Effects of fatty acids on mitochondria: implications for cell death

Fatty acids have prominent effects on mitochondrial energy coupling through at least three mechanisms: (i) increase of the proton conductance of the inner mitochondrial membrane; (ii) respiratory inhibition; (iii) opening of the permeability transition pore (PTP). Furthermore, fatty acids physically interact with membranes and possess the potential to alter their permeability; and they are also excellent respiratory substrates that feed electrons into the respiratory chain. Due to the complexity of their actions, the effects of fatty acids on mitochondrial function in situ are difficult to predict. We have investigated the mitochondrial and cellular effects of fatty acids of increasing chain length and degree of unsaturation in relation to their potential to affect mitochondrial function in situ and to cause cell death. We show that saturated fatty acids have little effect on the mitochondrial membrane potential in situ, and display negligible short-term cytotoxicity for Morris Hepatoma 1C1 cells. The presence of double bonds increases both the depolarizing effects and the cytotoxicity, but these effects are offset by the hydrocarbon chain length, so that more unsaturations are required to observe an effect as the hydrocarbon chain length is increased. With few exceptions, depolarization and cell death are due to opening of the PTP rather than to the direct effects of fatty acids on energy coupling.     

Ion movements during energy-linked mitochondrial structural changes

The structure of isolated rat liver mitochondria has been observed in the electron microscope following incubation of the mitochondria in vitro under a variety of conditions. The results show that ultrastructural changes are only associated with the energization and deenergization of isolated mitochondria if the composition of the incubation medium permits ion movements in or out of the matrix. The mechanism of energy coupling does not appear to depend on these major mitochondrial structural changes. The addition of low levels of valinomycin greatly increases the rate at which the matrix compartment swells and shrinks on energization and deenergization even at low K⁺ concentrations.     

Mitochondrial uncoupling protein-2 deficiency protects steatotic mouse hepatocytes from hypoxia/reoxygenation

Steatotic livers are sensitive to ischemic events and associated ATP depletion. Hepatocellular necrosis following these events may result from mitochondrial uncoupling protein-2 (UCP2) expression. To test this hypothesis, we developed a model of in vitro steatosis using primary hepatocytes from wild-type (WT) and UCP2 knockout (KO) mice and subjected them to hypoxia/reoxygenation (H/R). Using cultured hepatocytes treated with emulsified fatty acids for 24 h, generating a steatotic phenotype (i.e., microvesicular and broad-spectrum fatty acid accumulation), we found that the phenotype of the WT and UCP2 KO were the same; however, cellular viability was increased in the steatotic KO hepatocytes following 4 h of hypoxia and 24 h of reoxygenation; Hepatocellular ATP levels decreased during hypoxia and recovered after reoxygenation in the control and UCP2 KO steatotic hepatocytes but not in the WT steatotic hepatocytes; mitochondrial membrane potential in WT and UCP2 KO steatotic groups was less than control groups but higher than UCP2 KO hepatocytes. Following reoxygenation, lipid peroxidation, as measured by thiobarbituric acid reactive substances, increased in all groups but to a greater extent in the steatotic hepatocytes, regardless of UCP2 expression. These results demonstrate that UCP2 sensitizes steatotic hepatocytes to H/R through mitochondrial depolarization and ATP depletion but not lipid peroxidation.     

Rapid release of Mg(2+) from liver mitochondria by nonesterified long-chain fatty acids in alkaline media

Long-chain fatty acids induce a rapid release of Mg(2+) from both energized and nonenergized rat liver mitochondria suspended at pH 8 in isotonic saline but not sucrose media. The effect is observed only with fatty acids that possess protonophoric activity. The most active saturated fatty acids are myristic and palmitic, while the most active unsaturated acids are oleic, linolenic, and arachidonic. The rate of Mg(2+) release drastically decreases with decreasing medium pH to 7.2-7.6. However, at those pH values this rate is doubled by energization of mitochondria with respiratory substrates. Mg(2+) release is accompanied by cyclosporin A-insensitive large-amplitude swelling of mitochondria. This swelling is similar to that produced by the divalent metal ionophore A23187 and is interpreted as being due to activation of the inner membrane anion channel, the K(+) uniporter, and the K(+)/H(+) exchanger. In energized mitochondria, both swelling and Mg(2+) release are blocked by the exogenous K(+)/H(+) exchanger nigericin. It is proposed that fatty acids under conditions of alkaline mitochondrial matrix activate latent Mg(2+)-sensitive ion-conducting pathways in the inner mitochondrial membrane, which mediate swelling and Mg(2+) release. It is hypothesized that fatty acids activate an intrinsic Mg(2+)/H(+) exchanger that is related to, or identical with, the K(+)/H(+) exchanger.     

Fatty acids decrease mitochondrial generation of reactive oxygen species at the reverse electron transport but increase it at the forward transport

Long-chain nonesterified ("free") fatty acids (FFA) can affect the mitochondrial generation of reactive oxygen species (ROS) in two ways: (i) by depolarisation of the inner membrane due to the uncoupling effect and (ii) by partly blocking the respiratory chain. In the present work this dual effect was investigated in rat heart and liver mitochondria under conditions of forward and reverse electron transport. Under conditions of the forward electron transport, i.e. with pyruvate plus malate and with succinate (plus rotenone) as respiratory substrates, polyunsaturated fatty acid, arachidonic, and branched-chain saturated fatty acid, phytanic, increased ROS production in parallel with a partial inhibition of the electron transport in the respiratory chain, most likely at the level of complexes I and III. A linear correlation between stimulation of ROS production and inhibition of complex III was found for rat heart mitochondria. This effect on ROS production was further increased in glutathione-depleted mitochondria. Under conditions of the reverse electron transport, i.e. with succinate (without rotenone), unsaturated fatty acids, arachidonic and oleic, straight-chain saturated palmitic acid and branched-chain saturated phytanic acid strongly inhibited ROS production. This inhibition was partly abolished by the blocker of ATP/ADP transfer, carboxyatractyloside, thus indicating that this effect was related to uncoupling (protonophoric) action of fatty acids. It is concluded that in isolated rat heart and liver mitochondria functioning in the forward electron transport mode, unsaturated fatty acids and phytanic acid increase ROS generation by partly inhibiting the electron transport and, most likely, by changing membrane fluidity. Only under conditions of reverse electron transport, fatty acids decrease ROS generation due to their uncoupling action.     

Does diaminobenzidine demonstrate prostaglandin synthetase? A study on polyunsaturated fatty acid-induced DAB oxidation in sheep vesicular glands and rabbit kidney medulla.

A method histochemical localization of prostaglandin synthetase using DAB, potassium cyanide and polyunsaturated fatty acid has been revised. The arachidonic acid-induced DAB oxidation observed in the secretory epithelium of sheep vesicular glands and in collecting tubules as well as intersititial cells of rabbit kidney medulla was found to be insensitive to antiinflammatory cyclooxygenase (formerly referred as prostaglandin synthetase) inhibitors, such as indomethacin, aspirin, mefenamic acid and paracetamol, whereas aminotriazole caused complete inhibition of the reaction. Furthermore, DAB was oxidized in the presence of polyunsaturated fatty acids inconvertible to prostaglandins (linoleic and linolenic acid) as well as in the presence of H2O2--in the latter case reaction possessed identical features with that induced by fatty acids. Ultrastructurally, the reaction product was localized on the membranes of nuclear envelope and endoplasmic reticulum. On the ground of the results obtained a hypothesis is presented, that the polyunsaturated fatty acid-induced DAB oxidation is due to a peroxidatic activity of the investigated tissues. Possible relations between such peroxidatic activity and prostaglandin biosynthesis are discussed.     

Fatty acids decrease mitochondrial generation of reactive oxygen species at the reverse electron transport but increase it at the forward transport

Long-chain nonesterified (“free”) fatty acids (FFA) can affect the mitochondrial generation of reactive oxygen species (ROS) in two ways: (i) by depolarisation of the inner membrane due to the uncoupling effect and (ii) by partly blocking the respiratory chain. In the present work this dual effect was investigated in rat heart and liver mitochondria under conditions of forward and reverse electron transport. Under conditions of the forward electron transport, i.e. with pyruvate plus malate and with succinate (plus rotenone) as respiratory substrates, polyunsaturated fatty acid, arachidonic, and branched-chain saturated fatty acid, phytanic, increased ROS production in parallel with a partial inhibition of the electron transport in the respiratory chain, most likely at the level of complexes I and III. A linear correlation between stimulation of ROS production and inhibition of complex III was found for rat heart mitochondria. This effect on ROS production was further increased in glutathione-depleted mitochondria. Under conditions of the reverse electron transport, i.e. with succinate (without rotenone), unsaturated fatty acids, arachidonic and oleic, straight-chain saturated palmitic acid and branched-chain saturated phytanic acid strongly inhibited ROS production. This inhibition was partly abolished by the blocker of ATP/ADP transfer, carboxyatractyloside, thus indicating that this effect was related to uncoupling (protonophoric) action of fatty acids. It is concluded that in isolated rat heart and liver mitochondria functioning in the forward electron transport mode, unsaturated fatty acids and phytanic acid increase ROS generation by partly inhibiting the electron transport and, most likely, by changing membrane fluidity. Only under conditions of reverse electron transport, fatty acids decrease ROS generation due to their uncoupling action.     

Does diaminobenzidine demonstrate prostaglandin synthetase?

Summary A method histochemical localization of prostaglandin synthetase using DAB, potassium cyanide and polyunsaturated fatty acid has been revised. The arachidonic acid-induced DAB oxidation observed in the secretory epithelium of sheep vesicular glands and in collecting tubules as well as interstitial cells of rabbit kidney medulla was found to be insensitive to antiinflammatory cyclooxygenase (formerly referred as prostaglandin synthetase) inhibitors, such as indomethacin, aspirin, mefenamic acid and paracetamol, whereas aminotriazole caused complete inhibition of the reaction. Furthermore, DAB was oxidized in the presence of polyunsaturated fatty acids inconvertible to prostaglandins (linoleic and linolenic acid) as well as in the presence of H₂O₂ — in the latter case reaction possessed identical features with that induced by fatty acids. Ultrastructurally, the reaction product was localized on the membranes of nuclear envelope and endoplasmic reticulum. On the ground of the results obtained a hypothesis is presented, that the polyunsaturated fatty acid-induced DAB oxidation is due to a peroxidatic activity of the investigated tissues. Possible relations between such peroxidatic activity and prostaglandin biosynthesis are discussed.     

Spatial Reorganization of Saccharomyces cerevisiae Enolase To Alter Carbon Metabolism under Hypoxia

Hypoxia has critical effects on the physiology of organisms. In the yeast Saccharomyces cerevisiae, glycolytic enzymes, including enolase (Eno2p), formed cellular foci under hypoxia. Here, we investigated the regulation and biological functions of these foci. Focus formation by Eno2p was inhibited temperature independently by the addition of cycloheximide or rapamycin or by the single substitution of alanine for the Val22 residue. Using mitochondrial inhibitors and an antioxidant, mitochondrial reactive oxygen species (ROS) production was shown to participate in focus formation. Focus formation was also inhibited temperature dependently by an SNF1 knockout mutation. Interestingly, the foci were observed in the cell even after reoxygenation. The metabolic turnover analysis revealed that [U-¹³C]glucose conversion to pyruvate and oxaloacetate was accelerated in focus-forming cells. These results suggest that under hypoxia, S. cerevisiae cells sense mitochondrial ROS and, by the involvement of SNF1/AMPK, spatially reorganize metabolic enzymes in the cytosol via de novo protein synthesis, which subsequently increases carbon metabolism. The mechanism may be important for yeast cells under hypoxia, to quickly provide both energy and substrates for the biosynthesis of lipids and proteins independently of the tricarboxylic acid (TCA) cycle and also to fit changing environments.     

Effects of hypoxia and reoxygenation on adult rat heart cell metabolism

Isolated adult rat heart cells were used to study the effects of oxygen deprivation followed by reoxygenation upon myocardial metabolism. Calcium-tolerant nonbeating myocytes were incubated for 5, 30, or 60 min under 100% oxygen or 100% nitrogen and then rinsed with oxygenated buffer. Substrate oxidation was studied by incubating the cells with 14C-labeled glucose, pyruvate, or octanoate and determining the rates of 14CO2 production from the individual substrates. After 5 min of hypoxia, metabolism of glucose, as assessed by glucose oxidation and lactate production, was significantly depressed. Pyruvate and octanoate oxidation were unaltered. Oxygen consumption was also unchanged by short-term hypoxia and reoxygenation. With reoxygenation after 30 min of oxygen deprivation, more exaggerated changes in glucose metabolism were noted as well as a depression in pyruvate oxidation and unaltered octanoate oxidation. Oxidation of octanoate was slightly depressed after 60 min of hypoxia. Cell viability assessed after reoxygenation was not significantly altered until 60 min of oxygen deprivation. The results indicate that cytosolic changes occur after short periods of hypoxia followed by reoxygenation, whereas mitochondrial function is more resistant to damage inflicted by hypoxia and reoxygenation.     

Arachidonic and cis-unsaturated fatty acids induce selective platelet substrate phosphorylation through activation of cytosolic protein kinase C

The ability of arachidonic acid and other fatty acids to induce phosphorylation of endogenous substrates and the role of protein kinase C in mediating these effects were examined. In a cell-free cytosolic system derived from human platelets, arachidonic, oleic, and other cis-unsaturated fatty acids induced a dose-dependent phosphorylation of several endogenous substrates. These substrates form a subset of phorbol ester-induced phosphorylations. Multiple lines of evidence suggested the direct involvement of protein kinase C in mediating fatty acid-induced phosphorylations. These observations suggest that arachidonic acid and other unsaturated fatty acids are capable of activating protein kinase C in a physiologic environment resulting in the phosphorylation of multiple endogenous substrates.     

Neonatal hypoxic hyperlipidemia in the rat: effects on aldosterone and corticosterone synthesis in vitro

Neonatal hypoxia increases aldosterone production and plasma lipids. Because fatty acids can inhibit aldosterone synthesis, we hypothesized that increases in plasma lipids restrain aldosteronogenesis in the hypoxic neonate. We exposed rats to 7 days of hypoxia from birth to 7 days of age (suckling) or from 28 to 35 days of age (weaned at day 21). Plasma was analyzed for lipid content, and steroidogenesis was studied in dispersed whole adrenal glands untreated and treated to wash away lipids. Hypoxia increased plasma cholesterol, triglycerides, and nonesterified fatty acids in the suckling neonatal rat only. Washing away lipids increased aldosterone production in cells from 7-day-old rats exposed to hypoxia, but not in cells from normoxic 7-day-old rats or from normoxic or hypoxic 35-day-old rats. Addition of oleic or linolenic acid to washed cells inhibited both aldosterone and corticosterone production, although cells from hypoxic 7-day-old rats were less sensitive. We conclude that hypoxia induces hyperlipidemia in the suckling neonate and that elevated nonesterified fatty acids inhibit aldosteronogenesis.     

Acute administration of 3,5-diiodo-L-thyronine to hypothyroid rats stimulates bioenergetic parameters in liver mitochondria

The role of 3,5-diiodo-L-thyronine (T₂), initially considered only a 3,3′,5-triiodo-L-thyronine (T₃) catabolite, in the bioenergetic metabolism is of growing interest. In this study we investigated the acute effects (within 1 h) of T₂ administration to hypothyroid rats on liver mitochondria fatty acid uptake and β-oxidation rate, mitochondrial efficiency (by measuring proton leak) and mitochondrial oxidative damage (by determining H₂O₂ release). Fatty acid uptake into mitochondria was measured assaying carnitine palmitoyl transferase (CPT) I and II activities, and fatty acid β-oxidation using palmitoyl-CoA as a respiratory substrate. Mitochondrial fatty acid pattern was defined by gas-liquid chromatography. In hypothyroid + T₂ vs hypothyroid rats we observed a raise in the serum level of nonesterified fatty acids (NEFA), in the mitochondrial CPT system activity and in the fatty acid β-oxidation rate. A parallel increase in the respiratory chain activity, mainly from succinate, occurs. When fatty acids are chelated by bovine serum albumin, a T₂-induced increase in both state 3 and state 4 respiration is observed, while, when fatty acids are present, mitochondrial uncoupling occurs together with increased proton leak, responsible for mitochondrial thermogenesis. T₂ administration decreases mitochondrial oxidative stress as determined by lower H₂O₂ production. We conclude that in rat liver mitochondria T₂ acutely enhances the rate of fatty acid β-oxidation, and the activity of the downstream respiratory chain. The T₂-induced increase in proton leak may contribute to mitochondrial thermogenesis and to the reduction of oxidative stress. Our results strengthen the previously reported ability of T₂ to reduce adiposity, dyslipidemia and to prevent liver steatosis.