Non-lipid Substrates Research Articles

Empagliflozin Lowers Mitochondrial Respiration and Skeletal Muscle Sphingolipid Content in Female Mice Fed Western Diet

Sodium-glucose cotransporter-2 (SGLT2) inhibitors are a frontline treatment for type 2 diabetes due to their ability to attenuate hyperglycemia (via glucosuria). Mounting evidence indicates SGLT2 inhibitors can also alter mitochondrial metabolism, enhance reliance upon fat oxidation and attenuate fat accumulation in a growing list of tissues. Nevertheless, the impact of SGLT2 inhibition on skeletal muscle remains largely unexplored. The objective of this study was to test the hypothesis that SGLT2 inhibition would promote enhanced mitochondrial capacity for fat oxidation and lower accumulation of harmful lipids in skeletal muscle of diet-induced obese mice. Female C57BL/6J mice, aged 26 weeks, were provided ad libitum access to either low-fat diet (LFD, n=9) or western diet (WD) for 8-weeks. WD mice were then randomized to 4-weeks of diet supplemented with 10mg/kg (0.01%) empagliflozin (WD+Emp, n=10) or untreated control (WD, n = 10). Dual-energy x-ray absorptiometry and glucose tolerance tests were performed in all groups pre- and post-empagliflozin treatment. At week 12, mitochondria were isolated from quadriceps muscle and measured by high-resolution respirometry using octanoyl-carnitine+malate (OCM) and other non-lipid substrates. Intramyocellular lipids were assessed using liquid chromatography, tandem mass spectrometry. As expected, WD mice had greater body weight and fat mass, as well as impaired glucose tolerance, when compared with LFD mice (all P<0.05). Four weeks of empagliflozin treatment improved glucose tolerance in WD+Emp mice (P<0.01 for week 12 vs. 8), despite no differences in body weight or fat mass compared with pre-treatment (both P>0.05 for week 12 vs. 8). Lipid-supported mitochondrial respiration and overall lipid content (e.g., triacylglycerols, select sphingolipids and ceramides) were greater in skeletal muscle from WD compared with LFD mice (all comparisons P<0.05). Such effects of WD on muscle lipid metabolism were largely reversed with empagliflozin treatment. There was overall main effect of lower mitochondrial respiration in WD+Emp compared with WD (P<0.001), and a tendency for lower concentration of numerous sphingolipid and ceramide species (mean difference = 9-34%). We interpret this as preliminary evidence that empagliflozin can reverse some WD-induced adaptations to skeletal muscle lipid metabolism, which may contribute to the improvement in glucose control.

The Influence of High‐Fat Feeding and Exercise Training on Substrate‐Specific Mitochondrial Respiration in Skeletal Muscle of Female Mice

Objective High-fat diet (HFD) induced obesity is associated with excessive lipid supply which drives remodeling of skeletal muscle mitochondria. Exercise stimulates mitochondrial respiration but it is not clear if such adaptations are specific to lipids. Our objective was to determine if HFD-induced obesity would change the sensitivity and capacity for lipid oxidation, and if exercise modifies mitochondrial-specific respiration during HFD. Hypothesis We hypothesized that HFD-induced obesity would increase the sensitivity (intrinsic function) and capacity for lipid respiration while exercise would further augment overall capacity for skeletal muscle mitochondrial substrate oxidation in female mice. Methods We used a 2x2 designof diet and exercise in 10-week old female C57BL/6J mice (n=13-15 per group). Mice consumed diet (either 10% fat or 60% HFD) for 4 weeks then performed 8 weeks of treadmill training for 1 hour per day, 5 days per week or remained sedentary while continuing diet. HFD increased body-weight (P<0.01) by 4 weeks and fat mass measured by dual-energy x-ray absorptiometry at week 12 (P<0.01). We confirmed glucose and insulin intolerance at week 4 of HFD with no change after 8 weeks of treadmill training (P=0.435). Mice rested 24 hours prior to tissue collection. We isolated mitochondria from fresh quadriceps then performed high-resolution respirometry. We measured lipid sensitivity (Km) and capacity (Vmax) using titrations of octanoyl-carnitine (F-linked). Glutamate-succinate were used to determine non-lipid substrate oxidative phosphorylation capacity (N- and S-linked). Primary outcomes were analyzed using 2-way ANOVA (diet x exercise) with Tukey's multiple comparisons (α=0.05). Results The Km for lipid substrates was not different between diet (-7.7%, P=0.66) or exercise (+20.3%, P=0.26) groups. Absolute Vmax was 38% higher in HFD (P=0.018 versus low fat). There was a tendency for 27% higher Vmax after exercise (P=0.074 versus sedentary). For non-lipid substrates, HFD had 23% greater absolute N-linked respiration (P=0.04) and 19% greater S-linked respiration (P=0.02); however, no effects of exercise were detected. Normalizing respiration to mitochondrial protein content eliminated the HFD effects in all substrates. Conclusion We provide evidence that mitochondrial adaptations to HFD, in female mice, are driven primarily by increases in protein abundance rather than intrinsic factors as the normalization of respiration to mitochondrial protein content eliminated HFD effects for increased substrate respiration and there were no differences to lipid sensitivity between diet or exercise. Exercise did not appear to alter the mitochondrial respiratory response to HFD.

Substrate-Specific Respiration of Isolated Skeletal Muscle Mitochondria after 1 h of Moderate Cycling in Sedentary Adults.

Skeletal muscle mitochondria have dynamic shifts in oxidative metabolism to meet energy demands of aerobic exercise. Specific complexes oxidize lipid and nonlipid substrates. It is unclear if aerobic exercise stimulates intrinsic oxidative metabolism of mitochondria or varies between substrates. We studied mitochondrial metabolism in sedentary male and female adults (n = 11F/4M) who were free of major medical conditions with mean ± SD age of 28 ± 7 yr, peak aerobic capacity of 2.0 ± 0.4 L·min-1, and body mass index of 22.2 ± 2 kg·m-2. Biopsies were collected from the vastus lateralis muscle on separate study days at rest or 15 min after exercise (1 h cycling at 65% peak aerobic capacity). Isolated mitochondria were analyzed using high-resolution respirometry of separate titration protocols for lipid (palmitoylcarnitine, F-linked) and nonlipid substrates (glutamate-malate, N-linked; succinate S-linked). Titration protocols distinguished between oxidative phosphorylation and leak respiration and included the measurement of reactive oxygen species emission (H2O2). Western blotting determined the protein abundance of electron transfer flavoprotein (ETF) subunits, including inhibitory methylation site on ETF-β. Aerobic exercise induced modest increases in mitochondrial respiration because of increased coupled respiration across F-linked (+13%, P = 0.08), N(S)-linked (+14%, P = 0.09), and N-linked substrates (+17%, P = 0.08). Prior exercise did not change P:O ratio. Electron leak to H2O2 increased 6% increased after exercise (P = 0.06) for lipid substrates but not for nonlipid. The protein abundance of ETF-α or ETF-β subunit or inhibitory methylation on ETF-β was not different between rest and after exercise. In sedentary adults, the single bout of moderate-intensity cycling induced modest increases for intrinsic mitochondrial oxidative phosphorylation that was consistent across multiple substrates.

Western Diet And Exercise Training Increase Mitochondrial Lipid Respiration In Male But Not Female Mice

High-fat feeding in mice is a common model to investigate mitochondrial lipid metabolism during diet-induced obesity. Our previous work demonstrates 60% high-fat diet stimulates mitochondrial lipid respiration yet such fat content is higher than a typical western diet (~35% fat with higher sugar). Aerobic exercise training also stimulates mitochondrial lipid respiration, but the interaction with western diet is not clear. PURPOSE: We determined mitochondrial oxidative function in response to western diet in the absence and presence of concurrent exercise training. We hypothesized western diet would induce greater mitochondrial lipid respiration that would be further enhanced with exercise training. METHODS: Male and female C57BL/6J mice (n=5-7 per group for each sex) ate either western diet (WD) or low-fat diet (LFD) for 4 weeks, with a group of WD mice randomized to perform concurrent treadmill training (WD+Ex). At week 4, dual-energy x-ray absorptiometry was used to measure body composition and in-vivo substrate oxidation was assessed using metabolic cage indirect calorimetry. Ex-vivo mitochondrial oxidation was measured via high-resolution respirometry using isolated mitochondria from quadriceps muscles collected 36-hours after final exercise session (or rest). Respiration protocols included lipid (octanoyl-carnitine+malate) and non-lipid (glutamate+succinate) substrates. RESULTS: Among sedentary mice, WD had higher body weight and fat mass than LFD (P<0.0001), but only in males. WD had lower in-cage respiratory exchange ratio than LFD (P<0.05) regardless of sex, indicating greater whole-body reliance on lipids. In males, WD+Ex stimulated mitochondrial lipid respiration more so than WD alone (P<0.05). Females had no significant changes in mitochondrial lipid respiration. Non-lipid supported mitochondrial respiration was not significantly altered by WD or WD+Ex regardless of sex. CONCLUSION: In agreement with our hypothesis, WD stimulated lipid-specific mitochondrial respiration that further increased with Ex, but only in male mice. There were apparent sex differences such that females were protected against WD-induced weight gain alongside limited changes in mitochondrial lipid respiration.

Hepatic de novo lipogenesis is suppressed and fat oxidation is increased by omega-3 fatty acids at the expense of glucose metabolism

ObjectiveIncreased hepatic de novo lipogenesis (DNL) is suggested to be an underlying cause in the development of nonalcoholic fatty liver disease and/or insulin resistance. It is suggested that omega-3 fatty...

Mannanase activity produced through fermentation of coconut flour at various pH by Aspergilus niger

Coconut flour is one of agriculture waste product, containing high concentration of mannan polysaccharide. This waste product can be used as a substrate to produce mannanase enzyme through fermentation by using inoculum of Aspergilus niger. The aim of the study was to produce mannanase enzyme with high activity from the non-lipid and non-protein coconut flour. The coconut flour was extracted by using 1M NaOH obtain free-protein coconut flour. The non protein coconut flour was then extracted by using hexane to release lipid. The non-protein and non-lipid substrates were then fermented with Aspergilus niger. A completely randomized design was used in this study with various pH (pH 6 7 8 and 9). The result of the study indicated that pH 9 of the substrate produce the highest mannanase activity, being 0.0313 U/ml. In conclusion, optimal activity of mannanase was reached when the pH of substrate was 9.

Mitochondrial Function in High-Fat Fed and Exercise-Trained Mice Unable to Induce Autophagy

Impaired mitochondrial lipid respiration is implicated in obesity-induced insulin resistance but can be enhanced with exercise training. Autophagy is involved in remodeling the mitochondrial proteome and may be required for mitochondrial and whole-body adaptations in lipid respiration. We used a diet-induced obesity model of C57BL/6J mice and investigated the requirement of autophagy for mitochondrial adaptations to 8 weeks of treadmill exercise training using transgenic mice that cannot induce autophagy with exercise (BCL) compared to wild type controls (WT, n=3-5). High fat (HF) fed mice had lower glucose tolerance at 4 weeks compared to low fat (LF, ∼35%, P&amp;lt;0.01), regardless of genotype. After 8 weeks of exercise, LF-BCL mice had similar glucose tolerance to LF-WT mice. However, the HF-BCL mice had lower glucose tolerance compared to HF-WT mice (∼19%, P&amp;lt;0.01) suggesting a blunted improvement in glucose tolerance following training in the absence of autophagy. At week 12, mitochondria were isolated from quadriceps for respirometry. Oxidative phosphorylation capacity was normalized to electron transport system capacity. Lipid supported respiration (octanoyl-carnitine) was higher in the HF mice compared to LF (∼17%, P&amp;lt;0.01) and the lack of autophagy activation had no effect. Glutamate-malate-succinate supported respiration was lower in BCL mice compared to WT (∼11%, P&amp;lt;0.05), but only on a LF diet suggesting the requirement of autophagy for improved mitochondrial function with exercise may vary between substrates. Whole body respiratory exchange ratio was measured during 24 hours of fasting and all mice shifted to lipid oxidation with no difference between diet or genotype (P = 0.11). Collectively, skeletal muscle mitochondrial lipid oxidation was greater with HF, despite glucose intolerance. The activation of autophagy with exercise was not required for mitochondrial lipid adaptations or whole body metabolic flexibility, but may be required for non-lipid substrates. Disclosure S.E. Ehrlicher: None. H.D. Stierwalt: None. S.A. Newsom: None. M.M. Robinson: None.

Exploring the nutritional basis of starvation resistance in Drosophila melanogaster

Summary Food limitation is the most common environmental challenge faced by animals and the capacity of animals to survive prolonged periods of starvation is linked to their diet and nutritional status. The fruit flies of the genus Drosophila have been widely used to study the ecology and evolution of starvation resistance (SR), but the nutritional basis of their resistance to starvation has not yet been fully explored. We have taken a nutritionally explicit approach to investigate the quantitative and qualitative effects of nutrition on SR in Drosophila melanogaster using state‐space geometric models of nutrition. Experimental flies were given ad libitum access to artificial diets differing in concentrations and ratios of protein and carbohydrate for 5 days before they were assayed for starvation time, body composition and life‐history parameters. The main objectives of this study were to determine the most critical nutritional factor of SR and to understand the utilization patterns of endogenous metabolic fuels during starvation in Drosophila. Starvation resistance in Drosophila was greatly influenced by the dietary protein : carbohydrate (P : C) ratio, but neither by the caloric content of the diet nor by carbohydrate alone. SR was strongest at the lowest P : C ratio and decreased with rising P : C ratio. When fed on low P : C diets, Drosophila not only deposited more lipids, but also lowered the minimum body weight required for tolerating starvation. During starvation, Drosophila exhibited a dramatic transition in the utilization of endogenous fuels. In the early phase of starvation, Drosophila exclusively used nonlipid substrates, but switched to the late phase during which they mobilized lipids to maintain life under starvation. Our results highlight the important role that nutrition plays in determining the phenotypic expression of SR in Drosophila and provide broad implications for understanding starvation responses in other species, including humans. This study introduces how the recent advances in nutritional physiology can offer new insights into the ecology and evolution of starvation responses.

High fat intake with monounsaturated fatty acids, but not n6 polyunsaturated fatty acid, increases activity of key mitochondrial oxidative enzymes in the heart

Evidence suggests that a high fat intake may be beneficial for cardiovascular health, though optimal dietary fatty acid composition is unclear. In this study, we compare the effects of a standard low fat diet (STD, 15% energy from fat) to high fat diets (40% energy from fat) comprised of monounsaturated fat (MUFA, oleate (18:1)), n6 polyunsaturated fat (PUFA, linoleic acid (18:2n6)), or saturated fat (SAT, palmitate and stearate (16:0 and 18:0)). Male rats (n=13–14/group) were fed the STD diet until 16 weeks of age and then assigned to dietary treatment for 15 weeks. Cardiac function was assessed by echocardiography and left ventricle (LV) pressure measurements and was unaffected by diet. Mitochondria were isolated from the LV and show no impact of dietary treatment on mitochondrial yield or respiration with lipid and nonlipid substrates. Mitochondrial oxidative enzyme activity in whole tissue dramatically increased with MUFA compared to STD. Citrate synthase activity increased from 105±15 μmol/g/min in STD to 157±14 in MUFA (P&lt;0.05 vs STD and PUFA), with no significant effect in PUFA (107±9) or SAT (130±11). Similar changes occurred with aconitase and long chain acyl‐CoA dehydrogenase activities. In summary, a high fat diet did not adversely impact cardiac function, increased activity of mitochondrial oxidative enzymes of the TCA cycle and fatty acid oxidation, and did not affect mitochondrial function.

Increased mitochondrial substrate sensitivity in skeletal muscle of patients with type 2 diabetes

Mitochondrial respiration has been linked to insulin resistance. We studied mitochondrial respiratory capacity and substrate sensitivity in patients with type 2 diabetes (patients), and obese and lean control participants. Mitochondrial respiration was measured in permeabilised muscle fibres by respirometry. Protocols for respirometry included titration of substrates for complex I (glutamate), complex II (succinate) and both (octanoyl-carnitine). Myosin heavy chain (MHC) composition, antioxidant capacity (manganese superoxide dismutase [MnSOD]), citrate synthase activity and maximal oxygen uptake (VO2) were also determined. Insulin sensitivity was determined with the isoglycaemic-hyperinsulinaemic clamp technique. Insulin sensitivity was different (p < 0.05) between the groups (patients<obese controls<lean controls). MnSOD was lower in patients than in lean controls. MHC I content was lowest in patients (37 ± 11% [mean ± SE] vs 53 ± 6% and 56 ± 4%) vs obese controls and lean controls, respectively. VO2 was highest in lean controls (40 ± 3 ml min(-1) kg(-1) [mean ± SE]) compared with patients (25 ± 2) and obese controls (27 ± 2). Mitochondrial content (citrate synthase) was higher (p < 0.05) in lean controls than in patients and obese controls. When normalised for mitochondrial content by citrate synthase, mitochondrial respiratory capacity was similar in all groups. However, the half maximal substrate concentration (C(50)) for complex I was significantly lower (p = 0.03) in patients (1.1 ± 0.2 mmol/l [mean ± SE]) than in obese (2.0 ± 0.3) and lean (1.8 ± 0.3) controls. Likewise, C(50) for complex II was lower (p = 0.02) in patients (3.5 ± 0.2 mmol/l [mean ± SE]) than in obese controls (4.1 ± 0.2), but did not differ from that in lean controls (3.8 ± 0.4). Substrate sensitivity for octanoyl-carnitine did not differ between groups. Increased mitochondrial substrate sensitivity is seen in skeletal muscle from type 2 diabetic patients and is confined to non-lipid substrates. Respiratory capacity per mitochondrion is not decreased with insulin resistance.

Flipping and flopping-lipids on the move

The rapid movement of polar lipids from one membrane leaflet to the other is facilitated by lipid flippases or translocases. Although their activity was first observed over 30 years ago, the structures, physiological roles, and molecular mechanisms of this group of proteins remain enigmatic. Lipid flippases maintain membrane lipid asymmetry, and in eukaryotes they are also intimately involved in membrane budding and vesicle trafficking. The ATP-dependent flippases are members of well-characterized protein families, whose other members transport nonlipid substrates across cell membranes. The P(4)-type ATPases carry out the inward translocation of phospholipids, and various ABC transporters are involved in outward lipid movement. The ATP-independent flippases move lipid substrates in both directions between membrane leaflets. With only a few exceptions, the molecular identity of these proteins is still unknown, despite their involvement in key biosynthetic pathways in both bacteria and eukaryotes. This review provides an overview of the different classes of flippases, and summarizes recent progress in their identification and functional characterization. The possible mechanisms of action of lipid flippases are discussed, and future directions explored.

Inhibition of Autotaxin by Lysophosphatidic Acid and Sphingosine 1-Phosphate

Autotaxin (ATX) or nucleotide pyrophosphatase/phosphodiesterase 2 (NPP2) is an NPP family member that promotes tumor cell motility, experimental metastasis, and angiogenesis. ATX primarily functions as a lysophospholipase D, generating the lipid mediator lysophosphatidic acid (LPA) from lysophosphatidylcholine. ATX uses a single catalytic site for the hydrolysis of both lipid and non-lipid phosphodiesters, but its regulation is not well understood. Using a new fluorescence resonance energy transfer-based phosphodiesterase sensor that reports ATX activity with high sensitivity, we show here that ATX is potently and specifically inhibited by LPA and sphingosine 1-phosphate (S1P) in a mixed-type manner (Ki approximately 10(-7) M). The homologous ecto-phosphodiesterase NPP1, which lacks lysophospholipase D activity, is insensitive to LPA and S1P. Our results suggest that, by repressing ATX activity, LPA can regulate its own biosynthesis in the extracellular environment, and they reveal a novel role for S1P as an inhibitor of ATX, in addition to its well established role as a receptor ligand.

Bupivacaine inhibits acylcarnitine exchange in cardiac mitochondria.

The authors previously reported that secondary carnitine deficiency may sensitize the heart to bupivacaine-induced arrhythmias. In this study, the authors tested whether bupivacaine inhibits carnitine metabolism in cardiac mitochondria. Rat cardiac interfibrillar mitochondria were prepared using a differential centrifugation technique. Rates of adenosine diphosphate-stimulated (state III) and adenosine diphosphate-limited (state IV) oxygen consumption were measured using a Clark electrode, using lipid or nonlipid substrates with varying concentrations of a local anesthetic. State III respiration supported by the nonlipid substrate pyruvate (plus malate) is minimally affected by bupivacaine concentrations up to 2 mM. Lower concentrations of bupivacaine inhibited respiration when the available substrates were palmitoylcarnitine or acetylcarnitine; bupivacaine concentration causing 50% reduction in respiration (IC50 +/- SD) was 0.78+/-0.17 mM and 0.37+/-0.03 mM for palmitoylcarnitine and acetylcarnitine, respectively. Respiration was equally inhibited by bupivacaine when the substrates were palmitoylcarnitine alone, or palmitoyl-CoA plus carnitine. Bupivacaine (IC50 = 0.26+/-0.06 mM) and etidocaine (IC50 = 0.30+/-0.12 mM) inhibit carnitine-stimulated pyruvate oxidation similarly, whereas the lidocaine IC50 is greater by a factor of roughly 5, (IC50 = 1.4+/-0.26 mM), and ropivacaine is intermediate, IC50 = 0.5+/-0.28 mM. Bupivacaine inhibits mitochondrial state III respiration when acylcarnitines are the available substrate. The substrate specificity of this effect rules out bupivacaine inhibition of carnitine palmitoyl transferases I and II, carnitine acetyltransferase, and fatty acid beta-oxidation. The authors hypothesize that differential inhibition of carnitine-stimulated pyruvate oxidation by various local anesthetics supports the clinical relevance of inhibition of carnitine-acylcarnitine translocase by local anesthetics with a cardiotoxic profile.

Characterization of the antioxidant effects of melatonin and related indoleamines in vitro.

The antioxidant and possible pro-oxidant effects of melatonin and related indoleamines (tryptophan, serotonin, N-acetylserotonin, and 5-methoxytryptamine) were studied in vitro. In two model membrane systems, i.e., phospholipid liposomes and rat erythrocytes, lipid peroxidation induced by Fe2+ and H2O2, respectively, were reduced by the tested indoleamines except for tryptophan. The 5-hydroxy-indoleamines, serotonin, and N-acetylserotonin exhibited pro-oxidant actions in the bleomycin assay by reducing Fe3+ to Fe2+, which leads to DNA damage. The 5-methoxy-indoleamines, melatonin and 5-methoxytryptamine, were devoid of any pro-oxidant actions in this assay. Serotonin, but not N-acetylserotonin, scavenged the superoxide anion. None of the indoleamines tested had any reactivity towards H2O2. All the indoleamines, including tryptophan, were, however, shown to react with hypochlorous acid. These findings support an antiperoxidative and antioxidant action of melatonin which is devoid of pro-oxidant effect on non-lipid substrates.

Phospholipid metabolism in the cochlea: Differences between base and apex

Phospholipid-derived second messenger systems are one of the primary means for the transduction of extracellular signals to intracellular effector sites. We have investigated the longitudinal distribution of phospholipid metabolism in the guinea pig cochlea because of increasing evidence that the apex and base process auditory signals differently. Phospholipid metabolism was assayed by measuring the incorporation of radioactive phosphate ( 32P i) into lipids of the organ of Corti and the lateral wall tissues (stria vascularis and spiral ligament). 32P-labeling of total phospholipids was higher in the apex than the base, and individual phospholipids exhibited a tissue-specific base/apex distribution. Phosphatidylinositol was the most abundant of the labeled lipids in all tissues except the basal lateral wall, where phosphatidylinositol and phosphatidylcholine were labeled to a similar extent. Experiments on the availability of [ 32P]-ATP and other non-lipid substrates (inositol, choline, and cytidine) suggested that the base/apex distribution of phospholipid metabolism is based on differences in enzymatic activities. Additional evidence for this is an increased hydrolysis of phosphoinositides in the apex. The base/apex distribution of lipid metabolism suggests that physiological and pathological mechanisms involving phospholipids differ between the turns of the cochlea.

Elevated hepatic mitochondrial oxidative capacities in cold exposed rats

1. 1. Hepatic mitochondrial oxidative capacities were studied in rats exposed to cold for periods ranging from 5 to 15 days. The mitochondria obtained in this study were well coupled as shown by RCR and ADP/O ratios. 2. 2. The liver mitochondria of cold exposed rats showed significantly increased respiratory rates (ng atoms of oxygen consumed min −1 mg prot −1), starting from day 10 of cold exposure, using lipid and non-lipid substrates. 3. 3. For non-lipid substrates, the elevated respiratory rates found in the mitochondria could indicate an increased capacity to oxidize these substrates. For the lipid substrate, on the other hand, an enhanced oxidation through Krebs-cycle of a part of acetyl-CoA otherwise utilized to form ketone bodies, could also occur. 4. 4. Taken together the results suggest that, during cold exposure, liver mitochondria could participate in cold adaptation mechanisms, by improving ATP production.

Importance of endogenous substrates for cultured adult rat cardiac myocytes

In Ca-tolerant adult cardiomyocytes the contribution of endogenous substrates (glycogen, tri- and diacylglycerol) to oxidative substrate metabolism was investigated. After 4 h in culture medium (M 199 plus 4% fetal calf serum) the cellular triacylglycerol content is 3.6-fold higher than in fresh myocardium and reflects the free fatty acid composition of the medium. When triacylglycerol is degraded, all long-chain fatty acids are hydrolysed at equal rates. In these quiescent cells, the activity of pyruvate dehydrogenase is low (10% of full activity, in Tyrode solution with 5 mM glucose). Up to 30% of full pyruvate dehydrogenase activity, the contribution of non-lipid substrates (glycogen, glucose, lactate and pyruvate) to oxidative energy production is correlated to pyruvate dehydrogenase activity. At 5 mM mediumconcentration, glucose, lactate and pyruvate share in energy production the proportions of 15, 36 and 50%, whereas endogenous lipolysis accounts for 78, 61 and 46%. It is concluded that these quiescent cardiomyocytes represent cardiac metabolism in a basal state in which the preference for fatty acids, especially from endogenous lipids, is very pronounced. The utilization of endogenous substrates therefore has to be considered in all studies investigating the oxidative metabolism of these isolated cells.

Elevated hepatic mitochondrial and peroxisomal oxidative capacities in fed and starved adult obese (ob/ob) mice.

Hepatic mitochondrial and peroxisomal oxidative capacities were studied in young (4-5 weeks old) and adult (6-9 months old) lean and obese ob/ob mice that were fed or starved for 24 or 48 h. The adult obese mice showed elevated capacity for mitochondrial oxidation (ng-atoms of O consumed/min per mg of protein) of lipid and non-lipid substrates, with the exception of pyruvate + malate, and elevated activities of citrate synthase and total carnitine palmitoyltransferase. Oxidative rates and enzyme activities were not affected by starvation of lean or obese mice, and both males and females responded similarly. Peroxisomal palmitoyl-CoA oxidation (nmol/min per mg of peroxisomal protein) was also increased in livers of adult obese mice and did not change with starvation. In young mice, hepatic mitochondrial and peroxisomal oxidative capacities in lean and obese mice were comparable. The increased mitochondrial and peroxisomal oxidative capacities appear to develop with maturation in obese ob/ob mice.

Effect of Diet and Starvation on Hepatic Mitochondrial Function in the Rat

The purpose of the present study was to assess the effect of diet and starvation on mitochondrial function. Diet did not affect hepatic mitochondrial oxidation of lipid or nonlipid substrates when expressed as nanogram atoms of oxygen consumed per minute per milligram mitochondrial protein. Furthermore, diet did not affect mitochondrial palmitoylcarnitine utilization rate, mitochondrial ketoacid production, or mitochondrial carnitine palmitoyltransferase specific activity. When the data for palmitoylcarnitine utilization were expressed per gram liver, the rates were significantly lower in rats fed the high fat diet of Schemmel et al. (J. Nutr. 110: 1041–1048, 1970). DNA content (milligrams/gram) indicated that cell size rather than cell number contributed to greater total liver weight in rats fed the Schemmel high fat diet. In both stock diet-fed controls and rats fed the Schemmel high fat diet, mitochondrial protein per gram liver decreased with duration of starvation. Mitochondrial carnitine palmitoyltransferase was unchanged by diet or starvation. Thus, neither diet nor diet followed by starvation altered hepatic mitochondrial capacity to oxidize lipid substrates.

Free fatty acid metabolism of the human heart at rest.

Myocardial substrate metabolism was studied in 13 subjects at the time of diagnostic cardiac catheterization by means of palmitic acid-(14)C infusion with arterial and coronary sinus sampling. Two subjects were considered free of cardiac pathology and all, with one exception, demonstrated lactate extraction across the portion of heart under study. Data for this single lactate-producing subject were treated separately.The fractional extraction of (14)C-labeled free fatty acids (FFA) (44.4+/-9.5%) was nearly twice that of unlabeled FFA (23.2+/-7.8%) and raised the possibility of release of FFA into the coronary sinus. FFA uptake, based on either the arterial minus coronary sinus concentration difference or the FFA-(14)C fractional extraction, was directly proportional to the arterial FFA concentration. Gas-liquid chromatography failed to demonstrate selective handling of any individual FFA by the heart. Fractional oxidation of FFA was 53.5+/-12.7%, accounting for 53.2+/-14.4% of the heart's oxygen consumption while nonlipid substrates accounted for an additional 30.0+/-17.3%. Determinations of both labeled and unlabeled triglycerides suggested utilization of this substrate by the fasting human heart. Direct measurement of FFA fractional oxidation as well as FFA uptake, exclusive of possible simultaneous FFA release, would appear necessary in studies concerned with human myocardial FFA metabolism.