Bezafibrate activation of PPAR drives disturbances in mitochondrial redox bioenergetics and decreases the viability of cells from patients with VLCAD deficiency

Very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency is the most common inborn long-chain fatty acid oxidation (FAO) disorder. VLCAD deficiency is characterized by distinct phenotypes. The severe phenotypes are potentially life-threatening and affect the heart or liver, with a comparatively milder phenotype characterized by myopathic symptoms. There is an unmet clinical need for effective treatment options for the myopathic phenotype. The molecular mechanisms driving the gradual decrease in mitochondrial function and associated alterations of muscle fibers are unclear.The peroxisome proliferator-activated receptor (PPAR) pan-agonist bezafibrate is a potent modulator of FAO and multiple other mitochondrial functions and has been proposed as a potential medication for myopathic cases of long-chain FAO disorders. In vitro experiments have demonstrated the ability of bezafibrate to increase VLCAD expression and activity. However, the outcome of small-scale clinical trials has been controversial.We found VLCAD deficient patient fibroblasts to have an increased oxidative stress burden and deranged mitochondrial bioenergetic capacity, compared to controls. Applying heat stress under fasting conditions to bezafibrate pretreated patient cells, caused a marked further increase of mitochondrial superoxide levels. Patient cells failed to maintain levels of the essential thiol peptide antioxidant glutathione and experienced a decrease in cellular viability. Our findings indicate that chronic PPAR activation is a plausible initiator of long-term pathogenesis in VLCAD deficiency. Our findings further implicate disruption of redox homeostasis as a key pathogenic mechanism in VLCAD deficiency and support the notion that a deranged thiol metabolism might be an important pathogenic factor in VLCAD deficiency.     

Abnormal mitochondrial bioenergetics and heart rate dysfunction in mice lacking very-long-chain acyl-CoA dehydrogenase

Mitochondrial very-long-chain acyl-CoA dehydrogenase (VLCAD) deficiency is associated with severe hypoglycemia, cardiac dysfunction, and sudden death in neonates and children. Sudden death is common, but the underlying mechanisms are not fully understood. We report on a mouse model of VLCAD deficiency with a phenotype induced by the stresses of fasting and cold, which includes hypoglycemia, hypothermia, and severe bradycardia. The administration of glucose did not rescue the mice under stress conditions, but rewarming alone consistently led to heart rate recovery. Brown adipose tissue (BAT) from the VLCAD-/- mice showed elevated levels of the uncoupling protein isoforms and peroxisome proliferator-activated receptor-alpha. Biochemical assessment of the VLCAD(/- mice BAT showed increased oxygen consumption, attributed to uncoupled respiration in the absence of stress. ADP-stimulated respiration was 23.05 (SD 4.17) and 68.24 (SD 6.3) nmol O2.min(-1).mg mitochondrial protein(-1) for VLCAD+/+ and VLCAD-/- mice, respectively (P < 0.001), and carbonyl cyanide p-trifluoromethoxyphenylhydrazone-stimulated respiration was 35.9 (SD 3.6) and 49.3 (SD 9) nmol O2.min(-1).mg mitochondrial protein(-1) for VLCAD+/+ and VLCAD-/- mice, respectively (P < 0.20), but these rates were insufficient to protect them in the cold. We conclude that disturbed mitochondrial bioenergetics in BAT is a critical contributing factor for the cold sensitivity in VLCAD deficiency. Our observations provide insights into the possible mechanisms of stress-induced death in human newborns with abnormal fat metabolism and elucidate targeting of specific substrates for particular metabolic needs.     

Fasting-induced oxidative stress in very long chain acyl-CoA dehydrogenase-deficient mice

Hepatopathy and hepatomegaly as consequences of prolonged fasting or illnesses are typical clinical features of very long chain acyl-CoA dehydrogenase (VLCACD) deficiency, the most common long-chain fatty acid β-oxidation defect. Supplementation with medium-chain triglycerides (MCTs) is an important treatment measure in these defects, in order to supply sufficient energy. Little is known about the pathogenetic mechanisms leading to hepatopathy. Here, we investigated the effects of prolonged fasting and an MCT diet on liver function. Wild-type (WT) and VLCAD knockout mice were fed with either a regular long-chain triglyceride diet or an MCT diet for 5 weeks. In both groups, we determined liver and blood lipid contents under nonfasting conditions and after 24 h of fasting. Expression of genes regulating peroxisomal and microsomal oxidation pathways was analyzed by RT-PCR. In addition, glutathione peroxidase and catalase activities, as well as thiobarbituric acid reactive substances, were examined. In VLCAD knockout mice fed with a long-chain triglyceride diet, fasting is associated with excessive accumulation of liver lipids, resulting in hepatopathy and strong upregulation of peroxisomal and microsomal oxidation pathways as well as antioxidant enzyme activities and thiobarbituric acid reactive substances. These effects were even evident in nonfasted mice fed with an MCT diet, and were particularly pronounced in fasted mice fed with an MCT diet. This study strongly suggests that liver damage in fatty acid oxidation defects is attributable to oxidative stress and generation of reactive oxygen species as a result of significant fat accumulation. An MCT diet does not prevent hepatic damage during catabolism and metabolic derangement.     

Fasting promotes functional changes in liver mitochondria

Overnight fasting of rodents is commonly adopted in protocols to obtain isolated liver mitochondria, but the effects of fasting itself on mitochondrial function are poorly characterized. In this study we show that overnight fasting (15?h) promotes a shift in the liver mitochondrial bioenergetic profile, with a reduction in ADP-stimulated and maximal respiration, lower membrane potentials and lower resistance to Ca²⁺-induced mitochondrial permeability transition. Short term fasting (4?h) promoted similar changes, suggesting that this is a physiological shift in mitochondrial function associated with fasting, but not torpor. Our results suggest that the widely adopted liver mitochondrial isolation technique using fasted animals should be reconsidered, and also uncover physiological changes in bioenergetic function associated to nutritional status.     

Proteomic profiling of cardiomyopathic tissue from the aged mdx model of Duchenne muscular dystrophy reveals a drastic decrease in laminin,nidogen and annexin

The majority of patients afflicted with Duchenne muscular dystrophy develop cardiomyopathic complications, warranting large‐scale proteomic studies of global cardiac changes for the identification of new protein markers of dystrophinopathy. The aged heart from the X‐linked dystrophic mdx mouse has been shown to exhibit distinct pathological aspects of cardiomyopathy. In order to establish age‐related alterations in the proteome of dystrophin‐deficient hearts, cardiomyopathic tissue from young versus aged mdx mice was examined by label‐free LC‐MS/MS. Significant age‐dependent alterations were established for 67 proteins, of which 28 proteins were shown to exhibit a lower abundance and 39 proteins were found to be increased in their expression levels. Drastic changes were demonstrated for 17 proteins, including increases in Ig chains and transferrin, and drastic decreases in laminin, nidogen and annexin. An immunblotting survey of young and old wild‐type versus mdx hearts confirmed these proteomic findings and illustrated the effects of natural aging versus dystrophin deficiency. These proteome‐wide alterations suggest a disintegration of the basal lamina structure and cytoskeletal network in dystrophin‐deficient cardiac fibres, increased levels of antibodies in a potential autoimmune reaction of the degenerating heart, compensatory binding of excess iron and a general perturbation of metabolic pathways in dystrophy‐associated cardiomyopathy.     

Requirement of PPARalpha in maintaining phospholipid and triacylglycerol homeostasis during energy deprivation

The peroxisome proliferator-activated receptor alpha (PPARalpha) has been implicated as a key control of fatty acid catabolism during the cellular fasting. However, little is known regarding changes of individual fatty acids in hepatic triacylglycerol (TG) and phospholipid (PL) as a result of starvation. In the present work, the effects of 72 h fasting on hepatic TG and PL fatty acid profiles in PPARalpha-null (KO) mice and their wild-type (WT) counterparts were investigated. Our results indicated that mice deficient in PPARalpha displayed hepatomegaly and hypoketonemia following 72 h starvation. Histochemical analyses revealed that severe fatty infiltration was observed in the livers of KO mice under fasted conditions. Furthermore, 72 h fasting resulted in a 2.8-fold higher accumulation of hepatic TG in KO mice than in WT mice fasted for the same length of time. Surprisingly, the total hepatic PL contents in fasted KO mice decreased by 45%, but no significant change in hepatic PL content was observed in WT mice following starvation. Gas chromatographic analysis indicated that KO mice were deprived of arachidonic (20:4n-6) and docosahexaenoic (22:6n-3) acids during fasting. Taken together, these results show that PPARalpha plays an important role in regulation of fatty acid metabolism as well as phospholipid homeostasis during energy deprivation.     

Clear correlation of genotype with disease phenotype in very-long-chain acyl-CoA dehydrogenase deficiency.

Very-long-chain acyl-CoA dehydrogenase (VLCAD) catalyzes the initial rate-limiting step in mitochondrial fatty acid beta-oxidation. VLCAD deficiency is clinically heterogenous, with three major phenotypes: a severe childhood form, with early onset, high mortality, and high incidence of cardiomyopathy; a milder childhood form, with later onset, usually with hypoketotic hypoglycemia as the main presenting feature, low mortality, and rare cardiomyopathy; and an adult form, with isolated skeletal muscle involvement, rhabdomyolysis, and myoglobinuria, usually triggered by exercise or fasting. To examine whether these different phenotypes are due to differences in the VLCAD genotype, we investigated 58 different mutations in 55 unrelated patients representing all known clinical phenotypes and correlated the mutation type with the clinical phenotype. Our results show a clear relationship between the nature of the mutation and the severity of disease. Patients with the severe childhood phenotype have mutations that result in no residual enzyme activity, whereas patients with the milder childhood and adult phenotypes have mutations that may result in residual enzyme activity. This clear genotype-phenotype relationship is in sharp contrast to what has been observed in medium-chain acyl-CoA dehydrogenase deficiency, in which no correlation between genotype and phenotype can be established.     

Mitochondrial long chain fatty acid β-oxidation in man and mouse

Several mouse models for mitochondrial fatty acid β-oxidation (FAO) defects have been developed. So far, these models have contributed little to our current understanding of the pathophysiology. The objective of this study was to explore differences between murine and human FAO. Using a combination of analytical, biochemical and molecular methods, we compared fibroblasts of long chain acyl-CoA dehydrogenase knockout (LCAD^−/−), very long chain acyl-CoA dehydrogenase knockout (VLCAD^−/−) and wild type mice with fibroblasts of VLCAD-deficient patients and human controls. We show that in mice, LCAD and VLCAD have overlapping and distinct roles in FAO. The absence of VLCAD is apparently fully compensated, whereas LCAD deficiency is not. LCAD plays an essential role in the oxidation of unsaturated fatty acids such as oleic acid, but seems redundant in the oxidation of saturated fatty acids. In strong contrast, LCAD is neither detectable at the mRNA level nor at the protein level in men, making VLCAD indispensable in FAO. Our findings open new avenues to employ the existing mouse models to study the pathophysiology of human FAO defects.     

Native Size-Exclusion Chromatography–Based Mass Spectrometry Reveals New Components of the Early Heat Shock Protein 90 Inhibition Response Among Limited Global Changes

The molecular chaperone heat shock protein 90 (HSP90) works in concert with co-chaperones to stabilize its client proteins, which include multiple drivers of oncogenesis and malignant progression. Pharmacologic inhibitors of HSP90 have been observed to exert a wide range of effects on the proteome, including depletion of client proteins, induction of heat shock proteins, dissociation of co-chaperones from HSP90, disruption of client protein signaling networks, and recruitment of the protein ubiquitylation and degradation machinery—suggesting widespread remodeling of cellular protein complexes. However, proteomics studies to date have focused on inhibitor-induced changes in total protein levels, often overlooking protein complex alterations. Here, we use size-exclusion chromatography in combination with mass spectrometry (SEC-MS) to characterize the early changes in native protein complexes following treatment with the HSP90 inhibitor tanespimycin (17-AAG) for 8 h in the HT29 colon adenocarcinoma cell line. After confirming the signature cellular response to HSP90 inhibition (e.g., induction of heat shock proteins, decreased total levels of client proteins), we were surprised to find only modest perturbations to the global distribution of protein elution profiles in inhibitor-treated HT29 cells at this relatively early time-point. Similarly, co-chaperones that co-eluted with HSP90 displayed no clear difference between control and treated conditions. However, two distinct analysis strategies identified multiple inhibitor-induced changes, including known and unknown components of the HSP90-dependent proteome. We validate two of these—the actin-binding protein Anillin and the mitochondrial isocitrate dehydrogenase 3 complex—as novel HSP90 inhibitor-modulated proteins. We present this dataset as a resource for the HSP90, proteostasis, and cancer communities (https://www.bioinformatics.babraham.ac.uk/shiny/HSP90/SEC-MS/), laying the groundwork for future mechanistic and therapeutic studies related to HSP90 pharmacology. Data are available via ProteomeXchange with identifier PXD033459.     

Impaired mitochondrial fatty acid oxidation and insulin resistance in aging: novel protective role of glutathione

Aging is associated with impaired fasted oxidation of nonesterified fatty acids (NEFA) suggesting a mitochondrial defect. Aging is also associated with deficiency of glutathione (GSH), an important mitochondrial antioxidant, and with insulin resistance. This study tested whether GSH deficiency in aging contributes to impaired mitochondrial NEFA oxidation and insulin resistance, and whether GSH restoration reverses these defects. Three studies were conducted: (i) in 82‐week‐old C57BL/6 mice, the effect of naturally occurring GSH deficiency and its restoration on mitochondrial ¹³C₁‐palmitate oxidation and glucose metabolism was compared with 22‐week‐old C57BL/6 mice; (ii) in 20‐week C57BL/6 mice, the effect of GSH depletion on mitochondrial oxidation of ¹³C₁‐palmitate and glucose metabolism was studied; (iii) the effect of GSH deficiency and its restoration on fasted NEFA oxidation and insulin resistance was studied in GSH‐deficient elderly humans, and compared with GSH‐replete young humans. Chronic GSH deficiency in old mice and elderly humans was associated with decreased fasted mitochondrial NEFA oxidation and insulin resistance, and these defects were reversed with GSH restoration. Acute depletion of GSH in young mice resulted in lower mitochondrial NEFA oxidation, but did not alter glucose metabolism. These data suggest that GSH is a novel regulator of mitochondrial NEFA oxidation and insulin resistance in aging. Chronic GSH deficiency promotes impaired NEFA oxidation and insulin resistance, and GSH restoration reverses these defects. Supplementing diets of elderly humans with cysteine and glycine to correct GSH deficiency could provide significant metabolic benefits.     

Fasting induces ketoacidosis and hypothermia in PDHK2/PDHK4-double-knockout mice

The importance of PDHK (pyruvate dehydrogenase kinase) 2 and 4 in regulation of the PDH complex (pyruvate dehydrogenase complex) was assessed in single- and double-knockout mice. PDHK2 deficiency caused higher PDH complex activity and lower blood glucose levels in the fed, but not the fasted, state. PDHK4 deficiency caused similar effects, but only after fasting. Double deficiency intensified these effects in both the fed and fasted states. PDHK2 deficiency had no effect on glucose tolerance, PDHK4 deficiency produced only a modest effect, but double deficiency caused a marked improvement and also induced lower insulin levels and increased insulin sensitivity. In spite of these beneficial effects, the double-knockout mice were more sensitive than wild-type and single-knockout mice to long-term fasting, succumbing to hypoglycaemia, ketoacidosis and hypothermia. Stable isotope flux analysis indicated that hypoglycaemia was due to a reduced rate of gluconeogenesis and that slightly more glucose was converted into ketone bodies in the double-knockout mice. The findings establish that PDHK2 is more important in the fed state, PDHK4 is more important in the fasted state, and survival during long-term fasting depends upon regulation of the PDH complex by both PDHK2 and PDHK4.     

Prolonged QT interval and lipid alterations beyond β-oxidation in very long-chain acyl-CoA dehydrogenase null mouse hearts

Patients with very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency frequently present cardiomyopathy and heartbeat disorders. However, the underlying factors, which may be of cardiac or extra cardiac origins, remain to be elucidated. In this study, we tested for metabolic and functional alterations in the heart from 3- and 7-mo-old VLCAD null mice and their littermate counterparts, using validated experimental paradigms, namely, 1) ex vivo perfusion in working mode, with concomitant evaluation of myocardial contractility and metabolic fluxes using (13)C-labeled substrates under various conditions; as well as 2) in vivo targeted lipidomics, gene expression analysis as well as electrocardiogram monitoring by telemetry in mice fed various diets. Unexpectedly, when perfused ex vivo, working VLCAD null mouse hearts maintained values similar to those of the controls for functional parameters and for the contribution of exogenous palmitate to β-oxidation (energy production), even at high palmitate concentration (1 mM) and increased energy demand (with 1 μM epinephrine) or after fasting. However, in vivo, these hearts displayed a prolonged rate-corrected QT (QTc) interval under all conditions examined, as well as the following lipid alterations: 1) age- and condition-dependent accumulation of triglycerides, and 2) 20% lower docosahexaenoic acid (an omega-3 polyunsaturated fatty acid) in membrane phospholipids. The latter was independent of liver but affected by feeding a diet enriched in saturated fat (exacerbated) or fish oil (attenuated). Our finding of a longer QTc interval in VLCAD null mice appears to be most relevant given that such condition increases the risk of sudden cardiac death.     

Proteomic interrogation of HSP90 and insights for medical research

Introduction: Heat shock protein 90 (HSP90) regulates protein homeostasis in eukaryotes. As a ‘professional interactor’, HSP90 binds to and chaperones many proteins and has both housekeeping and disease-related functions but its regulation remains in part elusive. HSP90 complexes are a target for therapy, notably against cancer, and several inhibitors are currently in clinical trials. Proteomic studies have revealed the vast interaction network of HSP90 and, in doing so, the extent of cellular processes the chaperone takes part in, especially in yeast and human cells. Furthermore, small-molecule inhibitors were used to probe the global impact of its inhibition on the proteome.

Areas covered: We review here recent HSP90-related interactomics and total proteome studies and their relevance for research on cancer, neurodegenerative and pathogen diseases.

Expert commentary: Proteomics experiments are our best chance to identify the context-dependent global proteome of HSP90 and thus uncover and understand its disease-specific biology. However, understanding the complexity of HSP90 will require multiple complementary, quantitative approaches and novel bioinformatics to translate interactions into ordered functional networks and pathways. Developing therapies will necessitate more knowledge on HSP90 complexes and networks with disease relevance and on total proteome changes induced by their perturbation. Most work has been done in cancer, thus a lot remains to be done in the context of other diseases.     

Effects of fasting on muscle mitochondrial energetics and fatty acid metabolism in Ucp3(-/-) and wild-type mice

Uncoupling protein-3 (UCP3) is a mitochondrial carrier protein of as yet undefined physiological function. To elucidate characteristics of its function, we studied the effects of fasting on resting metabolic rate, respiratory quotient, muscle Ucp3 expression, and mitochondrial proton leak in wild-type and Ucp3(-/-) mice. Also analyzed were the fatty acid compositions of skeletal muscle mitochondria in fed and fasted Ucp3(-/-) and wild-type mice. In wild-type mice, fasting caused significant increases in Ucp3 (4-fold) and Ucp2 (2-fold) mRNA but did not significantly affect mitochondrial proton leak. State 4 oxygen consumption was not affected by fasting in either of the two groups. However, protonmotive force was consistently higher in mitochondria of Ucp3(-/-) animals (P = 0.03), and fasting further augmented protonmotive force in Ucp3(-/-) mice; there was no effect in wild-type mitochondria. Resting metabolic rates decreased with fasting in both groups. Ucp3(-/-) mice had higher respiratory quotients than wild-type mice in fed resting states, indicating impaired fatty acid oxidation. Altogether, results show that the fasting-induced increases in Ucp2 and Ucp3 do not correlate with increased mitochondrial proton leak but support a role for UCP3 in fatty acid metabolism.     

AMP‐activated protein kinase deficiency exacerbates aging‐induced myocardial contractile dysfunction

Aging is associated with myocardial dysfunction although the underlying mechanism is unclear. AMPK, a key cellular fuel sensor for energy metabolism, is compromised with aging. This study examined the role of AMPK deficiency in aging‐associated myocardial dysfunction. Young or old wild‐type (WT) and transgenic mice with overexpression of a mutant AMPK α₂ subunit (kinase dead, KD) were used. AMPK α isoform activity, myocardial function and morphology were examined. DCF and JC‐1 fluorescence probes were employed to quantify reactive oxygen species (ROS) and mitochondrial membrane potential (ΔΨm), respectively. KD mice displayed significantly reduced α₂ but not α₁ AMPK isoform activity at both ages with a greater effect at old age. Aging itself decreased α₁ isoform activity. Cardiomyocyte contractile function, intracellular Ca²⁺ handling, and SERCA2a levels were compromised with aging, the effects of which were exacerbated by AMPK deficiency. H&E staining revealed cardiomyocyte hypertrophy with aging, which was more pronounced in KD mice. TEM micrographs displayed severe disruption of mitochondrial ultrastructure characterized by swollen, irregular shape and disrupted cristae in aged KD compared with WT mice. Aging enhanced ROS production and reduced ΔΨm, the effects of which were accentuated by AMPK deficiency. Immunoblotting data depicted unchanged Akt phosphorylation and a significant decrease in mitochondrial biogenesis cofactor PGC‐1α in aged groups. AMPK deficiency but not aging decreased the phosphorylation of ACC and eNOS. Expression of membrane Glut4 and HSP90 was decreased in aged KD mice. Moreover, treatment of the AMPK activator metformin attenuated aging‐induced cardiomyocyte contractile defects. Collectively, our data suggest a role for AMPK deficiency in aging‐induced cardiac dysfunction possibly through disrupted mitochondrial function and ROS production.     

Targeted deletion of both thymidine phosphorylase and uridine phosphorylase and consequent disorders in mice

Thymidine phosphorylase (TP) regulates intracellular and plasma thymidine levels. TP deficiency is hypothesized to (i) increase levels of thymidine in plasma, (ii) lead to mitochondrial DNA alterations, and (iii) cause mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). In order to elucidate the physiological roles of TP, we generated mice deficient in the TP gene. Although TP activity in the liver was inhibited in these mice, it was fully maintained in the small intestine. Murine uridine phosphorylase (UP), unlike human UP, cleaves thymidine, as well as uridine. We therefore generated TP-UP double-knockout (TP(-/-) UP(-/-)) mice. TP activities were inhibited in TP(-/-) UP(-/-) mice, and the level of thymidine in the plasma of TP(-/-) UP(-/-) mice was higher than for TP(-/-) mice. Unexpectedly, we could not observe alterations of mitochondrial DNA or pathological changes in the muscles of the TP(-/-) UP(-/-) mice, even when these mice were fed thymidine for 7 months. However, we did find hyperintense lesions on magnetic resonance T(2) maps in the brain and axonal edema by electron microscopic study of the brain in TP(-/-) UP(-/-) mice. These findings suggested that the inhibition of TP activity caused the elevation of pyrimidine levels in plasma and consequent axonal swelling in the brains of mice. Since lesions in the brain do not appear to be due to mitochondrial alterations and pathological changes in the muscle were not found, this model will provide further insights into the causes of MNGIE.