Peroxisomal Pathway Research Articles

Concerted Action of Leptin in Regulation of Fatty Acid Oxidation in Skeletal Muscle and Liver

Central action of leptin on food intake and energy expenditure is integrated with leptin's peripheral action modulating the fatty acid and glucose metabolism and preventing the accumulation of lipids in nonadipose tissues. However, exact mechanism(s) of the leptin's action in the peripheral tissues has not yet been fully elucidated. Therefore, we investigated the effect of a single intravenous injection of leptin on palmitoyl-CoA and palmitoyl-carnitine oxidation rate in liver and skeletal muscle followed by measurements of the carnitine-palmitoyl transferase 1 (CPT1) activity and activities of ss-oxidation enzymes in mitochondria (acyl-CoA dehydrogenase) and in peroxisomes (acyl-CoA oxidase) of rats. Animals were euthanized and tissues and serum harvested 15 min, 1 hour, 3 hours and 6 hours after leptin administration. Intravenous leptin injection increased mitochondrial palmitoyl-CoA oxidation rate in both liver (95%; P<0.025) and skeletal muscle (2.7-fold; P<0.05). This was paralleled by lowering hepatic (-156%; P<0.001) and skeletal muscle (-191%; P<0.001) triglyceride content. Leptin-induced elevation of palmitoyl-CoA oxidation rate in liver was paralleled by increased CPT1 activity (52%; P<0.05) and ss-oxidation capacity (52%; P<0.05). Lack of the leptin's effect on the CPT1-activity in muscle (20%; p=0.09) suggests the existence of an alternative pathway for increasing the palmitoyl-CoA-oxidation rate bypassing the CPT1 regulatory step. Interestingly, leptin stimulated the overall ss-oxidation capacity in muscle by 69% (P=0.027). This may indicate to an involvement of mitochondrial acyl-CoA dehydrogenases as well as of peroxisomal fat catabolism. Taken together, we showed that leptin acutely increases palmitoyl-CoA oxidation rate in liver and in skeletal muscle, which was associated with tissue specific effect on the CPT1 activity as well as on the downstream enzymes of fatty acid oxidation pathways in rat mitochondria and peroxisomes. Tangible evidence for the leptin-induced increase of fatty acid catabolism was provided by a lowered skeletal muscle and hepatic lipid deposition.

Ubiquitination of the peroxisomal import receptor Pex5p is required for its recycling

Pex5p, which is the import receptor for peroxisomal matrix proteins harboring a type I signal sequence (PTS1), is mono- and polyubiquitinated in Saccharomyces cerevisiae. We identified Pex5p as a molecular target for Pex4p-dependent monoubiquitination and demonstrated that either poly- or monoubiquitination of the receptor is required for the ATP-dependent release of the protein from the peroxisomal membrane to the cytosol as part of the receptor cycle. Therefore, the energy requirement of the peroxisomal import pathway has to be extended by a second ATP-dependent step, namely receptor monoubiquitination.

PPARα mediates the effects of the pesticide methyl thiophanate on liver of the lizard Podarcis sicula

The majority of environmental pollutants are potential peroxisomal proliferators which include a heterogeneous group of compounds known to determine massive peroxisomal proliferation and hepatocarcinogenesis in rodents. Peroxisomal proliferation is accompanied by the induction of the peroxisomal fatty acid β-oxidation pathway mediated by a class of transcription factors named peroxisome proliferators activated receptors (PPARs). This phenomenon demonstrated also in ectotherm animals after exposition to environmental pollutants may be utilized as biomarker in environmental impact studies. In the present work we have tested the sensitivity to methyl thiophanate (TM) of the lizard Podarcis sicula in order to propose a biological model for monitoring the ecotoxicological effects of this pesticide on terrestrial sentinel species. The data obtained demonstrate that exposition to sub-lethal concentrations of TM leads to hepatocellular morphological changes and glycogen depletion, apoptosis, as well as probable peroxisomal proliferation attested by the increase of acyl-CoA oxidase (AOX). This effect seems to be mediated by the concomitant increase of PPARα. On the basis of these results we propose that also in Podarcis sicula, as just proposed for aquatic organisms, peroxisomal proliferation and AOX increase may be considered new biomarkers to evaluate pollution by organic compound in terrestrial environments.

Pyrroloquinoline quinone nutritional status and parameters important to mitochondrial function

Pyrroloquinoline quinone (PQQ) deficiency influences the mitochondrial content of heart and liver (Stites T et al., J Nutr., 2006) and impacts the metabolism of lysine and other amino acids oxidized via mitochondrial and peroxisomal pathways (Bauerly K et al., Biochim Biophys Acta, 2006). Because of mitochondria’s central role in metabolism, we investigated the effect of PQQ deficiency on mitochondriogenesis. Herein are data for serum glucose and fatty acids (FA) in rats fed amino-acid based diets with (2 mg PQQ/Kg diet) or devoid of PQQ for 3–5 weeks in post-weanling rats (fed and/or fasted states). Serum glucose, free fatty acids (FFA), RQ, triglycerides (TG), sphingomyelin (SM), total energy expenditure (EE) per metabolic body size (Wt3/4), relative mitochondrial amount, and expression of serine palmitoyl transferase (SPT) were estimated. Mitochondria content decreased 20% in PQQ deficient rats. Although no differences in serum glucose and FFA levels were observed (PQQ+ vs. PQQ−, fed or fasted), EE was significantly lower in PQQ deficiency. In the fed state, EE was positively correlated with mitochondrial amount and less in PQQ deficiency. Composition of FFA, TG, and SM were altered with PQQ deficiency, suggesting disturbances in long-chain FA metabolic pathways; serum TG were elevated while serum SM tended to be lower with corresponding decreases in liver SPT activity and mRNA levels in deficient rats. These data are consistent with our previous observations regarding PQQ status, mitochondrial function and associated activities. Funded in part by Mitsubishi, Inc., CA Vitamin Settlement Fund, and NDC.

Paradoxical HDL‐C reduction during rosiglitazone and fibrate treatment

Dyslipidaemia in Type 2 diabetes mellitus (T2DM) is one of the major contributors in the pathogenesis of atherosclerosis. Thiazolidinediones (TZD), a class of drugs used in the treatment of T2DM, also modify lipids, especially lowering serum triglycerides and raising high-density lipoprotein cholesterol (HDL-C). We describe five patients taking rosiglitazone and a fibrate who showed a paradoxical fall in HDL-C, which would have been missed if HDL-C had not been routinely monitored. This could have had a major impact in increasing the cardiovascular risk in these patients. Our five patients showed marked variation in both the decrease in serum HDL-C (50-89%) and also in the time taken for recovery of HDL-C after withdrawal of rosiglitazone (between 5 and 20 weeks). Apolipoprotein A1 mirrored the drop in HDL-C in four of the five patients but in one subject this was not seen, suggesting the possibility of multiple mechanisms leading to the phenomenon described, perhaps involving HDL metabolism. Improvements in glycaemic control with rosiglitazone (absolute HbA(1c) reduction between 0.6 and 3.0%) were seen in four of our patients. This suggests that the peroxisomal proliferator-activated receptor gamma signalling pathways relevant to glucose homeostasis were intact. As atherosclerosis is associated with a decrease in the HDL-C level, our observations reinforce the message that HDL-C should be measured before and after the commencement of rosiglitazone and also on increasing the dosage

Localization of the pre-squalene segment of the isoprenoid biosynthetic pathway in mammalian peroxisomes

Previous studies have indicated that the early steps in the isoprenoid/cholesterol biosynthetic pathway occur in peroxisomes. However, the role of peroxisomes in cholesterol biosynthesis has recently been questioned in several reports concluding that three of the peroxisomal cholesterol biosynthetic enzymes, namely mevalonate kinase, phosphomevalonate kinase, and mevalonate diphosphate decarboxylase, do not localize to peroxisomes in human cells even though they contain consensus peroxisomal targeting signals. We re-investigated the subcellular localization of the cholesterol biosynthetic enzymes of the pre-squalene segment in human cells by using new stable isotopic techniques and data computations with isotopomer spectral analysis, in combination with immunofluorescence and cell permeabilization techniques. Our present findings clearly show and confirm previous studies that the pre-squalene segment of the cholesterol biosynthetic pathway is localized to peroxisomes. In addition, our data are consistent with the hypothesis that acetyl-CoA derived from peroxisomal beta-oxidation of very long-chain fatty acids and medium-chain dicarboxylic acids is preferentially channeled to cholesterol synthesis inside the peroxisomes without mixing with the cytosolic acetyl-CoA pool.

Relationships between the functional PPARα Leu162Val polymorphism and obesity, type 2 diabetes, dyslipidaemia, and related quantitative traits in studies of 5799 middle-aged white people

Peroxisome proliferator-activated receptor-α (PPARα) is a nuclear receptor capable of regulating the expression of genes involved in peroxisomal and mitochondrial β-oxidation pathways. The common Leu162Val polymorphism in the gene encoding PPARα has inconsistently shown association with quantitative traits related to obesity, type 2 diabetes, and dyslipidaemia. We genotyped the Leu162Val polymorphism in 1383 patients with type 2 diabetes and 4401 control subjects with normal glucose tolerance (NGT) without showing any association between diabetes and genotype. In addition, the Leu162Val polymorphism was not associated with WHO-defined obesity or dyslipidaemia in case-control settings involving 961 obese and 2563 lean subjects and 1399 dyslipidaemic and 4399 normolipidaemic subjects, respectively. Quantitative trait studies of metabolic variables were carried out in 5799 middle-aged, treatment-naïve subjects showing a difference in fasting serum triglyceride concentrations among homozygous Val-carriers (Leu/Leu + Leu/Val, n = 5782, 1.33 ± 1.35 mmol/l vs. Val/Val, n = 17, 2.22 ± 2.4 mmol/l, p = 0.007). Similarly, Val/Val was associated with increased fasting serum total cholesterol concentrations ( p = 0.01). In conclusion, in a relative large-scale study of middle-aged whites we found no evidence of association between the PPARα Leu162Val polymorphism and obesity or type 2 diabetes. If replicated, the Val162Val variant may, however, confer an increase in fasting levels of serum lipids.

Current Pharmacological Treatment of Nonalcoholic Fatty Liver

Nonalcoholic fatty liver disease (NAFLD) is a frequent and potentially progressive chronic liver disease that occurs in subjects who do not abuse alcohol. NAFLD is often associated with obesity, metabolic syndrome and insulin resistance and its more aggressive form, nonalcoholic steatohepatitis (NASH) is a major cause of cryptogenic cirrhosis. NAFLD/NASH are commonly detected because of elevated serum aminotransferase levels, ultrasonographic fatty liver and, at liver histology, steatosis, inflammation, and occasionally fibrosis that may progress to cirrhosis. No established treatment exists for this potentially serious disorder. Current management of NAFLD/NASH is largely conservative and includes diet regimen, aerobic exercise, and interventions towards the associated metabolic abnormalities. The main concern is therefore to decrease liver steatosis and its progression toward steatohepatitis and fibrosis, and the risk of "cryptogenic" cirrhosis. Among the most promising medications, weight reducing drugs, insulin sensitizers and lipid-lowering agents, antioxidants, bile salts, co-factors increasing the mitochondrial transport of fatty acids are being considered. Among them, thiazolidinediones are the most promising drug family that act by activating PPARgamma nuclear receptors and by regulating both microsomal and peroxisomal lipid oxidative pathways. Pharmacological treatment of obesity and probiotics should be considered as potential therapeutic options. In this review, after summarizing the general background on fatty liver, the most current and attractive pharmacological approaches to the problem of NAFLD/NASH are discussed.

Multiple contributions of peroxisomal metabolic function to fungal pathogenicity in Colletotrichum lagenarium.

In Colletotrichum lagenarium, which is the causal agent of cucumber anthracnose, PEX6 is required for peroxisome biogenesis and appressorium-mediated infection. To verify the roles of peroxisome-associated metabolism in fungal pathogenicity, we isolated and functionally characterized ICL1 of C. lagenarium, which encodes isocitrate lyase involved in the glyoxylate cycle in peroxisomes. The icl1 mutants failed to utilize fatty acids and acetate for growth. Although Icl1 has no typical peroxisomal targeting signals, expression analysis of the GFP-Icl1 fusion protein indicated that Icl1 localizes in peroxisomes. These results indicate that the glyoxylate cycle that occurs inside the peroxisome is required for fatty acid and acetate metabolism for growth. Importantly, in contrast with the pex6 mutants that form nonmelanized appressoria, the icl1 mutants formed appressoria that were highly pigmented with melanin, suggesting that the glyoxylate cycle is not essential for melanin biosynthesis in appressoria. However, the icl1 mutants exhibited a severe reduction in virulence. Appressoria of the icl1 mutants failed to develop penetration hyphae in the host plant, suggesting that ICL1 is involved in host invasion. The addition of glucose partially restored virulence of the icl1 mutant. Heat shock treatment of the host plant also enabled the icl1 mutants to develop lesions, implying that the infection defect of the icl1 mutant is associated with plant defense. Together with the requirement of PEX6 for appressorial melanization, our findings suggest that peroxisomal metabolic pathways play functional roles in appressorial melanization and subsequent host invasion steps, and the latter step requires the glyoxylate cycle.

PPARβ/δ Regulates Paneth Cell Differentiation Via Controlling the Hedgehog Signaling Pathway

All 4 differentiated epithelial cell types found in the intestinal epithelium derive from the intestinal epithelial stem cells present in the crypt unit, in a process whose molecular clues are intensely scrutinized. Peroxisome proliferator-activated receptor beta (PPARbeta) is a nuclear hormone receptor activated by fatty acids and is highly expressed in the digestive tract. However, its function in intestinal epithelium homeostasis is understood poorly. To assess the role of PPARbeta in the small intestinal epithelium, we combined various cellular and molecular approaches in wild-type and PPARbeta-mutant mice. We show that the expression of PPARbeta is particularly remarkable at the bottom of the crypt of the small intestine where Paneth cells reside. These cells, which have an important role in the innate immunity, are strikingly affected in PPARbeta-null mice. We then show that Indian hedgehog (Ihh) is a signal sent by mature Paneth cells to their precursors, negatively regulating their differentiation. Importantly, PPARbeta acts on Paneth cell homeostasis by down-regulating the expression of Ihh, an effect that can be mimicked by cyclopamine, a known inhibitor of the hedgehog signaling pathway. We unraveled the Ihh-dependent regulatory loop that controls mature Paneth cell homeostasis and its modulation by PPARbeta. PPARbeta currently is being assessed as a drug target for metabolic diseases; these results reveal some important clues with respect to the signals controlling epithelial cell fate in the small intestine.

Molecular organization of peroxisomal enzymes: Protein–protein interactions in the membrane and in the matrix

The β-oxidation of fatty acids in peroxisomes produces hydrogen peroxide (H 2O 2), a toxic metabolite, as a bi-product. Fatty acids β-oxidation activity is deficient in X-linked adrenoleukodystrophy (X-ALD) because of mutation in ALD-gene resulting in loss of very long chain acyl-CoA synthetase (VLCS) activity. It is also affected in disease with catalase negative peroxisomes as a result of inactivation by H 2O 2. Therefore, the following studies were undertaken to delineate the molecular interactions between both the ALD-gene product (adrenoleukodystrophy protein, ALDP) and VLCS as well as H 2O 2 degrading enzyme catalase and proteins of peroxisomal β-oxidation. Studies using a yeast two hybrid system and surface plasmon resonance techniques indicate that ALDP, a peroxisomal membrane protein, physically interacts with VLCS. Loss of these interactions in X-ALD cells may result in a deficiency in VLCS activity. The yeast two-hybrid system studies also indicated that catalase physically interacts with l-bifunctional enzyme ( l-BFE). Interactions between catalase and l-BFE were further supported by affinity purification, using a catalase-linked resin. The affinity bound 74-kDa protein, was identified as l-BFE by Western blot with specific antibodies and by proteomic analysis. Additional support for their interaction comes from immunoprecipitation of l-BFE with antibodies against catalase as a catalase- l-BFE complex. siRNA for l-BFE decreased the specific activity and protein levels of catalase without changing its subcellular distribution. These observations indicate that l-BFE might help in oligomerization and possibly in the localization of catalase at the site of H 2O 2 production in the peroxisomal β-oxidation pathway.

Peroxisomal cholesterol biosynthesis and Smith-Lemli-Opitz syndrome

Smith-Lemli-Opitz syndrome (SLOS), caused by 7-dehydrocholesterol-reductase (DHCR7) deficiency, shows variable severity independent of DHCR7 genotype. To test whether peroxisomes are involved in alternative cholesterol synthesis, we used [1- 14C]C24:0 for peroxisomal β-oxidation to generate [1- 14C]acetyl-CoA as cholesterol precursor inside peroxisomes. The HMG-CoA reductase inhibitor lovastatin suppressed cholesterol synthesis from [2- 14C]acetate and [1- 14C]C8:0 but not from [1- 14C]C24:0, implicating a peroxisomal, lovastatin-resistant HMG-CoA reductase. In SLOS fibroblasts lacking DHCR7 activity, no cholesterol was formed from [1- 14C]C24:0-derived [1- 14C]acetyl-CoA, indicating that the alternative peroxisomal pathway also requires this enzyme. Our results implicate peroxisomes in cholesterol biosynthesis but provide no link to phenotypic variation in SLOS.

Pharmacological Therapy of Inherited Disease.

Pharmacological agents with widely differing known modes of action can induce HbF production in vivo and in vitro. Recent data from this laboratory and others suggests that common pathways of action may be responsible for HbF induction by disparate drugs. A second therapeutic conundrum is that some of these agents seem to induce cell-type specific responses which may be useful in a variety of unrelated diseases. For example butyrate compounds (BU) show promise in treating X-linked adrenoleukodystropy (XALD), cystic fibrosis, sickle cell disease (SCD), inflammatory bowel disease, cancer, and inborn errors of metabolism to name a few. These observations suggest that certain drugs may initiate a common set of cellular responses that are read out differently depending upon the cellular context. In this study we tested various pharmacologic agents for their ability to ameliorate disease phenotypes and identified a common set of cellular responses. Although all agents were cytotoxic at high dosage, we studied effects of non-toxic low dosages. We examined two divergent inherited disorders, XALD and SCD. Cultured skin fibroblasts from XALD patients and knockout mice, and XALD mouse tissues were utilized in XALD studies. All agents studied [BU, trichostatin A (TSA), and hydroxyurea (HU)] normalized the elevated levels of very long-chain fatty acids (VLCFA) characteristic of XALD. These agents also induced peroxisome proliferation by a mechanism that requires the up-regulation of PEX 11α and is distinct from the well known PPARα peroxisomal proliferation pathway. Using a fluorescent probe for mitochondrial mass (Mitotraker), we also demonstrated a 2–3 fold increase in mitochondrial mass following treatment with these drugs. Using mitochondrial inhibitors (antimycin A), mitochondrial mutations, and auxotrophic growth we further demonstrated that the ability of these drugs to normalize VLCFA levels in XALD was dependent on the increase in mitochondrial mass and/or activity.Using cellular model systems relevant to SCD including CD34 selected cell cultures derived from SCD patients and K562 cells, a HbF induction model, we demonstrate that HU, BU, TSA, 5-azacytadine and hemin stimulate HbF induction as indicated by an increase in F-containing cells. Interestingly, as in the ALD fibroblasts, these agents also stimulate mitochondrial proliferation 2–3 fold in these cell systems as assayed by Mitotraker staining and induce peroxisomal biogenesis. Antimycin A treatment of the K562 cells using concentrations that had no significant effect on cell division, viability or baseline HbF production blunted the ability of these agents to cause mitochondrial expansion and completely inhibited the ability of these agents to induce HbF. Taken together these studies demonstrate that common cellular responses (mitochondrial biogenesis and peroxisomal proliferation) occur in diverse cell types in response to treatment with disparate therapeutic agents. Furthermore, this common response appears to be necessary for the ultimate effect of disease amelioration by these drugs, specifically correction of VLCFA levels in ALD fibroblasts and HbF induction in erythroid cells.

Yeast Pex14p Possesses Two Functionally Distinct Pex5p and One Pex7p Binding Sites

Current evidence favors a cycling receptor model for the import of peroxisomal matrix proteins. The yeast Pex14 protein together with Pex13p and Pex17p form the docking subcomplex at the peroxisomal membrane and interact in this cycle with both soluble import receptors Pex5p and Pex7p. In a first step of a structure-function analysis of Saccharomyces cerevisiae Pex14p, we mapped its binding sites with both receptors. Using the yeast two-hybrid system and pull-down assays, we showed that Pex5p directly interacts with two separate regions of ScPex14p, amino acid residues 1-58 and 235-308. The latter binding site at the C terminus of ScPex14p overlaps with a binding site of Pex7p at amino acid residues 235-325. The functional assessment of these two binding sites of ScPex14p with the peroxisomal targeting signal receptors indicates that they have distinct roles. Deletion of the N-terminal 58 amino acids caused a partial defect of matrix protein import in pex14delta cells expressing the Pex14-(59-341)-p fragment; however, it did not lead to a pex phenotype. In contrast, truncation of the C-terminal 106 amino acids of ScPex14p completely blocked this process. On the basis of these and other published data, we propose that the C terminus of Pex14p contains the actual docking site and discuss the possibility that the N terminus could be involved in a Pex5p-Pex14p association inside the peroxisomal membrane.

Molecular Identification and Characterization of the Arabidopsis Δ3,5,Δ2,4-Dienoyl-Coenzyme A Isomerase, a Peroxisomal Enzyme Participating in the β-Oxidation Cycle of Unsaturated Fatty Acids

Degradation of unsaturated fatty acids through the peroxisomal beta-oxidation pathway requires the participation of auxiliary enzymes in addition to the enzymes of the core beta-oxidation cycle. The auxiliary enzyme delta(3,5),delta(2,4)-dienoyl-coenzyme A (CoA) isomerase has been well studied in yeast (Saccharomyces cerevisiae) and mammals, but no plant homolog had been identified and characterized at the biochemical or molecular level. A candidate gene (At5g43280) was identified in Arabidopsis (Arabidopsis thaliana) encoding a protein showing homology to the rat (Rattus norvegicus) delta(3,5),delta(2,4)-dienoyl-CoA isomerase, and possessing an enoyl-CoA hydratase/isomerase fingerprint as well as aspartic and glutamic residues shown to be important for catalytic activity of the mammalian enzyme. The protein, named AtDCI1, contains a peroxisome targeting sequence at the C terminus, and fusion of a fluorescent protein to AtDCI1 directed the chimeric protein to the peroxisome in onion (Allium cepa) cells. AtDCI1 expressed in Escherichia coli was shown to have delta(3,5),delta(2,4)-dienoyl-CoA isomerase activity in vitro. Furthermore, using the synthesis of polyhydroxyalkanoate in yeast peroxisomes as an analytical tool to study the beta-oxidation cycle, expression of AtDCI1 was shown to complement the yeast mutant deficient in the delta(3,5),delta(2,4)-dienoyl-CoA isomerase, thus showing that AtDCI1 is also appropriately targeted to the peroxisome in yeast and has delta(3,5),delta(2,4)-dienoyl-CoA isomerase activity in vivo. The AtDCI1 gene is expressed constitutively in several tissues, but expression is particularly induced during seed germination. Proteins showing high homology with AtDCI1 are found in gymnosperms as well as angiosperms belonging to the Monocotyledon or Dicotyledon classes.

Glycolate metabolism in green algae

Using 14C‐labelled substrates, the succession of the single steps in the glycolate metabolism was investigated in Mougeotia scalaris and Eremosphaera viridis, which, within the group of green algae, are representatives of the evolutionary lines of Charophyta and Chlorophyta, respectively. In both algae the same metabolites are formed as in higher plants, although in Eremosphaera, which in contrast to Mougeotia does not possess leaf peroxisomes, all reactions are exclusively mitochondrial. Concomitant with the oxidation of glycolate, the synthesis of ATP was demonstrated in Eremosphaera. Formation of tartronic semi‐aldehyde or other products different from those in land plants could not be demonstrated in either of these algae. Excretion of glycolate by Mougeotia and Eremosphaera is enhanced by decreasing the CO2 concentration as well as by increasing the light intensity, but is completely stopped about 14 h later. Whereas increasing enzyme activities of the glycolate pathway apparently reduces glycolate excretion in Mougeotia, activation of CO2 pumps seems to be the dominant reaction to prevent glycolate excretion in Eremosphaera. Mesostigma viride is one of the phylogenetically oldest algae in the group of Charophyceae. As this alga has already been demonstrated to contain microbodies with enzymes of leaf peroxisomes, the peroxisomal glycolate pathway must have originated at a very early stage. Surprisingly, the organelles from Mesostigma contain also the glyoxysomal marker enzyme isocitrate lyase suggesting these microbodies to be prototypes from which both glyoxysomes and leaf peroxisomes evolved.

The Peroxisomal Proliferator-Activated Receptor-Alpha Regulatory Pathway is Activated in Patients with Severe Idiopathic Dilated Cardiomyopathy

The Peroxisomal Proliferator-Activated Receptor-Alpha Regulatory Pathway is Activated in Patients with Severe Idiopathic Dilated Cardiomyopathy

Further lowering of muscle lipid oxidative capacity in obese subjects after biliopancreatic diversion.

A reduced lipid oxidative capacity is considered a risk factor for the development of obesity, but a further impairment of lipid oxidative capacity is observed after weight loss. We aimed to define the mechanisms underlying this phenomenon in skeletal muscle and in particular to study the mitochondrial and peroxisomal lipid oxidative pathways. Thus we measured intramyocellular triglyceride content (IMTG) and the expression of genes of lipid oxidation [peroxisome proliferator-activated receptor-alpha, carnitine palmitoyltransferase 1B, and acyl-coenzyme A (acyl-CoA) oxidase 1] and synthesis (acetyl-CoA carboxylase B) using RT-PCR analysis in muscle biopsies of morbidly obese patients before and after biliopancreatic diversion. Weight reduction significantly decreased IMTG while increasing insulin sensitivity, measured by euglycemic hyperinsulinemic clamp. Moreover, an increase in glucose and a decline in lipid oxidation, as assessed by respiratory chamber, were observed. Weight loss reduced the expression of peroxisome proliferator-activated receptor-alpha (-46.7%), carnitine palmitoyltransferase 1B (-43.1%), acyl-CoA oxidase 1 (-37.8%), and acetyl-CoA carboxylase B (-48.7%). Our results indicate that a defect of both peroxisomal and mitochondrial oxidative pathways at the muscular level may contribute to the reduced fat oxidation in obese subjects after biliopancreatic diversion. They also suggest that a depression of the de novo lipogenesis may account for IMTG depletion.

Mevalonate kinase is a cytosolic enzyme in humans.

In the past decade several reports have appeared which suggest that peroxisomes play a central role in isoprenoid/cholesterol biosynthesis. These suggestions were based primarily on the reported finding of several of the enzymes of the presqualene segment of the biosynthetic pathway in peroxisomes. More recently, however, conflicting results have been reported raising doubt about the postulated role of peroxisomes in isoprenoid biosynthesis, at least in humans. In this study we have studied the subcellular localisation of human mevalonate kinase (MK) using a variety of biochemical and microscopical techniques. These include conventional subcellular fractionation studies, digitonin permeabilisation studies, immunofluorescence microscopy and immunocytochemistry. We exclusively found a cytosolic localisation of both endogenous human MK (human fibroblasts, liver and HEK293 cells) and overexpressed human MK (human fibroblasts, HEK293 cells and CV1 cells). No indication of a peroxisomal localisation was obtained. Our results do not support a central role for peroxisomes in isoprenoid biosynthesis.

Correction of an enzyme trafficking defect in hereditary kidney stone disease in vitro.

In normal human hepatocytes, the intermediary-metabolic enzyme alanine:glyoxylate aminotransferase (AGT) is located within the peroxisomes. However, in approx. one-third of patients suffering from the hereditary kidney stone disease primary hyperoxaluria type 1, AGT is mistargeted to the mitochondria. AGT mistargeting results from the synergistic interaction between a common P11L (Pro11-->Leu) polymorphism and a disease-specific G170R mutation. The polymorphism generates a functionally weak mitochondrial targeting sequence, the efficiency of which is increased by the mutation. The two substitutions together, but not in isolation, inhibit AGT dimerization, highlighting the different structural requirements of the peroxisomal and mitochondrial protein-import machineries. In the present study, we show that treatments known to increase the stability of proteins non-specifically (i.e. lowering the temperature from 37 to 30 degrees C or by the addition of glycerol) completely normalize the intracellular targeting of mutant AGT expressed in transfected COS cells. On the other hand, treatments known to decrease protein stability (e.g. increasing the temperature from 37 to 42 degrees C) exacerbate the targeting defect. Neither of the treatments affects the relative efficiencies of the peroxisomal and mitochondrial protein-import pathways intrinsically. Results are discussed in the light of the known structural requirements of the two protein trafficking pathways and the formulation of possible treatment strategies for primary hyperoxaluria type 1.