Year of grant: 2018 Research Area: Heilsa Project type: Ph.d.-verkætlan Project title: Nýtslan av fitisýrum og umsetningurin av karnitini í vøddakyknum hjá sjúklingum við CTD. Grant number: 0346 Project manager: Rannvá Dahl Institution/company: Københavns Universitet og Landssjúkrahúsið Other participants: The National Hospital Faroe Islands, Faroese Food and Veterinary Agency, University of Copenhagen, National University Hospital (Rigshospitalet) Project period: Planned: 01/04/2019-31/03/2022 Total budget: kr. 1.876.800 Grant from the FRC in DKK: kr. 1.136.000 Project description: Original For several decades multiple cases of sudden death in young Faroese individuals with untreated carnitine transporter deficiency (CTD) have been reported. CTD is a disorder influencing fatty acid β- oxidation by a lack of the carnitine transporter(OCTN2). Skeletal muscle and cardiac muscle utilize fatty .acids as an energy source and low body carnitine levels in these tissues can lead to skeletal muscle myopathy and lethal cardiac arrhythmia. Most CTD patients are being treated with L-Carnitine,however preliminary unpublished data shows that skeletal muscle carnitine concentration of Faroese treated CTD patents is 20 fold lower than normal and this raises the question if the β-oxidation in CTD patients might be impaired even though they are being treated. The regulation of OCTN2 is not fully understood, however hyperinsulinaemia has been associated with increased muscle carnitine content in healthy humans. Human experiments CTD patients with the severe genotype N32S/N32S (n=10), CTD patients with N32S/haplotype (n=10) and healthy control subjects (n=10) will be recruited. The subjects/patients will come to the laboratory on two occasions. The patients/controls will come to the laboratory on two occasions. On the first occasion they will have their health examined. The second visit to the laboratory will be divided into 3 sub studies: STUDY 1: Mitochondrial and metabolic alterations in CTD skeletal muscle Analysis will be done on the muscle in order to answer the following key questions: 1) Is mitochondrial function impaired in CTD skeletal muscle? 2) If so, do the levels of muscle Carnitine inversely correlate to the degree of mitochondrial dysfunction? 3) What is the effect of carnitine deficiency on the metabolic state of skeletal muscle fibers? STUDY 2: Regulation of skeletal muscle carnitine uptake by insulin and, effect of co-administration of insulin and Carnitine on skeletal muscle carnitine levels. The results generated by this study will give us insight on the regulation of OCTN2 and potential alterations found in CTD skeletal muscle. Furthermore, the hypothesis that the insulin-induced upregulation of carnitine transport into skeletal muscle could be explained by OCTN2 intracellular redistribution will be tested. Furthermore, the results will show if the effect of insulin on skeletal muscle carnitine uptake is mediated by OCTN2 and if so, if it is impaired in CTD patients. These results are needed in order to potentially use insulin to enhance the effect of Carnitine supplementation on CTD patients. STUDY 3: Insulin induced enhancement of carnitine uptake in skin fibroblasts In this study analysis will be done on skin biopsies. By culturing fibroblasts from the subjects: 1) The carnitine transport over the plasmalemma can be measured. This will allow for the investigation of the effect that insulin has on carnitine uptake in fibroblasts from CTD patients and healthy controls. 2) The intracellular OCTN2 distribution and potential re-distribution in response to insulin and/or carnitine will be measured. The results generated by this study will give insight on the insulin induced regulation of OCTN2 and a potential deviation found in CTD fibroblasts Final Background: Primary carnitine deficiency (PCD) is an autosomal recessive disorder characterized by a lack of functional carnitine transporters OCTN2 (Organic Cation/Carnitine Transporter 2), which has been linked to several cases of sudden death in young Faroese individuals. It causes low carnitine levels and can present with cardiac complications as well as skeletal muscle symptoms including muscular weakness and fatigue. Patients are treated with L-carnitine, and even while receiving treatment, skeletal muscle carnitine levels are only approximately 7% of normal. The regulation of the carnitine transporter, OCTN2 is not fully understood, but a combination of hypercarnitinaemia with hyperinsulinemia upregulates skeletal muscle carnitine uptake and OCTN2 mRNA expression. Objective: The question arises as to whether hyperinsulinemia increases the efficiency of L-carnitine supplementation in PCD. The primary aim was to investigate the regulatory mechanisms behind insulin-induced carnitine uptake, and whether the combination of hypercarnitinaemia and hyperinsulinaemia increases skeletal muscle carnitine levels in patients with PCD. Additionally, we wanted to examine if the lifelong low muscle carnitine has caused any mitochondrial and metabolic alterations in patients with PCD. Method: Nine patients with PCD (homozygous for the c.95 A > G, P.N32S mutation), taking L-carnitine supplementation and nine healthy controls matched to age and body mass index (BMI) participated in the study (Study I, II). A six hour hyperinsulinemic clamp was supplemented with infusion of L-carnitine the last five hours. Skeletal muscle biopsies were collected before and after the clamp and carnitine content was measured. Mitochondrial function was measured as mitochondrial respiratory capacity and skeletal muscle H2O2 production in permeabilized muscle fibers. An attempt was done to measure mitochondrial respiratory capacity in intact fibroblast cell, but without useful results (fibroblast-pilot study). Confocal microscopy was used to access regulation of OCTN2 positive vesicles due to insulin stimulation and to measure intramuscular lipid distribution. Results and discussion: We found that the combination of hypercarnitinaemia with hyperinsulinemia did not increase skeletal muscle total carnitine levels significantly for neither patients with PCD (P = 0.28) nor controls (P = 0.053). Indicating that the attempt to increases the efficiency of L-carnitine supplementation in PCD did not succeed. The results from confocal microscopy suggested that insulin regulates skeletal muscle carnitine uptake by stimulating OCTN2 recruitment from intracellular storages to the plasma membrane in controls, a mechanism that seems to be impaired in patients with PCD. Even though, the regulatory mechanism seems to be impaired there are other mechanisms associated with OCTN2 recruitment to plasma membrane, such as muscle contraction. Which current study did not investigated. Furthermore, we found the mitochondrial respiratory capacity, in permeabilized muscle fibers, to be similar between groups, as well as the H2O2 emission and intramuscular lipid distribution. Indicating that the reduced levels of muscle carnitine, reported in patients with PCD (taking L-carnitine) do not seem to have damaged the mitochondrial complexes of skeletal muscle and that even though carnitine has been found to have antioxidant properties, the levels of H2O2 were not increased in skeletal muscle of patients with PCD. Project status: Liðug Project output: Ph.d.-ritgerð vard 16. apríl. Heitið: Insulin stimulation of L-carnitine uptake in skeletal muscle in patients with primary carnitine deficiency and the metabolic state of the skeletal muscle << Back |
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