Effects of Triheptanoin on Mitochondrial Respiration and Glycolysis in Cultured Fibroblasts from Neutral Lipid Storage Disease Type M (NLSD-M) Patients

{"Background": "Neutral Lipid Storage Disease Type M (NLSD-M) is an ultra-rare recessive disorder caused by mutations in the PNPLA2 gene encoding adipose triglyceride lipase (ATGL), leading to lipid accumulation in tissues and severe skeletal and cardiac muscle damage. No therapy currently exists, and the pathogenesis is incompletely understood. Mitochondria generate up to 90% of cellular ATP through fatty acid \u03b2-oxidation. The authors hypothesized that impaired ATGL function depletes TCA cycle intermediates, creating an energy deficit that could be addressed through anaplerotic therapy. Triheptanoin (Dojolvi\u00ae) is an FDA-approved odd-chain medium-chain triglyceride that produces both acetyl-CoA and propionyl-CoA (converted to succinyl-CoA), providing anaplerotic substrates to replenish TCA cycle intermediates, and has shown benefit in long-chain fatty acid oxidation disorders.", "Methods": "Fibroblasts were obtained via skin biopsy from five NLSD-M patients (NLSD 1-5) of the Italian NLSD Group and three controls. Patients had varying clinical severity (very mild to severe) and diverse ATGL mutations including c.24G>C/c.516C>A, c.177T>G/c.577A>T, c.659delT, and c.45_47del. Cells were cultured in DMEM high-glucose medium and treated with triheptanoin at 25 \u00b5M for one week before Seahorse analysis. Mitochondrial function was assessed using the Seahorse XFe96 analyzer with the Mito Stress Test, measuring OCR under basal conditions and after oligomycin (2 \u00b5M), FCCP (1 \u00b5M), and rotenone/antimycin A (0.5 mM). Glycolysis was measured via the Glycolysis Rate Assay with ECAR detection. Cell viability was assessed by MTT assay at 24, 48, and 72 hours with 12.5 or 25 \u00b5M triheptanoin. Intracellular triacylglycerol content was quantified colorimetrically before and after 9-day treatment, and lipid droplets visualized by Nile Red immunofluorescence. Statistical analysis used Mann-Whitney and Student's t-tests.", "Key Results": "Triheptanoin significantly increased all OXPHOS parameters in NLSD-M fibroblasts: basal respiration rose from 35\u00b15 to 49\u00b110 pmol/min/\u00d710^4 cells (p=0.03 vs untreated NLSD-M DMSO); maximal respiration increased from 95\u00b135 to 158\u00b158 pmol/min/\u00d710^4 cells; spare respiratory capacity increased from 31\u00b129 to 108\u00b150 pmol/min/\u00d710^4 cells; ATP production rose from 30\u00b15 to 39\u00b19 pmol/min/\u00d710^4 cells. Individual patient analyses showed significant increases in basal respiration (p=0.0003-0.03), maximal respiration (p=0.0001-0.007), and spare respiratory capacity (p=0.0001-0.03) across all five patients. When excluding NLSD 4 (complete ATGL deficiency), differences in maximal respiration and SRC became significant. Normal control fibroblasts showed no significant changes in any OXPHOS parameter. Basal glycolysis significantly increased in NLSD 2 (50\u00b17 to 112\u00b127, p=0.004), NLSD 3 (68\u00b137 to 175\u00b155, p=0.02), NLSD 4 (135\u00b131 to 206\u00b115, p=0.0001), and NLSD 5 (60\u00b114 to 78\u00b113, p=0.03); NLSD 1 showed increased compensatory glycolysis (529\u00b196 to 755\u00b184, p=0.004). Cell viability significantly increased at 48 hours in NLSD 2 with 12.5 \u00b5M triheptanoin (p\u22640.05), and at 72 hours in NLSD 2 and 3 with both doses (p\u22640.05). However, triheptanoin did not decrease TAG content or lipid droplet size/number after 9-day treatment, with cells showing slightly increased TAG accumulation.", "Clinical Implications": "These findings support triheptanoin as a potential adjunctive metabolic therapy for NLSD-M by providing anaplerotic substrates that bypass the metabolic block caused by ATGL deficiency, replenishing TCA cycle intermediates through propionyl-CoA conversion to succinyl-CoA. The enhanced mitochondrial respiration and glycolysis suggest improved cellular bioenergetic capacity without affecting the underlying lipid storage defect. Patients with residual ATGL enzymatic activity (missense mutations) showed greater benefit than those with complete loss-of-function mutations, indicating genotype may predict treatment response. The lack of effect on TAG accumulation confirms triheptanoin does not substitute for ATGL lipolytic function and would need to be combined with other therapeutic approaches. Limitations include the small sample size inherent to this ultra-rare disease, the in vitro design, and the inability to directly translate findings to clinical practice, though results provide proof-of-concept warranting further investigation."}