ESPEN 2020 Abstract Submission

Topic: Vitamins, antioxidants and minerals

Abstract Submission Identifier: ESPEN20-ABS-1201

IMPAIRMENT OF VITAMIN E INTESTINAL SECRETION IN PRIMARY HYPOBETALIPOPROTEINEMIAS: MECHANISTICS STUDIES IN A CELL MODELS

C. Bordat^*, ^{1, 2}, M. Nowicki ¹, C. Halimi ¹, E. Reboul ¹, N. Peretti ²

¹Aix-Marseille Université, INRA, INSERM, C2VN, Marseille, ²Laboratoire Carmen, Université Lyon 1, INRA, INSA, CENS, CRNH, Lyon, France

Rationale: Chylomicron retention disease (CMRD) is a rare recessive inborn disorders of lipoprotein metabolism due to mutations in the Microsomal Triglyceride Transfer Protein (MTTP) gene. Intestinal malabsorption of lipids and fat-soluble vitamins (A, D, E, K) due to the lack of secretion of chylomicrons causes neurological complications. Supplementation in large doses is essential to prevent them. However, serum vitamin E levels are never fully restored. To understand these persistent low levels, previous studies were performed on Mttp-KO mice. Data showed a reduced intestinal vitamin E accumulation that was not consistent with the lipid accumulation observed.

To investigate the mechanisms underlying this phenomenon, we created a model of MTTP-knockdown-Caco-2/TC7 cells using the CRISPR/Cas9 technique.

Methods: Vitamin E assays were conducted on human enterocyte-like MTTP-KO Caco- 2/TC7 cells produced using the CRISPR/Cas9 genome-editing system. Cellular model was first created by genome engineering and its relevance was investigated before performing absorption and secretion assays. DNA sequencing by Sanger method was performed to confirm single-nucleotide mutation. Absence of MTTP in the cell clone selected was confirmed by Wester-Blot. Mutated clone was then cultured on transwell permeable supports and incubated with vitamin E-enriched micelles. Apical and basolateral media were collected and analyzed by HPLC to quantify vitamin E absorption and secretion by MTTP-KO cells.

Results: DNA sequencing confirmed the existence of a mutation in the MTTP gene introduced by CRSIPR/cas9. Protein expression assays by Western Blot confirmed the absence of MTTP. The results on vitamin E assays show a non-disturbed vitamin E absorption (275,40 vs. 242,26 nmol/ml in control cells) but a reduced basolateral secretion (0,8 vs 11,2 nmol/ml in control cells). No vitamin E degradation was observed in our cell culture conditions.

Conclusion: MTTP knock-down in Caco2 cells led to a drastic decrease in vitamin E basolateral secretion in Caco-2 TC7 cells. Further studies are needed to understand whether a degradation of vitamin E can occur in conditions mimicking those found in vivo, and to open new therapeutic perspectives for these patients.

Disclosure of Interest: None Declared

Keywords: Chylomicron retention disease, Vitamin E