CRISPR editing of the PPARGC1A Gly482ser (rs8192678) polymorphism in human white adipose cells shows differential effects on mitochondrial function and adipogenesis.

Summary, in English

Background and aims: PPARGC1A encodes PGC-1α (peroxisome proliferator-activated receptor γ coactivator 1-α), a central regulator of energy metabolism and mitochondrial function. A common polymorphism in PPARGC1A (rs8192678, C/T, Gly482Ser) has been associated with obesity and related metabolic disorders, but no published functional studies have investigated direct allele-specific effects in adipocyte biology. Materials and methods: We used CRISPR-Cas9 to perform allele switching (C-to-T or T-to-C) at rs8192678 in isogenic human pre-adipocyte white adipose tissue (hWAT) cell line; we then evaluated the allelic effects at rs8192678 on adipogenic differentiation and mitochondrial function. Accordingly, single-cell clones were expanded and screened to obtain homozygous T/T (482Ser) and C/C (482Gly) isogenic cell populations. The effect of the allele editing on white adipocyte differentiation and on mitochondrial function was then studied in three cell populations of the respective genotype. In ongoing experiments, CRISPR/Cas9 was also used to append a luciferase tag to C/C and in T/T cells. The luciferase will be used as a reporter for the endogenously expressed PGC-1 protein stability, and will therefore provide insights into mechanisms by which rs8192678 alleles affect PGC-1 activity. Results (see figure): At the end of the differentiation protocol the C/C adipocytes were apparently less Oil-Red-O positive than T/T adipocytes under optical microscopy, they had 78.5% lower triglyceride content (p<0.0001, n=9), and lower expression of adipogenic markers (all markers p<0.0001, n=3). Furthermore, C/C adipocytes had lower mitochondrial content (p<0.001, n=9), which coincided with decreased oxygen consumption rate (OCR) at basal (p<0.0001, n=3) and maximal respiration (p<0.0001, n=3). Also, C/C adipocytes had lower ATP-linked OCR (p<0.0001, n=3). Conclusion: Our data showcases discriminatory causal effects of the two rs8192678 alleles in adipocytes. The C allele confers lower PPARGC1A expression, and consequential impaired adipocyte differentiation, at least in part due to disrupted mitochondrial biosynthesis and function. Our study is the first to give experimental insights into the molecular mechanisms behind observational epidemiological studies the Gly482Ser variant and obesity and metabolic disorders