Adipose tissue (AT) plays a central role in integrating energy metabolism and glucose homeostasis. It is the major site of fatty acid storage as triglycerides and is bodys largest cholesterol pool. In AT cholesterol is mostly found in its free, non-esterified form. There is accumulating evidence that cholesterol imbalance in AT is closely associated with adipocyte dysfunction and obesity-mediated metabolic complications, including low levels of high-density lipoprotein cholesterol and insulin resistance. Low levels of methyl donor S-adenosylmethionine (SAMe) has been shown to activate SREBP-1 and lipogenesis. Similarly, vitamins B12 and folate regulate the levels of SAMe and homocysteine. As homocysteine has been shown to induce ER stress, we investigated the effect of B12/folate on cholesterol synthesis and ER stress in adipocytes.
Human pre-adipocyte cell line CHUB-S7 was differentiated in various B12/folate concentrations: i) control: (B12 500 nM, folate-6 μM); ii) normal B12/high folate: (B12 500 nM, folate-15, 30, 60 μM). iii) Low B12/high folate: (B12 0.15 nM, folate-6, 15, 30, 60 μM) and iv) No B12/high folate: (B12 0 nM, folate-0, 30, 60 μM). DNA, RNA and protein was extracted from the differentiated adipocytes and used for investigation of cholesterol biosynthesis pathway using microarray, real-time PCR and western blotting. Conditioned media was used for homocysteine analysis by HPLC.
Microarray analysis led to identification of cholesterol biosynthesis and ER stress pathways, altered due to B12/folate imbalance. Validation by real-time-PCR confirmed that compared to normal B12/folate levels, the genes involved in cholesterol biosynthesis, regulation and ER stress were up-regulated in both B12 deficient conditions (P<0.05). Protein expression of SREBP-1 and SREBP-2 were also increased in the same conditions including levels of total cholesterol and homocysteine (all P<0.05).
Our data provides a novel mechanism that adipocytes subjected to inappropriate levels of B12/folate exhibit increased cholesterol biosynthesis and ER stress, thus potentially predisposing adipocyte dysfunction.
Declaration of funding: George Eliot NHS Trust Diabetes Research Fund.