Intermittent Hypoxia Stimulates Lipolysis, But Inhibits Differentiation and De Novo Lipogenesis in 3T3-L1 Cells.

Background: Exposure to intermittent hypoxia (IH) may play a role in the development of metabolic impairments in the context of obstructive sleep apnea syndrome, probably by elevated plasma levels of free fatty acids. Employing gas-permeable cultureware to grow differentiated human and mouse adipocytes in vitro, we directly studied the effects of pericellular oxygen fluctuations on key adipocyte metabolic functions-spontaneous lipolytic rates, triglyceride accumulation, de novo lipogenesis, and expression of adipocyte-specific marker genes. Materials and Methods: 3T3-L1 fibroblasts and human subcutaneous preadipocytes were differentiated under conditions that induced repetitive pericellular-oxygen cycles IH between 1% O2 (5 min) and 16% O2 (5 min), continuously for 14 days or under control conditions. Chemicals were used to inhibit the flux of acetyl-CoA from glycolysis (alfa-cyano-4-hydroxy cinnamate) or the tricarboxylic acid cycle (SB204990), or to stimulate the flux of acetyl-CoA from pyruvate to the lipogenic pool. Lipolytic rate, intracellular lipids, and expression of adipocyte differentiation markers were assessed and t-test or ANOVA were used to find significant differences. Results: The rate of lipolysis increased by 211% in 3T3-L1 cells and by 39% in obese human adipocytes. Exposure to IH reduced intracellular lipid stores by 37% and reduced the expression of adipocyte differentiation markers. Pharmacological stimulation or inhibition of de novo lipogenesis did not modify the intracellular lipid content under IH. Conclusions: Pericellular oxygen fluctuations directly stimulated lipolysis, but did not increase de novo lipogenesis from endogenous substrates. Similarly, IH hampered adipocyte differentiation from precursors.

The Effect of Hypoxia and Metformin on Fatty Acid Uptake, Storage, and Oxidation in L6 Differentiated Myotubes.

Metabolic impairments associated with obstructive sleep apnea syndrome (OSA) are linked to tissue hypoxia, however, the explanatory molecular and endocrine mechanisms remain unknown. Using gas-permeable cultureware, we studied the chronic effects of mild and severe hypoxia on free fatty acid (FFA) uptake, storage, and oxidation in L6 myotubes under 20, 4, or 1% O2. Additionally, the impact of metformin and the peroxisome proliferator-activated receptor (PPAR) ß/δ agonist, called GW501516, were investigated. Exposure to mild and severe hypoxia reduced FFA uptake by 37 and 32%, respectively, while metformin treatment increased FFA uptake by 39% under mild hypoxia. GW501516 reduced FFA uptake under all conditions. Protein expressions of CD36 (cluster of differentiation 36) and SCL27A4 (solute carrier family 27 fatty acid transporter, member 4) were reduced by 17 and 23% under severe hypoxia. Gene expression of UCP2 (uncoupling protein 2) was reduced by severe hypoxia by 81%. Metformin increased CD36 protein levels by 28% under control conditions and SCL27A4 levels by 56% under mild hypoxia. Intracellular lipids were reduced by mild hypoxia by 18%, while in controls only, metformin administration further reduced intracellular lipids (20% O2) by 36%. Finally, palmitate oxidation was reduced by severe hypoxia, while metformin treatment reduced non-mitochondrial O2 consumption, palmitate oxidation, and proton leak at all O2 levels. Hypoxia directly reduced FFA uptake and intracellular lipids uptake in myotubes, at least partially, due to the reduction in CD36 transporters. Metformin, but not GW501516, can increase FFA uptake and SCL27A4 expression under mild hypoxia. Described effects might contribute to elevated plasma FFA levels and metabolic derangements in OSA.

Adipogenesis, lipogenesis and lipolysis is stimulated by mild but not severe hypoxia in 3T3-L1 cells.

In-vitro investigation of the effects of hypoxia is limited by physical laws of gas diffusion and cellular O2 consumption, making prolonged exposures to stable O2 concentrations impossible. Using a gas-permeable cultureware, chronic effects of mild and severe hypoxia on triglyceride accumulation, lipid droplet size distribution, spontaneous lipolysis and gene expression of adipocyte-specific markers were assessed. 3T3-L1 cells were differentiated under 20%, 4% or 1% O2 using a gas-permeable cultureware. Triglyceride accumulation, expression of genes characteristic for advanced adipocyte differentiation and involvement of key lipogenesis enzymes were assessed after exposures. Lipogenesis increased by 375% under mild hypoxia, but dropped by 43% in severe hypoxia. Mild, but not severe, hypoxia increased formation of large lipid droplets 6.4 fold and strongly induced gene expression of adipocyte-specific markers. Spontaneous lipolysis increased by 488% in mild, but only by 135% in severe hypoxia. Inhibition of ATP-dependent citrate lyase suppressed hypoxia-induced lipogenesis by 81% and 85%. Activation of HIF inhibited lipogenesis by 59%. Mild, but not severe, hypoxia stimulates lipolysis and promotes adipocyte differentiation, probably through excess of acetyl-CoA originating from tricarboxylic acid cycle independently of HIF activation.