The molecular mechanisms linking obstructive sleep apnea syndrome (OSA) to obesity and the development of metabolic diseases are still poorly understood. The role of hypoxia (a characteristic feature of OSA) in excessive fat accumulation has been proposed. The present study investigated the possible effects of hypoxia (4% oxygen) on de novo lipogenesis by tracking the major carbon sources in differentiating 3T3-L1 adipocytes. Gas-permeable cultuware was employed to cultivate 3T3-L1 adipocytes in hypoxia (4%) for 7 or 14 days of differentiation. We investigated the contribution of glutamine, glucose or acetate using 13C or 14C labelled carbons to the newly synthesized lipid pool, changes in intracellular lipid content after inhibiting citrate- or acetate-dependent pathways and gene expression of involved key enzymes. The results demonstrate that, in differentiating adipocytes, hypoxia decreased the synthesis of lipids from glucose (44.1 ± 8.8 to 27.5 ± 3.0 pmol/mg of protein, p < 0.01) and partially decreased the contribution of glutamine metabolized through the reverse tricarboxylic acid cycle (4.6% ± 0.2-4.2% ± 0.1%, p < 0.01). Conversely, the contribution of acetate, a citrate- and mitochondria-independent source of carbons, increased upon hypoxia (356.5 ± 71.4 to 649.8 ± 117.5 pmol/mg of protein, p < 0.01). Further, inhibiting the citrate- or acetate-dependent pathways decreased the intracellular lipid content by 58% and 73%, respectively (p < 0.01) showing the importance of de novo lipogenesis in hypoxia-exposed adipocytes. Altogether, hypoxia modified the utilization of carbon sources, leading to alterations in de novo lipogenesis in differentiating adipocytes and increased intracellular lipid content.

• MeSH
• Acetates * metabolism   pharmacology   MeSH
• Cell Differentiation * drug effects   MeSH
• 3T3-L1 Cells * MeSH
• Citric Acid Cycle MeSH
• Glucose * metabolism   MeSH
• Glutamine * metabolism   MeSH
• Cell Hypoxia MeSH
• Lipids biosynthesis   MeSH
• Lipogenesis * drug effects   MeSH
• Lipid Metabolism drug effects   MeSH
• Mice MeSH
• Adipocytes * metabolism   drug effects   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Acetates * MeSH
• Glucose * MeSH
• Glutamine * MeSH
• Lipids MeSH

Obstructive sleep apnea syndrome, characterized by repetitive episodes of tissue hypoxia, is associated with several metabolic impairments. Role of fatty acids and lipids attracts attention in its pathogenesis for their metabolic effects. Parallelly, hypoxia-induced activation of reverse tricarboxylic acid cycle (rTCA) with reductive glutamine metabolism provides precursor molecules for de novo lipogenesis. Gas-permeable cultureware was used to culture L6-myotubes in chronic hypoxia (12%, 4% and 1% O2) with 13C labelled glutamine and inhibitors of glutamine uptake or rTCA-mediated lipogenesis. We investigated changes in lipidomic profile, 13C appearance in rTCA-related metabolites, gene and protein expression of rTCA-related proteins and glutamine transporters, glucose uptake and lactate production. Lipid content increased by 308% at 1% O2, predominantly composed of saturated fatty acids, while triacylglyceroles containing unsaturated fatty acids and membrane lipids (phosphatidylcholines, phosphatidylethanolamines, phosphatidylinositol) decreased by 20-70%. rTCA labelling of malate, citrate and 2-hydroxyglutarate increased by 4.7-fold, 2.2-fold and 1.9-fold in 1% O2, respectively. ATP-dependent citrate lyase inhibition in 1% O2 decreased lipid amount by 23% and increased intensity of triacylglyceroles containing unsaturated fatty acids by 56-80%. Lactate production increased with hypoxia. Glucose uptake dropped by 75% with progression of hypoxia from 4% to 1% O2. Protein expression remained unchanged. Altogether, hypoxia modified cell metabolism leading to lipid composition alteration and rTCA activation.

• Keywords
• L6 myotubes, glutamin, hypoxia, lipids, obstructive sleep apnea, reverse TCA,
• MeSH
• Citric Acid Cycle * genetics   MeSH
• Hypoxia metabolism   MeSH
• Muscle Fibers, Skeletal metabolism   MeSH
• Humans MeSH
• Fatty Acids * metabolism   MeSH
• Fatty Acids, Unsaturated metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Fatty Acids * MeSH
• Fatty Acids, Unsaturated MeSH

Obstructive sleep apnoea (OSA) is associated with type 2 diabetes mellitus (T2DM). However, mechanisms mediating association between these two conditions remain unclear. This study investigated, whether the OSA-associated changes in adipose tissue lipolysis might contribute to impaired glucose homeostasis in patient with T2DM. Thirty-five matched subjects were recruited into three groups: T2DM + severe OSA (T2DM + OSA, n = 11), T2DM with mild/no OSA (T2DM, n = 10) and healthy controls (n = 14). Subcutaneous abdominal adipose tissue microdialysis assessed spontaneous, epinephrine- and isoprenaline-stimulated lipolysis. Glucose metabolism was assessed by intravenous glucose tolerance test. Spontaneous lipolysis was higher in the T2DM + OSA compared with the T2DM (60.34 ± 23.40 vs. 42.53 ± 10.16 μmol/L, p = 0.013), as well as epinephrine-stimulated lipolysis (236.84 ± 103.90 vs. 167.39 ± 52.17 µmol/L, p < 0.001). Isoprenaline-stimulated lipolysis was unaffected by the presence of OSA (p = 0.750). The α2 anti-lipolytic effect was decreased in T2DM + OSA by 59% and 315% compared with T2DM and controls (p = 0.045 and p = 0.007, respectively). The severity of OSA (AHI) was positively associated with spontaneous (p = 0.037) and epinephrine-stimulated (p = 0.026) lipolysis. The α2-adrenergic anti-lipolytic effect (p = 0.043) decreased with increasing AHI. Spontaneous lipolysis was positively associated with Insulin resistance (r = 0.50, p = 0.002). Epinephrine-stimulated lipolysis was negatively associated with the Disposition index (r = - 0.34, p = 0.048). AHI was positively associated with Insulin resistance (p = 0.017) and negatively with the Disposition index (p = 0.038). Severe OSA in patients with T2DM increased adipose tissue lipolysis, probably due to inhibition of the α2-adrenergic anti-lipolytic effect. We suggest that dysregulated lipolysis might contribute to OSA-associated impairments in insulin secretion and sensitivity.

• MeSH
• Epinephrine administration & dosage   MeSH
• Diabetes Mellitus, Type 2 complications   epidemiology   metabolism   pathology   MeSH
• Glucose metabolism   MeSH
• Homeostasis physiology   MeSH
• Insulin metabolism   MeSH
• Insulin Resistance physiology   MeSH
• Isoproterenol administration & dosage   MeSH
• Middle Aged MeSH
• Humans MeSH
• Lipolysis drug effects   genetics   MeSH
• Sleep Apnea, Obstructive complications   epidemiology   metabolism   pathology   MeSH
• Aged MeSH
• Adipose Tissue drug effects   metabolism   MeSH
• Check Tag
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Epinephrine MeSH
• Glucose MeSH
• Insulin MeSH
• Isoproterenol MeSH

Many compounds have the potential to harm pancreatic beta-cells; organochlorine pollutants belong to those compounds. In this work, we aimed to find markers of acute toxicity of p,p'-DDT exposure among proteins expressed in NES2Y human pancreatic beta-cells employing 2-D electrophoresis. We exposed NES2Y cells to a high concentration (150 μM, LC96 after 72 hours) of p,p'-DDT for 24 and 30 hours and determined proteins with changed expression using 2-D electrophoresis. We have found 22 proteins that changed their expression. They included proteins involved in ER stress (GRP78, and endoplasmin), mitochondrial proteins (GRP75, ECHM, IDH3A, NDUS1, and NDUS3), proteins involved in the maintenance of the cell morphology (EFHD2, TCPA, NDRG1, and ezrin), and some other proteins (HNRPF, HNRH1, K2C8, vimentin, PBDC1, EF2, PCNA, biliverdin reductase, G3BP1, FRIL, and HSP27). The proteins we have identified may serve as indicators of p,p'-DDT toxicity in beta-cells in future studies, including long-term exposure to environmentally relevant concentrations.

• MeSH
• Electrophoresis, Gel, Two-Dimensional MeSH
• Insulin-Secreting Cells cytology   drug effects   metabolism   MeSH
• Biomarkers metabolism   MeSH
• Cell Line MeSH
• Endoplasmic Reticulum Chaperone BiP MeSH
• DDT toxicity   MeSH
• Mass Spectrometry MeSH
• Humans MeSH
• Proteomics methods   MeSH
• Gene Expression Regulation drug effects   MeSH
• Cell Survival drug effects   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Biomarkers MeSH
• Endoplasmic Reticulum Chaperone BiP MeSH
• DDT MeSH
• HSPA5 protein, human MeSH Browser

Hypoxia is characterized as insufficient oxygen delivery to tissues and cells in the body and is prevalent in many human physiology processes and diseases. Thus, it is an attractive state to experimentally study to understand its inner mechanisms as well as to develop and test therapies against pathological conditions related to hypoxia. Animal models in vivo fail to recapitulate some of the key hallmarks of human physiology, which leads to human cell cultures; however, they are prone to bias, namely when pericellular oxygen concentration (partial pressure) does not respect oxygen dynamics in vivo. A search of the current literature on the topic revealed this was the case for many original studies pertaining to experimental models of hypoxia in vitro. Therefore, in this review, we present evidence mandating for the close control of oxygen levels in cell culture models of hypoxia. First, we discuss the basic physical laws required for understanding the oxygen dynamics in vitro, most notably the limited diffusion through a liquid medium that hampers the oxygenation of cells in conventional cultures. We then summarize up-to-date knowledge of techniques that help standardize the culture environment in a replicable fashion by increasing oxygen delivery to the cells and measuring pericellular levels. We also discuss how these tools may be applied to model both constant and intermittent hypoxia in a physiologically relevant manner, considering known values of partial pressure of tissue normoxia and hypoxia in vivo, compared to conventional cultures incubated at rigid oxygen pressure. Attention is given to the potential influence of three-dimensional tissue cultures and hypercapnia management on these models. Finally, we discuss the implications of these concepts for cell cultures, which try to emulate tissue normoxia, and conclude that the maintenance of precise oxygen levels is important in any cell culture setting.

• Keywords
• animal model, cell culture, hypoxia, in vitro model, normoxia, oxygen concentration, partial pressure, pericellular oxygen,
• MeSH
• Cell Culture Techniques methods   MeSH
• Cell Physiological Phenomena * MeSH
• Hypoxia * MeSH
• Oxygen metabolism   MeSH
• Humans MeSH
• Feasibility Studies MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Oxygen MeSH

Saturated fatty acids (FAs) induce apoptosis in the human pancreatic NES2Y β-cell line while unsaturated FAs have nearly no detrimental effect. Moreover, unsaturated FAs are capable of inhibiting the pro-apoptotic effect of saturated FAs. Hypoxia is also known to have deleterious effects on β-cells function and viability. In the present study, we have tested the modulatory effect of hypoxia on the effect of FAs on the growth and viability of the human pancreatic NES2Y β-cells. This study represents the first study testing hypoxia effect on effects of FAs in pancreatic β-cells as well as in other cell types. We showed that hypoxia increased the pro-apoptotic effect of saturated stearic acid (SA). Endoplasmic reticulum stress signaling seemed to be involved while redistribution of FA transporters fatty acid translocase/cluster of differentiation 36 (FAT/CD36) and fatty acid-binding protein (FABP) do not seem to be involved in this effect. Hypoxia also strongly decreased the protective effect of unsaturated oleic acid (OA) against the pro-apoptotic effect of SA. Thus, in the presence of hypoxia, OA was unable to save SA-treated β-cells from apoptosis induction. Hypoxia itself had only a weak detrimental effect on NES2Y cells. Our data suggest that hypoxia could represent an important factor in pancreatic β-cell death induced and regulated by FAs and thus in the development of type 2 diabetes mellitus.

• Keywords
• ER stress, NES2Y, apoptosis, caspases, fatty acid transporters, fatty acids, hypoxia, hypoxia-inducible factor 1α, pancreatic β-cells,
• MeSH
• Insulin-Secreting Cells metabolism   MeSH
• Biomarkers MeSH
• Cell Line MeSH
• Hypoxia metabolism   MeSH
• Caspases metabolism   MeSH
• Humans MeSH
• Fatty Acids metabolism   MeSH
• Cell Proliferation MeSH
• Signal Transduction drug effects   MeSH
• Endoplasmic Reticulum Stress MeSH
• Cell Survival MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Biomarkers MeSH
• Caspases MeSH
• Fatty Acids MeSH