Dysregulation of the expression and secretion of inflammation-related adipokines by hypoxia in human adipocytes

Abstract

The effect of hypoxia, induced by incubation under low (1%) oxygen tension or by exposure to CoCl(2), on the expression and secretion of inflammation-related adipokines was examined in human adipocytes. Hypoxia led to a rapid and substantial increase (greater than sevenfold by 4 h of exposure to 1% O(2)) in the hypoxia-sensitive transcription factor, HIF-1alpha, in human adipocytes. This was accompanied by a major increase (up to 14-fold) in GLUT1 transporter mRNA level. Hypoxia (1% O(2) or CoCl(2)) led to a reduction (up to threefold over 24 h) in adiponectin and haptoglobin mRNA levels; adiponectin secretion also decreased. No changes were observed in TNFalpha expression. In contrast, hypoxia resulted in substantial increases in FIAF/angiopoietin-like protein 4, IL-6, leptin, MIF, PAI-1 and vascular endothelial growth factor (VEGF) mRNA levels. The largest increases were with FIAF (maximum 210-fold), leptin (maximum 29-fold) and VEGF (maximum 23-fold); these were reversed on return to normoxia. The secretion of IL-6, leptin, MIF and VEGF from the adipocytes was also stimulated by exposure to 1% O(2). These results demonstrate that hypoxia induces extensive changes in human adipocytes in the expression and release of inflammation-related adipokines. Hypoxia may underlie the development of the inflammatory response in adipocytes, leading to obesity-associated diseases.

Fig. 1

Time course of quantitative changes in HIF-1α mRNA level (a), leptin mRNA level (b) and HIF-1α protein (c) during the differentiation and development of human adipocytes. Confluent human preadipocytes (day 0) were differentiated and cultured for up to 14 days. Relative mRNA levels were normalised to human POLR2A at day 0 (or day 2 when no signal was evident at day 0). Total protein concentration of HIF-1α in cell lysates was measured by ELISA. Results are means±SE (n = 3)

Fig. 2

Effect of CoCl₂ at two dose levels on HIF-1α protein concentration (a) and on the mRNA levels of HIF-1α, GLUT1 and adipokines (b) in human adipocytes. Differentiated human adipocytes at day 15 were incubated in medium containing 75 or 200 μM CoCl₂ for 24 h. Total HIF-1α protein and mRNA level of adipokines were measured as described in Fig. 1. Results are means±SE (n = 6). In a,**P < 0.01, ***P < 0.001 compared with controls; in b †P < 0.001 compared with controls. Open bars, controls; shaded bars, 75 and 200μm CoCl₂ (light and dark bars, respectively)

Fig. 3

Time course of effects of CoCl₂ (100 μM) on total HIF-1α protein level (a and b) and GLUT1 mRNA levels (c) in human adipocytes. Cells were cultured and treated as in Fig. 2. Results are means±SE (n = 4). **P < 0.01, ***P < 0.001 compared with 0-h controls

Fig. 4

Time course of effects of CoCl₂ (100 μM) on adipokine mRNA levels of FIAF, IL-6, leptin, MCP-1, MIF, PAI-1, TNF-α and VEGF (a) and adiponectin, haptoglobin and adipsin (b) in human adipocytes. Relative mRNA levels. Cells were cultured and treated as in Fig. 2. Results are means±SE (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001 compared with 0-h controls

Fig. 5

Time course of effects of hypoxia (1% O₂) on total HIF-1α protein level (a, b) and GLUT1 mRNA (c) levels in human adipocytes. Differentiated adipocytes at day 15 post-induction were exposed to 1% O₂ for up to 24 h; some cells were transferred back to normoxic conditions (21% O₂) for 1 or 16 h after 8 h of hypoxia (8- to 1- and 8- to 16-h groups). Results are means±SE (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001 compared with controls

Fig. 6

Time course of effects of hypoxia (1% O₂) on adipokine gene expression in human adipocytes.Relative mRNA levels of FIAF, IL-6, leptin, MIF, PAI-1, VEGF (a) and adiponectin, haptoglobin, MCP-1, adipsin, TNF-α (b). Cells were cultured as in Fig. 5; some cells were transferred back to normoxic conditions (21% O₂) for 1 or 16 h after 8 h of hypoxia (8- to 1- and 8- to 16-h groups). Results are means±SE (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001 compared with controls

Fig. 7

Time course of effects of hypoxia (1% O₂) on the release of adipokines from human adipocytes. Adiponectin secretion (a) and IL-6, leptin, MIF and VEGF release (b–e) after exposure to hypoxia. Adipokine concentration was measured by ELISA in the medium of cells cultured as in Fig. 6; some cells were transferred back to normoxic conditions (21% O₂) after 8 h of hypoxia and the adipokine concentration then measured 16 h later (8- to 16-h group). Values are means±SE (n = 6). *P < 0.05, **P < 0.01, ***P < 0.001 compared with controls