Figure 1.
In vitro differentiated primary human adipocytes.
A. Adipocyte differentiation: Representative light micrographs of human visceral adipocytes at various stages of differentiation with and without AdipoRed staining. Similar morphology and rates of differentiation were observed in adipocytes derived from subcutaneous adipose tissue. B. Adipogenic transcription: QRTPCR data comparing transcript levels in mature human visceral adipocytes relative to undifferentiated visceral SVF referent. Transcript levels of adipogenic genes PPAR-γ, fatty acid synthase (FAS), ATGL, and SREBP1c, as well as the rate-limiting HBS enzyme GFAT, markedly increased over the course of adipocyte differentiation. Ordinate is fold change in transcript level in mature adipocytes relative to undifferentiated SVF referent; asterisk: p<0.050, paired t-test, comparing transcript levels in mature adipocytes and SVF referent; data from adipocytes from n = 6 obese subjects.
Figure 2.
HBS is up-regulated during differentiation, and hypoxia inhibits HBS in human adipocytes.
A. HBS increases during adipocyte differentiation: Representative Western blot comparing OGlcNAc levels measured by RL-2 antibody staining of protein lysates from undifferentiated visceral SVF and mature visceral adipocytes after 14 days of differentiation, with matched blot for same protein lysates probed with actin-specific antibody. Mean fold increase with differentiation determined by densitometry and normalized to actin levels = 2.00, SEM = 0.24, p = 0.025; paired t-test, n = 4 obese subjects. B. Hypoxia inhibits HBS in adipocytes: Representative Western blot comparing OGlcNAc levels measured by RL-2 antibody staining of protein lysates from mature visceral adipocytes cultured for 6 hrs in normoxic or hypoxic (N, H) conditions, with matched blot for same protein lysates probed with actin-specific antibody. Mean fold decrease with hypoxic culture relative to normoxic culture determined by densitometry and normalized to actin levels = 0.84, SEM = 0.04, p = 0.024, paired t-test, n = 8 obese subjects. C. Hypoxia inhibits HBS in adipocytes: Representative immunofluoresence microscopy comparing OGlcNAc levels measured by RL-2 antibody staining in mature visceral adipocytes cultured for 6 hrs in normoxic or hypoxic (N, H) conditions. RL-2-Green; nuclear stain: blue; mean fold decrease with hypoxic culture relative to normoxic culture referent determined by quantification of RL-2 immunfluoresence Z-stack microscopy signal normalized to cell number = 0.80, SEM = 0.04, p = 0.021, paired t-test,; n = 6 obese subjects. D. Hypoxia inhibits transcription of HBS and adipogenic genes in adipocytes: QRTPCR data comparing transcript levels in mature visceral adipocytes cultured in hypoxic conditions for 72 hours compared with matched visceral adipocytes cultured in normoxic conditions. Hypoxia decreased transcript levels of lipogenic genes PPAR-g and FAS, and the rate-limiting HBS enzyme GFAT. Hypoxia had no effect on transcript levels of ATGL, SREBP1c, or the non-rate-limiting HBS enzyme OGT. Ordinate: fold difference in transcript levels in cells cultured in N, H conditions; asterisk: p<0.050, paired t-test, comparing transcript levels in adipocytes cultured in N, H conditions; n = 7 obese subjects.
Figure 3.
Hypoxia attenuates and HBS promotes adipocyte lipogenesis.
A. Hypoxia inhibits adipocyte lipogenesis during differentiation: Lipogenesis measured by uptake of Adipo-Red reagent during visceral adipocyte differentiation in N, H conditions. Ordinate: Adipo-Red staining intensity determined by spectrometry normalized to cell number; asterisk: p<0.050, paired t-test, comparing N, H conditions; n = 6 obese subjects. Similar results were observed with subcutaneous adipocytes which demonstrated no quantitative difference in lipid accumulation at any time point c/w visceral adipocytes (data not shown). B. Azaserine inhibits lipogenesis in normoxic conditions: Visceral adipocytes were differentiated in normoxic conditions in the presence (Aza) or absence (media) of azaserine, a small molecule inhibitor of HBS. Azaserine inhibited lipogenesis over the course of adipocyte differentiation. Ordinate: AdipoRed staining intensity in arbitrary spectrophotometry units normalized to cell number; asterisk: p<0.050, paired t-test, comparing media, Aza arms; n = 7 obese subjects. C. Glucosamine slightly increases adipocyte lipogenesis in hypoxic conditions: Visceral adipocytes were differentiated in hypoxic conditions in the presence (Glc) or absence (media) of glucosamine, an HBS substrate and promoter of HBS. Glucosamine slightly increased adipocyte lipogenesis in hypoxic conditions. Ordinate: AdipoRed staining intensity in arbitrary spectrophotometry units normalized to cell number; asterisk: p<0.050, paired t-test, comparing media, Glc arms; n = 13 obese subjects.
Figure 4.
Hypoxia induces basal lipolysis in human adipocytes independent of HBS.
A. Hypoxia induces basal lipolysis in human adipocytes: Mature visceral (VAT) or subcutaneous (SAT) adipocytes were cultured in N, H conditions for 24 hrs, then glycerol release measured in supernatants with a spectrophotometric assay kit. Hypoxia induced basal lipolysis in visceral and subcutaneous adipocytes. Ordinate: glycerol concentration (mg/ml); asterisk: p<0.050, paired t-test, comparing N, H conditions; n = 10 obese subjects. B. Hypoxia does not regulate b-adrenergic stimulated lipolysis in VAT adipocytes: Mature VAT adipocytes were cultured in N, H conditions for 24 hrs with 3 mM isoproterenol then glycerol release measured in supernatants with a spectrophotometric assay kit. Hypoxia had no effect on isoproterenol-stimulated lipolysis in adipocytes. Ordinate: glycerol concentration (mg/ml); asterisk: p<0.050, paired t-test, comparing N, H conditions; n = 10 obese subjects. C. HBS does not regulate basal lipolysis in human adipocytes: Mature adipocytes were cultured in normoxic conditions for 24 hrs in the presence or absence of the HBS-inhibitor azaserine and supernatants studied for glycerol release. Ordinate: glycerol concentration (mg/ml); no statistically significant difference between media, azaserine arms for VAT or SAT adipocytes; n = 10 obese subjects.
Figure 5.
HBS regulates FAO in visceral but subcutaneous adipocytes independent of HBS.
A. Hypoxia induces FAO in VAT but not SAT adipocytes: Mature visceral (VAT) or subcutaneous (SAT) adipocytes were cultured in N, H conditions for 24 hrs, then pulsed with 3H-palmitate and release of 3H2O into supernatants studied with scintillation counting. Ordinate: counts per minute normalized to cell number; p<0.050, paired t-test, comparing N, H conditions; n = 11 obese subjects. B. HBS does not regulate fatty acid oxidation in human adipocytes: Mature adipocytes were cultured in normoxic conditions for 24 hrs in the presence (aza) or absence (media) of azaserine, pulsed with 3H-palmitate, and release of 3H2O into supernatants studied with scintillation counting. Ordinate: counts per minute normalized to cell number; no statistically significant difference between media, azaserine arms for VAT or SAT adipocytes; n = 11 obese subjects.
Figure 6.
A model for hypoxia/HBS-mediated adipocyte overflow.
HBS induces lipogenesis (LG) and inhibits lipolysis (LP) promotes FAO in VAT but not SAT adipocytes. Hypoxia also inhibits HBS. HBS in turn promotes LG and inhibitis LP in VAT but not SAT adipocytes. The net effect of hypoxia is to inhibit LG and induce LP. This shifts lipid metabolism towards LP, inhibiting adipocyte lipid storage and buffering capacity, increasing free fatty acid (FFA) release, and thus promoting systemic lipotoxicity. Depot-specific differences in the magnitude and direction of these responses add complexity.