Quantitative secretome and glycome of primary human adipocytes during insulin resistance

Abstract

Adipose tissue is both an energy storage depot and an endocrine organ. The impaired regulation of the secreted proteins of adipose tissue, known as adipocytokines, observed during obesity contributes to the onset of whole-body insulin resistance and the pathobiology of type 2 diabetes mellitus (T2DM). In addition, the global elevation of the intracellular glycosylation of proteins by O-linked β-N-acetylglucosamine (O-GlcNAc) via either genetic or pharmacological methods is sufficient to induce insulin resistance in both cultured cells and animal models. The elevation of global O-GlcNAc levels is associated with the altered expression of many adipocytokines. We have previously characterized the rodent adipocyte secretome during insulin sensitive and insulin resistant conditions. Here, we characterize and quantify the secretome and glycome of primary human adipocytes during insulin responsive and insulin resistant conditions generated by the classical method of hyperglycemia and hyperinsulinemia or by the pharmacological manipulation of O-GlcNAc levels. Using a proteomic approach, we identify 190 secreted proteins and report a total of 20 up-regulated and 6 down-regulated proteins that are detected in both insulin resistant conditions. Moreover, we apply glycomic techniques to examine (1) the sites of N-glycosylation on secreted proteins, (2) the structures of complex N- and O-glycans, and (3) the relative abundance of complex N- and O-glycans structures in insulin responsive and insulin resistant conditions. We identify 91 N-glycosylation sites derived from 51 secreted proteins, as well as 155 and 29 released N- and O-glycans respectively. We go on to quantify many of the N- and O-glycan structures between insulin responsive and insulin resistance conditions demonstrating no significant changes in complex glycosylation in the time frame for the induction of insulin resistance. Thus, our data support that the O-GlcNAc modification is involved in the regulation of adipocytokine secretion upon the induction of insulin resistance in human adipocytes.

Keywords: Adipocytokine; Glycomics; Insulin resistance; N-linked; O-GlcNAc; O-linked; Shotgun proteomics; Tandem mass spectrometry; Type 2 diabetes.

Figure 1

Detection of O-GlcNAc levels in primary human adipocytes. Global O-GlcNAc levels are elevated in three insulin resistant conditions generated by low glucose plus PUGNAc (LG + PUGNAc), low glucose plus GlcNAcstatin (LG + GlcNAcstatin), or high glucose plus chronic insulin exposure (HG + INS).

Figure 2

Schematic flow diagram of the experimental procedure. (A) Primary human adipocytes are treated with insulin responsive (LG, normoglycemic) or two insulin resistance generating conditions (HG + INS and LG + PUGNAc). (B) Identification and quantification of the secretory proteome and N-linked glycosylation site-mapping from the conditioned media of treated primary human adipocytes (C) Characterization and quantification of N- and O-linked glycans from a whole protein extract of treated primary human adipocytes.

Figure 3

The functional categories of the primary human adipocyte secretome. The biological function analysis was determined for each protein based on the Ingenuity Pathway Analysis software (Ingenuity Systems).

Figure 4

Orthogonal validation of proteomic quantification. Equal amounts of conditioned media from primary human adipocytes was immunoblotted with antibodies against SPARC or Chitinase-3-like protein 1.

Figure 5

Characterization of the primary human adipocyte glycome by MS/MS and TIM scan. (A) Upper left panel: a full FTMS spectrum of the N-linked glycan mixture, lower left panel: the characterization of a biantennary complex N-linked glycan structure by MS/MS fragmentation, right panel: a list of predominant N-linked glycans. (B) Upper left panel: a full FTMS spectrum of the O-linked glycan mixture, upper right panel: the characterization of a core 2 O-linked glycan structure by MS/MS fragmentation, lower panel: a list of predominant O-linked glycans. (pink star): Xyl, (red inverted triangle): Fuc, (blue circle): Glc, (green circle): Man, (yellow circle): Gal, (blue square): GlcNAc, (yellow square): GalNAc, and (violet diamond): NeuAc.

Figure 6

Relative quantification of the primary human adipocytes glycome during insulin resistance using ¹³C/¹²C labeling. (A) Upper left panel: a full FTMS spectrum of ¹³C/¹²C labeled N-linked glycans in HGINS and LG, upper right panel: a FTMS spectrum to calculate ¹³C/¹²C ratios from the sum of isotopic peak areas between the isotopic pairs, lower panel: a list of relative ratios for HGINS and LG for predominant N-linked glycans. (B) Upper left panel: a full FTMS spectrum of ¹³C/¹²C labeled O-linked glycans in LGPUG and LG, upper right panel: a FTMS spectrum to calculate ¹³C/¹²C ratios from the sum of isotopic peak areas between the isotopic pairs, lower panel: a list of relative ratios for LGPUG and LG for predominant O-linked glycans.