Identification of Ganglioside GM3 Molecular Species in Human Serum Associated with Risk Factors of Metabolic Syndrome

<div><p>Serum GM3 molecular species were quantified in 125 Japanese residents using tandem mass spectrometry multiple reaction monitoring. Individuals were categorized by the presence or absence of metabolic disease risk factors including visceral fat accumulation, hyperglycemia and dyslipidemia. A total of 23 GM3 molecular species were measured, of these, eight were found to be significantly elevated in individuals with visceral fat accumulation and metabolic disease, defined as the presence of hyperglycemia and dyslipidemia. All of the GM3 molecular species were composed of the sphingoid base sphingosine (d18:1 (Δ4)) and, interestingly, six of the eight elevated GM3 molecular species contained a hydroxylated ceramide moiety. The hydroxylated GM3 species were, in order of decreasing abundance, d18:1-h24:0 ≈ d18:1-h24:1 > d18:1-h22:0 » d18:1-h20:0 > d18:1-h21:0 > d18:1-h18:1. Univariate and multiple linear regression analyses were conducted using a number of clinical health variables associated with obesity, type 2 diabetes, metabolic disease, atherosclerosis and hypertension. GM3(d18:1-h24:1) was identified as the best candidate for metabolic screening, proving to be significantly correlated with intima-media thickness, used for the detection of atherosclerotic disease in humans, and a number of metabolic disease risk factors including autotaxin, LDL-c and homeostatic model assessment insulin resistance (HOMA-IR).</p></div

GM3 molecular species levels in human serum.

<p>Twelve most abundant (A) and eleven least abundant (B) GM3 molecular species detected in serum of patients with visceral fat accumulation (VFA) (n = 39), VFA with hyperglycemia (n = 15), VFA with dyslipidemia (n = 28) and VFA with both hyperglycemia and dyslipidemia (n = 17) compared with healthy lean control individuals (n = 26). Species were determined using LC-MS/MS MRM. Data are reported as means ± SD. * <i>P</i> ≤ 0.05, ** <i>P</i> ≤ 0.01, *** <i>P</i> ≤ 0.001, **** <i>P</i> ≤ 0.0001 metabolic risk factor groups vs. control; Mann-Whitney unpaired test.</p

Association between metabolic disease risk factors and GM3(d18:1-h24:1).

<p>Correlation of GM3(d18:1-h24:1) with fasting blood glucose (A), insulin (B), HOMA-IR (C), HbA1c (D), total cholesterol (E), LDL-c (F), autotaxin (G) and mean IMT (H). Spearman’s rank correlation was used to access correlation between GM3(d18:1-h24:1) and metabolic disease risk factors. All correlations were deemed significant with <i>P</i> values below 0.05.</p

Association between metabolic disease risk factors and total GM3.

<p>Correlation of total GM3 with fasting blood glucose (A), insulin (B), HOMA-IR (C), HbA1c (D), total cholesterol (E), LDL-c (F), autotaxin (G) and mean IMT (H). Spearman’s rank correlation was used to access correlation between total GM3 and metabolic disease risk factors. Correlations of fasting blood glucose, HbA1c, total cholesterol, LDL-c and mean IMT with total GM3 were deemed significant with <i>P</i> values below 0.05.</p

The sum of GM3 molecular species.

<p>Total GM3 (A) and total hydroxylated GM3 (B) detected in serum of patients with visceral fat accumulation (VFA) (n = 39), VFA with hyperglycemia (n = 15), VFA with dyslipidemia (n = 28) and VFA with both hyperglycemia and dyslipidemia (n = 17) compared with healthy lean control individuals (n = 26). Species were determined using LC-MS/MS MRM. Data are reported as means ± SD. * <i>P</i> ≤ 0.05, ** <i>P</i> ≤ 0.01, *** <i>P</i> ≤ 0.001, **** <i>P</i> ≤ 0.0001 metabolic risk factor groups vs. control; Mann-Whitney unpaired test.</p

Subject Characteristics.

<p>Means ± SE are presented. SBP, systolic blood pressure; DBP, diastolic blood pressure; TG, triglycerides; HOMA-IR and β, homeostatic model assessment insulin resistance and beta-cell function, respectively.</p><p>ns P > 0.05,</p><p>* P ≤ 0.05,</p><p>** P ≤ 0.01,</p><p>*** P ≤ 0.001,</p><p>**** P ≤ 0.0001</p><p>Subject Characteristics.</p

Serum GM3 Molecular Species Multivariate Analysis.

<p>Clinical variables found to be significantly (<i>P</i> < 0.05) correlated with 12 most abundant GM3 molecular species by multiple regression analysis. LPC, lysophosphatidylcholine; SM, sphingomyelin; EF, ejection fraction; BP, blood pressure; ATX, autotaxin; CRP, C-reactive protein; HOMA-IR, homeostatic model assessment insulin resistance; AST, aspartate aminotransferase; ALT, alanine aminotransferase; TG, triglyceride; CR, Creatinine; PLT, platelets; Hb, hemoglobin.</p><p>Risk factors associated with</p><p>* obesity,</p><p>** metabolic disease,</p><p>*** atherosclerosis,</p><p>**** hypertension and</p><p>***** nephropathy.</p><p>Serum GM3 Molecular Species Multivariate Analysis.</p

Association between metabolic disease risk factors and total hydroxylated GM3.

<p>Correlation of total hydroxylated GM3 molecular species with fasting blood glucose (A), insulin (B), HOMA-IR (C), HbA1c (D), total cholesterol (E), LDL-c (F), autotaxin (G) and mean IMT (H). Spearman’s rank correlation was used to access correlation between total hydroxylated GM3 molecular species and metabolic disease risk factors. All correlations were deemed significant with <i>P</i> values below 0.05.</p

The relative abundance of d18:1-h24:1 molecular species.

<p>Percent of d18:1-h24:1 relative to d18:1–24:1 in GM3, sphingomyelin and ceramide in individuals with metabolic disease (A). The average amounts of d18:1-h24:1 (B) and the sum of the 12 most abundant molecular species (C) of GM3, sphingomyelin and ceramide in six microliters of human serum from individuals with metabolic disease. Data are reported as means ± SD. **** <i>P</i> ≤ 0.0001 sphingomyelin and ceramide vs. GM3; Mann-Whitney unpaired test.</p