Advanced glycation end-products regulate extracellular matrix-adipocyte metabolic crosstalk in diabetes

Abstract

The adipose tissue extracellular matrix (ECM) regulates adipocyte cellular metabolism and is altered in obesity and type 2 diabetes, but mechanisms underlying ECM-adipocyte metabolic crosstalk are poorly defined. Advanced glycation end-product (AGE) formation is increased in diabetes. AGE alter tissue function via direct effects on ECM and by binding scavenger receptors on multiple cell types and signaling through Rho GTPases. Our goal was to determine the role and underlying mechanisms of AGE in regulating human ECM-adipocyte metabolic crosstalk. Visceral adipocytes from diabetic and non-diabetic humans with obesity were studied in 2D and 3D-ECM culture systems. AGE is increased in adipose tissue from diabetic compared to non-diabetic subjects. Glycated collagen 1 and AGE-modified ECM regulate adipocyte glucose uptake and expression of AGE scavenger receptors and Rho signaling mediators, including the DIAPH1 gene, which encodes the human Diaphanous 1 protein (hDia1). Notably, inhibition of hDia1, but not scavenger receptors RAGE or CD36, attenuated AGE-ECM inhibition of adipocyte glucose uptake. These data demonstrate that AGE-modification of ECM contributes to adipocyte insulin resistance in human diabetes, and implicate hDia1 as a potential mediator of AGE-ECM-adipocyte metabolic crosstalk.

Figure 1

AGE are increased in diabetic human adipose tissue. (a) Representative fluorescence microscopy images and (b) quantified mean relative fluorescence intensity units (RFU) of AGE expression in human DM and NDM VAT and SAT. Bars with different letters indicate P < 0.050 in multiple comparison analysis. (c,d) Association of VAT or SAT AGE fluorescence intensity and serum HbA1c percentage for entire cohort. Linear model b value (b), standard error (SE) and P-values shown are unadjusted and adjusted for age and sex. VAT: n = 21 NDM, 19 DM subjects; SAT: n = 13 NDM, 12 DM subjects.

Figure 2

Glycated collagen 1 regulates adipocyte insulin signaling and Rho and AGE scavenger receptor expression. Preadipocytes from human VAT were differentiated into mature adipocytes in 2D culture +/− unglycated or glycated recombinant human collagen 1 (UC1, GC1), then studied with glucose uptake assay or RT-qPCR. (a) Glucose uptake in non-stimulated (basal) or insulin-stimulated conditions. Ordinate: mean glucose uptake measured by ³H-2D-glucose in cell lysates (cpm) normalized to cell lysate protein concentration (mg/ml); bars with different letters indicate P < 0.050; n = 15 NDM, 15 DM subjects. (b) Gene expression studied with RT-qPCR. Ordinate: mean fold difference in transcript level in GC1 arm relative to UC1 arm referent = 1; *P < 0.050, comparing transcript levels in GC1 arm vs. UC1 arm; n = 12 NDM, 12 DM subjects.

Figure 3

AGE-modified adipose tissue ECM regulates adipocyte cellular metabolism. (a) Strategy for creation of ECM-adipocyte cultures, including macroscopic photographs and scanning electron micrographs. (b) Quantified mean fluorescence of VAT treated with indicated glucose or mannitol concentrations for 72 h, with or without PNGase for final 24 h of treatment. Bars with different letters indicate P < 0.050. (c) Basal and insulin-stimulated glucose uptake in preadipocytes from visceral adipose tissue of DM and NDM obese subjects differentiated into mature adipocytes in disease-matched (DM or NDM) ECM prepared from tissues treated with 17 mM (Low) or 100 mM (High) glucose or 100 mM mannitol, +/− PNGase. Ordinates: mean glucose uptake measured by ³H-2D-glucose in cell lysates (cpm) normalized to cell lysate protein concentration (mg/ml). Bars with different letters indicate P < 0.050; n = 29 NDM, 25 DM subjects.

Figure 4

hDia1 regulates AGE-mediated ECM-adipocyte metabolic crosstalk in 3D-ECM culture. Preadipocytes were differentiated in vitro into mature adipocytes +/− (a) the hDia1 small molecule inhibitor SMIFH2 or (b) AGE receptor (RAGE) antagonist or isotype control antibodies in disease-matched (NDM or DM) ECM prepared from visceral adipose tissue treated with Low (17 mM) or High (100 mM) glucose, then studied with glucose uptake assay without (basal) or with insulin stimulation. Ordinates: mean ³H-2D-glucose uptake (cpm) normalized to cell lysate protein concentration (mg/ml). Bars with different letters indicate P < 0.050; n = 22 NDM,16 DM subjects. (c) A simplified model for AGE regulation of adipocyte glucose uptake.