Markers of remodeling in subcutaneous adipose tissue are strongly associated with overweight and insulin sensitivity in healthy non-obese men

In this study on healthy men without metabolic disease we observed that: (1) gene expression in subcutaneous AT of MMP-9, TIMP-1 and PAI-1, reflecting ECM remodeling, strongly associated with the amount of abdominal AT, assessed by CT, (2) the expression of these remodeling markers was strongly associated with glucometabolic variables including insulin sensitivity and BMI, (3) measured in the circulation, only PAI-1 correlated to the amount of AT and to the corresponding gene expression, and (4) PAI-1 was also the only marker measured in the circulation which correlated significantly to any of the glucometabolic variables. Interestingly, when excluding the subset of subjects having a family history of diabetes, these associations were even strengthened.

It is well known that VAT is inflamed, especially in obesity and in subjects with the metabolic syndrome and in patients with atherosclerotic coronary artery disease, and is capable of secreting large amount of pro-inflammatory and atherogenic substances28 . Although the genes in our study were measured in subcutaneous AT, similar associations were observed in all compartments i.e. sSAT, dSAT and VAT, suggesting the genes measured in subcutaneous AT to reflect an ongoing overall remodeling of the AT. MMP-9 has previously, in a small study, been shown to be higher expressed in VAT in obese compared to lean individuals29, but also to be reduced in subcutaneous AT after weight loss in obese patient with metabolic syndrome30. We have recently published data from the present population, were we observed strong associations between the amount of AT and gene expression of the pro-inflammatory cytokine interleukin-18 and the NLR family pyrin domain containing-3 inflammasome (NLRP3) and further between these inflammatory markers and the glucometabolic state31. Taken together with the present findings, it seems that elevated amount of AT in non-obese healthy individuals, is accompanied by increased upregulation of inflammation and remodeling, and further reflected in the glucometabolic state, as also shown in obese subjects32.

Gene expression of MMP-9 and PAI-1 correlated significantly to all the measured glucometabolic variables, including insulin sensitivity assessed by glucose-clamp, waist circumference and BMI. A similar pattern was observed for TIMP-1, which, in addition to be an inhibitor of MMPs, also has been shown associated with tissue remodeling33. The results were strengthened when dichotomizing the glucometabolic levels at median values, showing MMP-9 expression especially, to be significantly higher with less favorable values for all the glucometabolic variables. Our results are supportive to a study on non-diabetic subject, which also showed reduction in subcutaneous AT MMP-9 expression with improved insulin sensitivity after treatment with insulin-sensitizing drugs32. In line with our results, it was recently also shown that expression of MMP-9 and TIMP-1 in AT from healthy overweight and obese individuals were associated with BMI, and especially MMP-9 was also strongly related to glucose clamp assessed insulin sensitivity, independent of BMI34. The authors discusses and concluded that the remodeling process together with adipogenesis in AT are the initial processes leading to reduced insulin sensitivity. This hypothesis is even strengthened by our additional findings of the strong associations with the amount of AT assessed by CT and further, that MMP-9 expression was high in individuals with poor insulin sensitivity, either high or low BMI. With our cross-sectional approach any course or consequence cannot be discussed, however, as shown (Supplementary Table 2), increased fat mass in all compartments associated with lower GDR, i.e. that fat mass may be the driver of reduced GDR, and that the ECM modifiers have only indirect relationship to GDR, via increased fat mass.

GALECTIN 3 expression did not correlate to BMI, but was inversely related to insulin sensitivity, insulin and C-peptide, although vanished after Bonferroni correction. This is to some degree in line with results from an animal model showing that GALECTIN 3 deficiency mice fed on a high fat diet develop increased VAT and increased fasting glucose, HbA1c and insulin resistance, indicating GALECTIN 3 to be protective against T2DM and obesity35. In another study increased accumulation of advanced glycation end products (AGEs) in the pancreatic islets was shown with GALECTIN 3 deficiency, suggesting GALECTIN 3 also to play a protective role against AGE related injury36.

The only significant association found between the measured markers in circulation and the corresponding gene expression in AT was for PAI-1, which is in line with others studies, suggesting AT to be a major source of circulating PAI-137,38. These findings are further supported in our study by PAI-1 being the only circulatory marker that also correlated to the amount of abdominal AT, in all compartments. PAI-1 was also the only circulatory marker that associated significantly to the glucometabolic variables, which is in line with accepted knowledge39. Thus, our hypotheses that the circulating levels of the biomarkers would mirror the corresponding genes expressed in AT and the amount of AT, were only very limited fulfilled.

The lack of correlation between circulating MMP-9, TIMP-1 and GALECTIN 3 and their levels of gene expression in AT as well as the amount of AT, indicates that AT is not the main source for these circulatory levels, which also has been suggested by others30, and enlargement of AT per se seems thus not to affect the levels of these biomarkers in the circulation. It should also be emphasized that although the remodeling markers were upregulated at the gene level, not all proteins undergo the post-transcriptional changes mandatory to function as an active protein40. Nevertheless, these results indicate that dysregulation in AT, shown by genetic upregulation of the remodeling markers, results in a non-beneficial glucometabolic state, in our healthy non-obese individuals. This hypothesis might be due to reduced storage capacity and reduced/dysfunctional glucose receptors, as also suggested by others41,42,43,44. It may, however, also be vice versa, that a dysregulated glucometabolism may result in non-beneficial AT remodeling.

Although some previous studies have shown associations between obesity, remodeling of AT, systemic inflammation and insulin resistance, our results contribute to elucidate early changes in AT also in healthy individuals, to be strongly association with insulin resistance and overweight. This is further underscored by the findings that those having a family history of diabetes, thus, probably already having more disturbed glucometabolism, no such associations were found. The number of subjects in this group was small and the results have to be confirmed in other studies.

Limitations

Our study is limited by its cross-sectional design and that only healthy men are included, and the results can therefore not be generalized. It nevertheless, supports experimental studies in this field45. A limitation is also that we used subcutaneous adipose tissue samples as sampling from other anatomic areas was not feasible. The results can therefore not be directly translated to adipose tissue in general. The markers investigated selected out of the complex process of remodeling, involving a number of proteins including collagens, can be discussed. The selection was based to elucidate corresponding proteins at a measurable level in the circulation, as indicated in our hypothesis. It should also be emphasized that our results occur from adipose tissue, not distinguished between adipocytes and macrophages.

Strengths in our study are CT-measurement of AT amount in different compartments, and the measure of insulin sensitivity performed by glucose clamp technique.

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