Obesity is a clear risk factor for metabolic, cardiovascular and oncological diseases 2, 5. In particular, the prevalence of T2D increases with increasing BMI. Hence, SO subjects have a higher risk than moderately obese individuals 19. However, there are 7- 28.3% SO subjects, who do not develop T2D 20. Most of the studies investigated the metabolic abnormalities associated with the more common moderate obesity, while much fewer data is available for morbid obese patients 4.
Here, we have shown, for the first time, that in SO subjects: i) circulating IL-9, IL-13 and MIP-1β are associated to T2D; ii) in the progression of glucose tolerance impairment, inflammation is detectable in SAT earlier than in serum; iii) SAT-derived MSCs from IGT are already educated to secrete more TNF-α; iiii) quercetin is able to control MSC inflammation.
Obesity and T2D are characterized by systemic low-grade inflammation with higher levels of both pro-inflammatory and anti-inflammatory cytokines 10, 21–23. However, conflicting data and discrepancies about the composition of the cytokinome exist. First, we have observed that SO subjects affected by T2D displayed higher levels of serum IL-9, IL-13 and MIP-1β, as compared to IGT.
IL-9 is a pleiotropic inflammatory cytokine also implicated in pancreatic β-cell destruction in T1D 24, 25. MIP-1β, also known as chemokine CC motif ligand 4 -CCL4, is a potent macrophage attractant, which acts through the binding to CCR5, a transmembrane receptor involved in infection, inflammation and cancer 26. At variance, IL-13 is an anti-inflammatory cytokine that promotes polarization of macrophages toward a M2 phenotype: it contributes to epicardial adipose tissue remodeling and increases glucose uptake and metabolism in skeletal muscle 27, 28. Previous studies have detected increased levels of IL-9, IL-13 and MIP-1β in serum of obese individuals compared with lean controls 21–23. Other studies in T2D have also reported an increase of MIP-1β, a reduction or no change of IL-13 and no difference of IL-9 compared to NGT subjects 21, 23, 26, 27, 29. However, at our knowledge, no data are available for T2D and IGT morbid obese individuals. In this population, the increase of IL-9 and MIP-1β is consistent with their possible involvement in β-cell damage and macrophage recruitment, respectively, and contribute to the progression from IGT toward T2D. On the other side, IL-13 increase may be due to a compensatory mechanism. Indeed, all T2D individuals enrolled in the study were either newly diagnosed or with a short duration of disease, also for their relatively young age. Moreover, no significant differences were detected in the systemic levels of cytokines of SO individuals with IGT, as compared to those with NGT.
AT has a central role in the association between obesity and T2D and is now considered a prominent target for new pharmacological approaches, including adipokine-based therapeutics 8. Excess glucose, lipids and other nutrients lead to an impaired AT with altered plasticity and imbalanced production of adipokines and pro-inflammatory factors, which sustain a local low-grade inflammation that is a crucial determinant for T2D and obesity complications 5, 11, 30. We have provided evidence that SAT and SAT-derived isolated cells of SO IGT and T2D patients expressed significantly higher levels of TNF-α compared to NGT patients, with no detectable differences between T2D and IGT. TNF-α is a well-known inflammatory cytokine, with a pivotal role in the onset and progression of insulin resistance 9. TNF-α levels are increased in AT and plasma of obese individuals, and its expression is positively related to BMI with no significant relationship between fat cell size and mRNA levels 9, 31–33. In addition to TNF-α, we have found that adipocytes from IGT SO people released higher levels of IL-1β and IP-10, other key cytokines in obesity and in etiology of T2D 34. TNFα and IL-1β work together to both initiate and propagate the inflammatory process, also inducing the expression of multiple cytokines, among which IP-10 35, 36. IP-10, also known as CXCL10, has been shown to play a deleterious role in obesity as potential inhibitor of AT angiogenesis 37. Thus, our data suggest that TNF-α, IL-1β and IP-10 are not only adipocytokines associated to obesity, but also molecules with a crucial role in the early phases of impairment of glucose metabolism in SO subjects.
SAT from obese subjects contains a dysfunctional pool of MSCs with impaired adipogenesis 38, 39. We have previously shown that a truncated isoform of PPAR (PPARΔ5), which acts as a dominant-negative isoform by reducing PPARγ activity, positively correlates – whereas canonical PPARG does not - with BMI in overweight or obese and T2D patients, possibly contributing to impaired adipogenesis 40. Furthermore, an increased ratio between PPARΔ5 and canonical isoforms has been recently reported in a in vitro generated model of hypertrophic-like adipocytes, also displaying increasing IL-6 secretion, whereas only a modest reduction of canonical PPARG expression was observed 41. Here, we detected similar levels of canonical PPARG in SAT biopsies of SO individuals with different glucose tolerance conditions, as well as similar levels of lipid accumulation in differentiated adipocytes. Thus, most likely, the difference in cytokine release does not relate to a different adipocyte differentiation program.
Notably, we found that MSCs from IGT subjects are already primed to express and secrete more TNFα, compared to MSCs from NGT subjects. High glucose exposure of MSCs leads to impaired cell functions, upregulation of inflammatory genes (IL-1 β, CCL5, IL-8, MCP-1) and alteration in DNA methylation 38, 42, 43. Epigenetic modifications represent a common mechanism through which both genetic and environmental exposures impact on the susceptibility to T2D 44. It has been shown that distinct methylations of the promoter regulate TNF-α expression 45, 46. It could be hypothesized that impaired glucose levels may imprint specific epigenetic signature on MSCs, upregulating TNF-α production, which in turn contributes to impaired glucose tolerance. However, further studies are necessary to elucidate the molecular mechanisms underlining increased TNF-α levels in IGT- derived MSCs.
To date, a special attention for both prophylactic and therapeutic interventions in T2D is directed to diet nutrients, able to regulate the metabolism and influence the consumer's health 47. Quercetin is a nutraceutical compound with anti-inflammatory and antioxidant effects both in vivo and in vitro. Possible mechanisms of actions include inhibition of COX-2, NF-kB and MAPK pathways, post-translational modifications and elimination of senescent cells 47–49. Here, we have shown that quercetin ameliorates the local low-grade inflammation by reducing the expression of TNF-α and the release of a number of inflammatory cytokines in MSCs isolated from IGT and T2D morbid obese subjects. Moreover, it increases the release of G-CSF, a cytokine able to counteract the activation of major inflammatory cytokines, including TNF-α, whose anti-obesity effects have been shown in animal models with diabetes and obesity 50.