In the present study we have used a proteomic approach to compare, for the first time, subcutaneous and visceral fat from the same patient with type 3 obesity and with or without T2DM as well as their serum proteome.
Obesity is an important risk factor for cardiovascular diseases (CVD). However, a protective role of the adipose tissue in certain clinical conditions has been described, giving rise to the “obesity paradox”, defined as the duality behavior of the adipose tissue . Until now, most of the studies carried out on AT have been performed by using non-invasive imaging technologies such as MRI, X-ray or ultrasound [19–21] as well as in different animal models. However, some limitations appear when animal findings are translated to human studies [22, 23]. Concerning human studies, there are a few studies that have compared the proteomic profile of both SAT and VAT of the same individual, but results have been quite different. Pérez-Pérez et. al.  found an increased expression of metabolic related proteins in VAT compared with SAT, but Insener et. al.  did not reproduce similar findings. Additionally, obesity is associated with the development of insulin resistance and type-2 diabetes mellitus (T2DM), although not all obese patients become diabetic or insulin resistant. Therefore, serum and adipose tissue composition, or adipose tissue location might differ in obese patients with or without any other metabolic complication [20, 25]. The proteomic signature of VAT from diabetic and non-diabetic patients was previously described but a little number of changes was reproduced among the different groups . However SAT proteomic signature in diabetic and non-diabetic has not been investigated.
VAT associates with higher metabolic complications than SAT  and accordingly, it is not surprising to see powerful changes on visceral adipose tissue compared with subcutaneous tissue, as a result of diabetes. Likewise, changes on SAT are related to the endocrine system, whereas changes in VAT are linked to metabolic disease, supporting the involvement of VAT in metabolic disorders in comparison with that of SAT. Thus, an altered metabolic and inflammatory state was observed both in adipose tissue and in serum suggesting a coordinated effect probably caused by the altered protein secretion from adipocites to the bloodstream. Among all the identified proteins in AT, only Hemopexin (HPX), actin related protein-2 (Arp2) and 14-3-3-protein beta/alpha (YWHAB) were reduced in VAT compared with the SAT. HPX and Arp2 are both only modified in the diabetic group, and both associated with adipocyte differentiation and development [28, 29] suggesting adipocyte dysfunction in VAT of diabetic patients. On the other hand, a relative increased expression in VAT compared with SAT, was observed in many other proteins, highlighting the presence of two proteins, kazrin and N(G)-dimethylarginine dimethylaminohydrolase 2 (DDAH2).
Kazrin was initially identified bounded to the N-terminal domain of the cytolinker periplakin . Multiple functions have been reported, including regulation of desmosome assembly , epidermal differentiation by regulation of keratinocytes adhesion and differentiation , but its association with visceral adiposity was unknown, in fact, kazrin had never before been detected in any adipose tissue. Desmosomes are intercellular junctions of epithelia and cardiac muscle. Disruption of desmosomal adhesion implicates human health problems, mutations in genes encoding desmosomal proteins have been related with arrhythmogenic right ventricular dysplasia, a heart muscle disease characterized by life-threatening arrhythmias and increased risk of sudden heart failure . The presence of Kazrin in AT could indicate the degree of AT dystrophy.
By contrast, DDAH2, protein narrowly related with insulin secretion , was previously detected in VAT, but not in SAT, and therefore, no one has previously compare the expression of this protein in different fat depots. The presence of these two proteins in AT underwrites the need to further investigate adipose cells function and regulation that is highly dependent on fat location
Besides its undoubted link to metabolic disorders, obesity has also a direct effect in oxidative stress that is present in countless diseases. This might be one potential connector of obesity with related disorders such as T2DM and cardiovascular diseases [34, 35]. Indeed, diets rich on carbohydrates induce oxidative stress and inflammation state in obese individuals . Also, Murri et al have previously described the effect of DM in the visceral oxidative proteome . They described an antioxidant state in the visceral fat of diabetic patients that could act as a defense against the metabolic stress induced by insulin resistance, diabetes and hyperglycaemia. In line with these, we aimed to compare the differential oxidative stress among the different fat depots, and we observed an up-regulation of different oxidation related proteins in VAT compared with SAT, including antioxidant proteins as Paroxiredoxin-6 , gluthatione-S Transferase (GSTP1) , and the glutathione related protein disulfide isomerase-3 (PDIA3) , supporting the statement that visceral adipose tissue from diabetic patients could try to compensate the characteristic oxidative stress that comes up with the impaired glucose handling. However, not only oxidation-related proteins were up-regulated in VAT but also proteins related with coagulation, inflammation and cholesterol efflux indicating that VAT is a much more active fat depot than SAT. Besides the differential adipose tissue protein composition, we also observe changes at serum levels, related both to obesity and DM. Obesity is known to be associated with a low-grade pro-inflammatory state and many inflammatory adipokines are altered in the secretome of obese patients compared with lean subjects [39, 40]. This also happens with the proteins secreted by the adipose tissue, adipokines such Leptin, Adiponectin and Visfatin among others, are dysregulated as a response of fat accumulation . Thus, as it could be expected, the pro-inflamatory adipokines Leptin, Adipsin, Insulin, C-Peptide, Chitinase 3-like 1, Tumor necrosis factor receptor 1 (TNF-R1), and TNF-ligand superfamily member 13 (TNFSF13 or APRIL) were found in higher levels in the serum profile of the obese group compared with lean subjects and osteocalcin, which improves insulin resistance by decreasing inflammation , was found in lower levels in the obese patients group. A narrow relation between insulin resistance, DM and inflammation is also generally accepted . Therefore, a coordinated pro-inflammatory state is observed not only in the adipose tissue, but also in the serum proteins as a response to obesity and diabetes. However, serum protein composition is more complex than just adipose tissue secreted proteins, being also affected by the secretion of other cytokines produced by other organs such as the pancreas, the liver, and the hypothalamus  and cells from the cardiovascular system.
At circulating level, we have observed significant higher levels of the gut derived incretin hormones, specifically the obese patients showed higher levels of Ghrelin, GIP, GLP-1 and Glucagon. Incretin hormones have an important role in glucose homeostasis . In fact, T2DM patients show a decreased bioavailability of these proteins. In previous studies, incretins have showed an anti-inflammatory, antioxidant and anti-apoptotic effect and they are major players on appetite and satiety, being also widely related with obesity . Interestingly, serum levels of PAI-1, an inhibitor of fibrinolysis usually up-regulated in obesity  were not much affected in our patients. This could be caused by the hypocaloric diet prior bariatric surgery received by the patients that could have affected their metabolic profile. However, as expected, the weight loss of those patients (around the 5 to 8% of the total body mass) was not enough to achieve a total improvement on the serum profile of type 3 obese subjects, and the down-regulation of Adiponectin, protein negatively correlated with body mass  and the up-regulation of adipsin and leptin, both proteins usually increased with body mass gain , were observed in the serum proteome of this type 3 obese individuals.
Not only obesity, but also diabetes affects the serum proteome. However, unlike in adipose tissue composition where metabolic, oxidation, coagulation and inflammatory proteins were altered, mainly inflammatory cytokines as IL-8, TNFSF13 B, TNFR1 and TNFR2  and the metalloproteinase MMP-3 were affected in the serum of diabetic patients, independently on the presence of obesity. This pro-inflammatory state is however accompanied with the increase of insulin in DM patients and adipsin levels in obese diabetic patients. Adipsin, which is a member of the complement system, is also associated with the protection of β-cells and therefore positively related to insulin levels .