Introduction to the Context of the Study
Adipose tissue is linked to induction of insulin resistance, with different fat depots having different impact on health. Indeed, the distribution of body fat appears to be more important than the total amount of fat. Abdominal and, in particular, visceral adipose tissue has been strongly associated with the development of insulin resistance, type 2 diabetes as well as dyslipidaemia, hypertension and cardiovascular disease. Moreover, surgical removal of visceral fat leads to a metabolic improvement in insulin-resistant humans and rodents.
Over-nutrition and obesity are suggested to impose a number of stress signals on adipose tissue. These signals include inflammatory and metabolic stress, oxidative stress (an imbalance between oxidant generation and antioxidant defence mechanisms), as well as endoplasmic reticulum (ER) stress characterized by a functional overload with newly synthesized proteins and/or misfolded proteins (Rudich et al. 2007). All these stress signals are heavily interconnected. In particular, inflammatory and oxidative stress may lead to ER stress and vice versa, suggesting that adipose tissue in obesity is exposed to multidimensional stress. Importantly, it has been shown that multiple stress signals lead to phosporylation-dependent activation of certain kinases, such as stress-activated protein kinase c-Jun kinase (JNK) and inhibitory -κB kinase (IKK). Mitogen-activated protein kinases (MAPKs), of which several has been shown to play a role in insulin resistance and diabetes, including p38 MAPK (Carlson et al. 2003), c-Jun N-terminal kinase (JNK) (Sourris et al. 2009), as well as IKK (inhibitor of kappaB kinase)/NFkappaB pathway (Ruan and Pownall, 2009).
Interestingly, the molecular insights into the depot-specific stress signalling in adipose tissue, as well as its effects on insulin resistance, are not fully understood. It has been postulated that the differences are associated with a combination of anatomical, metabolic and endocrinological factors. Visceral adipocytes produce more angiotensinogen, interleukin 6 (IL-6) and PAI-1 in comparison with subcutaneous adipocytes (Wajchenberg et al. 2002). Furthermore, in vitro experiments on primary human preadipocytes indicated that omental adipocytes have an increased beta-adrenoceptor-mediated lipolysis mostly due to differences in the coupling of beta-adrenoceptor subtypes to G-proteins (Dicker et al. 2009). Klöting and co-authors (2009) have recently shown that omental and subcutaneous adipose tissue have different patterns of microRNA (miRNA) expression, which may contribute to intrinsic differences between the fat depots. At the level of protein expression, a large-scale proteomic analysis of omental and subcutaneous fat depots revealed differences in expression of 43 proteins, mostly involved in glucose and lipid metabolism, lipid transport, protein synthesis and protein folding, but also in response to stress and inflammation ( Pérez-Pérez et al. 2009). Importantly, the differences in gene expression, adiponectin secretion as well as insulin signalling are retained in subcatenous and visceral adipocytes differentiated in vitro from precursor stromal cells (Perrini et al. 2008).
Considering the clear differences between omental and subcutaneous fat depots, as well as reported effect that omental adipocytes can exert on subcutaneous adipocytes and other tissues with respect to insulin action (Lundgren et al. 2004), it is of primary importance to understand in detail the molecular mechanisms underlying this phenomenon. The study by Bashan and co-authors focuses on analysis of depot-specific changes in MAPK (p38 MAPK, JNK1 and ERK1) as well as IKK signalling, and associated alterations in insulin signalling.
In the study by Bashan and co-authors several biochemical and molecular biology techniques were used to determine the cell signalling events within paired samples of omental (OM) and abdominal-sc adipose tissue obtained from patients undergoing abdominal surgery for gastric banding, weight reduction, or exploratory. The patient group was very well characterized, including body mass index (BMI), fasting insulin and glucose levels, blood pressure as well as triglyceride and HDL cholesterol levels analysis.
Authors used standard Western blotting to examine expression of a panel of cell signalling proteins. The assay was done to the high standard, using actin as a loading control, and finally densitometry analysis to allow presentation of the results in a quantitative manner. Importantly, western blot analysis was performed 2 to 4 times on the same samples. This ensures that the technical variation in the results can be detected and minimized. It is a good practise to publish the exact catalogue numbers of the antibodies used, which has not been done in this particular paper.
Standard procedures of quantitative RT-PCR were used to study gene expression. Authors performed a relative quantification, using 18S as a house-keeping gene internal control. Although often several internal controls are used to ensure that there are no changes in the expression of house-keeping genes, 18S is commonly considered as the best possible internal control. Alternatively, the authors could have used primers for beta actin, as they did show using western blot analysis that the expression of actin remains constant between OM and sc fat. Still, the authors do not report what was the RNA equivalent used in the qRT-PCR reactions, and also failed to describe the chemistry used to detect the accumulation of PCR product.
Importantly, all the results were analysed for statistical significance, and clearly marked with p values. The statistical analyses were performed both for gene and protein expression experiments, as well as for comparison of clinical characteristics.
Summary of the Findings and the General Context of the Study
In their study Bashan and co-authors report that the expression of all the investigated kinases (p38MAPK, JNK1, ERK2 and IKKbeta) is higher in human omental adipose tissue (OM) as compared with subcutaneous adipose tissue. The increased expression of the kinases was detected at both protein and mRNA levels. Importantly, omental fat exhibited an increased activation of p38MAPK, JNK1 and IKKβ, as revealed by western blot analysis with antibodies recognizing phosphorylated forms of the proteins of interest. Of utmost importance, Bashan et al. observed that obesity is associated with increased p38 MAPK and JNK1 protein expression and phosphorylation, indicative of enhanced activation of these kinases. No significant differences were detected in the activation ERK2 or IKKβ between obese and lean subjects. Interestingly, these results are in conceptual agreement with data published earlier by Hirosumin and co-authors, who observed an increased JNK activity in adipose tissue in dietary and genetic (ob/ob) models of obesity (Hirosumi et al. 2002). Moreover, since the publication of Bashan’s study, several other papers have confirmed the findings.
The most important question with regards to the clinical significance of stress-signalling pathway activation in omental adipose tissue is whether the kinase activity correlates with clinical parameters. Bashan et al. indicate that phosphorylation of p38 MAPK is associated with insulin resistance and triglyceride levels, and possibly with BMI. Interestingly, the authors suggest that phosphorylation of JNK1 does not have as significant effect on these clinical parameters. It is worth noting that discrepancy exists between the studies. For example, phosphorylation of JNK1/2 in adipose tissue is reportedly not associated with obesity although there is a strong association with insulin sensitivity (Sourris et al. 2009). However, the abovementioned conclusion was based on experiments performed on subcutaneous adipose tissue.
The correlation between p38 MAPK phosphorylation in omental adipose tissue and clinical parameters in obese subjects is certainly a very promising finding. However, molecular and cellular signalling that links the activity of p38 MAPK should be dissected. Indeed, the same group has recently conducted a study to assess the signalling events upstream of p38 MAPK and JNK in adipose tissue. Interestingly, these experiments identified an activated stress-sensing pathway, consisting of the MAP3 kinase Ask1 and MAP2 kinases MKK4,3/6 (Blüher et al. 2009). More direct experimental evidence on the role of p38 MAPK in omental adipose tissue, as well as its transcriptional and posttranscriptional regulation is required. Finally, apart from western blot analysis of phosphorylation status, a direct assay to assess the activity of p38 MAPK should be employed.
Studies utilizing primary human tissues have obvious limitations of low sample numbers. However, as differences between subcutaneous and visceral adipocytes are retained upon their differentiation in vitro, more detailed molecular and mechanistic analysis could be performed using human cell lines. Experiments using cells cultured in vitro, as well as animal models, could help in dissecting the critical role of p38 MAPK in adipose tissue, for example by using pharmacological inhibitors (eg. PD169316), knock-down strategies (siRNAs or shRNAs), or a knock-out approach. In particular, the role of p38 MAPK in insulin-mediated glucose uptake by adipose tissue remains controversial. It has been recently suggested that transcriptional activity of p38 MAPK regulates the expression of peroxisome proliferator-activated receptor (PPAR)gamma, peroxisome proliferator activator receptor-gamma coactivator 1 (PGC-1), and tricarboxylic acid cycle and oxidative phosphorylation mitochondrial genes (Aouadi et al. 2006; Crunkhorn et al. 2007). Similarly, JNK1 has been shown to regulate hepatic insulin signalling, mitochondrial biogenesis, fatty acid oxidation, oxidative phosphorylation, and TCA cycle (Yang and Trevillyan, 2008).
Understanding of the exact mechanism by which p38 MAPK leads to insulin resistance will hopefully assist in the development of targeted therapies. Inhibition of p38 MAPK as such would have multiple effects on plethora of cell signalling pathways. Therefore, it is of primary importance to identify down-stream targets of p38 MAPK that are directly linked with altered insulin-signalling pathways.