Dysregulation of the heat shock response is a crucial event that leads to diabetes through the development of insulin resistance. However, the contribution of obesity to this dysregulation in humans has not yet been investigated. In the current study, we profiled the expression pattern of various components of the heat shock response in lean and obese subjects and established the possible correlations with various physical, clinical and biochemical parameters of the study population. We also investigated whether physical exercise could restore the expression of proteins showing differential expression between lean and obese subjects. The main findings of this investigation are: 1) obesity increased significantly the expression of the majority HSPs investigated while it had no effect on the expression of HSP-27 and reduced significantly the expression of DNAJB3-HSP-40, 2) the expression levels of HSP-72 in obese subjects are linked to the diabetes status of the study population, and 3) a 3-months physical exercise protocol was sufficient to restore the abnormal expression of HSPs with a concomitant decrease in the inflammatory response.
To the best of our knowledge, this is the first study that investigated simultaneous expression of components of the HSR in non-diabetic human obese subjects using adipose tissue as well as evaluating their response to physical exercise. It is worth noting that the observed expression pattern of the HSR between lean and obese subjects was not related to gender as both males and females have shown the same trend at mRNA and protein levels (data not shown).
The fact that we observed a similar pattern in the expression of the HSR in PBMCs and the adipose tissue indicate that HSR dysregulation is not only limited to the subcutaneous adipose tissue and thus PBMCs could be an interesting surrogate target for follow up studies to understand the mechanisms leading to dysregulated expression of the HSR by obesity.
It is well established that in obesity, the uncontrolled inflammatory reaction and impairment of the host defense system, plays an important role in the inhibition of insulin receptor signaling cascade and as a direct consequence, disruption of systemic metabolic homeostasis that leads to T2D . Recently, it was proposed that T2D is the result of a metabolic paradigm in which metabolic inflammation; insulin resistance and impairment of the HSR work in a vicious cycle [34, 35]. Our findings that investigated the sole contribution of obesity on the status of the heat shock response indicate that the defect in HSR observed previously in obesity-induced insulin resistance and diabetes [5, 6, 18] is more complex than what was initially thought and it presumably involves different mechanism of action.
The activation of the HSR is a crucial step required to maintain normal homeostasis in response to physiological and patho-physiological damages. It is well known that exposure to a preconditioning stress increases a cell's tolerance to subsequent stress, and this effect has been shown to be partially due to an increase in HSP synthesis; particularly HSP-27, HSP-60 and HSP-72 following the preconditioning stress . Increased content of HSPs is thought to restore cellular homeostasis and remodelling, and protect against further cellular stress and damage in vulnerable tissue organs [37, 38]. In the context of obesity, the observed dysregulation of the HSR can be suggested as a physiological response to alleviate metabolic stress in key tissue organs such as the adipose tissue and muscles. It is well established that obesity is associated with activation of JNK stress kinase [31, 39] and enhanced inflammatory response as monitored by the endogenous levels of IL-6, TNF-α, RANTES and CCR5 receptor in the adipose tissue . Thus, the up-regulation of the HSPs in obese subjects is presumably required to mitigate the inflammatory response and to alleviate various forms of metabolic stress response induced by obesity. It is not however impossible that the observed increase in the expression of HSPs in the current study could be a compensation for reduced expression of DNAJB3.
Several studies showed that interventions leading to induction of the HSR or chaperone therapy such as heat therapy [18, 25], transgenic mice or liposomal delivery of HSPs , electrical therapy , physical exercise  and pharmacological drugs [27, 28] were associated with beneficial outcomes as monitored by improved glucose homeostasis, enhanced insulin sensitivity, reduction of visceral adiposity and suppression of the chronic inflammatory state [16, 41–43]. Nevertheless, it is worth noting that most of these studies investigated the status of the HSR in human and animal models of obesity-induced insulin resistance and diabetes and they mainly focused on the expression of HSP-72 and HSP-25/27 in skeletal muscles. In agreement with those studies, we found that HSP-72 expression levels in the subcutaneous adipose tissue were attenuated in a diabetic obese group as compared to non-diabetic obese subjects. In non-diabetics obese subjects, our data indicated that HSP-72 was increased in obese subjects as compared to lean subjects. The mechanisms underlying the differential regulation of HSP-72 in diabetics and non-diabetic obese subjects are still unknown. In line with this, previous studies have report discrepancy in the expression levels of HSP-72 depending on the tissues [44, 45].
Another important aspect that was investigated in the current study is the effect of physical exercise on the expression pattern of components of HRS in obese subjects. Physical exercise is one of the highly recommended strategies to reduce weight and to improve the clinical outcomes in obese and diabetic patients although the exact molecular mechanisms underlying these beneficial effects are not yet well established. Our findings indicated that a 3-month aerobic exercise was sufficient to restore the expression of the HSR in obese subjects to normal levels observed in lean subjects. Under the same conditions, the endogenous levels of RANTES and its CCR5 receptor , IL-6 and TNF-α in the adipose tissue of obese subjects were significantly reduced by physical exercise (Figure 4). It is worth noting that the exercise affected similarly the expression pattern of the HSR in both male and female obese subjects (data not shown). Some previous studies have investigated the effect of a short time “acute” exercise on the expression of HSPs in males and females and the results were not consistent. For example, Gillum et al.  reported the effect of a single acute exercise reported that HSP-72 was more increased in men than women, whereas Njemini et al.  reported no difference in HSP expression between genders. Nevertheless, up to the best of our knowledge no report is available on a gender-linked effect of long-term exercise on the expression of HSP in human.
Our current findings are however contrasting previous studies that reported increased expression of HSPs by physical exercise [6, 18, 38, 41, 48]. One of the possible reasons for this discrepancy is that these investigations focused on acute effect of exercise on transient expression of HSPs either immediately after [6, 18, 41, 48] or within 7 days of exercise using a single-session exercise . The different behavior of HSPs from the adipose tissue in our study as compared to those previous studies could also be due to the non-damaging and long-term nature of our physical exercise protocol. Indeed, exercise is considered as a stress factor in particular when it is intensive and the type of exercise strongly influences the levels of circulating HSp-72 (eHSP-72) in blood for example . For instance, in treadmill or downhill running aerobic exercise increased several times both cellular HSP-72 and blood eHSP-72 during and immediately after exercise [50, 51]. In contrast, elbow flexion eccentric exercise doesn’t increase eHSP-72 . The transient increase in HSPs could be assigned to the elevation of temperature in the tissues during exercise as temperature is critical for the activation of HSR in exercising mammals [53–55]. As the adipose tissue, in particular the subcutaneous one, is superficial and has less vascularisation, its temperature increase will be limited compared to other organs. For example the temperature of muscles and core body at the end of 45 min moderate treadmill exercise exceeded 39°C . Thus, the observed elevated and long-lasting HSP levels in the cells may have better benefits, as compared to only transient effect, for the body to cope with the low-grade inflammation characterising obesity. Other authors have already suggested, in agreement with our current study, that HSP are expressed and induced by exercise in a tissue-specific manner [38, 56].
In summary, we demonstrated here that obesity triggers differential regulation of various components of the HSR in obese non-diabetic subjects and a 3-month physical exercise was sufficient to restore the normal expression of HSPs in the adipose tissue with concomitant attenuation in the inflammatory response. This suggests that the in non-diabetic obese human the body is still able to cope with the obesity-related cellular stress via an upregulation of key cytoprotective HSPs in adipose tissue and PBMCs. Given the well documented decrease of HSPs in insulin-resistant and diabetic subjects, it might be interesting to enhance those proteins in non-diabetic obese and sustain their over-expression to prevent the development of further metabolic disorders including diabetes. Further studies are warranted to examine the expression levels of HSPs in visceral adipose tissue and other organs of non-diabetic obese before suggesting precise biological significance of increased HSPs expression due to obesity and the beneficial effects of physical exercise.