Abdelkrim Khadir

Proteomics Analysis of Human Obesity Reveals the Epigenetic Factor HDAC4 as a Potential Target for Obesity

Sedentary lifestyle and excessive energy intake are prominent contributors to obesity; a major risk factors for the development of insulin resistance, type 2 diabetes and cardiovascular diseases. Elucidating the molecular mechanisms underlying these chronic conditions is of relevant importance as it might lead to the identification of novel anti-obesity targets. The purpose of the current study is to investigate differentially expressed proteins between lean and obese subjects through a shot-gun quantitative proteomics approach using peripheral blood mononuclear cells (PBMCs) extracts as well as potential modulation of those proteins by physical exercise. Using this approach, a total of 47 proteins showed at least 1.5 fold change between lean and obese subjects. In obese, the proteomic profiling before and after 3 months of physical exercise showed differential expression of 38 proteins. Thrombospondin 1 (TSP1) was among the proteins that were upregulated in obese subjects and then decreased by physical exercise. Conversely, the histone deacetylase 4 (HDAC4) was downregulated in obese subjects and then induced by physical exercise. The proteomic data was further validated by qRT-PCR, Western blot and immunohistochemistry in both PBMCs and adipose tissue. We also showed that HDAC4 levels correlated positively with maximum oxygen consumption (VO2 Max) but negatively with body mass index, percent body fat, and the inflammatory chemokine RANTES. In functional assays, our data indicated that ectopic expression of HDAC4 significantly impaired TNF-α-dependent activation of NF-κB, establishing thus a link between HDAC4 and regulation of the immune system. Together, the expression pattern of HDAC4 in obese subjects before and after physical exercise, its correlation with various physical, clinical and metabolic parameters along with its inhibitory effect on NF-κB are suggestive of a protective role of HDAC4 against obesity. HDAC4 could therefore represent a potential therapeutic target for the control and management of obesity and presumably insulin resistance.

DNAJB3/HSP-40 cochaperone is downregulated in obese humans and is restored by physical exercise.

Obesity is a major risk factor for a myriad of disorders such as insulin resistance and diabetes. The mechanisms underlying these chronic conditions are complex but low grade inflammation and alteration of the endogenous stress defense system are well established. Previous studies indicated that impairment of HSP-25 and HSP-72 was linked to obesity, insulin resistance and diabetes in humans and animals while their induction was associated with improved clinical outcomes. In an attempt to identify additional components of the heat shock response that may be dysregulated by obesity, we used the RT2-Profiler PCR heat shock array, complemented with RT-PCR and validated by Western blot and immunohistochemistry. Using adipose tissue biopsies and PBMC of non-diabetic lean and obese subjects, we report the downregulation of DNAJB3 cochaperone mRNA and protein in obese that negatively correlated with percent body fat (P = 0.0001), triglycerides (P = 0.035) and the inflammatory chemokines IP-10 and RANTES (P = 0.036 and P = 0.02, respectively). DNAJB positively correlated with maximum oxygen consumption (P = 0.031). Based on the beneficial effect of physical exercise, we investigated its possible impact on DNAJB3 expression and indeed, we found that exercise restored the expression of DNAJB3 in obese subjects with a concomitant decrease of phosphorylated JNK. Using cell lines, DNAJB3 protein was reduced following treatment with palmitate and tunicamycin which is suggestive of the link between the expression of DNAJB3 and the activation of the endoplasmic reticulum stress. DNAJB3 was also shown to coimmunoprecipiate with JNK and IKKβ stress kinases along with HSP-72 and thus, suggesting its potential role in modulating their activities. Taken together, these data suggest that DNAJB3 can potentially play a protective role against obesity.

Physical Exercise Reduces the Expression of RANTES and Its CCR5 Receptor in the Adipose Tissue of Obese Humans

RANTES and its CCR5 receptor trigger inflammation and its progression to insulin resistance in obese. In the present study, we investigated for the first time the effect of physical exercise on the expression of RANTES and CCR5 in obese humans. Fifty-seven adult nondiabetic subjects (17 lean and 40 obese) were enrolled in a 3-month supervised physical exercise. RANTES and CCR5 expressions were measured in PBMCs and subcutaneous adipose tissue before and after exercise. Circulating plasma levels of RANTES were also investigated. There was a significant increase in RANTES and CCR5 expression in the subcutaneous adipose tissue of obese compared to lean. In PBMCs, however, while the levels of RANTES mRNA and protein were comparable between both groups, CCR5 mRNA was downregulated in obese subjects (P < 0.05). Physical exercise significantly reduced the expression of both RANTES and CCR5 (P < 0.05) in the adipose tissue of obese individuals with a concomitant decrease in the levels of the inflammatory markers TNF-α, IL-6, and P-JNK. Circulating RANTES correlated negatively with anti-inflammatory IL-1ra (P = 0.001) and positively with proinflammatory IP-10 and TBARS levels (P < 0.05). Therefore, physical exercise may provide an effective approach for combating the deleterious effects associated with obesity through RANTES signaling in the adipose tissue.

Map kinase phosphatase dusp1 is overexpressed in obese humans and modulated by physical exercise

Chronic low-grade inflammation and dysregulation of the stress defense system are cardinal features of obesity, a major risk factor for the development of insulin resistance and diabetes. Dual-specificity protein phosphatase 1 (DUSP1), known also as MAP kinase phosphatase 1 (MKP1), is implicated in metabolism and energy expenditure. Mice lacking DUSP1 are resistant to high-fat diet-induced obesity. However, the expression of DUSP1 has not been investigated in human obesity. In the current study, we compared the expression pattern of DUSP1 between lean and obese nondiabetic human subjects using subcutaneous adipose tissue (SAT) and peripheral blood mononuclear cells (PBMCs). The levels of DUSP1 mRNA and protein were significantly increased in obese subjects with concomitant decrease in the phosphorylation of p38 MAPK (p-p38 MAPK) and PGC-1α and an increase in the levels of phospho-JNK (p-JNK) and phospho-ERK (p-ERK). Moreover, obese subjects had higher levels of circulating DUSP1 protein that correlated positively with various obesity indicators, triglycerides, glucagon, insulin, leptin, and PAI-1 (P < 0.05) but negatively with V̇O(2max) and high-density lipoprotein (P < 0.05). The observation that DUSP1 was overexpressed in obese subjects prompted us to investigate whether physical exercise could reduce its expression. In this study, we report for the first time that physical exercise significantly attenuated the expression of DUSP1 in both the SAT and PBMCs, with a parallel increase in the expression of PGC-1α and a reduction in the levels of p-JNK and p-ERK along with attenuated inflammatory response. Collectively, our data suggest that DUSP1 upregulation is strongly linked to adiposity and that physical exercise modulates its expression. This gives further evidence that exercise might be useful as a strategy for managing obesity and preventing its associated complications.

Immunohistochemical profiling of the heat shock response in obese non-diabetic subjects revealed impaired expression of heat shock proteins in the adipose tissue

BackgroundObesity is characterized by a chronic low-grade inflammation and altered stress responses in key metabolic tissues. Impairment of heat shock response (HSR) has been already linked to diabetes and insulin resistance as reflected by decrease in heat shock proteins (HSPs) expression. However, the status of HSR in non-diabetic human obese has not yet been elucidated. The aim of the current study was to investigate whether obesity triggers a change in the HSR pattern and the impact of physical exercise on this pattern at protein and mRNA levels.MethodsTwo groups of adult non-diabetic human subjects consisting of lean and obese (n = 47 for each group) were enrolled in this study. The expression pattern of HSP-27, DNAJB3/HSP-40, HSP-60, HSC-70, HSP72, HSP-90 and GRP-94 in the adipose tissue was primarily investigated by immunohistochemistry and then complemented by western blot and qRT-PCR in Peripheral blood mononuclear cells (PBMCs). HSPs expression levels were correlated with various physical, clinical and biochemical parameters. We have also explored the effect of a 3-month moderate physical exercise on the HSPs expression pattern in obese subjects.ResultsObese subjects displayed increased expression of HSP-60, HSC-70, HSP-72, HSP-90 and GRP-94 and lower expression of DNAJB3/HSP-40 (P < 0.05). No differential expression was observed for HSP-27 between the two groups. Higher levels of HSP-72 and GRP-94 proteins correlated positively with the indices of obesity (body mass index and percent body fat) and circulating levels of IFN-gamma-inducible protein 10 (IP-10) and RANTES chemokines. This expression pattern was concomitant with increased inflammatory response in the adipose tissue as monitored by increased levels of Interleukin-6 (IL-6), Tumor necrosis factor-α (TNF-α), and RANTES (P < 0.05). Physical exercise reduced the expression of various HSPs in obese to normal levels observed in lean subjects with a parallel decrease in the endogenous levels of IL-6, TNF-α, and RANTES.ConclusionTaken together, these data indicate that obesity triggers differential regulation of various components of the HSR in non-diabetic subjects and a 3-month physical moderate exercise was sufficient to restore the normal expression of HSPs in the adipose tissue with concomitant attenuation in the inflammatory response.

DNAJB3/HSP-40 cochaperone improves insulin signaling and enhances glucose uptake in vitro through JNK repression.

Heat shock response (HSR) is an essential host-defense mechanism that is dysregulated in obesity-induced insulin resistance and type 2 diabetes (T2D). Our recent data demonstrated that DNAJB3 was downregulated in obese human subjects and showed negative correlation with inflammatory markers. Nevertheless, DNAJB3 expression pattern in diabetic subjects and its mode of action are not yet known. In this study, we showed reduction in DNAJB3 transcript and protein levels in PBMC and subcutaneous adipose tissue of obese T2D compared to obese non-diabetic subjects. Overexpression of DNAJB3 in HEK293 and 3T3-L1 cells reduced JNK, IRS-1 Ser-307 phosphorylation and enhanced Tyr-612 phosphorylation suggesting an improvement in IRS-1 signaling. Furthermore, DNAJB3 mediated the PI3K/AKT pathway activation through increasing AKT and AS160 phosphorylation. AS160 mediates the mobilization of GLUT4 transporter to the cell membrane and thereby improves glucose uptake. Using pre-adipocytes cells we showed that DNAJB3 overexpression caused a significant increase in the glucose uptake, possibly through its phosphorylation of AS160. In summary, our results shed the light on the possible role of DNAJB3 in improving insulin sensitivity and glucose uptake through JNK repression and suggest that DNAJB3 could be a potential target for therapeutic treatment of obesity-induced insulin resistance.

Physical exercise alleviates ER stress in obese humans through reduction in the expression and release of GRP78 chaperone

BACKGROUND AND OBJECTIVES Perturbation of the endoplasmic reticulum (ER) homeostasis has emerged as one of the prominent features of obesity and diabetes. This occurs when the adaptive unfolded protein response (UPR) fails to restore ER function in key metabolic tissues. We previously reported increased inflammation and impaired heat shock response (HSR) in obese human subjects that were restored by physical exercise. Here, we investigated the status of ER stress chaperone; glucose-regulated protein 78 (GRP78) and its downstream UPR pathways in human obese, and their modulation by a supervised 3-month physical exercise. METHODS Subcutaneous adipose tissue (SAT) and blood samples were collected from non-diabetic adult human lean (n=40) and obese (n=40, at baseline and after 3months of physical exercise). Transcriptomic profiling was used as a primary screen to identify differentially expressed genes and it was carried out on SAT samples using the UPR RT(2) Profiler PCR Array. Conventional RT-PCR, immunohistochemistry, immunofluorescence, Western blot and ELISA were used to validate the transcriptomic data. Correlation analyses with the physical, clinical and biochemical outcomes were performed using Pearsons rank correlation coefficient. RESULTS Levels of GRP78 and its three downstream UPR arms; activating transcription factor-6 (ATF6), inositol-requiring enzyme-1α (IRE1α) and protein kinase RNA-like endoplasmic reticulum kinase (PERK) were increased in obese subjects. More interestingly, higher levels of circulating GRP78 protein were found in obese compared to lean subjects which correlated negatively with maximum oxygen uptake (VO2 Max) but positively with high-sensitivity C-reactive protein (hsCRP) and obesity indicators such as BMI, percentage body fat (PBF) and waist circumference. GRP78 increased secretion in obese was further confirmed in vitro using 3T3-L1 preadipocyte cells under ER stress. Finally, we showed that physical exercise significantly attenuated the expression and release of GRP78 with a concomitant reduction in the phosphorylation of IRE1α and eukaryotic initiation factor-2α (eIF2α). CONCLUSION Our results suggest that physical exercise alleviates ER stress in human obese through attenuation of GRP78 signaling network.

Functional Foods and Lifestyle Approaches for Diabetes Prevention and Management

Functional foods contain biologically active ingredients associated with physiological health benefits for preventing and managing chronic diseases, such as type 2 diabetes mellitus (T2DM). A regular consumption of functional foods may be associated with enhanced anti-oxidant, anti-inflammatory, insulin sensitivity, and anti-cholesterol functions, which are considered integral to prevent and manage T2DM. Components of the Mediterranean diet (MD)—such as fruits, vegetables, oily fish, olive oil, and tree nuts—serve as a model for functional foods based on their natural contents of nutraceuticals, including polyphenols, terpenoids, flavonoids, alkaloids, sterols, pigments, and unsaturated fatty acids. Polyphenols within MD and polyphenol-rich herbs—such as coffee, green tea, black tea, and yerba maté—have shown clinically-meaningful benefits on metabolic and microvascular activities, cholesterol and fasting glucose lowering, and anti-inflammation and anti-oxidation in high-risk and T2DM patients. However, combining exercise with functional food consumption can trigger and augment several metabolic and cardiovascular protective benefits, but it is under-investigated in people with T2DM and bariatric surgery patients. Detecting functional food benefits can now rely on an “omics” biological profiling of individuals’ molecular, genetics, transcriptomics, proteomics, and metabolomics, but is under-investigated in multi-component interventions. A personalized approach for preventing and managing T2DM should consider biological and behavioral models, and embed nutrition education as part of lifestyle diabetes prevention studies. Functional foods may provide additional benefits in such an approach.

Proteomics Approaches towards Early Detection and Diagnosis of Cancer

Early stage detection of cancer is the key to provide a better outcome for therapeutic intervention. Most routine screening and diagnosis tools for cancer lack sufficient sensitivity and/or specificity and sometimes they are invasive. Proteomic technologies hold recently great promise in the search of new clinical biomarkers for the early detection and diagnosis of cancer as well as the discovery of new therapeutic targets from accessible bio-specimens. They also have the potential for contributing to the better understanding of cancer biology and helping in making the right therapeutic decisions for patients. Whereas some proteomic approaches, such as the ones used for identifying proteins and analyzing their interaction and function, are well established, others, such as protein expression profiling for biomarker discovery and validation, are still suffering from robustness and reproducibility issues before being able to have their clinical applications in cancer. In this perspective, we attempt here to first, briefly summarize the various mass spectrometry (MS)-based proteomic approaches and techniques used in cancer studies, then we discuss the complexity and the critical steps in handling and processing biological samples, and finally, we focus on the most promising strategies and techniques which may shorten the way from bench to bedside in cancer proteomics. These include; directed proteomics approaches, such as targeting disease tissues and subcellular compartments, analysis of post-translational modifications of selected subsets of proteins, selective reaction monitoring quantitation, and ‘omics’ integration strategy. MS-based imaging of tissue biopsies and Surface Plasmon Resonance techniques coupled to MS will be also discussed as they emerged recently as promising applications for biomarker discovery and validation.

Gender-Specific Association of Oxidative Stress and Inflammation with Cardiovascular Risk Factors in Arab Population

Background. The impact of gender difference on the association between metabolic stress and cardiovascular disease (CVD) remains unclear. We have investigated, for the first time, the gender effect on the oxidative and inflammatory stress responses and assessed their correlation with classical cardiometabolites in Arab population. Methods. A total of 378 adult Arab participants (193 females) were enrolled in this cross-sectional study. Plasma levels of CRP, IL-6, IL-8, TNF-α, ROS, TBARs, and PON1 were measured and correlated with anthropometric and cardiometabolite parameters of the study population. Results. Compared to females, males had significantly higher FBG, HbA1c, TG, and blood pressure but lower BMI, TC, and HDL (P < 0.05). After adjustment for BMI and WC, females had higher levels of ROS, TBARS, and CRP (P < 0.001) whereas males had increased levels of IL-8, IL-6, and TNF-α (P < 0.05). Moreover, after adjustment for age, BMI, and gender, the levels of TNF-α, IL-6, and ROS were associated with central obesity but not general obesity. Conclusion. Inflammation and oxidative stress contribution to CVD risk in Arab population linked to gender and this risk is better reflected by central obesity. Arab females might be at risk of CVD complications due to increased oxidative stress.