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

Abubaker J, Tiss A, Abu-Farha M, Al-Ghimlas F, Al-Khairi I, Baturcam E, Cherian P, Elkum N, Hammad M, John J, Kavalakatt S, Khadir A, Warsame S, Dermime S, Behbehani K, Dehbi M - PLoS ONE (2013)

Bottom Line: DNAJB positively correlated with maximum oxygen consumption (P = 0.031).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.

View Article: PubMed Central - PubMed

Affiliation: Biomedical Research Department, Dasman Diabetes Institute, Kuwait, Kuwait.

ABSTRACT
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 RT(2)-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.

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Obesity triggers a downregulation of DNAJB3 protein.(A) Total proteins were extracted from PBMC of lean (n = 4) and obese (n = 4) non-diabetic participants and subjected to western blot using the indicated antibodies. The bands were quantified as described in materials and methods and the relative intensity was determined after correction with actin that was used as internal control to monitor loading efficiency. The data are presented at the bottom as fold changes compared to lean group. The blots shown are representative of at least three independent experiments with consistent results. (B) Immunohistochemical staining using subcutaneous adipose biopsies from lean (n = 4) and obese (n = 11) non-diabetic participants. Aperio software was used to quantify positive staining (indicated by arrows) and the values are illustrated at the bottom as fold changes compared to lean. As negative control (NC) for the experiment, the primary antibodies were omitted. * P<0.05 as determined using student's t-test.
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pone-0069217-g002: Obesity triggers a downregulation of DNAJB3 protein.(A) Total proteins were extracted from PBMC of lean (n = 4) and obese (n = 4) non-diabetic participants and subjected to western blot using the indicated antibodies. The bands were quantified as described in materials and methods and the relative intensity was determined after correction with actin that was used as internal control to monitor loading efficiency. The data are presented at the bottom as fold changes compared to lean group. The blots shown are representative of at least three independent experiments with consistent results. (B) Immunohistochemical staining using subcutaneous adipose biopsies from lean (n = 4) and obese (n = 11) non-diabetic participants. Aperio software was used to quantify positive staining (indicated by arrows) and the values are illustrated at the bottom as fold changes compared to lean. As negative control (NC) for the experiment, the primary antibodies were omitted. * P<0.05 as determined using student's t-test.

Mentions: In order to validate the RT-PCR data on these members of the Hsp-40 at the protein level, we performed Western blot analysis on PBMCs and immunohistochemical (IHC) analysis on adipose tissue from selected subjects. As shown in Figure 2A, Western blot analysis performed on lean and obese subjects (n = 4 for each group) showed a major reduction in the expression of DNAJB3 protein in obese subjects (P<0.05). Under the same conditions, we were unable to validate the expression of DNAJC5B and DNAJB7 (data not shown). In contrast to DNAJB3, the expression levels of HSC-70 and HSP-90 were increased in obese subjects (Fig. 2A). Consistent with Western blot analysis, IHC studies on adipose tissue biopsies isolated from lean (n = 4) and obese (n = 11) subjects indicated a significant reduction of DNAJB3 (P<0.05) and an increase in the expression of HSP-70 and HSP-90 (Fig. 2B). Hence both Western blot and IHC data were in complete agreement with each other and they fully support the gene expression data obtained on dnajb3, Hsp-70 and Hsp-90.


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

Abubaker J, Tiss A, Abu-Farha M, Al-Ghimlas F, Al-Khairi I, Baturcam E, Cherian P, Elkum N, Hammad M, John J, Kavalakatt S, Khadir A, Warsame S, Dermime S, Behbehani K, Dehbi M - PLoS ONE (2013)

Obesity triggers a downregulation of DNAJB3 protein.(A) Total proteins were extracted from PBMC of lean (n = 4) and obese (n = 4) non-diabetic participants and subjected to western blot using the indicated antibodies. The bands were quantified as described in materials and methods and the relative intensity was determined after correction with actin that was used as internal control to monitor loading efficiency. The data are presented at the bottom as fold changes compared to lean group. The blots shown are representative of at least three independent experiments with consistent results. (B) Immunohistochemical staining using subcutaneous adipose biopsies from lean (n = 4) and obese (n = 11) non-diabetic participants. Aperio software was used to quantify positive staining (indicated by arrows) and the values are illustrated at the bottom as fold changes compared to lean. As negative control (NC) for the experiment, the primary antibodies were omitted. * P<0.05 as determined using student's t-test.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3722167&req=5

pone-0069217-g002: Obesity triggers a downregulation of DNAJB3 protein.(A) Total proteins were extracted from PBMC of lean (n = 4) and obese (n = 4) non-diabetic participants and subjected to western blot using the indicated antibodies. The bands were quantified as described in materials and methods and the relative intensity was determined after correction with actin that was used as internal control to monitor loading efficiency. The data are presented at the bottom as fold changes compared to lean group. The blots shown are representative of at least three independent experiments with consistent results. (B) Immunohistochemical staining using subcutaneous adipose biopsies from lean (n = 4) and obese (n = 11) non-diabetic participants. Aperio software was used to quantify positive staining (indicated by arrows) and the values are illustrated at the bottom as fold changes compared to lean. As negative control (NC) for the experiment, the primary antibodies were omitted. * P<0.05 as determined using student's t-test.
Mentions: In order to validate the RT-PCR data on these members of the Hsp-40 at the protein level, we performed Western blot analysis on PBMCs and immunohistochemical (IHC) analysis on adipose tissue from selected subjects. As shown in Figure 2A, Western blot analysis performed on lean and obese subjects (n = 4 for each group) showed a major reduction in the expression of DNAJB3 protein in obese subjects (P<0.05). Under the same conditions, we were unable to validate the expression of DNAJC5B and DNAJB7 (data not shown). In contrast to DNAJB3, the expression levels of HSC-70 and HSP-90 were increased in obese subjects (Fig. 2A). Consistent with Western blot analysis, IHC studies on adipose tissue biopsies isolated from lean (n = 4) and obese (n = 11) subjects indicated a significant reduction of DNAJB3 (P<0.05) and an increase in the expression of HSP-70 and HSP-90 (Fig. 2B). Hence both Western blot and IHC data were in complete agreement with each other and they fully support the gene expression data obtained on dnajb3, Hsp-70 and Hsp-90.

Bottom Line: DNAJB positively correlated with maximum oxygen consumption (P = 0.031).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.

View Article: PubMed Central - PubMed

Affiliation: Biomedical Research Department, Dasman Diabetes Institute, Kuwait, Kuwait.

ABSTRACT
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 RT(2)-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.

Show MeSH
Related in: MedlinePlus