The dual nature of DNA damage response in obesity and bariatric surgery-induced weight loss

Abstract

This novel study applies targeted functional proteomics to examine tissues and cells obtained from a cohort of individuals with severe obesity who underwent bariatric surgery (BS), using a Reverse-Phase Protein Array (RPPA). In obese individuals, visceral adipose tissue (VAT), but not subcutaneous adipose tissue (SAT), shows activation of DNA damage response (DDR) markers including ATM, ATR, histone H2AX, KAP1, Chk1, and Chk2, alongside senescence markers p16 and p21. Additionally, stress-responsive metabolic markers, such as survivin, mTOR, and PFKFB3, are specifically elevated in VAT, suggesting both cellular stress and metabolic dysregulation. Conversely, peripheral blood mononuclear cells (PBMCs), while exhibiting elevated mTOR and JNK levels, did not present significant changes in DDR or senescence markers. Following BS, unexpected increases in phosphorylated ATM, ATR, and KAP1 levels, but not in Chk1 and Chk2 nor in senescence markers, were observed. This was accompanied by heightened levels of survivin and mTOR, along with improvement in markers of mitochondrial quality and health. This suggests that, following BS, pro-survival pathways involved in cellular adaptation to various stressors and metabolic alterations are activated in circulating PBMCs. Moreover, our findings demonstrate that the DDR has a dual nature. In the case of VAT from individuals with obesity, chronic DDR proves to be harmful, as it is associated with senescence and chronic inflammation. Conversely, after BS, the activation of DDR proteins in PBMCs is associated with a beneficial survival response. This response is characterized by metabolic redesign and improved mitochondrial biogenesis and functionality. This study reveals physiological changes associated with obesity and BS that may aid theragnostic approaches.

Fig. 1. DDR and senescence markers are activated in adipose tissue of obese individuals.

Box plots resulting from comparison of RPPA profiles of biopsies of subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) of severely obese patients (OB-SAT and OB-VAT) and normal-weight (NW) controls (a–h). The plots represent the distribution of RPPA intensity values (median ± SD). Statistical comparisons were performed with Wilcoxon rank sum test as non-parametric test and t-test as parametric test for not-paired samples (black brackets). Paired samples were compared using Wilcoxon signed-rank test as non-parametric test and t-test as parametric test (blue brackets). Statistical significance is coded with an asterisk according to the level of significance (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Fig. 2. Metabolic markers of obesity are elevated in adipose tissue of obese individuals.

Box plots resulting from comparison of the RPPA profiles of biopsies of subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) of severely obese patients (OB-SAT and OB-VAT) and normal-weight (NW) controls (a-e). The plots represent the distribution of RPPA intensity values (median ± SD). The mtDNA/nDNA ratio was measured by digital droplet PCR (copy/µl) (f). Statistical comparisons were performed with Wilcoxon rank sum test as non-parametric test and t-test as parametric test for not-paired samples (black brackets). Paired samples were compared using Wilcoxon signed-rank test as non-parametric test and t-test as parametric test (blue brackets). Statistical significance is coded with an asterisk according to the level of significance (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Fig. 3. DDR markers are activated in PBMCs after bariatric surgery.

Box plots resulting from the comparison of the RPPA profiles of peripheral blood mononuclear cells (PBMCs) of severely obese patients before (T0) and six (T6), and 12 (T12) months after bariatric surgery and normal-weight (NW) controls. a–h The plots represent the distribution of RPPA intensity values (mean ± SD), and statistical comparisons between OB and NW subjects were performed using the Wilcoxon rank sum test for non-normal data and t-test for normal distributed data. For the comparison at the different times, we used two tests for paired samples: Wilcoxon signed-rank test as non-parametric test and t-test as parametric test. Statistical significance is coded with an asterisk according to the level of significance (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Fig. 4. Metabolic redesign occurs in PBMC after bariatric surgery.

Box plots resulting from the comparison of RPPA profiles of peripheral blood mononuclear cells (PBMCs) of severely obese patients before (T0) and six (T6), and 12 (T12) months after bariatric surgery and their normal-weight (NW) controls. a–e The plots represent the distribution of RPPA intensity values (mean ± SD), and statistical comparisons were performed using the Wilcoxon rank sum test for the comparison between OB and NW subjects. For the comparison at the different times, we used two tests for paired samples: Wilcoxon signed-rank test as non-parametric test and t-test as parametric test. Statistical significance is coded with an asterisk according to the level of significance (*p ≤ 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Fig. 5. Bariatric surgery restores systemic mitochondrial homeostasis.

Scatter plots resulting from the analysis of mitochondrial markers in whole blood, PBMCs and plasma samples by RT-PCR and ddPCR. Whole blood PGC1-α (a), DRP-1 (b) and SIRT3 (c) expression levels (mean ∓ SD) of severely obese patients before (T0) and six (T6) and twelve (T12) months after BS. Ratio between mitochondrial and nuclear DNA (mtDNA/nDNA) (mean ± SD) in PBMCs (d). Circulating cell-free mtDNA (ccf-mtDNA) levels in plasma samples before and after bariatric surgery (e). Telomere length (TL) by RT-PCR in PBMCs expressed in arbitrary units (A.U.) (f). Statistics were calculated using a two-tailed Student’s t-test (α = 0.05) if the requirements of normal distribution (Shapiro-Wilk test) were met. Otherwise, the Mann-Whitney test was performed. Non-parametric statistics (Wilcoxon signed-rank test) or paired t-tests were used to compare the levels of repeated measurements T0, T6, and T12. Statistical significance is coded with an asterisk according to the level of significance (*p ≤ 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Fig. 6. A hypothetical model: the dual role of DNA damage response in obesity.

Under excessive caloric intake, hypertrophic expansion of white adipose tissue leads to adipocyte dysfunction, necrosis and fibrosis leading to low-grade systemic inflammation. At the molecular level, we speculate that DNA damage accumulation, chronic DDR activation, cellular senescence and mitochondrial dysfunction in visceral adipocytes concur to the release of autocrine and paracrine signals. These signals contribute to the development of a senescence-associated secretory phenotype (SASP), which is a systemic maladaptive homeostasis. Within the bloodstream, PBMCs in their quiescent state accumulate DNA damage without DDR activation. Following massive weight loss post-bariatric surgery, PBMCs shift toward an active state, characterized by cell proliferation, DDR activation associated with DNA repair and recovery of mitochondrial homeostasis. This adaptive cell survival response potentially contributes to the beneficial health effects of bariatric surgery. M1, M2: macrophages 1 and 2.