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. 2020 Oct 26;12(20):20001-20023.
doi: 10.18632/aging.104154. Epub 2020 Oct 26.

Systemic overexpression of C-C motif chemokine ligand 2 promotes metabolic dysregulation and premature death in mice with accelerated aging

Fedra Luciano-Mateo  1   2 , Noemí Cabré  1   2 , Gerard Baiges-Gaya  1   2 , Salvador Fernández-Arroyo  1   2 , Anna Hernández-Aguilera  2 , Elisabet Elisabet Rodríguez-Tomàs  2 , Meritxell Arenas  3 , Jordi Camps  1   2 , Javier A Menéndez  4   5 , Jorge Joven  1   2   6
Affiliations

Systemic overexpression of C-C motif chemokine ligand 2 promotes metabolic dysregulation and premature death in mice with accelerated aging

Fedra Luciano-Mateo et al. Aging (Albany NY). .

Abstract

Injection of tissues with senescent cells induces changes that mimic aging, and this process is delayed in mice engineered to eliminate senescent cells, which secrete proinflammatory cytokines, including C-C motif chemokine ligand 2 (Ccl2). Circulating levels of Ccl2 correlate with age, but the impact of Ccl2 on tissue homeostasis has not been established. We generated an experimental model by crossbreeding mice overexpressing Ccl2 with progeroid mice bearing a mutation in the lamin A (Lmna) gene. Wild-type animals and progeroid mice that do not overexpress Ccl2 were used as controls. Ccl2 overexpression decreased the lifespan of the progeroid mice and induced the dysregulation of glycolysis, the citric acid cycle and one-carbon metabolism in skeletal muscle, driving dynamic changes in energy metabolism and DNA methylation. This impact on cellular bioenergetics was associated with mitochondrial alterations and affected cellular metabolism, autophagy and protein synthesis through AMPK/mTOR pathways. The data revealed the ability of Ccl2 to promote death in mice with accelerated aging, which supports its putative use as a biomarker of an increased senescent cell burden and for the assessment of the efficacy of interventions aimed at extending healthy aging.

Keywords: C-C motif chemokine ligand 2; energy metabolism; one-carbon metabolism; progeria.

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Conflict of interest statement

CONFLICTS OF INTEREST: There are no conflicts of interest associated with this report.

Figures

Figure 1
Figure 1
Ccl2 significantly reduced the lifespan of mice with accelerated aging. Ccl2 overexpression was critical for (A) the differential progression in body weight with age, (B) development of features of accelerated aging, as shown in the representative photographs of mice at 20 weeks of age, and (C) reduced lifespan, as shown by Kaplan-Meier survival plots from the wild-type (n=20), LMNAG609G/+ (n=28) and LMNAG609G/+;CGCCL2+/- (n=34) mice. LMNAG609G/+;CGCCL2+/- and LMNAG609G/+ denote progeroid mice overexpressing and not overexpressing Ccl2, respectively.
Figure 2
Figure 2
Histological phenotypes were altered in the muscles of aged mice. The structural changes in quadriceps muscles are depicted in representative micrographs of tissue stained with hematoxylin and eosin and Sirius red staining and determined by immunohistochemical analysis of β-actin and F4/80 cells. Ccl2 overexpression accelerated the presence of histological alterations (A). Fibrosis was a prominent feature in the progeroid mice, but the relative amount of actin-stained cells was similar among strains, despite major differences in muscle weight. Interestingly, the number of F4/80-stained cells was significantly lower in the progeroid mice. We then used immunoblotting to examine (B) the expression of the markers of macrophage polarization, including cluster of differentiation (CD) 11 and 163, and tumor necrosis factor (TNFα) to indicate the relative anti-inflammatory activity, which was accompanied by lower expression of the major antioxidant enzyme paraoxonase 1 (PON1) in both progeroid strains. The LMNAG609G/+;CGCCL2+/- mice were compared with wild-type mice, as depicted by a p<0.05, and with LA +/- mice, as depicted by b p<0.05, according to the Mann–Whitney U tests (n=15 for each strain). LMNAG609G/+;CGCCL2+/- and LMNAG609G/+ denote progeroid mice overexpressing and not overexpressing Ccl2, respectively.
Figure 3
Figure 3
The impact of Ccl2 overexpression on the metabolic pathways associated with energy and 1-C metabolism. Metabolomic analysis of skeletal muscle from progeroid mice with (LMNAG609G/+;CGCCL2+/-) or without (LMNAG609G/+) increased Ccl2 levels revealed the metabolic impact of Ccl2 in accelerated aging. (A) From left to right, partial least square discriminant analysis (PLSDA), heatmap with hierarchical clustering and random forest analysis indicating that Ccl2 drove changes in energy and 1-C metabolism in the quadriceps muscles of the genetically modified mice. The levels of metabolites were distinct in both strains, and the variables of highest importance, according to the projection scores, were glucose, citrate, SAH and methionine. (B) Comparison of the levels of metabolite abundance from glycolysis and the CAC cycle in the quadriceps muscles were assessed by fold changes, as indicated in the legend, suggesting that Ccl2 decreased mitochondrial oxidative metabolism. (C) Dysregulation in the methionine cycle was, at least partially, the cause of the increased DNA methylcytosine levels in the LMNAG609G/+;CGCCL2+/- mice with respect to the controls and LMNAG609G/+ mice. Values are shown as the means ± SEM; ap< 0.05 with respect to the wild-type mice and b p<0.05 with respect to the LMNAG609G/+ mice, according to the Mann–Whitney U tests.
Figure 4
Figure 4
Ccl2 overexpression significantly altered the expression of the complexes related to oxidative phosphorylation and the relevant metabolic signaling pathways in the quadriceps muscle. Ccl2 overexpression exacerbated the metabolic reduction, mainly in mitochondria, (A) of the mice with accelerated aging and altered (B) the functioning of AMPK-mTOR-driven pathways. LMNAG609G/+;CGCCL2+/- and LMNAG609G/+ denote progeroid mice overexpressing and not overexpressing Ccl2, respectively. Values are shown as the means ± SEM; a p< 0.05 with respect to the wild-type mice and b p<0.05 with respect to the LMNAG609G/+ mice according to the Mann–Whitney U tests. Acronyms in (A) denote complex I or NADH/ubiquinone oxidoreductase, complex II or succinate dehydrogenase, complex III or cytochrome C reductase, complex IV or cytochrome C oxidase, complex V or ATP synthase, translocase of outer membrane (TOM20), and mitofusin 2 (Mfn2); acronyms in (B) phospho-tumor suppressor p53 (p53-pS15), tumor suppressor p53 (P53), phosphoinositide 3-kinase (PI3K), protein kinase B (AKT) and phospho-protein kinase B (AKT-pS473), phospho-ribosomal protein S6 (S6-pS235/236), ribosomal protein S6 (S6), AMPK activated protein kinase (AMPK), phospho-AMPK activated protein kinase (AMPK pT172), microtubule-associated proteins 1A/1B light chain 3B (LC3), p62 adaptor protein or sequestosome 1, and lysosome-associated membrane protein 2 (LAMP2A).
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