Elevated leptin levels induce inflammation through IL-6 in skeletal muscle of aged female rats

Ryo Tazawa, Kentaro Uchida, Hisako Fujimaki, Masayuki Miyagi, Gen Inoue, Hiroyuki Sekiguchi, Kosuke Murata, Ken Takata, Ayumu Kawakubo, Masashi Takaso, Ryo Tazawa, Kentaro Uchida, Hisako Fujimaki, Masayuki Miyagi, Gen Inoue, Hiroyuki Sekiguchi, Kosuke Murata, Ken Takata, Ayumu Kawakubo, Masashi Takaso

Abstract

Background: Chronic inflammation with aging contributes to sarcopenia. Previous studies have suggested that the accumulation of adipose tissue in skeletal muscle, referred to as intermuscular adipose tissue (IMAT), increases with age and is associated with inflammation. However, the mechanism governing ectopic inflammation in skeletal muscle due to aging is not fully understood. Leptin, an adipocytokine derived from adipose tissue, is an important mediator of inflammatory processes. We examined changes in leptin levels with age and whether leptin contributes to ectopic inflammation.

Methods: To evaluate ectopic inflammation in skeletal muscle, we measured alterations to the expression of inflammatory cytokine genes (Il1b, Il6, and Tnfa) and muscle break down-related gene (MuRF1 and Atrogin1) in the quadricep muscles of young (10 weeks) and aged (48 weeks) female rats using quantitative reverse-transcription polymerase chain reaction (Q-RT-PCR). Histological examination was performed to identify the extent of IMAT. Leptin mRNA and leptin protein expression were examined using Q-RT-PCR and enzyme-linked immunosorbent assay, respectively. The effect of leptin on the mRNA expression of Il1b, Il6, and Tnfa in quadricep muscle-derived cells was also examined by stimulating the cells with 0 (control), 1, or 10 μg/mL rat recombinant leptin using Q-RT-PCR.

Results: Aged rats had significantly higher Il6, MuRF1, and Atrogin1 but not Il1b and Tnfa, expression and greater levels of IMAT in their quadricep muscles than young rats. Aged rats also had significantly higher leptin expression and leptin protein concentration in their quadricep muscles than young rats. The addition of exogenous leptin to quadricep muscle-derived cells significantly increased the gene expression of Il1b and Il6 but not Tnfa.

Conclusions: Our results suggest that elevated leptin levels due to aging cause ectopic inflammation through IL-6 in the skeletal muscle of aged rats.

Keywords: Inflammation; Interleukin-6; Intermuscular adipose tissue; Leptin.

Conflict of interest statement

Ethics approval and consent to participate

All experimental protocols were approved by the Kitasato University School of Medicine Animal Care Committee (Permission number: 2018–085).

Consent for publication

Not applicable

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
Quantitative reverse-transcription polymerase chain reaction analysis of inflammatory cytokines and muscle breakdown-related gene expression in quadriceps muscles. Relative gene expression of inflammatory cytokines, Il6 (a), Il1b (b), Tnfa (c), and muscle breakdown-related gene Atrogin1 (d) and MuRF1 (e) in quadriceps muscle extracted from young (10 weeks) and aged (48 weeks) rats. Data represent mean ± SE (n = 10). * p < 0.05, between young and aged groups
Fig. 2
Fig. 2
Accumulation of adipose tissue in quadricep muscles. Representative micrographs of muscle sections stained with hematoxylin-eosin from young (10 weeks) and aged (48 weeks) rats. a young (× 40), (b) young (× 200), (c) aged (× 40), (d) aged (× 200). Black arrows indicate adipocytes. Scale bar = 100 μm
Fig. 3
Fig. 3
Leptin gene expression by quantitative reverse-transcription polymerase chain reaction analysis and leptin protein concentration by enzyme-linked immunosorbent assay in quadricep muscles. a Relative leptin gene expression in quadricep muscles extracted from young (10 weeks) and aged (48 weeks) rats. b Leptin protein concentration in quadriceps muscles from young (10 weeks) and aged (48 weeks) rats. Data indicate mean ± SE (n = 10). * p < 0.05, between young and aged groups
Fig. 4
Fig. 4
Effect of leptin on Il1b, Il6, and Tnfa mRNA expression. Effect of leptin on Il6 (a), Il1b (b), Tnfa (c) gene expression in cultured quadricep muscle-derived cells. Quadricep muscle-derived cells were stimulated with 0 (control), 1, or 10 μg/mL rat recombinant leptin. Gene expression in the leptin-stimulated groups was compared with that in the control group. Data indicate mean ± SE (n = 10). * p < 0.05, compared to control

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