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. 2020 Jun:107:154225.
doi: 10.1016/j.metabol.2020.154225. Epub 2020 Apr 7.

Sustained mitochondrial biogenesis is essential to maintain caloric restriction-induced beige adipocytes

Raja Gopal Reddy Mooli  1 , Dhanunjay Mukhi  1 , Mikayla Watt  1 , Lia Edmunds  1 , Bingxian Xie  1 , Joseph Capooci  1 , Matthew Reslink  1 , Chetachukwu Eze  1 , Amanda Mills  1 , Donna B Stolz  1 , Michael Jurczak  1 , Sadeesh K Ramakrishnan  2
Affiliations

Sustained mitochondrial biogenesis is essential to maintain caloric restriction-induced beige adipocytes

Raja Gopal Reddy Mooli et al. Metabolism. 2020 Jun.

Abstract

Background: Caloric restriction (CR) delays the onset of metabolic and age-related disorders. Recent studies have demonstrated that formation of beige adipocytes induced by CR is strongly associated with extracellular remodeling in adipose tissue, decrease in adipose tissue inflammation, and improved systemic metabolic homeostasis. However, beige adipocytes rapidly transition to white upon CR withdrawal through unclear mechanisms.

Materials and methods: Six-week old C57BL6 mice were fed with 40% CR chow diet for 6 weeks. Subsequently, one group of mice was switched back to ad libitum chow diet, which was continued for additional 2 weeks. Adipose tissues were assessed histologically and biochemically for beige adipocytes.

Results: Beige adipocytes induced by CR rapidly transition to white adipocytes when CR is withdrawn independent of parkin-mediated mitophagy. We demonstrate that the involution of mitochondria during CR withdrawal is strongly linked with a decrease in mitochondrial biogenesis. We further demonstrate that beige-to-white fat transition upon β3-AR agonist-withdrawal could be attenuated by CR, partly via maintenance of mitochondrial biogenesis.

Conclusion: In the model of CR, our study highlights the dominant role of mitochondrial biogenesis in the maintenance of beige adipocytes. We propose that loss of beige adipocytes upon β3-AR agonist withdrawal could be attenuated by CR.

Keywords: Beige adipocytes; Caloric restriction; Fission; Fusion; Mitochondrial biogenesis; Mitochondrial dynamics; Mitophagy.

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Conflict of interest statement

Declaration of competing interest The authors declare no conflict of interests.

Figures

Figure 1.
Figure 1.. CR-induced mitochondrial biogenesis promotes CKMT2-beige adipocytes.
Six-weeks old C57BL6 mice were fed with 40% less calories (CR) for 6 weeks compared to control mice who were fed with chow diet ad libitum (AL). (A) Body weight of the mice. (B) Intraperitoneal glucose tolerance test in CR mice. (C) H&E staining (top panel) and immunohistochemistry for TOM20 (bottom panel) in iWAT, magnification - 20X. (D) Immunoblots and densitometric analysis for the mitochondrial and OXPHOS proteins in the iWAT. (E) Transmission electron micrograph images in the iWAT (M=Mitochondria, LD=Lipid droplet, N=Nucleus), magnification - 20,000X. (F) Immunohistochemistry for CKMT2 in iWAT, magnification - 20X. (G) Western blot and densitometric analysis for CKMT2 in the iWAT. (H) Oxygen consumption in the iWAT assessed by Oroboros. (I) Respiratory exchange ratio (RER) assessed by indirect calorimetry in the CR mice (n=4 mice/group). (J) Immunoblots and densitometric analysis for proteins involved in mitochondrial biogenesis and dynamics in the WAT and iWAT of CR mice. Results are representative data from one cohort. Bar graphs are presented as mean SE ± SEM (*p < 0.05, **p < 0.01).
Figure 2:
Figure 2:. CR did not induce mitochondrial remodeling in the BAT, liver, and muscle.
(A) Immunoblots and densitometric analysis for mitochondrial proteins in the BAT. (B) mRNA levels of the mitochondrial genes in the BAT. The mRNA levels were normalized to that of 18S. (C) Immunoblots and densitometric analysis of the mitochondrial biogenesis and dynamics-related proteins in the BAT. (D) Immunoblots and densitometric analysis of the mitochondrial proteins in the liver. (E) Immunoblots and densitometric analysis of the mitochondrial biogenesis and dynamics-related proteins in the liver. (F) Immunoblots and densitometric analysis of the gluconeogenesis-related proteins in the liver. (G) Immunoblots and densitometric analysis of the mitochondrial proteins in the muscle. (H) Immunoblots and densitometric analysis of the mitochondrial biogenesis and dynamics-related proteins in the muscle.
Figure 3.
Figure 3.. Beige-to-white fat transition upon CR withdrawal is due to decrease in mitochondrial biogenesis.
(A) Schematic representation of the experimental design. Six-weeks old C57BL6 mice were fed with 40% calorie restricted chow diet (CR) for 6 weeks following which one group of mice were switched back to ad libitum chow diet and all the mice continued on their respective diets for additional 2 weeks. (B) Body weight of the mice. (C) Intraperitoneal glucose tolerance test (ipGTT) performed in CR→AL mice (n=3–5 mice/group). (D) H&E staining (top panel) and immunohistochemistry for TOM20 (bottom panel) in the iWAT, magnification - 20X. (E) Immunoblots and densitometric analysis for mitochondrial proteins in the iWAT. (F) H&E staining (top panel) and immunohistochemistry (bottom panel) performed on the iWAT of global females parkin2 KO (Park2−/−) and their littermates (Park2+/+) controls, magnification - 20X (n= 3–4 mice/group). (G) Immunoblots and densitometric analysis of the mitochondrial proteins in the iWAT of female Park2−/−mice. (H) Immunoblots and densitometric analysis of the mitochondrial biogenesis and dynamics-related proteins in the iWAT. (I) Immunoblots and densitometric analysis of the mitochondrial biogenesis and dynamics-related proteins in the iWAT of Park2−/−mice.
Figure 4.
Figure 4.. CR attenuates CL withdrawal-mediated loss of mitochondria in WAT.
(A) Immunoblots and densitometric analysis of the mitochondrial proteins, and (B) Mitochondrial biogenesis and dynamics and densitometric analysis of the iWAT of WT mice injected with CL (1 mg/kg) for 7 days following which one group of mice was withdrawn from CL injection and maintained for another additional 10 days (n=4–5 mice/group). (C–F) C57BL6 mice fed with 60% high-fat diet for 7 weeks were injected with CL (1 mg/kg) for 7 days with or without 40% CR, and then CL was withdrawn for 10 days while the mice were continued on their respective dietary regimen (CL-WDL or CL-WDL+CR). For the CL group, CL (1 mg/kg) was injected for 7 days in mice fed with high-fat diet ad libitum. (C) Fat mass and weight of organs. (D) H&E analysis (top panel) and TOM20 immunostaining (bottom panel) in iWAT, magnification - 20X. (E) Immunoblots and densitometric analysis of the mitochondrial proteins. (F) Western blot and densitometric analysis of proteins involved in mitochondrial biogenesis and dynamics in the iWAT. Results are representative data from one cohort. Bar graphs are presented as mean SE ± SEM (*p < 0.05, **p < 0.01).
Figure 5.
Figure 5.. CR maintains uncoupled respiration and improves metabolic homeostasis during CL withdrawal
(A–G) C57BL6 mice fed with 60% high-fat diet for 7 weeks were injected with CL (1 mg/kg) for 7 days with or without 40% CR, and then CL was withdrawn for 10 days while the mice were continued on their respective dietary regimen (CL-WDL or CL-WDL+CR). For the CL group, CL (1 mg/kg) was injected for 7 days in mice fed with high-fat diet ad libitum. (A) Immunohistochemistry, magnification - 20X and (B) Immunoblot and densitometric analysis of CKMT2 in the iWAT. (C) Immunohistochemistry, magnification-20X and (D) Immunoblot and densitometric analysis of UCP1 in the iWAT. (E) Immunoblot and densitometric analysis of the Oxphos proteins in the iWAT. (F) Oxygen consumption and proton leak assessed in the iWAT using Oroboros. (G) Intraperitoneal glucose tolerance test (left panel) and area under curve (AUC; right panel) (n= 4 mice/group). Bar graphs are presented as mean SE ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001).
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