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Lysosomal Cathepsin D contributes to cell death during adipocyte hypertrophy.

Eguchi A, Feldstein AE - Adipocyte (2013)

Bottom Line: In this report, we demonstrated that another key Cathepsin, Cathepsin D (CTSD), is also activated at the early stages of weight gain.In addition, activated CTSD induced proapoptotic protein activation.In conclusion, our data identify lysosomal CTSD as a potential key mediator of adipocyte cell death during weight gain and obesity.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics; University of California, San Diego; San Diego, CA USA.

ABSTRACT
Obesity has reached epidemic proportions in most of the western world. With obesity comes a variety of adverse health effects such as insulin resistance, dyslipidemia, hypertension, glucose intolerance, and hepatic steatosis. It has become clear that a state of low grade chronic inflammation, typically associated with obesity, and characterized by macrophage infiltration of adipose tissue (AT) and increased production of pro-inflammatory cytokines, plays a crucial role in the development of insulin resistance. The pathogenic mechanisms resulting in AT macrophage recruitment are under intense investigation and remain incompletely understood. We recently demonstrated that lysosomal permeabilization, and subsequent Cathepsin B (CTSB) activation, occurs at the early stages of high fat diet induced weight gain and is preceded by macrophage infiltration into hypertrophied AT resulting in adipocyte cell death through mitochondrial dysfunction. In this report, we demonstrated that another key Cathepsin, Cathepsin D (CTSD), is also activated at the early stages of weight gain. In addition, activated CTSD induced proapoptotic protein activation. In conclusion, our data identify lysosomal CTSD as a potential key mediator of adipocyte cell death during weight gain and obesity.

No MeSH data available.


Related in: MedlinePlus

Figure 1. CTSD activation associated with macrophage infiltration occurs in hypertrophied adipose tissue of diet-induced obese mice, and induces upregulatin of pro-apoptotic proteins. C57BL/6 male mice were fed high fat (HFAT) or standard low fat control (CTL) diet for 12 weeks. (A) Immunoblot analysis of CTSD, Bax, and Bid in epididymal adipose tissue. (B) Correlation graph between ratio of CTSD activation and epididymal adipose tissue weight. (C) Correlation graph between ratio of CTSD activation and mouse body weight. (D) Correlation graph between ratio of CTSD activation and Bax expression. (E) Correlation graph between ratio of CTSD activation and Bid expression. (F) Representative microphotograph of double immunofluorescence for expression of CTSD and active Bax in epididymal adipose section. (G) Quantification of F4/80-CD11b positive cells by flow cytometry analysis of epididymal adipose tissue. Data are presented as mean ± SEM, ***P < 0.001.
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Figure 1: Figure 1. CTSD activation associated with macrophage infiltration occurs in hypertrophied adipose tissue of diet-induced obese mice, and induces upregulatin of pro-apoptotic proteins. C57BL/6 male mice were fed high fat (HFAT) or standard low fat control (CTL) diet for 12 weeks. (A) Immunoblot analysis of CTSD, Bax, and Bid in epididymal adipose tissue. (B) Correlation graph between ratio of CTSD activation and epididymal adipose tissue weight. (C) Correlation graph between ratio of CTSD activation and mouse body weight. (D) Correlation graph between ratio of CTSD activation and Bax expression. (E) Correlation graph between ratio of CTSD activation and Bid expression. (F) Representative microphotograph of double immunofluorescence for expression of CTSD and active Bax in epididymal adipose section. (G) Quantification of F4/80-CD11b positive cells by flow cytometry analysis of epididymal adipose tissue. Data are presented as mean ± SEM, ***P < 0.001.

Mentions: Since CTSB has been shown to be an important initiator of cell death, we hypothesized that CTSD may also have a significant role during adipocyte hypertrophy associated with weight gain. To investigate whether CTSD is activated in the hypertrophied adipocytes, we placed wild-type mice on a HFAT, or control chow (CTL), diet for 12 weeks and examined CTSD activation in the epididymal adipose tissue (EAT) by immunoblotting. CTSD is synthesized as an inactive preprocathepsin D (43 kDa), which then gets cleaved and glycosylated to form a procathepsin D (46 kDa), which is then further cleaved to CTSD heavy chain (28 kDa) and light chain (15 kDa). As a result, the active form of CTSD (heavy chain) is clearly separated from CTSD precursor (prepro and pro) on the blot. The CTSD active form, as well as CTSD precursor, was significantly increased in the HFAT diet mice compared with the mouse fed a CTL diet (Fig. 1A, the upper column), showing a positive correlation of CTSD activation (ratio of active form to precursors) with epididymal fat weights (Fig. 1B; r = 0.77, P < 0.02) and total body weights (Fig. 1C; r = 0.9, P < 0.001). Furthermore, we examined the expression of Bax and Bid, key proapoptotic proteins that CTSD has been shown to cleave and activate, in the AT. The Bax and Bid expressions were significantly upregulated in the HFAT diet fed mice via immunoblotting (Fig. 1A, middle and lower columns). Notably, there is a significant positive correlation between CTSD activation and Bax expression levels (Fig. 1D; r = 0.9, P < 0.001) as well as Bid expression levels (Fig. 1E; r = 0.76, P < 0.02). Furthermore, the increase of CTSD expression and Bax activation were detected in the adipocytes of HFAT diet fed mice via immunofluorescence with anti-CTSD and anti-Bax (6A7) (Fig. 1F). To investigate the association of these events with macrophage infiltration, we isolated stromal vascular fraction (SVF) from EAT and analyzed the number of F4/80+-CD11b+ macrophages by flow cytometry. A significant amount of F4/80+-CD11b+ macrophages were infiltrated into the adipose tissue of the mice fed a HFAT diet (Fig. 1G).


Lysosomal Cathepsin D contributes to cell death during adipocyte hypertrophy.

Eguchi A, Feldstein AE - Adipocyte (2013)

Figure 1. CTSD activation associated with macrophage infiltration occurs in hypertrophied adipose tissue of diet-induced obese mice, and induces upregulatin of pro-apoptotic proteins. C57BL/6 male mice were fed high fat (HFAT) or standard low fat control (CTL) diet for 12 weeks. (A) Immunoblot analysis of CTSD, Bax, and Bid in epididymal adipose tissue. (B) Correlation graph between ratio of CTSD activation and epididymal adipose tissue weight. (C) Correlation graph between ratio of CTSD activation and mouse body weight. (D) Correlation graph between ratio of CTSD activation and Bax expression. (E) Correlation graph between ratio of CTSD activation and Bid expression. (F) Representative microphotograph of double immunofluorescence for expression of CTSD and active Bax in epididymal adipose section. (G) Quantification of F4/80-CD11b positive cells by flow cytometry analysis of epididymal adipose tissue. Data are presented as mean ± SEM, ***P < 0.001.
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Figure 1: Figure 1. CTSD activation associated with macrophage infiltration occurs in hypertrophied adipose tissue of diet-induced obese mice, and induces upregulatin of pro-apoptotic proteins. C57BL/6 male mice were fed high fat (HFAT) or standard low fat control (CTL) diet for 12 weeks. (A) Immunoblot analysis of CTSD, Bax, and Bid in epididymal adipose tissue. (B) Correlation graph between ratio of CTSD activation and epididymal adipose tissue weight. (C) Correlation graph between ratio of CTSD activation and mouse body weight. (D) Correlation graph between ratio of CTSD activation and Bax expression. (E) Correlation graph between ratio of CTSD activation and Bid expression. (F) Representative microphotograph of double immunofluorescence for expression of CTSD and active Bax in epididymal adipose section. (G) Quantification of F4/80-CD11b positive cells by flow cytometry analysis of epididymal adipose tissue. Data are presented as mean ± SEM, ***P < 0.001.
Mentions: Since CTSB has been shown to be an important initiator of cell death, we hypothesized that CTSD may also have a significant role during adipocyte hypertrophy associated with weight gain. To investigate whether CTSD is activated in the hypertrophied adipocytes, we placed wild-type mice on a HFAT, or control chow (CTL), diet for 12 weeks and examined CTSD activation in the epididymal adipose tissue (EAT) by immunoblotting. CTSD is synthesized as an inactive preprocathepsin D (43 kDa), which then gets cleaved and glycosylated to form a procathepsin D (46 kDa), which is then further cleaved to CTSD heavy chain (28 kDa) and light chain (15 kDa). As a result, the active form of CTSD (heavy chain) is clearly separated from CTSD precursor (prepro and pro) on the blot. The CTSD active form, as well as CTSD precursor, was significantly increased in the HFAT diet mice compared with the mouse fed a CTL diet (Fig. 1A, the upper column), showing a positive correlation of CTSD activation (ratio of active form to precursors) with epididymal fat weights (Fig. 1B; r = 0.77, P < 0.02) and total body weights (Fig. 1C; r = 0.9, P < 0.001). Furthermore, we examined the expression of Bax and Bid, key proapoptotic proteins that CTSD has been shown to cleave and activate, in the AT. The Bax and Bid expressions were significantly upregulated in the HFAT diet fed mice via immunoblotting (Fig. 1A, middle and lower columns). Notably, there is a significant positive correlation between CTSD activation and Bax expression levels (Fig. 1D; r = 0.9, P < 0.001) as well as Bid expression levels (Fig. 1E; r = 0.76, P < 0.02). Furthermore, the increase of CTSD expression and Bax activation were detected in the adipocytes of HFAT diet fed mice via immunofluorescence with anti-CTSD and anti-Bax (6A7) (Fig. 1F). To investigate the association of these events with macrophage infiltration, we isolated stromal vascular fraction (SVF) from EAT and analyzed the number of F4/80+-CD11b+ macrophages by flow cytometry. A significant amount of F4/80+-CD11b+ macrophages were infiltrated into the adipose tissue of the mice fed a HFAT diet (Fig. 1G).

Bottom Line: In this report, we demonstrated that another key Cathepsin, Cathepsin D (CTSD), is also activated at the early stages of weight gain.In addition, activated CTSD induced proapoptotic protein activation.In conclusion, our data identify lysosomal CTSD as a potential key mediator of adipocyte cell death during weight gain and obesity.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics; University of California, San Diego; San Diego, CA USA.

ABSTRACT
Obesity has reached epidemic proportions in most of the western world. With obesity comes a variety of adverse health effects such as insulin resistance, dyslipidemia, hypertension, glucose intolerance, and hepatic steatosis. It has become clear that a state of low grade chronic inflammation, typically associated with obesity, and characterized by macrophage infiltration of adipose tissue (AT) and increased production of pro-inflammatory cytokines, plays a crucial role in the development of insulin resistance. The pathogenic mechanisms resulting in AT macrophage recruitment are under intense investigation and remain incompletely understood. We recently demonstrated that lysosomal permeabilization, and subsequent Cathepsin B (CTSB) activation, occurs at the early stages of high fat diet induced weight gain and is preceded by macrophage infiltration into hypertrophied AT resulting in adipocyte cell death through mitochondrial dysfunction. In this report, we demonstrated that another key Cathepsin, Cathepsin D (CTSD), is also activated at the early stages of weight gain. In addition, activated CTSD induced proapoptotic protein activation. In conclusion, our data identify lysosomal CTSD as a potential key mediator of adipocyte cell death during weight gain and obesity.

No MeSH data available.


Related in: MedlinePlus