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Table of Contents
ORIGINAL ARTICLE
Year : 2020  |  Volume : 63  |  Issue : 5  |  Page : 195-203

Acylated and unacylated ghrelin relieve cancer cachexia in mice through multiple mechanisms


1 Department of Gastrointestinal Surgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou, Fujian, China
2 Department of Anesthesiology, The First Affiliated Hospital of Fujian Medical University, Fuzhou, Fujian, China
3 Department of Dermatology, The First Affiliated Hospital of Fujian Medical University, Fuzhou, Fujian, China

Date of Submission29-Jul-2020
Date of Acceptance25-Sep-2020
Date of Web Publication27-Oct-2020

Correspondence Address:
Dr. Sizeng Chen
Department of Gastrointestinal Surgery, The First Affiliated Hospital of Fujian Medical University, 20 Chazhong Road, Fuzhou 350004, Fujian
China
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Source of Support: The work was supported by Joint Funds for the innovation of science and Technology, Fujian Province (Grant number: 2018Y9083)., Conflict of Interest: None


DOI: 10.4103/CJP.CJP_59_20

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  Abstract 

Cancer cachexia is a wasting syndrome resulting from decreased protein synthesis and increased protein degradation. Calpain-dependent cleavage of myofilament is the initial step of myofilament degradation and plays a critical role in muscle atrophy. Ghrelin is a multifunctional hormone known to improve protein synthesis and inhibit protein degradation. However, its mechanism of action is not fully understood. Here we investigated whether acylated ghrelin (AG) and unacylated ghrelin (UnAG) could protect against cancer cachexia in mice bearing CT26 colorectal adenocarcinoma. We found for the first time that both AG and UnAG could inhibit calpain activity in skeletal muscle of cancer cachectic mice. AG and UnAG also improved tumor-free body weight, grip strength, muscle mass, epididymal fat mass, and nutritional state in tumor-bearing (TB) mice. Moreover, AG and UnAG reduced serum tumor necrosis factor-± concentration, increased Akt activity, and downregulated atrogin-1 expression in TB mice. Our results may contribute to the development of an AG/UnAG-based therapy for cancer cachexia.

Keywords: Akt, calpain, cancer cachexia, ghrelin, muscle wasting


How to cite this article:
Zeng X, Chen P, Zhao L, Chen S. Acylated and unacylated ghrelin relieve cancer cachexia in mice through multiple mechanisms. Chin J Physiol 2020;63:195-203

How to cite this URL:
Zeng X, Chen P, Zhao L, Chen S. Acylated and unacylated ghrelin relieve cancer cachexia in mice through multiple mechanisms. Chin J Physiol [serial online] 2020 [cited 2020 Nov 29];63:195-203. Available from: https://www.cjphysiology.org/text.asp?2020/63/5/195/299251

Xianliang Zeng & Ping Chen contributed equally to this work.



  Introduction Top


Cancer cachexia is a multifactorial and often irreversible syndrome characterized by skeletal muscle wasting (with or without fat wasting), negative protein and energy balance, systemic inflammation, and a progressive functional impairment.[1] It occurs in nearly 85% of terminal cancer patients, and is responsible for approximately 20% of all cancer deaths.[2] Conventional nutritional support cannot fully reverse cancer cachexia, and no effective treatment has been reported.[3] Although the pathogenesis of cancer cachexia has been constantly researched in the past decade, it is still not well established even today. Skeletal muscle atrophy is the major physiological effect of cancer cachexia, which is the result of increased muscle proteolysis and decreased protein synthesis,[4] calpain system,[5] ubiquitin-proteasome pathway (UPP),[6] and Akt pathway,[7] all which have been reported as critical mechanisms of this protein metabolism disorder.

Calpain system consists of 15 calcium-activated cysteine proteases and includes two ubiquitously expressed members, calpain-1, and calpain-2, and one muscle-specific member, calpain-3.[8] Calpains are implicated in several diseases, including muscular dystrophy, diabetes, neurological disorders, and hematonosis.[9] In animals with muscle atrophy, active calpain can anchor to the Z disc of myofilament, disrupt its structural integrity, leading to the release of actin and myosin for ubiquitination and degradation.[5] Since the ubiquitin-proteasome cannot degrade intact myofilament, calpain-dependent disassembly of myofilament is considered as the initial step of myofilament degradation and plays a critical role in muscle atrophy. Furthermore, active calpain can inhibit Akt activity and activate ubiquitin E3 ligases to promote muscle wasting.[5],[10] Calpain can decrease the phosphorylation of Akt to inhibit Akt activity.[11] Akt can phosphorylate mTOR to increase its activity, which in turn activates p70S6K to promote muscle protein synthesis.[12] Akt can also phosphorylate FoxO3a to decrease its activation and nuclear entrance.[13],[14] Active FoxO3a can act on the Atrogin-1 and MuRF1 promoter and increases their expressions, which causes muscle atrophy.[10],[14],[15] Thus, calpain can act through Akt to decrease muscle protein synthesis and increase Atrogin-1 and MuRF1 expression, which promotes muscle protein degradation and induces muscle atrophy. A recent study[16] in our laboratory showed that calpain system was activated (as indicated by increased calpain/calpastatin ratio) and contributed to skeletal muscle degradation in cachexic tumor-bearing (TB) mice.

Ghrelin is a multifunctional 28-amino-acid gastric peptide hormone and exists in two different forms: acylated ghrelin (AG) and unacylated ghrelin (UnAG), respectively. The only difference in their structures is the octanoylated Ser 3 found in AG. Both AG and UnAG are synthesized in stomach cells from the same precursor and then secreted into blood serum.[17] Ghrelin receptors are widely expressed in skeletal muscle, adipose tissue, intestine, pancreas, and liver. Owing to this wide receptor distribution, ghrelin system participates in numerous biological functions, including skeletal muscle metabolism, pancreatic function, and hepatic function.[17],[18],[19] By increasing muscle protein synthesis and decreasing proteolysis, ghrelin inhibits cachexic muscle atrophy occurring due to chronic renal failure, thermal injury, cancer, and chemotherapy.[20] Studies further suggest that both AG and UnAG can activate the muscular Akt pathway[15],[21] and suppress ubiquitin E3 ligase activity.[17],[22]

However, it has never been investigated whether ghrelin can affect the calpain system in cancer cachexic skeletal muscle. In this study, we evaluated the therapeutic effect of AG and UnAG on a murine model of cancer cachexia, with special emphasis on the calpain system activity in skeletal muscle.


  Materials and Methods Top


Cell culture and animal models

CT26 colon carcinoma cells (ATCC, Manassas, VA, USA) were cultured in Dulbecco's Modified Eagle's Medium (Invitrogen; Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Gibco; New Zealand) and 1% penicillin-streptomycin (Invitrogen) in an atmosphere of 5% CO2 at 37°C.

BALB/c male mice (age 50 ± 7 days) were obtained from the Animal Center of the Chinese Academy of Sciences (Shanghai, China). Animals were individually housed in a temperature-controlled environment (22°C ± 1°C) on a 12 h light-dark cycle and fed ad libitum. All experiments were approved by the Institutional Animal Care Committee of the Fujian Medical University and were in compliance with the NIH Guidelines for Use and Care of Laboratory Animals (No. 2017015).

After 5 days of acclimation, 100 μl CT26 cells (5 × 106), or 100 μl PBS (control, NC group, n = 8) was injected subcutaneously into the dorsal side of mice (Day 1). Mice transplanted with tumors were randomly divided into three groups (n = 8 per group): mice received injection of saline (T + V group), mice received injection of AG group, and mice received injection of UnAG group. 0.8 mg/kg[23],[24] AG or UnAG (TOCRIS, 1465/2951) was injected intraperitoneally twice daily at 08:00 and 20:00 from Day 14, when the mice presented signs of cachexia and tumor volume were larger than 0.5 cm3,[24] and lasted for 14 consecutive days. Body weight, food intake and tumor volume were recorded daily before the a. m. injection. Tumor volume was calculated with the formula: tumor volume (cm3) = L × W2/2, where L is the length and W is the width.

Mice were anesthetized and sacrificed on day 28, 12 h after the last ghrelin injection. Blood was taken by heart puncture and clotted for 1 h, then centrifuged to collect the serum. Gastrocnemius, tibialis anterior, soleus, extensor digitalis longus, epididymal fats, and tumors were harvested and weighed.

Grip strength

Grip strength was assessed on day 1 (before tumor implantation) and on day 28, with a grip strength meter (Columbus Instruments, Columbus, OH, USA) as previously described.[25] Each mouse was tested 5 times, and the average maximum value was recorded.

Immunofluorescence staining

To determine the myofiber cross-sectional area (CSA), frozen 8 μm cross-section of the midbelly region of gastrocnemii were fixed with 4% paraformaldehyde, blocked with 5% goat serum and then incubated with anti-MHC antibody (Abcam; Cambridge, UK; ab91506, 1:200) overnight at 4°C. Next, sections were incubated with Alexa Fluor 594-conjugated goat anti-rabbit IgG (Abcam; ab150080, 1:500) for 1 h at room temperature and then mounted with Fluoroshield Mounting Medium containing DAPI (Abcam; ab104139). Images were acquired using an Olympus BX53 fluorescence microscope (Olympus Corporation; Tokyo, Japan) and were measured using ImageJ 1.51 software (NIH, Bethesda, MD, USA). At least 200 myofiber areas (x100 magnification) per muscle were measured.

Western blot analysis

Protein from gastrocnemius was isolated using tissue lysis buffer (Cell Signaling Technology; Danvers, MA, USA; #9803) supplemented with a protease inhibitor cocktail (Roche, 05892970001). Protein lysates were loaded onto 8%-15% SDS-PAGE gels and then transferred onto PVDF membranes. The membranes were incubated overnight at 4°C with a primary antibody and then incubated with a HRP-conjugated secondary antibody (Abcam, ab97051, 1:2000). The immunostained proteins were visualized by enhanced chemiluminescence reagents (GE2301; Gen-View Scientific; Arcade, NY, USA). Images were recorded with a ChemiDoc XRS + system (Bio-Rad; Hercules, CA, USA), and analyzed by Quantity One software (version 4.6.6; Bio-Rad, USA). The primary antibodies were listed as follows: Abcam: Anti-atrogin-1 (ab168372); Anti-MuRF1 (ab172479); Anti-calpain-1 (ab108400); Anti-calpain-2 (ab126600); Anti-calpastatin (ab28252); Anti-Akt (phospho S473, ab81283); Anti-Akt (ab179463); Anti-mTOR (phospho S2448, ab109268); Anti-mTOR (ab32028); Anti-GAPDH (ab181602); Santa Cruz Biotechnology: Anti-calpain-3 (sc-365277).

Calpain activity assay

A calpain activity assay kit (Biovision; Mountain View, CA, USA) was used to measure calpain activity, in samples of muscle tissue according to the manufacturer's instructions as previously described.[16] The assay plates were read using a SpectraMax M5 microplate reader (Molecular Devices; Sunnyvale, CA, USA).

ELISA

Mouse tumor necrosis factor (TNF) alpha ELISA Kit (Abcam, ab46105), Mouse interleukin-1 (IL-1) beta ELISA Kit (Boster; EK0394), and Mouse IL-6 ELISA Kit (Beyotime; Shanghai, China; PI326) were used to measure TNF-α, IL-1β, and IL-6 concentrations, respectively, in samples of serum according to the manufacturer's instructions. The assay plates were read using a SpectraMax M5 microplate reader (Molecular Devices; Sunnyvale, CA, USA).

Bioanalytical assays

Levels of serum total protein, albumin, glucose, and triglyceride were measured by Olympus AU2700 automated biochemistry analyzer (Olympus, Tokyo, Japan).

Statistical analysis

Data were shown as the mean ± standard deviation SPSS Statistics for Windows (IBM Corp.; Armonk, NY, USA; Version 21.0) was used for all statistical analysis. Statistical comparisons between groups were performed using one-way ANOVA followed by Tukey test when equal variances were assumed. When equal variances were not assumed, Dunnett's T3 test was applied. Two-sided P < 0.05 was considered statistically significant.


  Results Top


Tumor development

The tumors of CT26-inoculated mice were palpable by day 8 (approximately 0.5 cm in diameter). Tumor volume increased more rapidly from day 14 onward. Mice exhibited signs of cachexia, including poor physical activity, delayed responsiveness, and scruffy fur on day 14. Despite a progressive increase in tumor volume over time, no significant difference of tumor volume was detected among three TB groups [Figure 1]a.
Figure 1: Analysis of tumor size, body mass, tumor-free body mass, grip strength and epididymal fat mass in mice. Analyses of BALB/c mice injected subcutaneously with CT-26 cells inducing cachexia, or with PBS. Intraperitoneal injection of PBS, acylated ghrelin or unacylated ghrelin to the CT-26-tumor-bearing mice were performed. Tumor volume (a) and body mass (b) were recorded daily for 14 days, no significant differences were detected among the four groups, by one-way ANOVA followed by Tukey test. On day 28, the tumor was surgically excised and weighed, allowing calculation of the percentage change in tumor-free body mass from pre-inoculation (c); significant differences were detected between NC and T + V groups, by one-way ANOVA followed by Tukey test, F = 18.056, P < 0.001. Grip strength was assessed on day 1 before tumor implantation and day 28, allowing calculation of the percentage change in grip strength from pre-inoculation (d); significant differences were detected between T + V and NC groups, between T + V and AG/UnAG groups, by one-way ANOVA followed by Tukey test, F = 50.884, P < 0.001. The epididymal fat was excised and weighed on an analytical balance (e); significant differences were detected between T + V and NC groups, between T + V and AG/UnAG groups, by one-way ANOVA followed by Dunnett T3 test, F = 443.651, P < 0.001 (*P < 0.05, **P < 0.01, ***P < 0.001).

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Changes in body mass

Loss of body mass is a characteristic symptom of cancer cachexia. The body mass of NC group increased continuously during the study. The TB mice had a constant increase in body mass during the first 14 days, then the body mass remained steady from day 15 to day 28, despite a slight transient increase or decrease in some groups [Figure 1]b. There was no difference in body mass among the four groups during day 1 to day 8. After that, TB mice had a lower body mass compared with NC mice, however, was not statistically significant. Compared with T + V group, AG/UnAG group had higher body mass from day 16 to day 28, but was not statistically significant. No statistical difference in body mass was observed between AG group and UnAG group. The percentage change of tumor-free body mass from day 1 to day 28 in T + V group was significantly different from NC group [[Figure 1]c, P < 0.001]; AG/UnAG treatment ameliorated this change but was not statistically significant.

Changes in grip strength, muscle mass, and muscle fiber size

The percentage change of grip strength from day 1 to day 28 in T + V group was significantly different from NC group [[Figure 1]d, P < 0.001]; AG/UnAG administration ameliorated this change [[Figure 1]d, P < 0.001 and P = 0.009, respectively]. The hind limb muscles of T + V group mice were thinner compared with NC group, which was ameliorated by AG/UnAG administration [Figure 2]a and [Figure 2]b. The masses of gastrocnemius muscle and tibialis anterior muscle of T + V group were lower than NC group (-38% versus NC, -29% versus NC, respectively); AG/UnAG administration ameliorated these changes as well [Table 1]. No significant difference in extensor digitalis longus or soleus muscle mass was observed among the four groups [Table 1].
Figure 2: Effect of AG and UnAG on lower limb muscles and tumors. (a) Macroscopic view of tumor-free mice and tumor-bearing mice at the end of the experiment. (b) Macroscopic view of tumors and lower limbs excised from mice at the end of the experiment. (c) Immunofluorescence staining images of a middle cross-section of the midbelly region of gastrocnemii incubated with anti-MHC antibody (×100). Bar represents 50 μm. (d) The average CSA of myofibers in the gastrocnemii; significant differences were detected between T + V and NC groups, between T + V and AG/UnAG groups, by one-way ANOVA followed by Dunnett T3 test, F = 46.812, P < 0.001. (*P < 0.05, **P < 0.01, ***P < 0.001).

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Table 1: Masses of various muscle tissues in different mice groups (x±s, n=8)

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Significant loss of gastrocnemius muscle fiber CSA was observed in the T + V group as compared to the NC group [[Figure 2]c and [Figure 2]d, P < 0.001], which was ameliorated by AG/UnAG treatment [[Figure 2]c and [Figure 2]d, P < 0.001].

Changes in fat mass

In cancer cachexia, skeletal muscle atrophy is always accompanied by the loss of white adipose tissue (WAT) mass,[26] sometimes WAT is lost more rapidly than skeletal muscle.[27] Consistent with this finding, our results showed that epididymal fat mass of T + V group was reduced by 81% compared with health controls, and AG/UnAG administration ameliorated this reduction [[Figure 1]e, P < 0.001].

Calpain expression and activity

A recent study in our laboratory showed that TB increased calpain activity and decreased calpastatin expression in mice skeletal muscle.[16] Here, we performed western blot assay to investigate whether TB and AG/UnAG administration affected calpain and calpastatin expression in mice skeletal muscle. As shown in [Figure 3]a, [Figure 3]b and [Figure 3]f, calpain-1 expression and calpain-1/calpastatin ratio in T + V group were significantly higher compared with NC group (P < 0.01 and P < 0.05, respectively), and AG/UnAG partially alleviated the increase. Moreover, the calpain activity in T + V group was also increased compared with NC group (P < 0.001), which was alleviated by AG/UnAG administration [[Figure 3]g, P < 0.01]. No statistically significant change in calpain-2, calpain-3, or calpastatin expressions was observed among the four groups [Figure 3]a and [Figure 3]c, [Figure 3]d, [Figure 3]e.
Figure 3: Regulation of calpain system activity by tumor-bearing and AG/UnAG administration in gastrocnemii of mice. (a) Western blot of protein levels. (b) Quantification of calpain-1 was normalized to GAPDH. Significant differences were detected between T + V and NC groups, between T + V and AG/UnAG groups, by one-way ANOVA followed by Tukey test, F = 31.361, P < 0.001. (c) Quantification of calpain-2 was normalized to GAPDH. No significant differences were detected between four groups, by one-way ANOVA followed by Tukey test. (d) Quantification of calpain-3 was normalized to GAPDH. No significant differences were detected between four groups, by one-way ANOVA followed by Tukey test. (e) Quantification of calpastatin was normalized to GAPDH. No significant differences were detected between four groups, by one-way ANOVA followed by Tukey test. (f) Quantification of calpain-1/calpastatin ratio. Significant differences were detected between T + V and NC groups, between T + V and AG groups, by one-way ANOVA followed by Dunnett T3 test, F = 29.918, P < 0.001. (g) Quantification of calpain activity. Significant differences were detected between T + V and NC groups, between T + V and AG/UnAG groups, by one-way ANOVA followed by Tukey test, F = 23.979, P = 0.001. Data are represented as mean ± standard deviation (*P < 0.05, **P < 0.01, ***P < 0.001).

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Atrogin-1 and MuRF1 expression

Atrogin-1 and MuRF1 are two muscle-specific ubiquitin ligases that drive muscle protein degradation.[5] Here, we performed western blot assay to assess their expressions in muscle tissue. As shown in [Figure 4]a and b, the expression of atrogin-1 in TB mice was significantly higher compared with NC group, and AG/UnAG ameliorated this change. However, the expression of MuRF1 was not affected by TB or AG/UnAG administration [Figure 4]a and [Figure 4]c.
Figure 4: Regulation of ubiquitin E3 legases expression and Akt/mTOR pathway by tumor-bearing and AG/UnAG administration in gastrocnemii of mice. (a) Western blot of protein levels. (b) Quantification of atrogin-1 was normalized to GAPDH. Significant differences were detected between T + V and NC groups, between T + V and AG/UnAG groups, by one-way ANOVA followed by Tukey test, F = 23.981, P < 0.001. (c) Quantification of MuRF1 was normalized to GAPDH. No significant differences were detected between four groups, by one-way ANOVA followed by Tukey test. (d) Quantification of p-Akt was normalized to Akt. Significant differences were detected between T + V and NC groups, between T + V and AG/UnAG groups, by one-way ANOVA followed by Tukey test, F = 17.941, P = 0.001. (e) Quantification of p-mTOR was normalized to mTOR. No significant differences were detected between four groups, by one-way ANOVA followed by Tukey test. Data are represented as mean ± standard deviation (*P < 0.05, **P < 0.01, ***P < 0.001).

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Akt/mTOR pathway activity

The Akt/mTOR pathway is a critical anabolic pathway that promotes muscle protein synthesis. To assess the activity of Akt/mTOR pathway, we performed western blot assay to measure the Akt, p-Akt, mTOR, and p-mTOR level in skeletal muscle. As shown in [Figure 4]a and [Figure 4]d, p-Akt/Akt ratio of the T + V group was significantly lower compared with NC group; AG/UnAG attenuated this change. No statistically significant change in mTOR or p-mTOR levels was observed among the four groups [Figure 4]a and [Figure 4]e.

Changes in serum pro-inflammatory cytokines

In cancer cachexic animals, immune cells or tumors directly secrete pro-inflammatory cytokines such as TNF-α or IL-1 to induce the loss of skeletal muscle or adipose tissue.[26],[28] In this study, we also found that the serum TNF-α concentration in T + V group was significantly higher compared with NC group; AG/UnAG administration ameliorated this change [Figure 5]a. However, the serum concentration of IL-1β or IL-6 was not affected by TB or AG/UnAG administration [Figure 5]b and [Figure 5]c.
Figure 5: Concentrations of pro-inflammatory factors in the serum of mice. (a) ELISA for TNF-a concentrations in serum. Significant differences were detected between T + V and NC groups, between T + V and AG groups, by one-way ANOVA followed by Tukey test, F = 54.064, P < 0.001. (b) ELISA for IL-1β concentrations in serum. No significant differences were detected among the four groups, by one-way ANOVA followed by Tukey test. (c) ELISA for IL-6 concentrations in serum. No significant differences were detected among the four groups, by one-way ANOVA followed by Tukey test. Data are represented as mean ± standard deviation (*P < 0.05, **P < 0.01, ***P < 0.001).

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Changes in serum nutritional markers

To evaluate the effect of TB and AG/UnAG treatment on the nutritional status in mice, serum levels of total protein, albumin, glucose, and triglycerides were measured. Compared with NC group, T + V group had higher level of triglycerides and significantly lower levels of total protein, albumin, and glucose [[Table 2], P < 0.01, P < 0.05, P < 0.001 and P < 0.01, respectively], and AG/UnAG administration partially reversed these changes.
Table 2: Biochemical tests results in different mice groups (x±s, n=8)

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  Discussion Top


Cancer cachexia is a debilitating and life-threatening syndrome which is currently lacking effective treatment. Ghrelin is an anabolic hormone with anti-atrophic effects.[29] Ours is the first study which found that AG/UnAG could inhibit calpain activity in skeletal muscle of cancer cachectic mice. Moreover, AG/UnAG attenuated muscle and fat wasting in cancer cachectic mice through multiple mechanisms.

AG/UnAG administration attenuated the loss of tumor-free body mass induced by TB mice, indicating that TB mice benefited from AG/UnAG administration. In addition, no statistical difference in tumor volume was observed among the TB groups, suggesting that AG/UnAG has no adverse effects on tumor growth.

Cancer cachexia is invariably associated with skeletal muscle wasting and results in muscle weakness and fatigue.[30] In this study, we observed significant decrease in muscle mass, muscle fiber size and grip strength in TB groups compared with NC group. AG/UnAG administration partially reverse these changes, consistent with previous finding that both AG and UnAG could prevent cachectic muscle wasting.[20] These data indicated that AG/UnAG could inhibit cancer cachectic muscle atrophy and maintain muscle physiological function in mice. This finding might be important for cancer cachexia patients, since loss of muscle physiological function results in poor quality of life, and sometimes even causes lethal complications such as respiratory failure.

Calpain-dependent cleavage of myofilament is the initial step in myofilament degradation.[5],[26] Calpain system is activated in atrophic muscle of patient with cancer, sepsis, uremia or burn injuries.[31],[32],[33],[34] A recent study[16] in our laboratory showed that calpain system was activated and contributed to muscle degradation in cachectic tumor-bearing mice. Consistent with this finding, we found that calpain system was activated (as shown by increased calpain/calpastatin expression ratio and increased calpain activity) in TB mice. AG/UnAG administration inhibited this activation. These data indicated that AG/UnAG might inhibit calpain system activity to prevent muscle degradation in cancer cachectic mice.

Previous studies demonstrated that activated calpain system could up-regulate the expression of ubiquitin E3 ligases in atrophic muscle.[5],[35] Our data also support this finding: the expression of atrogin-1 was up-regulated in muscle of TB mice, and this upregulation was ameliorated by AG/UnAG administration, which is consistent with the trend of calpain system activity. These data indicated that AG/UnAG might act on calpain system to regulate the atrogin-1 expression. However, the expression of MuRF1 was not affected by TB or AG/UnAG administration. Further studies will be needed to clarify the specific linkage between the calpain system and ubiquitin E3 ligases.

The Akt/mTOR pathway is a critical anabolic pathway that promotes protein synthesis and inhibits protein degradation. Akt phosphorylates mTOR to increase its activity, which in turn activates p70S6K to promote muscle protein synthesis.[12] Both AG and UnAG were found to increase Akt activity to ameliorate muscle atrophy.[15] Consistent with this finding, we observed that AG/UnAG ameliorated the decrease of Akt activity in TB mice. However, the mTOR activity was not affected by TB or AG/UnAG administration. Previous studies indicated that Akt phosphorylated FoxO3a and inhibited its activation and nuclear entrance.[13],[14] Active FoxO3a acts on the atrogin-1 promoter and increases atrogin-1 expression, which causes muscle atrophy. Thus, instead of activating mTOR to promote protein synthesis, Akt might act through FoxO3a to inhibit protein degradation. Further studies will be needed to assess the FoxO3a activity in muscle.

In cancer cachexic animals and patients, immune cells or tumors directly secrete pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6 to induce muscle atrophy.[2],[26],[28] Previous studies showed that the concentrations of plasma TNF-α, IL-1β and IL-6 were elevated in cancer cachectic mice.[36],[37] The concentration of serum IL-1β was also elevated in cancer cachectic rat bearing methylcholanthrene sarcoma.[38] Based on those studies, we detected the concentrations of serum TNF-α, IL-1β, and IL-6 in our study. However, only the TNF-α concentration was elevated in our animal model. This may due to the different animal models used in each study, or the different experimental protocols in each study. Previous studies demonstrate that TNF-α can activate FoxO3a and NF-κB and increased their nuclear entrance to up-regulate atrogin-1 and MuRF1 expression in muscle, which promotes muscle proteolysis and induces muscle atrophy.[15],[22],[37] AG/UnAG were found to inhibit the secretion of TNF-α to prevent muscle atrophy.[36],[39] Consistent with these findings, our data showed that AG/UnAG ameliorated the increase of serum TNF-α concentration in TB mice, which indicated that besides inhibiting calpain system activity, AG/UnAG might also inhibit the secretion of TNF-α to prevent muscle atrophy in TB mice.

Loss of adipose tissue is always observed in cancer cachexia.[26] The consistent loss of WAT is the result of increased lipolysis and decreased lipogenesis.[40] It has been reported that ghrelin could ameliorate the loss of WAT by preventing the imbalance between lipogenesis and lipolysis.[23] Consistent with these findings, we observed that AG/UnAG ameliorated the loss of epididymal fat in TB mice. In addition, we observed that AG/UnAG attenuated the increase of serum triglyceride concentration in TB mice. Previous studies demonstrated that there is an interorgan communication between skeletal muscle and adipose tissue, which goes through an interplay between myokines and adipokines, and triglyceride is one of these adipokines.[26],[41],[42] Over lipolysis leads to the increase of triglyceride, which promotes muscle proteolysis. Inhibition of this lipolysis can prevent cancer-induced muscle atrophy.[43] Thus, AG/UnAG might ameliorate the WAT and muscle loss by decreasing the serum triglyceride concentration.

Lipoprotein lipase (LPL) is a key enzyme in triglyceride metabolism. It catalyzes the hydrolysis of triglycerides from circulating chylomicrons and very low-density lipoproteins; thereby plays an important role in triglyceride clearance from the blood stream, lipid utilization, and storage.[44] Decreased LPL activity results in increased serum triglyceride concentration and contributed to the loss of WAT in cancer cachexia.[40] It has been reported that ghrelin could prevent the cisplatin-induced decrease of LPL activity in mice.[23] Based on these studies, we hypothesize that AG/UnAG might act on LPL to decrease the serum triglyceride concentration and inhibit the loss of WAT in TB mice. However, we failed to detect the expression of LPL in mice vascular and muscle tissue. Further studies will be needed to investigate whether there is a linkage between AG/UnAG and LPL.

Cancer cachexia is a complicated syndrome with multi-organ metabolic changes.[26] In this study, we measured the serum levels of nutritional markers to evaluate the nutritional status of mice. We hypothesize that the serum levels of total protein, albumin and glucose were lower in cachectic mice compared with NC group, which were ameliorated by AG/UnAG. These data indicated that AG/UnAG could improve the poor nutritional state in cachectic mice. It is well known that cancer cachexia cannot be fully reversed by conventional nutritional support, and thus AG/UnAG administration together with nutritional support may be a new therapy for cancer cachexia.

In this study, to the best of our knowledge, we report for the first time that AG and UnAG could inhibit the activity of calpain system in cancer cachectic mice. However, the exact link between AG/UnAG and the calpain system is yet to be elucidated. Previous studies have shown that both AG and UnAG could directly act on skeletal muscle, and these two forms of ghrelin may have common high-affinity binding sites on skeletal myocytes.[15],[22],[45] However, the identity of this AG/UnAG receptor is yet to be established and requires further investigation.

In conclusion, our study showed that treating cachectic TB mice with AG or UnAG alleviated cachectic symptoms, improved nutritional status, inhibited muscle and adipose atrophy, and reduced serum TNF-α concentration. Moreover, AG/UnAG administration decreased calpain activity, inhibited atrogin-1 expression and increased Akt activity in skeletal muscle. AG/UnAG acted through multiple mechanisms to ameliorate cancer cachexia in mice. However, the exact mechanism of ghrelin in cancer cachexia amelioration is not fully elucidated and therefore, warrants further studies, our results may contribute to the development of an AG/UnAG-based therapy for cancer cachexia.

Financial support and sponsorship

The work was supported by Joint Funds for the innovation of science and Technology, Fujian Province (Grant number: 2018Y9083).

Conflicts of interest

There are no conflicts of interest.



 
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