Regulatory mechanisms involved in muscle and bone remodeling during refeeding in gilthead sea bream

Abstract

The tolerance of fish to fasting offers a model to study the regulatory mechanisms and changes produced when feeding is restored. Gilthead sea bream juveniles were exposed to a 21-days fasting period followed by 2 h to 7-days refeeding. Fasting provoked a decrease in body weight, somatic indexes, and muscle gene expression of members of the Gh/Igf system, signaling molecules (akt, tor and downstream effectors), proliferation marker pcna, myogenic regulatory factors, myostatin, and proteolytic molecules such as cathepsins or calpains, while most ubiquitin-proteasome system members increased or remained stable. In bone, downregulated expression of Gh/Igf members and osteogenic factors was observed, whereas expression of the osteoclastic marker ctsk was increased. Refeeding recovered the expression of Gh/Igf system, myogenic and osteogenic factors in a sequence similar to that of development. Akt and Tor phosphorylation raised at 2 and 5 h post-refeeding, much faster than its gene expression increased, which occurred at day 7. The expression in bone and muscle of the inhibitor myostatin (mstn2) showed an inverse profile suggesting an inter-organ coordination that needs to be further explored in fish. Overall, this study provides new information on the molecules involved in the musculoskeletal system remodeling during the early stages of refeeding in fish.

Figure 1

Relative gene expression of skeletal white muscle total igf1 (A), igf1a (B), igf1b (C), igf1c (D), igf2 (E), igf1ra (F), igf1rb (G), igfbp5 (H) and ghr1 and ghr2 (I) in gilthead sea bream during the fasting and refeeding experiment. The postprandial period is shown in grey and the time in hours. Data are shown as means ± SEM (n = 6). Letters indicates significant differences (p < 0.05) by one-way ANOVA, LSD and Tukey HSD test.

Figure 2

Relative gene expression of skeletal white muscle akt (A), tor (B), 70s6k (C) and 4ebp1 (D) and representative blot and densiometric analysis of the phosphorylation ratios of Akt (E) and Tor (F) in gilthead sea bream during the fasting and refeeding experiment. For the Western blots, the same membranes cropped in two were used to analyze Tor (top part) and Akt (bottom part). The phosphorylated forms were analyzed first and after stripping, the corresponding total forms were determined in the same membranes. The intensity of the phosphorylated form was normalized by its total form, and the intensity of each specific band was normalized by the total transferred protein for the corresponding well. The postprandial period is shown in grey and the time in hours. Data are shown as means ± SEM (n = 6). Letters indicates significant differences (p < 0.05) by one-way ANOVA, LSD and Tukey HSD test.

Figure 3

Relative gene expression of skeletal white muscle pax7 (A), pcna (B), myf5 (C), myod1 (D), myog (E), mrf4 (F), mstn2 (G), mlc2a (H) and mlc2b (I) in gilthead sea bream during the fasting and refeeding experiment. The postprandial period is shown in grey and the time in hours. Data are shown as means ± SEM (n = 6). Letters indicates significant differences (p < 0.05) by one-way ANOVA, LSD and Tukey HSD test.

Figure 4

Relative gene expression of skeletal white muscle capn1 (A), capn2 (B), capns1a and capns1b (C), capn3 (D), ctsda (E), ctsl (F), mafbx (G), murf1 (H) and ub and n3 (I) in gilthead sea bream during the fasting and refeeding experiment. The postprandial period is shown in grey and the time in hours. Data are shown as means ± SEM (n = 6). Letters indicates significant differences (p < 0.05) by one-way ANOVA, LSD and Tukey HSD test.

Figure 5

Representative blot and densiometric protein levels of skeletal white muscle Ctsd (A) and Ctsl (B) in gilthead sea bream during the fasting and refeeding experiment. Each protein was analyzed in cropped membranes of different Western blots along with other proteins (data not shown). The intensity of each specific band was normalized by the total transferred protein. The postprandial period is shown in grey and the time in hours. Data are shown as means ± SEM (n = 6). Letters indicates significant differences (p < 0.05) by one-way ANOVA, LSD and Tukey HSD test.

Figure 6

Relative gene expression of bone total igf1 (A), igf1a (B), igf1b (C), igf1c (D), igf1ra (E) and ghr1 and ghr2 (F) in gilthead sea bream during the fasting and refeeding experiment. The postprandial period is shown in grey and the time in hours. Data are shown as means ± SEM (n = 6). Letters indicates significant differences (p < 0.05) by one-way ANOVA, LSD and Tukey HSD test.

Figure 7

Relative gene expression of bone runx2 (A), fib1a (B), col1a1 (C), ocn (D), on (E), ctsk (F), mmp9 (G), myod2 (H) and mstn2 (I) in gilthead sea bream during the fasting and refeeding experiment. The postprandial period is shown in grey and the time in hours. Data are shown as means ± SEM (n = 6). Letters indicates significant differences (p < 0.05) by one-way ANOVA, LSD and Tukey HSD test.

Figure 8

Schematic representation of the proposed changes occurring during early refeeding in gilthead sea bream. The Gh/Igf system recovers the synthetizing role with the Gh plasma levels still elevated activating now the hepatic expression/secretion of igf1, in parallel with the progressive up-regulation of the anabolic system components (ghr1, igf1, igf2 and igfbp5b). This condition contributes to the activation of the myogenic (pax7, myf5, myod1 and mrf4) and osteogenic (runx2, fib1a, col1a1 and on) genes, while downregulates the proteolytic (mafbx and murf1) and osteoclastogenic (ctsk) genes in muscle and bone, respectively. This early stage of refeeding may require a fine regulation of the different molecules involved, being myostatin a good candidate for bone and muscle crosstalk to assure harmonic musculoskeletal growth. P: pituitary; L: liver; WM: white muscle; B: bone.