Previous studies regarding the nutritional influences on satellite cells have been focused on the changes of cell mitotic activity by determining DNA synthesis with incorporation of either 3H]thymidine[8, 10] or BrdU[8, 11, 12, 27]. Pax7 is also used as a specific marker for satellite cells in immunohistochemistry. These are classic methods for cell proliferation studies, which are more sensitive and accurate compared with the MTT assay. However, normal cell growth regulation not only includes proliferation but also apoptosis (programmed cell death). The MTT assay is a convenient and efficient method for establishing the number of living cells (viability), reflecting the ultimate balance between cell proliferation and apoptosis. Here, the morphological differences of the satellite cells revealed the significant impact of early-age intermittent feeding on cell proliferation and differentiation, while the changes in the ratio of Bax to Bcl-2 mRNA expression implicated changes of apoptotic potential of the satellite cells responding to nutrition restriction and restoration. This implies some strategies of feed restriction for higher feeding efficiency should be appropriately (for example, fasted one day per three days) and duly (for example, fasted at an older age). If not, the total number of satellite cells will be less for full muscle growth.
We found that 2 days re-feeding after 12 days of intermittent feeding was unable to restore completely the proliferation and differentiation capabilities of the satellite cells, as indicated by the cell morphology and viability detected with the MTT assay. This result adds to the previous findings that several days of re-feeding was able to completely reverse the depressed mitotic activity of the satellite cell caused by short-term (2–3 days) feed deprivation or fasting in chicks or turkeys[10, 12, 27, 28]. A delayed peak of satellite cell DNA synthesis and mitotic activity was observed after 2–3 days of re-feeding in chicks and 3 days re-feeding in young turkeys, which allows a complete restoration of satellite cell numbers. Difference in fasting strategies should take into account this divergence. For those starter diet withdrawal treatments, the fasting only lasted 2 or 3 days after hatching, when yolk residue still serves as an energy resource. It is possible that intermittent (skip a day) feeding for 2 weeks after hatching is more stringent compared with the short-term fasting in other studies, 2 days of re-feeding was not sufficient for restoring satellite cell numbers. Another possibility is the critical window during early post-hatch development for satellite cell proliferation. The first week after hatching is considered as the most active period for satellite cell proliferation and differentiation. Re-feeding occurring within this period may be more effective for a complete compensation, compared with the delayed re-feeding in this study. The most dramatic change in satellite cell activity may occur within the first week, yet the decreased satellite cell activity observed on Day 15 in this study reflects the cumulative effects of intermittent feeding in the first 2 weeks of post-hatch life. This also implicates a suited period for feed restriction and re-feeding that should be considered.
Previous findings have shown a role for apoptosis in muscle induced by under-nutrition[28–30], so here we also tested the expressions of apoptotic regulatory factors, Bax, a death-promoting molecule, and Bcl-2, a survival protein, in extracted satellite cells to explore the survival of them in different feed treatments[31, 32]. We noticed in our previous research that early feed restriction decreased the mRNA expression of Bcl-2 and increased the ratio of Bax mRNA/Bcl-2 mRNA in gastrocnemius muscle tissue at the end of 14 days of early-age feed restriction, but there was no difference in the evaluation of DNA ladder electrophoresis (data not published). However, no changes were found here in the mRNA levels of Bcl-2 and Bax in satellite cells of the three feed treatment groups, and there was no difference in Bax/Bcl-2 ratio between the RF and Con groups. It may be that the 14 days of alternate fasting did not induce apoptosis obviously or exhibited in these factors. We found a down-regulation of Bax/Bcl-2 ratio in the RF group compared with the IF group, suggesting satellite cell apoptosis was repressed by restoration of nutrition during re-feeding.
It is suggested that the GH/IGF-I system mediates the effect of nutritional state on satellite cells. Feed restriction induces a significant fall in circulating IGF-I[34, 35] and a rise in plasma GH, which could be restored to the normal levels by re-feeding. We reported previously that IF chickens expressed lower IGF-I and higher IGF-IR mRNA in the gastrocnemius muscle on Day 14. Satellite cells isolated from the muscle showed similar responses with lower GHR, IGF-1 and higher IGF-IR mRNA expression in the IF group. It was suggested that in chickens after hatching, hepatic gene expression of IGF-I is GH-dependent while muscular gene expression of IGF-I is independent of GH and GHR. However, it is unknown whether IGF-I expression in satellite cells is dependent on GHR. Here, expression of GHR and IGF-I in satellite cells exhibited a similar pattern in response to feed restriction and re-feeding, suggesting a possible regulatory link between these two genes.
The role of the GH/IGF-I axis in the regulation of avian muscle growth remains obscure. Growth hormone can promote skeletal muscle satellite cell proliferation in vitro[13, 14] and in vivo[40, 41], and modify GHR expression[13, 14]. Satellite cell proliferation was decreased in starved chicks along with a lower GHR gene expression, which were reversed with re-feeding. IGF-I stimulates the proliferation, and fusion of satellite cells in vitro[42–46]. However, IGF-I together with GH in culture showed no enhancement effect on DNA synthesis in chicken satellite cells. Since both myofibers and satellite cells are able to produce IGF-I, the effects of paracrine and autocrine IGF-I on satellite cell activity have to be considered, in addition to the role of endocrine IGF-I. Recently, mechano growth factor E (MGF-E), derived from an isoform of IGF-I, was reported to activate human muscle progenitor cells.
In addition to the GH/IGF system, thyroid hormones were suggested to be involved in mediating the effect of nutrition on satellite cell function[15, 49]. Subcutaneous injections of T4 in rats would stimulate the number of total satellite cells and satellite cells per muscle fiber, while satellite cell numbers extracted from the hypothyroid rats were fewer and less active in proliferation and differentiation at the start of culture. However, it is unclear how expression of the thyroid hormone receptor in satellite cells responds to nutritional status and thyroid hormone levels. We reported previously that serum concentrations of both T3 and T4 decreased with IF for 14 days in chicks. We observed a significant up-regulation of TRα mRNA expression in the IF group, which was completely restored with re-feeding. This up-regulation of TRα mRNA expression in satellite cells may represent a feedback regulation through decreased serum thyroid hormone levels. However, the TRα mRNA expression in satellite cells was not coinciding with the viability of satellite cells (Figures 2 and3). It is speculated that the thyroid hormone receptor activity, which determines the sensitivity of the satellite cells to T3, may be blunted. This speculation was supported in the T3 challenge test for satellite cells from the three different groups (Figure 5). Satellite cells from the IF group were insensitive to T3 while re-feeding partly restored the responsiveness of satellite cells to T3, although the viabilities were still significantly lower compared with the Con group at both basal and T3-stimulated conditions. It is likely that the up-regulation of TRα mRNA expression in the IF group represents a feedback mechanism of disrupted signaling of thyroid hormones on satellite cells.
In conclusion, long-term feed restriction (12–14 days of intermittent feeding) immediately after hatching impairs proliferation and differentiation capabilities of satellite cells, which could not be completely restored by 2 days of re-feeding. The disrupted satellite cell viability was associated with alterations in mRNA expression of the GH, IGF-I and thyroid hormone receptors, as well as the blunted sensitivity of satellite cells to T3. Therefore, the persistent retardation in myofiber hypertrophy caused by 14 days of intermittent feeding post-hatching reported previously can be explained by the decreased satellite cell proliferation and differentiation activity, lower serum T3 levels and the blunted sensitivity of satellite cells to T3. This suggests that long-term IF carried too early after hatching is not an ideal strategy for poultry meat production. RF partially reverses these effects, which indicates a moderate nutritional strategy for feed restriction if implemented early post-hatching.