Glutamine supplementation stimulates cell proliferation in skeletal muscle and cultivated myogenic cells of low birth weight piglets

Abstract

Muscle growth of low birth weight (LBW) piglets may be improved with adapted nutrition. This study elucidated effects of glutamine (Gln) supplementation on the cellular muscle development of LBW and normal birth weight (NBW) piglets. Male piglets (n = 144) were either supplemented with 1 g Gln/kg body weight or an isonitrogeneous amount of alanine (Ala) between postnatal day 1 and 12 (dpn). Twelve piglets per group were slaughtered at 5, 12 and 26 dpn, one hour after injection with Bromodeoxyuridine (BrdU, 12 mg/kg). Muscle samples were collected and myogenic cells were isolated and cultivated. Expression of muscle growth related genes was quantified with qPCR. Proliferating, BrdU-positive cells in muscle sections were detected with immunohistochemistry indicating different cell types and decreasing proliferation with age. More proliferation was observed in muscle tissue of LBW-GLN than LBW-ALA piglets at 5 dpn, but there was no clear effect of supplementation on related gene expression. Cell culture experiments indicated that Gln could promote cell proliferation in a dose dependent manner, but expression of myogenesis regulatory genes was not altered. Overall, Gln supplementation stimulated cell proliferation in muscle tissue and in vitro in myogenic cell culture, whereas muscle growth regulatory genes were barely altered.

Figure 1

Incorporation of BrdU in proliferating cells within muscle tissue. Immunohistochemical detection of BrdU-positive nuclei (green) and total nuclei (red, stained with propidium iodide) in a longissimus muscle cross section of a 12-day-old piglet. Arrows indicate proliferating cells of different types. MF muscle fibers, AD adipocytes, BV blood vessel, CT connective tissue.

Figure 2

Detection and quantification of proliferating cells in M. longissimus. (a–c) Immunohistochemical detection of BrdU-positive nuclei (green) and total nuclei (red) at 5, 12 and 26 dpn; (d) area percentage of total nuclei; (e) area percentage of BrdU-positive nuclei; (f) ratio of BrdU-positive nuclei to total nuclei; (g) number of total nuclei per mm² in a region comprising muscle fibers exclusively; (h) number of BrdU-positive nuclei per mm²; (i) ratio of BrdU-positive nuclei to total nuclei in M. longissimus of glutamine (GLN) or alanine (ALA) supplemented low birth weight (LBW) and normal birth weight (NBW) piglets at 5, 12 and 26 dpn. Values are presented as LSmeans and SE (n = 12 per group). Different uppercase letters (A-C) indicate significant differences among ages and different lowercase letters (a, b) indicate significant differences among groups within the same age (p ≤ 0.05, Tukey–Kramer test).

Figure 3

Relative mRNA abundances of genes in M. longissimus. Expressions of PAX7 (a), MYOD (b), MYF5 (c), MYOG (d), MSTN (e) and PPARGC1A (f) were analyzed in MLD of glutamine (GLN) or alanine (ALA) supplemented low birth weight (LBW) and normal birth weight (NBW) piglets at 5, 12 and 26 dpn (n = 12 per group). Values were normalized to YHWAZ and PPIA expression and are presented as LSmeans and SE. Different uppercase letters (A, B) indicate significant differences among ages (p ≤ 0.05, Tukey–Kramer test), ^#trend among groups within the same age (0.05 < p ≤ 0.1).

Figure 4

Cell indexes of proliferating myogenic cells. Cell indexes were recorded for myogenic cells with the xCELLigence RTCA-DP device. Cells, isolated from M. longissimus of low birth weight (LBW) and normal birth weight (NBW) piglets at 4 dpn, were supplemented with 0.5 mM Gln or Ala (a), 5 mM Gln or Ala (b), 10 mM Gln or Ala (c) and compared with myogenic cells without supplementation (no suppl.). The basic medium contained 0.28 mM Gln and Ala. All values are presented as Means and SD (standard deviations) of three repetitive wells.

Figure 5

Expression of muscle growth-related genes in proliferating porcine myogenic cells. (a) Representative microscopic images of cells of the same sample at day 2 and day 3 without supplementation or with 10 mM Gln or Ala. Scale bars represent 200 µm. (b) Relative mRNA abundances of PAX7, MYOD, MYOG and PPARGC1A in proliferating cells of low (LBW) or normal birth weight (NBW) piglets at day 2 and day 3 after seeding. Values were normalized to YHWAZ and PPIA expression and are presented as LSmeans and SE (n = 3 per group).

Figure 6

Expression of muscle growth-related genes in differentiating porcine myogenic cells. (a) Representative microscopic images of cells of the same sample at day 0 (day of differentiation induction), day 3 and day 6 without supplementation or with 10 mM Gln or Ala. Scale bars represent 200 µm. (b) Relative mRNA abundances of PAX7, MYOD, MYOG and PPARGC1A in myogenic cells from low (LBW) or normal birth weight (NBW) piglets at day 0, day 3 and day 6 after induction of differentiation. Values were normalized to YHWAZ and PPIA expression and are presented as LSmeans and SE (n = 3 per group). Different lowercase letters indicate significant differences among groups within the same time point, different uppercase letters indicate significant differences (p ≤ 0.05, Tukey–Kramer test) and ^#trend for differences (0.5 < p ≤ 0.1) among different time points.