Musclin a novel skeletal muscle-derived secretory factor
 |
Alignments of musclin amino acid
sequences for mouse, rat and human. Predicted signal
sequence; shaded box. The putative serine protease cleavage site,
KKKR; open box. The region homologous to mouse ANP, BNP and CNP;
hatched box. C, Secretion of musclin protein. The cell lysates and
culture medium from C2C12 myocytes infected by retrovirus carrying
Flag-tagged mouse musclin or GFP were immunoprecipitated, and
subjected to western blot analysis, using anti-Flag antibody. Mus;
musclin. D, Secretion of wild type and mutant musclin from HEK 293
cells. The culture medium from HEK 293 cells transfected with
pEF-BOS-WT musclin Flag (left lane) or pEFBOS-Mut musclin Flag
(76KKKR79-76A) (right lane) was subjected to
immunoprecipitation followed by western blotting using anti-Flag
antibody. |
In vivo expression of musclin
mRNA. A, Northern blot analysis of musclin expression in
mice. B, Quantitative RT-PCR analysis of musclin mRNA expression in
mice. C, Regulation of musclin mRNA expression by nutritional
status. Gastrocnemius muscles of mice fed ad libitum, fasted for 48
h, or fasted for 48 h and refed for 24 h (n=5, each) were used for
real-time RT-PCR. Inset: representative Northern blotting (n=2,
each). D, Musclin mRNA expression in insulin-deficient STZ mice.
Gastrocnemius muscles of STZ and control mice (n=5, each) were
subjected to real-time RT-PCR. The values were expressed relative to
the level of 36B4 mRNA. E, Musclin mRNA expression in KKAy mice.
Gastrocnemius muscles of 13-wk-old KKAy mice and age-matched
C57BL/6J mice (n=8, each) were used for real-time RT-PCR. The values
were normalized to the level of cyclophilin mRNA. Data are mean +-
SEM values. *p< 0.05, **p< 0.01.
Regulation of musclin mRNA expression by
various hormones, and effects of musclin on insulin-stimulated
glucose uptake and glycogen synthesis in C2C12 myocytes. A, Musclin
mRNA expression during myocyte differentiation. C2C12 cells were
harvested at indicated days after differentiation-induction for
real-time PCR. B-D, Regulation of musclin mRNA expression by insulin
(INS), IGF-1 (IGF), epinephrine (Epi), isoproterenol (Iso), and
forskolin (FSK) (real-time PCR). The expression level was expressed
relative to that of cyclophlin mRNA (n=3, each). E-F, Effect of
recombinant musclin on 2-DG uptake and glycogen synthesis. After 5-h
pre-treatment with Flag-peptide (0.5 ug/ml) as control or Flagtagged
musclin (0.5 ug/ml), 2-DG uptake (E) and glycogen synthesis (F) were
determined as described in Experimental Procedures. Data are mean+-
SEM values (n=6). **p<0.01. Similar results were obtained in two
other independent experiments.
