Automated Methods for the Analysis of Skeletal Muscle Fiber Size and Metabolic Type - International Review of Cell and Molecular Biology - page 278

et al., 2006 ). There is a growing recognition that muscle, adipose tissue, and
the pancreas communicate with each other via secreted factors ( Plomgaard
et al., 2012 ) and that mitochondrial dysfunction ( Martins et al., 2012 ) may be
involved. Also, once established, the insulin insensitivity can persist in unex-
pected ways; for example, muscle satellite cells isolated from obese individ-
uals differentiate into insulin-resistant myotubes, in vitro ( Houmard et al.,
2011 ), and the male offspring of mice fed a high caloric diet during preg-
nancy and lactation develop insulin insensitivity several months after
weaning to a standard diet ( Shelley et al., 2009 ). Increasing our understand-
ing of how metabolic processes in skeletal muscle contribute to diabetes will
become increasingly important as the frequency of obesity increases.
3. METHODS FOR CHARACTERIZING MUSCLE
FIBER SUBTYPES
3.1. Historical overview
Early methods of fiber typing utilized colorimetric staining methods to
detect fiber-specific enzyme activities in skeletal muscle tissue sections.
The histochemical assay of succinate-dehydrogenase (SDH) activity, which
correlates with the amount of mitochondria per fiber (also discussed in
Section 4 ), was one of the earliest methods utilized to distinguish fiber types
in skeletal muscle ( Wachstein and Meisel, 1955 ). The SDH technique was
further developed for fiber typing by Dubowitz and Pearse and adopted by
others in the 1960s to evaluate fiber-type composition ( Dubowitz and
Pearse, 1960; George and Talesara, 1961; Susheela and Walton, 1969;
Tsukamoto and Mori, 1966 ). When stained for SDH activity, Type
I fibers, which have the largest number of mitochondria, develop an intense
blue color. Type II fibers develop less color and can be further subdivided
into Type IIA and Type IIB fibers, which feature intermediate and the low-
est levels of SDH activity, respectively. Other approaches were considered
for fiber typing, including the assay of glycogen or lipid content (via Oil Red
O staining) and cytochrome c oxidase (COX) activity ( Engel, 1970 ); how-
ever, the metabolic character of the fibers changes in response to a variety of
developmental, nutritional, and pathological conditions, which challenges
the reliability of these methods for fiber-subtype determination ( Brooke
and Kaiser, 1970 ).
As it became clear that different myosin isoforms are preferentially
expressed by different fiber subtypes, an alternative method emerged based
upon assaying the ATPase activity of myosin (mATPase). Developed by