Biology Reference
In-Depth Information
differentiated mature adipocytes. The use of electroporation allows a high-efficiency
delivery of siRNA oligos and subsequent knockdown of specific gene expression.
A centrifugation-assisted infection with recombinant adenovirus, on the other hand,
enables robust overexpression of ectopic proteins. Most importantly, by combining
siRNA electroporation with adenovirus infection, simultaneous manipulation of
levels of two different proteins can be achieved in differentiated adipocytes. Through
subsequent analyses of lipase activity in cell extracts and fatty acid or glycerol
release from living cells, mutual interdependence between the two proteins in the
context of basal and hormone-stimulated adipocyte lipolysis can be evaluated.
INTRODUCTION AND RATIONALE
Adipose tissue or fat is loose connective tissue consisting mainly of adipocytes
(
Nakajima, Yamaguchi, Ozutsumi, & Aso, 1998; Sethi & Vidal-Puig, 2007
). There
are two types of adipose tissues: white adipose tissue (WAT) and brown adipose tissue
(BAT) (
Saely, Geiger, & Drexel, 2012
). While BAT consumes energy (
Cannon &
Nedergaard, 2004; Frontini & Cinti, 2010
), WAT is the main energy store in the body
of mammals. Upon food intake, excess dietary fatty acids (FAs) are esterified to
triacylglycerols (TGs) and stored in the lipid droplets of adipocytes (
Oller do
Nascimento, Ribeiro, & Oyama, 2009; Saely et al., 2012
). In response to cold expo-
sure, TG stores in brown adipocytes are mobilized via a hydrolytic process called
lipolysis to supply FAs for
-oxidation and subsequent heat production (
Cannon &
Nedergaard, 2004; Frontini & Cinti, 2010
). During chronic fasting or extended
physical exercise, lipolysis in white adipocytes is stimulated to release free FAs (FFAs)
into circulation for the purpose of providing energy to other peripheral tissues
(
Ahmadian, Wang, & Sul, 2010; Greenberg et al., 2011
;
Watt & Spriet, 2010
).
Although nonadipocyte cells are able to esterify FFAs into TGs and rehydrolyze
the latter upon demand, they do not secrete FFAs but instead utilize them locally
for ATP production and/or lipid synthesis. Thus, the provision of FFAs as systemic
energy substrate is a unique function of lipolysis in white adipocytes (
Lass,
Zimmermann, Oberer, & Zechner, 2011
). In obesity, alterations in lipolysis contribute
to elevated circulating FAs and the development of peripheral lipotoxicity and insulin
resistance (
Arner et al., 2008; Deng et al., 2012; Langin et al., 2005; Moro et al., 2009
).
Accordingly, the precise mechanisms for lipolytic regulation have attracted immense
research interest over the years.
Lipolysis in adipocytes is mediated by a complex proteome comprised of specific
acylglycerol lipases and their regulators (
Kolditz & Langin, 2010
). The adipose
lipases include adipose triglyceride lipase (ATGL), hormone-sensitive lipase
(HSL), and monoacylglycerol lipase (
Lass et al., 2011
). ATGL favors TG substrates
and catalyzes the rate-limiting first step of lipolysis. HSL, on the other hand, is be-
lieved to function as a diacylglycerol lipase
in vivo
(
Haemmerle et al., 2002; Osuga
et al., 2000; Schweiger et al., 2006; Zimmermann et al., 2004
). Research conducted
b
