4 Discussion
4.4 Lipolytic product release is elevated upon UCP1 ablation in brown but not in brite
Lipolysis represents the sequential hydrolysis of TAG stored in cell LDs and is catalyzed by ATGL, HSL and monoacylglycerol lipase (MGL) (Bolsoni-Lopes and Alonso-Vale, 2015).
Thereby, ATGL and HSL are responsible for more than 90% of TAG hydrolysis in adipose tissue (Schweiger et al., 2006). Lipolysis is a highly regulated process and occurs under non-stimulated, basal conditions as well as after hormonal stimulation. Adrenaline and NE stimulate lipolysis through activation of β-adrenergic receptors (β-ARs) that activate adenylate cyclase (AC). The subsequent rise in cyclic AMP (cAMP) activates protein kinase A (PKA), which phosphorylates hormone sensitive lipase (HSL) and LD-coating protein perilipin as well as the adipose trigyceride lipase (ATGL) co-activator CGI-58 (comparative gene indentification 58) to stimulate lipolysis. On the other hand, catecholamines can also inhibit lipolysis via the activation of α2-AR that mediate AC inhibition and therefore cAMP production. Insulin signaling leads to phosphodiesterase 3B (PDE3B) activation that degrades cAMP and hence reduces PKA activation (Degerman et al., 1997). Furthermore, insulin-induced antilipolytic effects are mediated via lactate and its receptor GPR81 (Ahmed et al., 2010; Liu et al., 2009). ATGL is mainly located in the LD of adipocytes and is considered as the main enzyme responsible for TAG hydrolysis, especially under basal
conditions, since it has direct access to the LDs. Nevertheless, ATGL activity is significantly increased by CGI-58 binding (Ahmadian et al., 2010; Bezaire and Langin, 2009; Yamaguchi et al., 2004).In brite and brown adipocytes, adrenergic UCP1 activation is largely dependent on ATGL rather than HSL and mice with lower expression of ATGL develop severe hypothermia when exposed to cold (Haemmerle et al., 2011; Li et al., 2014b)
FFA are required for fueling and activation of UCP1. Full activity of UCP1 requires an adequate lipolytic rate. Moreover, differences observed in the LD size distribution of brite adipocyte cultures might influence their lipolytic capacities. To investigate the influence of UCP1 abundance and activity on lipolysis, the lipolytic activity was assessed under basal and isoproterenol stimulated conditions in brite and brown adipocyte cultures. The concentrations of the lipolytic products FFA and glycerol were measured in the incubation medium. In the case of brown adipocytes, cells were pretreated with or without oligomycin.
Brite and brown adipocytes released glycerol and FFA in quantitatively significant amounts under basal conditions and especially after treatment with isoproterenol (Figure 13, Figure 33). The lipolytic capacities of 129SV/S6, C57BLB/6J UCP1+/+ and C57BLB/6J UCP1-/- cells were comparable and independent from UCP1 abundance or activity. Human hMADS cells, differentiated into brite adipocytes possess a higher lipolytic capacity together with enhanced FA oxidation and FA esterification (Barquissau et al., 2016).
Similar to the present work, the brite phenotype was induced by PPAR agonist treatment.
PPARγ and PPARα are important regulators of lipolysis. PPARγ activation with rosiglitazone stimulates basal and NE induced lipolysis by increasing the lipolytic potential, including increased ATGL and MGL gene expression in WAT (Festuccia et al., 2006).
Palmitoleic acid treatment increases lipolysis in vivo and in vitro under basal conditions and in response to isoproterenol by increasing phosphorylated ATGL and HSL in adipocyte via PPARα in WAT (Bolsoni-Lopes et al., 2013). Similar effects PPARγ and PPARα agonists were observed in 3T3L1 adipocytes (Bolsoni-Lopes et al., 2013; Goto et al., 2011; Kershaw et al., 2007). Consequently, PPARγ and PPARα mediated elevated lipolytic capacity does not require UCP1. In the present work, lipolytic capacity was assessed in brite adipocytes which were differentiated in the presence of rosiglitazone.
Thus, possible UCP1 mediated effects on lipolysis might have been blunted or overlaid by PPARγ agonist treatment. Rosiglitazone can also upregulate the lipolytic machinery of BAT by increasing expression of ATGL, CGI-58 and MGL (Festuccia et al., 2010).
The release of lipolytic products was not only independent from UCP1. The distinct lipid droplet size distribution observed in the basal state of brite adipocytes cultures (Figure 11) did also not cause differences in lipolytic activity. In general, a smaller LD size is assumed to be associated with an increased LD surface/volume ratio which facilitates the rapid lipid degradation and utilization for adaptive thermogenesis (Cinti, 2012). Adrenergic treatment and stimulation of lipolysis activates gradual fragmentation and dispersion of micro-lipid droplets (Brasaemle et al., 2004; Marcinkiewicz et al., 2006). FA can be quickly mobilized and transferred from LD to mitochondria (Rambold et al., 2015). Rapid relocalization of fatty acids to mitochondria is accomplished by direct transfer via contact sites (Welte, 2015; Zhang et al., 2016). Adrenergic induced LD fission processes may rapidly overcome the difference in basal lipid droplet size distribution that might be too small to affect lipolytic activity.
Brown adipocytes derived from 129SV/S1 UCP1+/+ and UCP1-/- mice showed a similar glycerol and FFA release under basal conditions, indicating a comparable basal lipolytic activity. However, after isoproterenol treatment adipocyte cultures from 129SV/S1 UCP1- /-mice released significantly more glycerol and FFA than cultures from UCP1+/+ mice. This discrepancy was even more pronounced, when cells were pretreated with oligomycin.
Pretreatment with oligomycin further elevated isoproterenol induced FFA release in UCP1- /- cells, while it caused a reduced FFA release in UCP1+/+ cells. Such an effect of oligomycin was not observed in basal FFA or glycerol concentrations. Thus, there is a negative correlation between UCP1 activity and isoproterenol induced release of FFA into the medium.
A lower release of FFA and glycerol can be explained by an alternative fate of these metabolites. FFA can be combusted in β-oxidation or reesterified with G3P to form TAG.
They can be also used for synthesis of other FA and lipids. Glycerol either can serve as energy substrate or can be transformed into G3P, a process that requires GK. GK activity is strongly increased in BAT by cold adaptation (Bertin, 1976). Moreover, during cold exposure glyceroneogenesis is substantially activated in BAT, contributing to G3P generation, which is mostly used to reesterify FFA (Moura et al., 2005). On the one hand UCP1 activity increases substrate oxidation with FFA being the primary fuel for thermogenesis (Ma and Foster, 1986; Nedergaard and Lindberg, 1982; Nicholls and Locke, 1984). On the other hand, brown adipocytes must maintain adequate intracellular TAG stores for constant fuel supply for NST, as observed in cold acclimated rodents, which show a marked increase in BAT TAG (Himms-Hagen, 1965; Moura et al., 2005) an
FA synthesis (Trayhurn, 1979, 1981). Thus, lipid catabolism and anabolism might occur simultaneously upon UCP1 activation and may explain the reduced release of lipolytic products in UCP1 expressing cells under isoproterenol treatment.