In addition to its important role as a lipid storage depot, adipocytes play a key role in the control of energy balance and insulin sensitivity through the secretion of various hormones. Several studies have documented that secretion of these proteins occurs by both constitutive and regulated mechanisms. For example, it is well documented that insulin stimulates the secretion of leptin [17
], adiponectin [48
], and adipsin [49
]. The specific intracellular compartmentalization and trafficking of these hormones is poorly understood. In this report, we examined and compared the intracellular secretory pathways of two critical adipocyte hormones, adiponectin and leptin.
Biochemical and fluorescence microscopy studies have previously demonstrated that neither leptin [51
] nor adiponectin [48
] localize intracellularly with the well-studied intracellular compartment containing the insulin-responsive glucose transporter Glut4. Furthermore, the precise intracellular steady-state distribution of leptin in adipocytes has remained somewhat controversial. While initial studies [17
] had reported that a substantial amount of leptin is found in the cortical endoplasmic reticulum of rat isolated adipocytes, other biochemical studies [38
] had found that the distribution of leptin-containing vesicles separated by equilibrium sucrose density centrifugation was not consistent with that of the localization of endoplasmic reticulum markers. To reconcile these differences and to investigate the intracellular trafficking pathways required for leptin secretion in adipocytes, we first compared the intracellular steady-state distribution of expressed leptin-HA with that of another adipokine, adiponectin-myc in 3T3L1 adipocytes. Here we report that these hormones have distinct steady state localizations, whereas adiponectin is localized within the TGN overlapping with the marker syntaxin6, leptin is predominantly localized in close proximity of the endoplasmic reticulum marker BIP. Thus our results support those found by Barr et.al. [17
] in rat adipocytes, and confirm that in the steady-state the majority of adiponectin and leptin are located in distinct intracellular compartments.
Despite displaying a distinct intracellular localization, treatment with brefeldin A, inhibited the secretion of both adipokines in either cultured adipocytes or rat isolated adipose cells (by 58% and 35% in 3T3L1 and 39% and 36% in rat adipocytes for adiponectin and leptin secretion respectively). This finding suggests that both adiponectin and leptin traffic via Golgi/TGN. Interestingly however, in both cell types, BFA did not completely inhibit the secretion of these adipokines, indicating that the short term release of these hormones occurs partially through a BFA insensitive mechanism, and most probably, a post-TGN compartment which we hypothesize could represent the site for regulated secretion.
GGA adaptor proteins are important for the formation of adiponectin-containing vesicles at the TGN of adipocytes [35
]. Since GGA proteins participate in the TGN to endosome traffic, we hypothesized that adiponectin secretion but not leptin secretion would involve the endosomal compartment(s). To test this hypothesis, we conducted biochemical experiments to selectively inactivate the endosomes of 3T3L1 adipocytes following the procedure first described by Livingstone et al., 1996 [40
]. We also adapted this technique to isolate rat adipocytes. To our knowledge this is the first time that this technique has been applied to these cells. With this technique we achieved a complete inactivation of the endosomal compartments in 3T3L1 cells and a substantial (60%) inactivation of the endosomes in isolated rat adipocytes. Several reasons could account for this difference. First, transferrin-HRP internalization may be slower or less efficient than in 3T3L1 cells or alternatively, rat adipocytes may contain a larger endosomal compartment than 3T3L1 adipocytes which may require higher amounts of Tfn-HRP and DAB to achieve a complete inactivation. Attempts to increase the time of capture of transferrin-HRP or DAB and H2
substrate concentrations did not result in an increase in endosome inactivation in these cells. Furthermore, higher concentrations of H2
resulted in decreased viability of adipocytes (not shown). Nevertheless, our findings in rat isolated adipocytes corroborated the results obtained in the cell line 3T3L1. Endosomal inactivation profoundly reduced the amount of adiponectin secreted while leptin release remained unaffected., indicating that adiponectin but not leptin secretion requires functional endosomes.
To identify the endosomal compartments involved in adiponectin secretion we used confocal immunofluorescence and secretion studies in 3T3L1 adipocytes expressing adiponectin-myc and GFP-tagged wild type and/or constitutively active/inactive mutants of the endosomal markers rab11 and rab5. Immunofluorescence studies revealed adiponectin-myc distributed in close proximity to the endogenous rab 11 and rab5 proteins. in addition rab11 was detected in precipitated adiponectin-containing vesicles. Furthermore, over expression of a constitutively active or inactive forms of either rab11 or the rab5 proteins, modestly but significantly, inhibited adiponectin-myc release in 3T3L1 cells without affecting leptin release. Taken together, these findings strongly suggest that intact endosomal compartments are required for adiponectin but not leptin secretion. In agreement with these results, Gould’s group recently reported that a mutant of rab11 (rab11S25N) inhibited both basal and insulin-stimulated adiponectin secretion in 3T3L1 cells [52
]. In line with this, our results in rat adipocytes strongly support the idea that insulin-stimulation of adiponectin release may be mediated via insulin-mediated activation of the endosomal membrane recycling. Similarly to adiponectin, adipsin, a serine protease released by adipocytes is also released via TGN to endosome traffic [50
]. While the acute effects of insulin on leptin secretion have been reported to act at the level of secretion [18
], our data support a model where insulin may increase secretion of adiponectin and leptin in different ways. It remains to be established whether a similar or distinct insulin intracellular signaling cascades are utilized to control secretion of each adipokine.
To further identify molecular regulators for leptin secretion we tested whether leptin secretion is dependent on Protein Kinase D1 activity, and enzyme that regulates the formation of TGN-derived vesicles en route to the plasma membrane [46
]. Over expression of a dominant interfering mutant PKD1 K618N exhibited a moderate but significant decrease in leptin release. This suggests the possibility that PKD1 may be involved in regulating at least partially, the trafficking of leptin-containing vesicles. Further experiments are required to test whether the PKD1 activity is required for the insulin-stimulated effect on leptin secretion.
Taken together, our results demonstrate that adiponectin and leptin are secreted through distinct intracellular trafficking pathways and suggest that adipocytes rely on different avenues for the constitutive and regulated secretion of these adipokines.