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  • SC 560 The following is the supplementary data


    The following is the supplementary data related to this article.
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    Introduction Adipose tissues contribute to systemic glucose and lipid homeostasis under both anabolic and catabolic conditions [1]. In white adipose tissue (WAT), the uptake and processing of dietary nutrients is stimulated by insulin in the postprandial state, enabling efficient storage of excess energy as triglycerides within lipid droplets. In the fasted state, lack of insulin signaling facilitates lipolysis by intracellular lipases to release fatty acids for e.g. VLDL production in the liver and ATP generation in muscles [1,2]. In contrast to WAT, energy substrates internalized by brown adipose tissue (BAT) are used for heat production upon cold-induced non-shivering thermogenesis, mediated primarily by uncoupling protein 1 (UCP1) [3]. Both thermogenic activation by cold-exposure as well as anabolic insulin signaling promote BAT uptake of glucose and lipids [[4], [5], [6], [7]]. Accordingly, systemic insulin resistance triggered by obesity leads to impaired glucose and lipid disposal into adipose tissues. These metabolic dysfunctions contribute to hyperglycemia and dyslipidemia and promote the development of type 2 diabetes and atherosclerosis that are associated with obesity [8,9]. BAT activation can correct these metabolic disturbances, and consequently the obesity-associated comorbidities [5,[10], [11], [12]]. The capacity for glucose uptake in adipocytes and skeletal muscle SC 560 is regulated by insulin-dependent redistribution of glucose transporter 4 (GLUT4) from intracellular endosomal vesicles to the plasma membrane. Mice with adipose tissue-specific GLUT4 deficiency or overexpression presented with impaired or enhanced glucose tolerance, respectively, underscoring the relevance of GLUT4 in adipocytes for systemic glucose homeostasis [13,14]. The insulin signaling cascade that initiates GLUT4 translocation to the cell surface depends on activation of the protein kinase B (AKT) pathway. Subsequently, GLUT4-containing vesicles are targeted to the plasma membrane in a process dependent on numerous proteins involved in signaling (e.g. the TBC1 domain family member 4 also known as AS160 [15]), protein recycling (e.g. syntaxin 6 and tumor suppressor candidate 5 [16,17]), vesicle tethering (e.g. tether, containing a UBX domain, for GLUT4 [18]) as well as vesicle fusion (e.g. vesicle-associated membrane proteins [19,20]). The same GLUT4-storage vesicles also contain low-density lipoprotein receptor related protein 1 (LRP1) [21], for which insulin-dependent transport to the plasma membrane has been described in hepatocytes and adipocytes [[22], [23], [24]]. Insulin-mediated LRP1 translocation in the liver is dependent on the phosphorylation of its distal NPxYxxL motif [23,25]. Notably, by binding to the unphosphorylated form of this motif, the protein phosphotyrosine interacting domain containing 1 (PID1) serves as an intracellular adaptor for LRP1 [23,26,27]. In addition, PID1 has been implicated to regulate glucose uptake via modulation of insulin signaling in adipocytes and muscle cells [[28], [29], [30], [31]]. We recently demonstrated that insulin signaling in hepatocytes disrupts PID1 binding to LRP1, which results in the translocation of LRP1 to the cell surface and thus the efficient lipoprotein receptor-mediated endocytosis in the postprandial state [23]. Based on these findings and the high co-expression of PID1 and LRP1 in adipose tissues [23], we propose that PID1 has a fundamental role in systemic glucose homeostasis by regulating sorting of GLUT4 vesicles via insulin-dependent trafficking of LRP1.
    Materials and methods
    Discussion PID1 is an intracellular adaptor protein for LRP1 with higher expression in adipose tissues of obese compared to lean subjects [26,39]. Recently, we have shown that PID1 controls clearance of pro-atherogenic lipoproteins into the liver by regulating cell surface abundance of LRP1 [23]. In addition to its role in hepatic lipoprotein metabolism, elevated PID1 expression was previously found to be associated with reduced glucose uptake in adipocytes and myotubes [31,40]. In the current study, we show that PID1 determines the cellular localization of both LRP1 and GLUT4 that are found in perinuclear compartments of primary adipocytes under non-stimulated, fasting conditions. As expected, insulin stimulation leads to the translocation of both proteins to the plasma membrane [21,24]. In Pid1 adipocytes and muscle, LRP1 and GLUT4 are targeted to the cell surface irrespective of insulin signaling. Notably, LRP1-deficiency also resulted in elevated cell surface levels of GLUT4 in primary brown adipocytes, supporting the concept that PID1-LRP1 interaction is involved in the intracellular trafficking of GSVs. Furthermore, we show the in vivo relevance of PID1 for glucose metabolism in global as well as adipocyte-specific PID1-deficient mice. The lack of PID1 did not alter insulin signaling and responsiveness in vivo, but resulted in an improved glucose tolerance and increased glucose disposal into GLUT4-expressing tissues despite insulin resistance. These data support the model (Fig. 6H–J) that in the basal state, PID1 interacts with the non-phosphorylated distal NPxY motif within the intracellular domain of LRP1 [23,41,42] to retain GLUT4-containing storage vesicles in perinuclear compartments of adipocytes (Fig. 6H). Insulin-mediated phosphorylation of the distal NPxY motif of LRP1 promotes the disintegration of the PID1-LRP1 complex. This process ultimately leads to the insulin-triggered targeting of GLUT4 to the cell surface to facilitate efficient glucose uptake in the postprandial situation (Fig. 6I). Consequently, the lack of PID1 resulted in the perpetual cell surface presence of GLUT4, stimulating glucose uptake even in insulin-resistant, diabetogenic conditions (Fig. SC 560 6J). The proposed mechanism is in line with our recent findings demonstrating that PID1 is involved in the hepatic clearance of pro-atherogenic lipoproteins [23], a process dependent on the insulin-mediated translocation of LRP1 to the cell surface [22]. Mechanistically, we showed that PID1 functions as an adaptor protein for LRP1 retaining the non-phosphorylated form of this receptor in intracellular storage compartments. In the postprandial phase, insulin signaling triggers the phosphorylation of the LRP1 - 60NPxYxxL66 motif, thereby disrupting the LRP1-PID1 complex ultimately leading to the vesicular transport of LRP1 to the plasma membrane [23].