Sac3 Is an Insulin-regulated Phosphatidylinositol 3,5-Bisphosphate Phosphatase: GAIN IN INSULIN RESPONSIVENESS THROUGH Sac3 DOWN-REGULATION IN ADIPOCYTES*

Insulin-regulated stimulation of glucose entry and mobilization of fat/muscle-specific glucose transporter GLUT4 onto the cell surface require the phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P₂) pathway for optimal performance. The reduced insulin responsiveness observed under ablation of the PtdIns(3,5)P₂-synthesizing PIKfyve and its associated activator ArPIKfyve in 3T3L1 adipocytes suggests that dysfunction of the PtdIns(3,5)P₂-specific phosphatase Sac3 may yield the opposite effect. Paradoxically, as uncovered recently, in addition to turnover Sac3 also supports PtdIns(3,5)P₂ biosynthesis by allowing optimal PIKfyve-ArPIKfyve association. These opposing inputs raise the key question as to whether reduced Sac3 protein levels and/or hydrolyzing activity will produce gain in insulin responsiveness. Here we report that small interfering RNA-mediated knockdown of endogenous Sac3 by ∼60%, which resulted in a slight but significant elevation of PtdIns(3,5)P₂ in 3T3L1 adipocytes, increased GLUT4 translocation and glucose entry in response to insulin. In contrast, ectopic expression of Sac3^WT, but not phosphatase-deficient Sac3^D488A, reduced GLUT4 surface abundance in the presence of insulin. Endogenous Sac3 physically assembled with PIKfyve and ArPIKfyve in both membrane and soluble fractions of 3T3L1 adipocytes, but this remained insulin-insensitive. Importantly, acute insulin markedly reduced the in vitro C8-PtdIns(3,5)P₂ hydrolyzing activity of Sac3. The insulin-sensitive Sac3 pool likely controls a discrete PtdIns(3,5)P₂ subfraction as the high pressure liquid chromatography-measurable insulin-dependent elevation in total [³H]inositol-PtdIns(3,5)P₂ was minor. Together, our data identify Sac3 as an insulin-sensitive phosphatase whose down-regulation increases insulin responsiveness, thus implicating Sac3 as a novel drug target in insulin resistance.Insulin simulation of glucose uptake in fat and muscle, which is mediated by the facilitative fat/muscle-specific glucose transporter GLUT4, is essential for maintenance of whole-body glucose homeostasis (1–7). In basal states GLUT4 is localized in the cell interior, cycling slowly between the plasma membrane and one or more intracellular compartments. Insulin action profoundly activates movements of preformed postendosomal GLUT4 storage vesicles toward the cell surface and their subsequent plasma membrane fusion, thereby increasing the rate of glucose transport >10-fold. Defective signaling/execution of GLUT4 translocation is considered to be a common feature in insulin resistance and type 2 diabetes (8, 9). However, the molecular and cellular regulatory mechanisms whereby insulin activates GLUT4 membrane dynamics and glucose transport are still not fully understood. More than 60 protein and phospholipid intermediate players are currently implicated in orchestrating the overall process (1–7). A central role is attributed to the highest phosphorylated member of the phosphoinositide (PI)³ family, i.e. phosphatidylinositol (PtdIns) (3,4,5)P₃ (3). PtdIns(3,4,5)P₃ is generated at the cell surface by the action of wortmannin-sensitive class 1A PI3K that is activated via the insulin-stimulated IR/IR receptor substrate signaling pathway. Inositol polyphosphate 5-phosphatases SHIP or SKIP and 3-phosphatase PTEN rapidly convert PtdIns(3,4,5)P₃ to PtdIns(3,4)P₂ and PtdIns(4,5)P₂, respectively, thereby terminating insulin signal through class 1A PI3K (10–13). The class 1A PI3K-opposing function of these lipid phosphatases has provided an appealing prospect that inhibition of their hydrolyzing activities could produce significant efficacy in the treatment of type 2 diabetes and obesity (14–16).It has recently become apparent that signals by other PIs act in parallel with that of PtdIns(3,4,5)P₃ in integrating the IR-issued signal with GLUT4 surface translocation (3, 4). One such signaling molecule is PtdIns(3,5)P₂, whose functioning as a positive regulator in 3T3L1 adipocyte responsiveness to insulin has been supported by several lines of experimental evidence. Thus, expression of dominant-negative kinase-deficient mutants of PIKfyve, the sole enzyme for PtdIns(3,5)P₂ synthesis (17, 18), inhibits insulin-induced gain of surface GLUT4 without noticeable aberrations of cell morphology (19). Likewise, reduction in the intracellular PtdIns(3,5)P₂ pool through siRNA-mediated PIKfyve depletion reduces GLUT4 cell-surface accumulation and glucose transport activation in response to insulin (20). Concordantly, loss of ArPIKfyve, a PIKfyve activator that physically associates with PIKfyve to facilitate PtdIns(3,5)P₂ intracellular production (21, 22), also decreases insulin-stimulated glucose uptake in 3T3L1 adipocytes (20). Combined ablation of PIKfyve and ArPIKfyve produces a greater decrease in this effect, correlating with a greater reduction in the intracellular PtdIns(3,5)P₂ pool (20). Finally, pharmacological inhibition of PIKfyve activity powerfully reduces the net insulin effect on glucose uptake (23). These observations indicate positive signaling through the PtdIns(3,5)P₂ pathway and suggest that arrested PtdIns(3,5)P₂ turnover might potentiate insulin-regulated activation of glucose uptake.Sac3, a product of a single-copy gene in mammals, is a recently characterized phosphatase implicated in PtdIns(3,5)P₂ turnover (24). Our observations in several mammalian cell types have revealed that Sac3 plays an intricate role in the PtdIns(3,5)P₂ homeostatic mechanism. It is a constituent of the PtdIns(3,5)P₂ biosynthetic PIKfyve-ArPIKfyve complex and facilitates the association of these two (24, 25). Intriguingly, only if the PIKfyve-ArPIKfyve-Sac3 triad (known as the “PAS complex”) is intact will the PIKfyve enzymatic activity be activated (25). Thus, Sac3 not only catalyzes PtdIns(3,5)P₂ turnover but also promotes PtdIns(3,5)P₂ synthesis by functioning as an adaptor for the efficient association of PIKfyve with, and activation by, ArPIKfyve (25). Given these two seemingly opposing inputs, a critical question is whether reduction in Sac3 protein levels or phosphatase activity would facilitate or mitigate insulin action on glucose uptake and GLUT4 translocation. We demonstrate here that reduced levels of Sac3 potentiate, whereas ectopic expression of active Sac3 phosphatase reduces insulin responsiveness of GLUT4 translocation and glucose transport in 3T3L1 adipocytes. Whereas insulin action does not affect the PIKfyve kinase-Sac3 phosphatase association, it markedly inhibits the Sac3 hydrolyzing activity. We suggest that increased PtdIns(3,5)P₂ local availability through Sac3 phosphatase inhibition links insulin signaling to its effect on GLUT4 vesicle dynamics and glucose transport.     

��-Defensins in Enteric Innate Immunity: FUNCTIONAL PANETH CELL ��-DEFENSINS IN MOUSE COLONIC LUMEN*

Paneth cells are a secretory epithelial lineage that release dense core granules rich in host defense peptides and proteins from the base of small intestinal crypts. Enteric α-defensins, termed cryptdins (Crps) in mice, are highly abundant in Paneth cell secretions and inherently resistant to proteolysis. Accordingly, we tested the hypothesis that enteric α-defensins of Paneth cell origin persist in a functional state in the mouse large bowel lumen. To test this idea, putative Crps purified from mouse distal colonic lumen were characterized biochemically and assayed in vitro for bactericidal peptide activities. The peptides comigrated with cryptdin control peptides in acid-urea-PAGE and SDS-PAGE, providing identification as putative Crps. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry experiments showed that the molecular masses of the putative α-defensins matched those of the six most abundant known Crps, as well as N-terminally truncated forms of each, and that the peptides contain six Cys residues, consistent with identities as α-defensins. N-terminal sequencing definitively revealed peptides with N termini corresponding to full-length, (des-Leu)-truncated, and (des-Leu-Arg)-truncated N termini of Crps 1–4 and 6. Crps from mouse large bowel lumen were bactericidal in the low micromolar range. Thus, Paneth cell α-defensins secreted into the small intestinal lumen persist as intact and functional forms throughout the intestinal tract, suggesting that the peptides may mediate enteric innate immunity in the colonic lumen, far from their upstream point of secretion in small intestinal crypts.Antimicrobial peptides (AMPs)² are released by epithelial cells onto mucosal surfaces as effectors of innate immunity (1–5). In mammals, most AMPs derive from two major families, the cathelicidins and defensins (6). The defensins comprise the α-, β-, and θ-defensin subfamilies, which are defined by the presence of six cysteine residues paired in characteristic tridisulfide arrays (7). α-Defensins are highly abundant in two primary cell lineages: phagocytic leukocytes, primarily neutrophils, of myeloid origin and Paneth cells, which are secretory epithelial cells located at the base of the crypts of Lieberkühn in the small intestine (8–10). Neutrophil α-defensins are stored in azurophilic granules and contribute to non-oxidative microbial cell killing in phagolysosomes (11, 12), except in mice whose neutrophils lack defensins (13). In the small bowel, α-defensins and other host defense proteins (14–18) are released apically as components of Paneth cell secretory granules in response to cholinergic stimulation and after exposure to bacterial antigens (19). Therefore, the release of Paneth cell products into the crypt lumen is inferred to protect mitotically active crypt cells from colonization by potential pathogens and confer protection against enteric infection (7, 20, 21).Under normal, homeostatic conditions, Paneth cells are not found outside the small bowel, although they may appear ectopically in response to local inflammation throughout the gastrointestinal tract (22, 23). Paneth cell numbers increase progressively throughout the small intestine, occurring at highest numbers in the distal ileum (24). Mouse Paneth cells express numerous α-defensin isoforms, termed cryptdins (Crps) (25), that have broad spectrum antimicrobial activities (6, 26). Collectively, α-defensins constitute approximately seventy percent of the bactericidal peptide activity in mouse Paneth cell secretions (19), selectively killing bacteria by membrane-disruptive mechanisms (27–30). The role of Paneth cell α-defensins in gastrointestinal mucosal immunity is evident from studies of mice transgenic for human enteric α-defensin-5, HD-5, which are immune to infection by orally administered Salmonella enterica sv. typhimurium (S. typhimurium) (31).The biosynthesis of mature, bactericidal α-defensins from their inactive precursors requires activation by lineage-specific proteolytic convertases. In mouse Paneth cells, inactive ∼8.4-kDa Crp precursors are processed intracellularly into microbicidal ∼4-kDa Crps by specific cleavage events mediated by matrix metalloproteinase-7 (MMP-7) (32, 33). MMP-7 null mice exhibit increased susceptibility to systemic S. typhimurium infection and decreased clearance of orally administered non-invasive Escherichia coli (19, 32). Although the α-defensin proregions are sensitive to proteolysis, the mature, disulfide-stabilized peptides resist digestion by their converting enzymes in vitro, whether the convertase is MMP-7 (32), trypsin (34), or neutrophil serine proteinases (35). Because α-defensins resist proteolysis in vitro, we hypothesized that Paneth cell α-defensins resist degradation and remain in a functional state in the large bowel, a complex, hostile environment containing varied proteases of both host and microbial origin.Here, we report on the isolation and characterization of a population of enteric α-defensins from the mouse colonic lumen. Full-length and N-terminally truncated Paneth cell α-defensins were identified and are abundant in the distal large bowel lumen.     

Differential Regulation of Glycogenolysis by Mutant Protein Phosphatase-1 Glycogen-targeting Subunits

PTG and G_L are hepatic protein phosphatase-1 (PP1) glycogen-targeting subunits, which direct PP1 activity against glycogen synthase (GS) and/or phosphorylase (GP). The C-terminal 16 amino residues of G_L comprise a high affinity binding site for GP that regulates bound PP1 activity against GS. In this study, a truncated G_L construct lacking the GP-binding site (G_Ltr) and a chimeric PTG molecule containing the C-terminal site (PTG-G_L) were generated. As expected, GP binding to glutathione S-transferase (GST)-G_Ltr was reduced, whereas GP binding to GST-PTG-G_L was increased 2- to 3-fold versus GST-PTG. In contrast, PP1 binding to all proteins was equivalent. Primary mouse hepatocytes were infected with adenoviral constructs for each subunit, and their effects on glycogen metabolism were investigated. G_Ltr expression was more effective at promoting GP inactivation, GS activation, and glycogen accumulation than G_L. Removal of the regulatory GP-binding site from G_Ltr completely blocked the inactivation of GS seen in G_L-expressing cells following a drop in extracellular glucose. As a result, G_Ltr expression prevented glycogen mobilization under 5 mm glucose conditions. In contrast, equivalent overexpression of PTG or PTG-G_L caused a similar increase in glycogen-targeted PP1 levels and GS dephosphorylation. Surprisingly, GP dephosphorylation was significantly reduced in PTG-G_L-overexpressing cells. As a result, PTG-G_L expression permitted glycogenolysis under 5 mm glucose conditions that was prevented in PTG-expressing cells. Thus, expression of constructs that contained the high affinity GP-binding site (G_L and PTG-G_L) displayed reduced glycogen accumulation and enhanced glycogenolysis compared with their respective controls, albeit via different mechanisms.Hepatic glycogen metabolism plays a central role in the maintenance of circulating plasma glucose levels under various physiological conditions. The rate-controlling enzymes in glycogen metabolism, glycogen synthase (GS)² and glycogen phosphorylase (GP), are subject to multiple levels of regulation, including allosteric binding of activators and inhibitors, protein phosphorylation, and changes in subcellular localization. GS is phosphorylated on up to 9 residues by a variety of kinases, although site 2 appears to be the most important regulator of hepatic GS (1). In contrast, GP is phosphorylated on a single N-terminal serine residue by phosphorylase kinase, which increases GP activity and its sensitivity to allosteric activators. Both GS and GP are in turn also regulated by protein phosphatases, most notably PP1. Although PP1 is a cytosolic protein, a family of five molecules has been reported that targets the enzyme to glycogen particles (2–7), whereas another two glycogen-targeting subunits have been putatively identified based on sequence homology (8). Published work has indicated that each targeting subunit confers differential regulation of PP1 activity by extracellular hormonal signals and/or intracellular changes in metabolites (9–11).Four PP1-glycogen-targeting proteins are expressed in rodent liver, although G_L and PTG/R5 have been most extensively studied (9, 12–15). G_L is present at higher levels in rat liver than PTG (12), but the expression of both proteins is subject to coordinate regulation by fasting/refeeding and insulin (12, 13). Previous studies indicated that the PTG-PP1 complex is primarily responsible for GP dephosphorylation and regulation of glycogenolysis (13, 16), whereas the G_L-PP1 complex preferentially mediates the activation of GS upon elevation of extracellular glucose (9, 13). However, the molecular mechanisms underlying these differential properties of PTG and G_L have not been completely defined.Both PTG and G_L directly bind to specific PP1 substrates involved in glycogen metabolism, albeit for different physiological reasons. The extreme C-terminal 16 amino acids of G_L comprises a unique, high affinity binding site for phosphorylated GP (GPa (17)), which has been further delineated to two critical tyrosine residues (18, 37). Interaction of PP1 with G_L reduces phosphatase activity against GPa (3). In turn, GPa binding to the G_L-PP1 complex potently inhibits phosphatase activity against GS in vitro (3, 19) and regulates glycogen-targeted PP1 activity in liver cells and extracts (20–22). PTG contains a single substrate-binding site that interacts with GS and GP (5, 23). In contrast to the regulatory role of the GPa binding to G_L, interaction of substrates with PTG increases PP1 activity against these proteins (24). Indeed, disruption of the substrate-binding site by point mutagenesis abrogated the ability of mutant PTG expression to increase cellular glycogen levels (23), indicating an important role for substrate binding to the PTG-PP1 complex.Previous work has comprehensively compared the metabolic impact of PTG versus G_L overexpression in hepatocytes and thus was not the goal of this study (9, 10). Instead, two novel PP1 targeting constructs were generated in which the high affinity GPa-binding site was removed from G_L or added to the C terminus of PTG. The effects of expressing wild-type and mutant constructs on GS and GP activities and on the regulation of glycogen metabolism by extracellular glucose were investigated using primary mouse hepatocytes.