Endocrine release of insulin principally controls glucose homeostasis. these metabolic phenotypes. Thus anchored signalling events that facilitate insulin secretion and glucose homeostasis may be set by AKAP150 associated phosphatase activity. gene has metabolic implications for glucose homeostasis. AKAP150 null mice secrete less insulin from -cells, yet display improved glucose handling because of increased insulin sensitivity in target tissues. These metabolically advantageous characteristics are retained in AKAP150PIX mice that lack a seven amino-acid sequence responsible for tethering PP2B. Hence p150 anchoring of PP2B is an hitherto unrecognized molecular and metabolic determinant that contributes to glucose homeostasis. Results AKAP150 organizes insulin secretion Membrane depolarization and the concurrent mobilization of Ca2+ and cAMP signalling cascades drive the regulated exocytosis of insulin granules (Hinke et al, 2004; Hiriart and Aguilar-Bryan, 2008; Seino et al, 2011). Although anchored PKA augments GLP-1 mediated insulin secretion (Lester et al, 1997), a question of broader significance is whether AKAPs facilitate nutrient-induced insulin release. Several RII binding proteins were detected in INS-1(832/13) insulinoma cells by overlay assay (Figure 1A). These included AKAP150 and AKAP220 (Figure 1B; Supplementary Figure S1A and (S)-10-Hydroxycamptothecin supplier B): two channel-associated AKAPs that integrate Ca2+ and cAMP signals (Gao et al, 1997; Yang et al, 2008). A gene-silencing screen evaluated the role of each AKAP in hormone secretion from INS-1 cells. Co-transfected human growth hormone (hGH) served as a marker of exocytosis (Supplementary Figure S1CCJ). Under basal conditions, insulin secretion was unaltered from AKAP150-depleted cells (Figure 1C and D; and locus were introduced to permit deletion of the entire open reading frame (Tunquist et al, 2008). Loss of AKAP150 protein from brain and islet extracts was initially confirmed by immunoblot (Figure 1G; Supplementary Figure S1KCM). Subsequent immunofluorescent analyses of paraffin-embedded pancreatic sections revealed that AKAP150 is present in wild-type islets but is not detected in equivalent samples from knockout animals (Figure 1H). At higher magnification, the AKAP150 signal was less prevalent in the non-insulin-positive islet periphery (Figure 1I). Moreover, only trace levels of AKAP150 were present in the -cell-derived line (TC1C6; Supplementary Figure S1N). Metabolic studies performed on matched male mice revealed that fasted serum insulin levels were reduced 27.48.1% in AKAP150KO mice compared to WT. After IP glucose injection, circulating serum insulin was 27.05.6% lower in AKAP150KO animals (Figure 1H; gene is deleted. Table 1 Islet area and -cell mass of AKAP150 transgenic mice AKAP150 coordinates Ca2+ and cAMP-stimulated insulin secretion from -cells Next, we monitored a range of cellular and molecular events associated with insulin secretion in primary islets from AKAP150KO mice. Static insulin release measurements confirmed that islets from both genotypes responded to glucose. However, less insulin (S)-10-Hydroxycamptothecin supplier was discharged from AKAP150 null islets (Figure 2A). Dynamic insulin release, evaluated by islet perifusion, also detected a modest reduction in the rate of insulin secretion from AKAP150KO islets. The peak secretory response to 11 mM glucose was 31.510.9% lower from KO islets, and reached statistical significance upon delivery of the potent insulin (S)-10-Hydroxycamptothecin supplier secretagogue forskolin (Figure 2B; (AKAP150fl/fl) were backcrossed onto mice (Postic et al, 1999) to conditionally delete the anchoring protein in insulin expressing cells (Figure 4A). Immunoblot analysis detected equivalent levels of AKAP150 in brain extracts from both (AKAP150KO) conditional deletion mice. (A) Schematic depicting the deletion of the floxed coding region in insulin expressing tissues by promoter-driven Cre-recombinase. … PKA anchoring to AKAP150 has little effect on glucose homeostasis Since each AKAP150-anchored enzyme influences distinct metabolic events, we investigated glucose homeostasis in mouse models where discrete elements of the AKAP150 signalling complex were disrupted. An amphipathic helix that binds the regulatory (R) subunits of PKA is a defining characteristic of AKAPs. This structural element is located between residues 705 and 724 of AKAP150. Mice expressing a form of the anchoring protein lacking this region (AKAP15036; Weisenhaus et al, 2010) are unable to anchor PKA but retain the ability to tether PP2B (Figure 5ACC; Supplementary Figure S5A and B). Metabolic profiling of matched male AKAP15036 mice selectively evaluated the contribution of anchored PKA to glucose homeostasis. Figure 5 Metabolic profiling of knock-in mice lacking the PKA binding domain of AKAP150. (A) Schematic depicting the insertion of a premature stop codon into the coding region of the locus (S)-10-Hydroxycamptothecin supplier to generate a truncated AKAP150 protein (AKAP15036) unable … An unanticipated outcome of these studies was that most aspects of glucose homeostasis are comparable between AKAP15036 and WT mice.