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Results: 7

1.
Figure 7

Figure 7. From: Anchored phosphatases modulate glucose homeostasis.

The AKAP150–PP2B interface. (A) Model of the molecular architecture of a membrane-associated AKAP150/(PP2B)2/PKA dimer. An AKAP79/150 homodimer coordinates four PP2B holoenzymes and two PKA holoenzymes localized to the plasma membrane. (B) The structure of the PP2B–PIAIIIT peptide complex and (C) magnified cartoon diagram of the PP2B-PIAIIIT-PP2B interface. Structural information and coordinates obtained from Gold et al (2011) and Li et al (2012) [PDB ID: 3LL8].

Simon A Hinke, et al. EMBO J. 2012 October 17;31(20):3991-4004.
2.
Figure 4

Figure 4. From: Anchored phosphatases modulate glucose homeostasis.

Metabolic profiling of Ins2-Cre+/−.AKAP5fl/fl (AKAP150βKO) conditional deletion mice. (A) Schematic depicting the deletion of the floxed AKAP5 coding region in insulin expressing tissues by Ins2 promoter-driven Cre-recombinase. (B) Immunoblot detection of AKAP150 from brain and isolated islets of Langerhans lysates of AKAP150fl/fl (control) and AKAP150βKO mice. (C) Plasma insulin concentrations from matched control and AKAP150βKO mice following overnight fasting and IP glucose (1.5 g/kg) injection. (D) Insulin content of acid extracted pancreata from control and AKAP150βKO mice. (E) Blood glucose profiles of control (grey) and AKAP150βKO (orange) mice during IP glucose tolerance testing (1.5 g/kg). (F) Blood glucose profiles of control (grey) and AKAP150βKO (orange) mice following IP injection of insulin (0.5 U/kg). Data represent mean±s.e.m. *P⩽0.05, **P⩽0.01, ***P⩽0.001. Immunoblots are representative of three independent experiments. Age-matched male mice were used in all experiments (control: 20.4±0.4 weeks, 29.5±0.6 g; AKAP150βKO: 20.4±0.4 weeks, 28.1±0.5 g). Figure source data can be found with the Supplementary data.

Simon A Hinke, et al. EMBO J. 2012 October 17;31(20):3991-4004.
3.
Figure 2

Figure 2. From: Anchored phosphatases modulate glucose homeostasis.

Cellular analysis of insulin release from isolated β-cells. (A) Measurement of glucose-dependent insulin release from isolated islets from WT and AKAP150KO mice. Data are normalized to total cell content (%TCC) of insulin. (B) Dynamic insulin secretion from perifused WT and AKAP150KO islets in response to different concentrations of glucose and forskolin. Integrated responses are shown in Supplementary Figure S2B and C. (C) Representative nifedipine-sensitive, whole-cell Ca2+ current traces from dissociated WT (grey) or AKAP150KO (green) β-cells. (D) Current–voltage relationship of ICa from WT (grey) or AKAP150KO (green) β-cells. (E) ICa voltage dependence of activation and steady-state inactivation from WT (grey) and AKAP150KO (green) β-cells. (F) Montage of images showing the time course of Ca2+ transients in WT and AKAP150KO β-cells in response to 11 mM glucose. Cells were loaded with Fluo-4 AM dye. (G) Fluo-4 AM imaging of glucose-induced intracellular [Ca2+] transients in WT (grey) and AKAP150KO (green) β-cells. (H) Amalgamated data of [Ca2+]i transients in WT and AKAP150KO β-cells after stimulation with 11 or 20 mM glucose. (I) Schematic representation of method used for measuring submembrane cAMP concentration (J, K). cAMP levels were measured using TIRF to detect PKA-catalytic subunit-YFP dissociation from membrane bound PKA-RII-CFP in dispersed β-cells. (J) Representative TIRF traces of the membrane cAMP oscillations in WT (grey) and AKAP150KO (green) islet cells in response to elevated glucose or forskolin. (K) Amalgamated TIRF detection of cAMP production at the membrane of WT (grey) and AKAP150KO (green) β-cells. Data represent mean±s.e.m. *P⩽0.05.

Simon A Hinke, et al. EMBO J. 2012 October 17;31(20):3991-4004.
4.
Figure 3

Figure 3. From: Anchored phosphatases modulate glucose homeostasis.

Glucose tolerance and peripheral insulin sensitivity in AKAP150KO mice. (A) Blood glucose profiles of WT and AKAP150KO mice during IP glucose (1.5 g/kg) tolerance test (IPGTT). Integrated glycemic responses are found in Supplementary Figure S3A. (B) Blood glucose profiles of WT (grey) and AKAP150KO (green) mice following IP injection of pyruvate (1.5 g/kg) into fasted animals as an index of gluconeogenesis. (C) Changes in blood glucose in response to IP injection of glucagon (25 nmol/kg) in fed WT (grey) and AKAP150KO (green) mice as an index of glycogenolysis. (D) Glycemic profiles of WT (grey) and AKAP150KO (green) mice following IP injection of recombinant insulin (0.5 U/kg). (E) Immunoblot detection of AKAP150, PKA-RII and GAPDH in gastrocnemius muscle, liver and epididymal fat pad homogenates from WT and AKAP150KO mice. (F) Immunoblot detection of active (P)Ser473-Akt, total Akt and GAPDH from gastrocnemius muscle homogenates from WT and AKAP150KO mice 15 min after IP saline or insulin (1.0 U/kg) injection. Two biological replicates are shown for each genotype and condition. Quantification of compiled results was performed by densitometry analysis of active (P)Ser473-Akt normalized to total respective protein level (G). (H) Immunoblot detection and (I) densitometry analysis of active (P)Ser473-Akt, total Akt and GAPDH from liver homogenates from WT and AKAP150KO mice as per (F, G). (J) Immunoblot analysis of active (P)Thr172-AMPK, total AMPK and GAPDH from skeletal muscle homogenates from WT and AKAP150KO mice. Two biological replicates are shown for each genotype and condition. Quantification of compiled results was performed by densitometry analysis of active (P)Thr172-AMPK normalized to total respective protein level (K). (L) Immunoblot detection and (M) densitometry analysis of (P)Thr172-AMPK, total AMPK and GAPDH from liver homogenates from WT and AKAP150KO mice as per (J, K). Data represent mean±s.e.m. *P⩽0.05, **P⩽0.01. Immunoblots are representative of three independent experiments; densitometry represents results from six individual animals of each genotype. Figure source data can be found with the Supplementary data.

Simon A Hinke, et al. EMBO J. 2012 October 17;31(20):3991-4004.
5.
Figure 6

Figure 6. From: Anchored phosphatases modulate glucose homeostasis.

Metabolic profiling of AKAP150 knock-in mice unable to anchor PP2B. (A) Schematic depicting deletion of 21 bases in the coding region of the AKAP5 locus to generate an internally truncated AKAP150 protein (AKAP150ΔPIX) unable to bind PP2B. (B) Immunoblot detection AKAP150 and AKAP150ΔPIX from brain and islets of Langerhans lysates. (C) Immunoprecipitation of AKAP150 and AKAP150ΔPIX complexes, and immunoblot detection of AKAP150 and co-immunoprecipitating PKA-RII and PP2BB. (D) Plasma insulin concentrations from matched WT and AKAP150ΔPIX mice following overnight fasting and IP glucose (1.5 g/kg) injection. (E) Insulin content of acid extracted pancreata from WT and AKAP150ΔPIX mice. (F) Measurement of glucose-stimulated insulin release (%TCC) from isolated WT and AKAP150ΔPIX mouse islets of Langerhans. (G) Dynamic insulin secretion from perifused WT and AKAP150ΔPIX islets in response to different concentrations of glucose and forskolin. Integrated responses are shown in Supplementary Figure S5C and D. (H) Blood glucose profiles of WT (grey) and AKAP150ΔPIX (purple) mice during IP glucose tolerance testing (1.5 g/kg). (I) Blood glucose profiles of WT (grey) AKAP150ΔPIX (purple) mice following IP injection of insulin (0.5 U/kg). (J) Immunoblot detection of active (P)Ser473-Akt, total Akt and GAPDH from gastrocnemius muscle homogenates from WT and AKAP150ΔPIX mice 15 min after IP saline or insulin (1.0 U/kg) injection. Two biological replicates are shown for each genotype and condition. Quantification of compiled results was performed by densitometry analysis of active (P)Ser473-Akt normalized to total respective protein level (K). Data represent mean±s.e.m. *P⩽0.05. Immunoblots are representative of three independent experiments; densitometry represents results from three individual animals of each genotype. Age-matched male mice were used in all experiments (WT: 18.3±0.7 weeks, 26.1±0.6 g; AKAP150ΔPIX: 19.1±0.6 weeks, 26.6±0.6 g). Figure source data can be found with the Supplementary data.

Simon A Hinke, et al. EMBO J. 2012 October 17;31(20):3991-4004.
6.
Figure 5

Figure 5. From: Anchored phosphatases modulate glucose homeostasis.

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 AKAP5 locus to generate a truncated AKAP150 protein (AKAP150Δ36) unable to anchor PKA. (B) Immunoblot detection AKAP150 and AKAP150Δ36 from brain and isolated islets of Langerhans lysates. (C) Immunoprecipitation of AKAP150 and AKAP150Δ36 complexes, and immunoblot detection of AKAP150 and co-immunoprecipitating PKA-RII and PP2BB. (D) Plasma insulin concentrations from matched WT and AKAP150Δ36 mice following overnight fasting and IP glucose (1.5 g/kg) injection. (E) Insulin content of acid extracted pancreata from WT and AKAP150Δ36 mice. (F) Measurement of glucose-stimulated insulin release (%TCC) from isolated WT and AKAP150Δ36 mouse islets of Langerhans. (G) Dynamic insulin secretion from perifused WT and AKAP150Δ36 islets in response to different concentrations of glucose and forskolin. Integrated responses are shown in Supplementary Figure S5C and D. (H) Blood glucose profiles of WT (grey) and AKAP150Δ36 (blue) mice during IP glucose tolerance testing (1.5 g/kg). (I) Blood glucose profiles of WT (grey) and AKAP150Δ36 (blue) mice following IP injection of insulin (0.5 U/kg). (J) Immunoblot detection of active (P)Ser473-Akt, total Akt and GAPDH from gastrocnemius muscle homogenates from WT and AKAP150Δ36 mice 15 min after IP saline or insulin (1.0 U/kg) injection. Two biological replicates are shown for each genotype and condition. Quantification of compiled results was performed by densitometry analysis of active (P)Ser473-Akt normalized to total respective protein level (K). Data represent mean±s.e.m. Immunoblots are representative of three independent experiments; densitometry represents results from three individual animals of each genotype. Age-matched male mice were used in all experiments (WT: 18.4±0.7 weeks, 26.9±0.6 g; AKAP150Δ36: 18.9±0.6 weeks, 25.8±0.5 g). Figure source data can be found with the Supplementary data.

Simon A Hinke, et al. EMBO J. 2012 October 17;31(20):3991-4004.
7.
Figure 1

Figure 1. From: Anchored phosphatases modulate glucose homeostasis.

Loss of AKAP150 supresses insulin secretion in vitro and in vivo. (A) Detection of AKAPs in INS-1(832/13) lysate by digoxigenin-labelled RII overlay in the presence of 50 μM scrambled peptide (lane 1) or 50 μM AKAPis PKA-anchoring disruptor peptide (lane 2). (B) Co-purification of AKAP220 (top) and AKAP150 (mid) with PKA-RII (bottom) from INS-1(832/13) lysate by cAMP-agarose affinity chromatography. Ethanolamine agarose was used as a control. (C) Immunoblot demonstrating gene silencing of AKAP150. Cells were co-transfected with human growth hormone and pSilencer or p150i, and used in secretion studies; see Supplementary Figure S1C for quantification. (D) Measurement of Glucose-, GLP-1- and (E) forskolin-induced insulin secretion from INS-1(832/13) cells co-transfected with pSilencer or p150i. (F) Schematic of AKAP150KO mice generation. Exon2 of the AKAP5 locus was flanked with loxP sites and mice were crossed onto a EIIa-Cre-deleter line to excise the AKAP5 coding region. (G) Immunoblot detection of AKAP150, PKA-RII and GAPDH loading control from brain and islets of Langerhans lysates from matched WT and AKAP150KO mice. (H) Immunofluorescence detection of AKAP150 (green), E-cadherin (red) and insulin (blue) from paraffin-embedded sections of wild-type and AKAP150 null mouse pancreas, and enlarged composite image (I). (J) Plasma insulin concentrations measured from fasted wild-type and AKAP150KO mice and following IP glucose (1.5 g/kg) injection. (K) Insulin content of acid extracted pancreata from WT (grey) and AKAP150KO (green) mice. (L, M) Depict representative 10 × images from WT and AKAP150KO pancreata (respectively) used for determination of islet area and β-cell mass (Table I) as per Materials and methods; sections are stained for insulin (blue), glucagon (green) and E-Cadherin (red). Data represent mean±s.e.m. *P⩽0.05, **P⩽0.01. Immunoblots are representative of 3–6 independent experiments. Age-matched male mice were used for all experiments (WT: 18.5±0.5 weeks, 29.1±0.5 g; AKAP150KO: 18.6±0.6 weeks, 28.3±0.5 g). Figure source data can be found with the Supplementary data.

Simon A Hinke, et al. EMBO J. 2012 October 17;31(20):3991-4004.

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