Phenotypic Variability in a Family with Acrodysostosis Type 2 Caused by a Novel PDE4D Mutation Affecting the Serine Target of Protein Kinase-A Phosphorylation

Abstract Glucagon-like peptide-1 (GLP-1)-based medicines have recently been widely used to treat type 2 diabetic patients, whereas adverse effects of nausea and vomiting have been documented. Inhibition of voltage-gated K+ channel subtype Kv2.1 in pancreatic β-cells has been suggested to contribute to mild depolarization and promotion of insulin release. This study aimed to determine whether the blockade of Kv2.1 channels potentiates the insulinotropic effect of GLP-1 agonists. Kv2.1 channel blocker guangxitoxin-1E (GxTx) and GLP-1 agonist exendin-4 at subthreshold concentrations, when combined, markedly increased the insulin release and cytosolic Ca2+ concentration ([Ca2+]i) in a glucose-dependent manner in mouse islets and β-cells. Exendin-4 at subthreshold concentration alone increased islet insulin release and β-cell [Ca2+]i in Kv2.1+/− mice. The [Ca2+]i response to subthreshold exendin-4 and GxTx in combination was attenuated by pretreatment with protein kinase A inhibitor H-89, indicating the protein kinase A dependency of the cooperative effect. Furthermore, subthreshold doses of GxTx and GLP-1 agonist liraglutide in combination markedly increased plasma insulin and improved glucose tolerance in diabetic db/db mice and NSY mice. These results demonstrate that a modest suppression of Kv2.1 channels dramatically raises insulinotropic potency of GLP-1-based drugs, which opens a new avenue to reduce their doses and associated adverse effects while achieving the same glycemic control in type 2 diabetes.

Download Full-text

Loss of SPEG Inhibitory Phosphorylation of Ryanodine Receptor Type-2 Promotes Atrial Fibrillation

Circulation ◽

10.1161/circulationaha.120.045791 ◽

2020 ◽

Vol 142 (12) ◽

pp. 1159-1172 ◽

Cited By ~ 3

Author(s):

Hannah M. Campbell ◽

Ann P. Quick ◽

Issam Abu-Taha ◽

David Y. Chiang ◽

Carlos F. Kramm ◽

...

Keyword(s):

Atrial Fibrillation ◽

Protein Kinase ◽

Sarcoplasmic Reticulum ◽

Protein Kinase A ◽

Western Blotting ◽

Knockout Mice ◽

Electric Stimulation ◽

Kinase A ◽

Increased Susceptibility

Background: Enhanced diastolic calcium (Ca 2+ ) release through ryanodine receptor type-2 (RyR2) has been implicated in atrial fibrillation (AF) promotion. Diastolic sarcoplasmic reticulum Ca 2+ leak is caused by increased RyR2 phosphorylation by PKA (protein kinase A) or CaMKII (Ca 2+ /calmodulin-dependent kinase-II) phosphorylation, or less dephosphorylation by protein phosphatases. However, considerable controversy remains regarding the molecular mechanisms underlying altered RyR2 function in AF. We thus aimed to determine the role of SPEG (striated muscle preferentially expressed protein kinase), a novel regulator of RyR2 phosphorylation, in AF pathogenesis. Methods: Western blotting was performed with right atrial biopsies from patients with paroxysmal AF. SPEG atrial knockout mice were generated using adeno-associated virus 9. In mice, AF inducibility was determined using intracardiac programmed electric stimulation, and diastolic Ca 2+ leak in atrial cardiomyocytes was assessed using confocal Ca 2+ imaging. Phosphoproteomics studies and Western blotting were used to measure RyR2 phosphorylation. To test the effects of RyR2-S2367 phosphorylation, knockin mice with an inactivated S2367 phosphorylation site (S2367A) and a constitutively activated S2367 residue (S2367D) were generated by using CRISPR-Cas9. Results: Western blotting revealed decreased SPEG protein levels in atrial biopsies from patients with paroxysmal AF in comparison with patients in sinus rhythm. SPEG atrial-specific knockout mice exhibited increased susceptibility to pacing-induced AF by programmed electric stimulation and enhanced Ca 2+ spark frequency in atrial cardiomyocytes with Ca 2+ imaging, establishing a causal role for decreased SPEG in AF pathogenesis. Phosphoproteomics in hearts from SPEG cardiomyocyte knockout mice identified RyR2-S2367 as a novel kinase substrate of SPEG. Western blotting demonstrated that RyR2-S2367 phosphorylation was also decreased in patients with paroxysmal AF. RyR2-S2367A mice exhibited an increased susceptibility to pacing-induced AF, and aberrant atrial sarcoplasmic reticulum Ca 2+ leak, as well. In contrast, RyR2-S2367D mice were resistant to pacing-induced AF. Conclusions: Unlike other kinases (PKA, CaMKII) that increase RyR2 activity, SPEG phosphorylation reduces RyR2-mediated sarcoplasmic reticulum Ca 2+ release. Reduced SPEG levels and RyR2-S2367 phosphorylation typified patients with paroxysmal AF. Studies in S2367 knockin mouse models showed a causal relationship between reduced S2367 phosphorylation and AF susceptibility. Thus, modulating SPEG activity and phosphorylation levels of the novel S2367 site on RyR2 may represent a novel target for AF treatment.

Download Full-text

GLP-1 Analog Liraglutide Enhances Proinsulin Processing in Pancreatic β-Cells via a PKA-Dependent Pathway

Endocrinology ◽

10.1210/en.2014-1218 ◽

2014 ◽

Vol 155 (10) ◽

pp. 3817-3828 ◽

Cited By ~ 8

Author(s):

Liang Wang ◽

Ye Liu ◽

Jin Yang ◽

Hejun Zhao ◽

Jing Ke ◽

...

Keyword(s):

Type 2 Diabetes ◽

Protein Kinase ◽

Protein Kinase A ◽

Pancreatic Β Cell ◽

Β Cells ◽

Β Cell ◽

Pancreatic Β Cells ◽

Proinsulin Processing ◽

Kinase A

Abstract Hyperproinsulinemia has gained increasing attention in the development of type 2 diabetes. Clinical studies have demonstrated that glucagon-like peptide-1 (GLP-1)-based therapies significantly decrease plasma proinsulin/insulin ratio in patients with type 2 diabetes. However, the underlying mechanism remains unclear. Prohormone convertase (PC)-1/3 and PC2 are primarily responsible for processing proinsulin to insulin in pancreatic β-cells. We have recently reported that Pax6 mutation down-regulated PC1/3 and PC2 expression, resulting in defective proinsulin processing in Pax6 heterozygous mutant (Pax6m/+) mice. In this study, we investigated whether and how liraglutide, a novel GLP-1 analog, modulated proinsulin processing. Our results showed that liraglutide significantly up-regulated PC1/3 expression and decreased the proinsulin to insulin ratio in both Pax6m/+ and db/db diabetic mice. In the cultured mouse pancreatic β-cell line, Min6, liraglutide stimulated PC1/3 and PC2 expression and lowered the proinsulin to insulin ratio in a dose- and time-dependent manner. Moreover, the beneficial effects of liraglutide on PC1/3 and PC2 expression and proinsulin processing were dependent on the GLP-1 receptor-mediated cAMP/protein kinase A signaling pathway. The same mechanism was recapitulated in isolated mouse islets. In conclusion, liraglutide enhanced PC1/3- and PC2-dependent proinsulin processing in pancreatic β-cells through the activation of the GLP-1 receptor/cAMP/protein kinase A signaling pathway. Our study provides a new mechanism for improvement of pancreatic β-cell function by the GLP-1-based therapy.

Download Full-text

Type 2 Iodothyronine Deiodinase Expression is Upregulated by the Protein Kinase A-Dependent Pathway and is Downregulated by the Protein Kinase C-Dependent Pathway in Cultured Human Thyroid Cells

Thyroid ◽

10.1089/105072501753210957 ◽

2001 ◽

Vol 11 (10) ◽

pp. 899-907 ◽

Cited By ~ 13

Author(s):

Yuko Imai ◽

Nagaoki Toyoda ◽

Akimasa Maeda ◽

Tamayo Kadobayashi ◽

Fangzheng Wang ◽

...

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Protein Kinase A ◽

Human Thyroid ◽

Iodothyronine Deiodinase ◽

Thyroid Cells ◽

Kinase C ◽

Dependent Pathway ◽

Kinase A

Download Full-text

Abstract 34: Apolipoprotein A-I Increases Insulin Secretion from β-cells via a Protein Kinase A Dependent Mechanism

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.32.suppl_1.a34 ◽

2012 ◽

Vol 32 (suppl_1) ◽

Author(s):

Blake J Cochran ◽

Kerry-Anne Rye

Keyword(s):

Type 2 Diabetes ◽

Insulin Secretion ◽

Protein Kinase ◽

Protein Kinase A ◽

Adenyl Cyclase ◽

Β Cells ◽

Pancreatic Β Cells ◽

Kinase A ◽

Dependent Mechanism

Introduction: The progression to hyperglycaemia in type 2 diabetes is marked by β-cell insulin secretory dysfunction and cell loss. We have previously demonstrated that apolipoprotein (apo) A-I, the major protein constituent of high density lipoproteins (HDL) increases insulin expression and secretion from β-cells. Clinical data also suggests that pharmacological elevation of HDL levels is associated with improved glycemic control in patients with type 2 diabetes. With the current interest in HDL raising therapeutics, defining the mechanism by which apoA-I acts on insulin secretion is of importance. Objective: To elucidate the cell signalling events responsible for increasing insulin secretion from pancreatic β-cells treated with lipid-free apoA-I. Methods: Ins-1E (rat insulinoma) cells were pre-treated for 30 min with the Protein kinase A (PKA) specific inhibitor H89 (20 μM), soluble and transmembrane adenyl cyclase specific inhibitors (KH7, 30 μM and 2’5’ dideoxyadenosine, 50 μM, respectively) or vehicle control, then incubated for 1 h with lipid-free apoA-I (final concentration 1 mg/mL) under both basal (2.8 mM) and high (25 mM) glucose conditions. The insulin concentration in the culture supernatants was determined by radioimmunoassay and the cells were either lysed for protein analysis by western blotting or treated with 0.1 M HCl for determining cAMP by enzyme immunoassay. Results: Incubation of Ins-1E cells with apoA-I increased insulin secretion up to 3-fold. This increase was no longer apparent when the cells were pre-treated with H89. Incubation with apoA-I increased cAMP accumulation in Ins-1E cells 2.5-fold. This increase was totally inhibited when the cells were pre-incubated with 2’5’ dideoxyadenosine but not by KH7, indicating that transmembrane adenyl cyclase(s) are responsible for this response. ApoA-I also activated the small GTPase Cdc42, which may link cell surface apoA-I receptors with transmembrane adenyl cyclases. Conclusion: ApoA-I increases insulin secretion from pancreatic β-cells via a PKA-dependent mechanism involving transmembrane, but not soluble, adenyl cyclases and possibly Cdc42. This provides a possible explanation of the clinical observations that increased HDL may be beneficial in type 2 diabetes.

Download Full-text