The Ca2+/calmodulin-dependent protein kinase IIa (Thr286Asp) transgenic mice: a novel mouse model of severe insulin-dependent diabetes

Research Article

Austin J Endocrinol Diabetes. 2014;1(1): 1002.

The Ca2+/calmodulin-dependent protein kinase IIa (Thr286Asp) transgenic mice: a novel mouse model of severe insulin-dependent diabetes

Hikari Suzuki1, Shin Takasawa2*, Isao Usui3, Yoko Ishii4, Ichiro Kato5, Hiroshi Okamoto6, Masashi Kobayashi1, Masakiyo Sasahara4 and Kazuyuki Tobe3

1Social Insurance Takaoka Hospital, 8–5 Fushikikofumoto–machi, Takaoka, Toyama, 933–0115 Japan

2Department of Biochemistry, Nara Medical University, 840 Shijo–cho, Kashihara, Nara, 634–8521 Japan

3First Department of Internal Medicine, University of Toyama, 2630 Sugitani, Toyama, 930–0194Japan

4Department of Pathology, University of Toyama, 2630 Sugitani, Toyama, 930–0194Japan

5Tonami Sunshine Hospitals, 575 Takanosu, Tonami, Toyama, 939–1335 Japan

6Tohoku University, 2–1, Seiryo–machi, Aoba–ku, Sendai, Miyagi, 980–8575 Japan

*Corresponding author: Shin Takasawa, Professor and Chairman, Nara Medical University, 840 Shijo-cho, Kashihara, Nara, 634-8521 Japan

Received: January 02, 2014; Accepted: January 17, 2014; Published: January 20, 2014;


Diabetes mellitus is the leading cause of blindness and end–stage renal disease. To understand the pathogenesis of diabetic complications, suitable animal models for this disease have been needed. The activation ofCa2+⁄ calmodulin–dependent protein kinase II (CaMKII) in pancreatic β–cells has been thought to play a central role in Ca2+–mediated insulin secretion. We generated transgenic mice over expressing the constitutively active–type CaMKIIα (Thr286Asp) in β–cells, which showed very high plasma glucose levels and exhibited the features of diabetic nephropathy and retinopathy. In cDNA microarray analysis osteopontin mRNA increased in CaMKIIα transgenic mice. In quantitative real–time RT–PCR analyses, not only M1 macrophage marker genes but also M2 macrophage marker genes were over expressed in renal cortex of CaMKIIα transgenic mice. The mice were crossed with conditional knockout mice in which platelet–derived growth factor receptor–β gene (Pdgfr–β) was deleted postnatal. The increased oxidative stress in the kidneys of the CaMKII α transgenic mice, which was shown by the increased urinary 8–hydroxydeoxyguanosine excretion and the increased expression of NAD (P) H oxidase 4, was decreased by Pdgfr–β deletion. The CaMKIIα (Thr286Asp) transgenic mice will be valuable as a novel model of severe insulin–dependent diabetes accompanied by an early progression of diabetic micro vascular complications.


ATP: Adenosine triphosphate; cADPR: Cyclic adenosine diphosphate–ribose; CaM: Calmodulin; CaMKII:Ca2+⁄calmodulindependent protein kinase II; CCR2: CCmotif receptor 2; CHI3l3: Chitinase 3–like 3; DM: Diabetes mellitus; ERG: Electroretinography; IL–1β: ?nterleukin–1β; IP3 : Inositol 1,4,5–trisphosphate; MCP–1:Monocyte chemotactic protein–1; MGL2: Macrophage galactose N–acetyl–galactosamine specific lectin 2; MRC1: Mannose receptor C–type1; NAD+: Nicotinamide adenine dinucleotide; NOS2: Nitric oxide synthase 2; NOX4: NAD(P)H oxidase 4; 8–OHdG: 8–hydroxydeoxyguanosine; PDGF: Platelet–derived growth factor; PDGFRs: Platelet–derived growth factor receptors; RyR: Ryanodinereceptor; TG: Transgenic; TNFα: ?umor necrosis factor α; WT: Wild type.

Introduction and Background

Diabetes mellitus (DM) is a disease characterized by hyperglycemia and is caused by absolute or relative insulin deficiency, sometimes associated with insulin resistance [1]. As a consequence of its micro vascular pathology, DM is the leading cause of blindness, end–stage renal disease, and a variety of neuropathies [2]. Approximately 30% of type 1 DM patients suffered from diabetic nephropathy eventually undergo renal dialysis or transplantation [3]. Nephropathy is thus alife–threatening complication of DM and is the leading cause of endstage renal disease in developed countries. The features of diabetic nephropathy include persistent albuminuria, a progressive decline in renal function, and histopathologically mesangial expansion followed by glomerulosclerosis [4]. However, the molecular mechanisms leading to end–stage renal disease in DM have not been fully understood.

Analysis and Interpretation

Mechanism of Insulin Secretion

Cyclic ADP–Ribose in Insulin Secretion

Mobilization ofCa2+ from intracellular stores in the endoplasmic reticulum is required for insulin secretion from pancreatic β–cells. Inositol 1,4,5–trisphosphate (IP3) is thought to be a second messenger for intracellular calcium mobilization, while in islet microsomes cyclic adenosine diphosphate–ribose (cADPR) induces mobilization ofCa2+. In the process of glucose metabolism, adenosine triphosphate (ATP) is generated. ATP induces cADPR formation from nicotinamide adenine dinucleotide (NAD+)by inhibiting the cADPR hydrolase activity of CD38.CD38 has enzymic activites of both cADPR synthesis from NAD+(ADP–ribosyl cyclase activity) and cADPR hydrolysis to produce ADP–ribose (cADPR hydrolase activity) [5]. cADPR functions as a second messenger forCa2+ mobilization fromendoplasmic reticulum for glucose–induced insulin secretion from pancreatic β–cells [6].

cADPR Requires Calmodulin–Dependent Protein Kinase II For IntracellularCa2+ Mobilization

In sea urchin eggs, calmodulin (CaM) directly interacts with the ryanodine receptor (RyR) to enhance the cADPR–mediatedCa2+ release [7]. In rat islets, CaM sensitized and activated the cADPRmediatedCa2+ release from islet microsomes. It is reported that cADPR–mediatedCa2+ mobilization for insulin secretion is achieved by the activatedCa2+/calmodulin–dependent protein kinaseII (CaMKII) not by the direct interaction of CaM andCa2+ release [8].

Glucose stimulation to pancreatic β cell induces cADPR accumulation in islets by inhibiting cADPR hydrolase activity of CD38 by ATP. Glucose stimulation also activates CaMKII, and the activated kinase phosphorylates RyR to sensitize theCa2+ channel for the cADPR signal.As shown in Figure 1, cADPR acts as a second messenger for intracellularCa2+ mobilization via RyR, and inducesinsulin secretion [8–10]. Thus, CaMKII is suggested to be essential kinase for glucose–induced insulin secretion.