CaMKII modulates β-cell Ca2+ homeostasis  

Inhibition of pancreatic beta-cell CaMKII reduces glucose-stimulated calcium influx and insulin secretion, impairing glucose tolerance

Prasanna K. Dadi1, Nicholas C. Vierra1, Alessandro Ustione1, David W. Piston1, Roger J. Colbran1,

David A. Jacobson1

1Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN 37232, USA

*Running Title: CaMKII modulates β-cell Ca2+ homeostasis

Keywords: CAM Kinase II; Islet; Insulin; L-type Ca2+ channel; Diabetes Background: Glucose activates CaMKII in -cells, how this influences glucose homeostasis has not been determined. Results: Inhibiting CaMKII in mouse -cells causes glucose intolerance by reducing Ca2+ entry and insulin secretion. Conclusions: CaMKII is a -cell Ca2+ sensor that amplifies secretagogue-induced Ca2+ entry and insulin secretion to maintain glucose homeostasis. Significance: This provides the first evidence that -cell CaMKII modulates glucose homeostasis under physiological and insulin resistant states. ABSTRACT Glucose stimulated insulin secretion (GSIS) from pancreatic β-cells is caused by Ca2+ entry via voltage-dependent Ca2+ channels (VDCCs). CaMKII is a key mediator and feedback regulator of Ca2+ signaling in many tissues, but its role in β-cells is poorly understood, especially in vivo. Here we report that mice with conditional inhibition of CaMKII in β-cells show significantly impaired glucose tolerance due to decreased GSIS. Moreover, β-cell CaMKII inhibition dramatically exacerbates glucose intolerance following exposure to a high fat diet (HFD). The impairment of islet GSIS by β-cell CaMKII inhibition is not accompanied by changes in either glucose metabolism or the activities of KATP and voltage-gated potassium channels. However, glucose-stimulated Ca2+ entry via VDCCs is reduced in islet β-cells with CaMKII inhibition, as well as in primary wild-type β-cells treated with a peptide inhibitor of CaMKII. The levels of basal β-cell cytoplasmic Ca2+ and of endoplasmic reticulum Ca2+ stores are also decreased by CaMKII inhibition. In addition, CaMKII inhibition suppresses

glucose-stimulated action potential firing frequency. These results reveal that CaMKII is a Ca2+ sensor with a key role as a feed-forward stimulator of β-cell Ca2+ signals that enhance GSIS under physiological and pathological conditions.

Insulin secretion from pancreatic β-cells is caused by glucose-stimulated Ca2+ entry. While the primary insulin secretory machinery is directly stimulated by Ca2+, protein phosphorylation modulates the coupling of Ca2+ entry to insulin secretion (1). Studies in insulin-secreting cell lines and in isolated -cells suggest that Ca2+ dependent kinases such as Ca2+/calmodulin-dependent protein kinase II (CaMKII) are key regulators of insulin secretion (2-5). However, the role of CaMKII in modulating GSIS in vivo under physiological and pathological conditions is poorly understood.

Glucose-stimulated activation of β-cell CaMKII temporally correlates with GSIS from isolated pancreatic islets (5). Secretagogue stimulation of Ca2+ entry activates β-cell CaMKII enhancing GSIS, whereas inhibition of CaMKII activity blunts islet GSIS (6-8). However, these acute in vitro studies do not address the role of CaMKII in modulating islet β-cell function and glucose homeostasis in vivo (2).

Here we investigated the physiological role of CaMKII in pancreatic β-cells using a transgenic mouse model that allows tetracycline-inducible inhibition of β-cell CaMKII activity. We find that inhibition of β-cell CaMKII significantly impairs glucose tolerance by reducing L-type Ca2+ channel (LTCC) facilitation, Ca2+ entry and insulin secretion. These studies reveal that β-cell CaMKII is an integral Ca2+ sensor in a positive feedback pathway that regulates cytosolic and endoplasmic reticulum (ER) Ca2+ levels to enhance insulin secretion.

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http://www.jbc.org/cgi/doi/10.1074/jbc.M114.562587The latest version is at JBC Papers in Press. Published on March 13, 2014 as Manuscript M114.562587

Copyright 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

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EXPERIMENTAL PROCEDURES

Mouse β-cell CaMKII inhibition-For β-cell specific CaMKII inhibition transgenic animals were utilized; these are a cross between C57BL/6 mice with a tetracycline transactivator expressed in β-cells via an insulin promoter (RIP-rtTA, Jackson labs 008250)(9) and C57BL/6 mice with a CaMKII pseudosubstrate inhibitor peptide fused to GFP under the expression of a tetracycline operator controlled promoter (EAC3I-GFP; Figure 1)(10). To induce expression of EAC3I-GFP these mice were treated with doxycycline (2mg/mL; RPI, Mount Prospect, IL) in the drinking water for either 11 or 14 days as indicated. Controls were RIP-rtTA animals or RIP-rtTA + tetO-GFP mice (Jackson labs 018913) as indicated, both were treated with doxycline identically to the EAC3I-GFP mice. For acute inhibition of islet-cell CAMKII, C57BL/6 islets were dispersed into 20 cell clusters and then treated with the cell permeable CAMKII inhibitor peptide, autocamtide-2 related Inhibitory Peptide II (AIP2), which is a Antennapedia transport peptide fused to AIP2 (EMD Millipore). Islet cells were incubated with 20 M AIP2 in 2mM glucose for 20 minutes immediately before Ca2+ imaging.

Mouse diets and glucose tolerance testing-Mice were placed either on a normal chow diet or a high fat diet (HFD, 60 kcal% fat; Research Diets inc.) and monitored for glucose tolerance. The glucose tolerance test (GTT) was performed as previously described by injecting 2mg/kg dextrose (animals on a normal chow diet) or 1mg/kg (animals on a HFD) and monitoring blood glucose at the indicated time points post glucose injection (11). The mice fed a normal chow diet were treated with doxycline at 6 weeks of age and GTT was performed at 7.5 weeks of age. A cohort of mice was also placed on a HFD at 3 weeks of age for 2 months when GTT was performed; at this time doxycycline was added to the drinking water for 14 days in the presence of the HFD following which another GTT was performed.

Mouse islet and β-cell isolation-Islets were isolated from pancreata of mice, using collagenase digestion and Ficoll gradients as previously described (12). Islets were plated or dissociated in 0.005% trypsin, placed on glass coverslips, and cultured for 16 hours in RPMI-1640 medium supplemented with 10% fetal calf

serum, concentrations of glucose specified, 100 IU ml−1 penicillin, and 100 mg ml−1 streptomycin. Dissociated β-cells were specifically used in all voltage clamp experiments recording Ca2+ currents. Beta-cells on the periphery of intact islets were recorded in current clamp mode in all of the membrane potential recordings. Cells and islets were maintained in a humidified incubator at 37◦C under an atmosphere of 95% air and 5% CO2.

Western Blot Analysis-Mouse Islets in groups of 50 were treated with 1 M ionomycin for 2 minutes. Protein extracts were prepared from islets by extraction with SDS loading buffer (1% SDS, 30 mmol/l Tris-HCl [pH 6.8], 5% β-mercaptoethanol, 5% glycerol, and 0.1% bromophenol blue) with protease and phosphatase inhibitors at 80°C for 10 min. After electrophoresis through a 4-12% denaturing polyacrylamide gel, proteins were prepared as a western blot on a nitrocellulose membrane (BioRad). Anti-phospho-synapsin antibody (Santa Cruz) or anti-GAPDH (Rockland Immunochemicals) was used to probe the membrane at 1:250 or 1:700 dilution respectively in PBS, 0.1% Tween, and 3% powdered dried milk followed by a horseradish peroxidase (HRP)-coupled secondary antibody (Jackson ImmunoResearch) at 1:5000 in the same solution. The membranes were washed in PBS containing 0.1% Tween between and after antibody incubations; HRP was illuminated using Pico Signal (Pierce) and exposed on Kodak X-Omat Blue film.

Perforated-patch electrophysiology-Patch electrodes (2–4 micro-ohms) loaded with solution containing (in mmol.l−1) 140 KCl, 1 MgCl2[H2O]6, 10 EGTA, 10 HEPES (pH 7.25 with KOH) and the pore-forming antibiotic amphotericin B (Sigma) were used to record islet attached β-cells (13). Islets were perfused with Krebs–Ringer-HEPES buffer containing (in mmol.l−1) 119 NaCl, 2 CaCl2, 4.7 KCl, 10 HEPES, 1.2 MgSO4, 1.2 KH2PO4, adjusted to pH 7.35 with NaOH, with the indicated concentrations of glucose and compounds. Cells on the periphery of islets were sealed in voltage clamp at −80 mV, and access was obtained over several minutes through perforations by amphotericin (13). After being switched to current clamp, cells that had a resting membrane voltage near −65 mV in 2mM glucose and fired voltage dependent Ca2+ channel (VDCC)

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dependent action potentials post glucose-stimulation were assumed to be β-cells.

Whole Cell Voltage Clamp Electrophysiological Recordings-Voltage-activated currents were recorded using whole-cell ruptured patch clamp with an Axopatch 200B amplifier and pCLAMP10 software (Molecular Devices, Sunnyvale, CA). For calcium channel recordings patch electrodes (2–4 micro-ohms) were loaded with intracellular solution containing (in mmol.l−1): 102 CsCl, 10 tetraethylamonium-Cl, 0.1 tolbutamide, 10 EGTA, 1 MgCl2, 3 Na2ATP, 5 HEPES, pH 7.4, adjusted with CsOH. Cells were perifused with a Krebs-Ringer buffer (KRB) containing (in mmol.l−1): 82 NaCl, 20 tetraethylamonium-Cl, 0.1 tolbutamide, 30 CaCl2, 5 CsCl, 1 MgCl2, 0.1 EGTA, 10 glucose, 5 HEPES, pH 7.4, adjusted with NaOH. For KATP channel recordings, patch electrodes were loaded with (in mmol.l−1): 0.1 MgATP, 140 KCl, 1 MgCl2, 10 HEPES. Cells were perifused with Krebs–Ringer-HEPES buffer with 14mM glucose and 20 mM tetraethylamonium-Cl. For KV channel recordings patch electrodes were loaded with (in mmol.l−1): MgATP, 140 KCl, 1 MgCl2, 10 HEPES. Cells were perifused with Krebs–Ringer-HEPES buffer with 14mM glucose and 100 M tolbutamide.

Measurement of Cytoplasmic Calcium- Islets were incubated (20 min. at 37°C) in KRB supplemented with 2 μM Fura-2 acetoxymethyl ester (Molecular Probes, Eugene, OR). Fluorescence imaging was performed using a Nikon Eclipse TE2000-U microscope equipped with an epifluorescence illuminator (SUTTER, inc.) a CCD camera (HQ2, Photometrics inc.) and Nikon Elements software (NIKON inc.). Cells were perifused at 37°C at a flow of 2 ml/min with appropriate KRB-based solutions that contained glucose concentrations and compounds specified in the figures. Relative Ca2+ concentrations were quantified every 5 seconds by determining the ratio of emitted fluorescence intensities at excitation wavelengths of 340 nm and 380 nm (F340/F380). The relative Ca2+ is plotted as the average FURA-2 ratio (F340/F380) of each experimental group +/- the SEM. The averaged area under the curve (AUC) measurement for FURA-2 ratios during the indicated time period (in minutes) was plotted as a bar graph. Data was

analyzed using Excel and GraphPad Prism software and compared by student t test.

Insulin secretion measurements-Mouse islets were allowed to recover following isolation overnight. For insulin measurements, islets were incubated with DMEM with 5.6 mM glucose overnight followed by treatment with the indicated glucose concentrations and analyzed for insulin content using an RIA or ELISA-based detection kit (ALPCO Diagnostics, Salem, NH), and data are presented as means ±S.E.M.. For static insulin secretion islets were incubated in 2mM glucose containing KRB for 1 hour followed by the indicated treatments and insulin was assayed by ELISA-based detection.

Islet Immunofluorescence Staining-Pancreas cubes from RIP-rtTA+EAC3I-GFP and RIP-rtTA animals were fixed in 2% paraformaldehyde, paraffin embedded and cut into 5 μM sections on a microtome. Islet β-cells were stained using insulin (Millipore) and glucagon (Sigma) antibodies at 1:300 in combination with fluorescein isothiocyanate-conjugated secondary antibodies (Jackson ImmunoResearch) together with DAPI nuclear stain (Invitrogen).

Islet NAD(P)H fluorescence Imaging- NAD(P)H fluorescence imaging was performed on a LSM780 confocal microscope (Zeiss Inc.), using a tunable Chameleon Ti:Sapphire laser (Coherent Inc.). Multiphoton excitation was obtained using a 710 nm wavelength. The emitted light was spectrally resolved using the λ-mode configuration of the LSM780, with a band-pass of 412 nm – 631 nm and a spectral resolution of 8.6 nm. The pixel dwell time was set at 50.4 µs, and the pixel size was 0.277 µm. We used a Fluor 40x oil objective, with NA 1.30 and dishes with a 1.5 glass bottom (Mattek). The NAD(P)H emission and the GFP emission were extracted by linear unmixing, using the ZEN software (Zeiss Inc.). The reference spectrum for NAD(P)H was obtained measuring the emission from a freshly made solution of β-NADH (Sigma, N8160) in Tris 0.01M, pH 8.5. The reference spectrum for GFP was measured from a solution of purified GFP in PBS, pH 7.4. The unmixed images were processed using Fiji software (14), to measure the average NAD(P)H intensity across the entire islet and just in the GFP expressing cells. During the imaging, the islets where maintained in KRBH (Krebs-Ringer bicarbonate/HEPES buffer), pH 7.40. The

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islets were kept at 37 °C, in a humidified atmosphere with 5% CO2, using the microscope stage incubator. The images were collected 8 minutes post glucose stimulus and 30 µm deep in the islet. RESULTS CaMKII activity modulates glucose tolerance by enhancing glucose-stimulated insulin secretion To investigate the physiological role of β-cell CaMKII during GSIS, we created a mouse model which allows tetracycline-inducible inhibition of CaMKII. These animals are a cross between mice with a reverse tetracycline transactivator expressed in β-cells via an insulin promoter (RIP-rtTA) and mice with a CaMKII pseudosubstrate inhibitor peptide fused to GFP under the control of a tetracycline operator (tetO) controlled promoter (EAC3I-GFP; Figure 1A)(10). Doxycycline treatment of these animals, for 1.5 weeks, induces expression of EAC3I-GFP specifically in pancreatic β-cells, which can be visualized with GFP fluorescence (Figure 1B). EAC3I-GFP expression occurs in a majority of -cells, resembling previously published reports on RIP-rtTA mice showing doxycycline induced expression of tetO-controlled transgenes in 68% to 80% of -cells (Figure 1B and 1C)(15,16). Beta-cell expression of EAC3I-GFP causes a significant reduction in Ca2+-induced serine-553 phosphorylation of islet synapsin1 compared with controls (RIP-rtTA islets), consistent with the effective inhibition of CaMKII activity by EAC3I-GFP expression (Figure 1D) (17). However, total pancreatic insulin levels were equivalent between mice with β-cell EAC3I-GFP expression and controls (RIP-rtTA pancreata; Figure 1E). Furthermore, β-cell EAC3I-GFP expression does not cause any changes in islet morphology, as β-cell size and insulin immunofluorescence were equivalent to controls (Figure 1F and G). Therefore, conditional β-cell expression of EAC3I-GFP is a good model for assessing the role of CaMKII during physiological insulin secretion.

The effect of -cell CaMKII inhibition on glucose homeostasis was assessed using intraperitoneal (i.p.) glucose tolerance tests. EAC3I-GFP expression in β-cells resulted in a significant impairment of glucose tolerance

compared with controls (RIP-rtTA mice; Figure 2A, n=13 per group). Insulin secretion assays comparing pancreatic islets isolated from control and EAC3I-GFP mice showed that inhibition of β-cell CaMKII suppresses insulin secretion (Figure 2B-D). Although there was no effect of CaMKII inhibition on basal insulin secretion with 5.6 mM glucose, which resembles blood glucose concentrations during fasting (Figure 2B and 2C), both first and second phase insulin secretion in response to 16.7 mM glucose were reduced in islets with β-cell CaMKII inhibition compared with controls (RIP-rtTA islets). Inhibiting β-cell CaMKII only significantly reduced GSIS at glucose concentrations above 7 mM, which resemble blood glucose levels (10 mM) reached postprandially (Figure 2D). In combination, these data show that CaMKII plays a key role in modulating glucose homeostasis and GSIS in vivo.

CaMKII inhibition does not affect glucose metabolism or -cell K+ channel activity

GSIS involves multiple steps that may be

affected by CaMKII inhibition. First, we examined the potential effect of CaMKII inhibition on -cell glucose metabolism by monitoring NAD(P)H fluorescence under varying glucose conditions (Figure 3). No significant difference in glucose- stimulated changes in NAD(P)H fluorescence was observed between intact islet -cells or whole islets that were obtained from mice with -cell expression of EAC3I-GFP or GFP alone (Figure 3A and 3B). Thus, -cell glucose metabolism is not affected by chronic CaMKII inhibition.

We next investigated potential effects of CaMKII inhibition on the two major -cell K+ channels, the delayed rectifier voltage-gated K+ channel (KV) and ATP-sensitive K+ channel (KATP). The voltage step induction of KV currents was equivalent in control (RIP-rtTA, Figure 4A) and CaMKII inhibited (EAC3I-GFP, Figure 4B) -cells, with similar overall current-voltage relationships (Figure 4C). Similarly, reducing intracellular ATP concentrations induced equivalent KATP currents in control (RIP-rtTA) and EAC3I-GFP expressing -cells in response to a voltage ramp (from -120 mV to 60 mV, Figure 4D). Thus, CaMKII inhibition has no detectable affect on K+ channel activities in cells.

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Consistent with these findings, insulin secretion induced by 20 mM potassium chloride in the presence of basal glucose, which bypasses cell glucose metabolism and K+ channels by directly depolarizing -cells, was significantly lower from EAC3I-GFP expressing islets compared to controls (Figure 2B and 2C). Taken together, these data indicate that CaMKII modulates GSIS downstream of glucose metabolism and K+ channels to modulate GSIS.

CaMKII modulates glucose-stimulated Ca2+ entry in β-cells

Although CaMKII inhibition reduces Ca2+-dependent insulin secretion induced by high glucose or direct depolarization (see above), there was no difference in insulin secretion from EAC3I-GFP expressing and control islets in response to the Ca2+ ionophore ionomycin (Figure 4E) (18), suggesting that CaMKII modulates β-cell Ca2+ homeostasis at some level. Consistent with this hypothesis, islet β-cells expressing EAC3I-GFP showed reduced basal Ca2+ levels compared to control islets (Figure 5). Moreover, the changes in Ca2+ following glucose stimulation were also affected: CaMKII inhibition shortened the delay in initiation of Ca2+ influx, but suppressed peak Ca2+ concentrations.

Acute CaMKII inhibition with a cell permeable inhibitor peptide, AIP2, in dispersed wild type C57/BL6 islet cell clusters also significantly affected Ca2+ homeostasis (Figure 5C and 5D). In contrast to the effects of chronic CaMKII inhibition (see above), acute CaMKII inhibition using AIP2 increased basal Ca2+ levels (2 mM glucose) and delayed the initiation of Ca2+ influx following glucose stimulation (Fig. 5C). However, acute CaMKII inhibition with AIP2 suppressed total glucose-induced increases in Ca2+, similar to the effects of chronic CaMKII inhibition. Notably, Ca2+ homeostasis in EAC3I-GFP β-cells treated with AIP2 was similar to EAC3I-GFP islet Ca2+ without AIP2 (Figure 5C), indicating that these acute changes in basal β-cell Ca2+ are specifically caused by inhibition of CaMKII activity. The somewhat different effects of chronic in vivo and acute in vitro CaMKII inhibition on β-cell Ca2+ homeostasis presumably reflect long-term adaptive responses to CaMKII inhibition that may be influenced by the in vivo

environment. Nevertheless, the peak glucose-stimulated increase in Ca2+ concentration was significantly reduced by both acute and chronic CaMKII inhibition.

CaMKII modulates ER Ca2+ storage in β-cells

CaMKII modulates ER Ca2+ uptake and

release in cardiomyocytes (19-23). Therefore, we investigated whether the alterations in basal Ca2+ levels following -cell CaMKII inhibition are due to changes in the intracellular Ca2+ stores. Beta-cell ER Ca2+ levels were determined by inhibiting the sarco/endoplasmic reticulum ATPase (SERCA) with thapsigargin in the absence of extracellular Ca2+ and measuring the resulting elevation of cytosolic Ca2+ (Figure 5G and 5H). We found that ER Ca2+ stores were significantly reduced in islets with chronic β-cell inhibition of CaMKII compared with controls (RIP-rtTA islets; Figure 5G and 5H). To confirm that the reduction in thapsigargin-stimulated ER Ca2+ release was due to diminished ER Ca2+ levels, islets were also stimulated with the insulin secretagogue carbachol (CCh) in the absence of extracellular Ca2+ to induce release of ER Ca2+ via IP3 receptors. ER Ca2+ release induced by CCh treatment was found to be significantly reduced in islets with β-cell CaMKII inhibition (EAC3I-GFP) compared to control islets (RIP-rtTA) (Figure 5I and 5J). Therefore, CaMKII regulates β-cell cytoplasmic Ca2+ at least in part through modulation of ER Ca2+ handling. CaMKII activity is required for β-cell L-type Ca2+ channel facilitation GSIS is driven by Ca2+ influx via LTCCs. CaMKII facilitates voltage-induced Ca2+ entry via LTCCs in myocytes and neurons by multiple mechanisms involving different LTCC subunits (24-29). Therefore, we tested if β-cell LTCCs also show voltage-induced facilitation that is dependent on CaMKII activity. Ca2+ currents were measured in response to a 100 ms voltage step to 30 mV before and after a strong positive voltage prepulse for 200 ms (160 mV, Figure 6A and 6B). Control β-cells (RIP-rtTA-cells) show significant Ca2+ channel facilitation following the positive voltage prepulse (current increase of 42.2±4.8%, n=19; Figure 6B and 6C), whereas β-cells with CaMKII

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inhibition show minimal voltage-induced Ca2+ channel facilitation (current increase of 11.1±3.7%, n=25; Figure 6B and 6C). This indicates that CaMKII activity facilitates β-cell Ca2+ channels following depolarization. To determine if this change in LTCC facilitation influenced β-cell Ca2+ currents, LTCC currents were recorded with voltage steps from -70 to +70 mV in 10 mV increments. To prevent Ca2+ induced changes in LTCC currents before the first recording, the whole-cell configuration was obtained with a constant holding potential of -80 mV in low glucose (2 mM) and only the first set of voltage step recordings for each cell was utilized for subsequent analysis. Interestingly, under these conditions, β-cells with CaMKII inhibition show significantly reduced Ca2+ currents in response to voltage steps between 0 and +40 mV when compared to control β-cells (RIP-rtTA-cells; Figure 6C and 6D). However, there is no change in VDCC activation or inactivation with CaMKII inhibition (Figure 6E and 6F). Thus, CaMKII enhances Ca2+ entry into β-cells via LTCCs. Glucose-stimulated activation of LTCCs is responsible for the upstroke of the action potential (AP), resulting in β-cell firing (30,31). Therefore, we measured mouse β-cell membrane potentials to test whether CaMKII modulation of LTCCs affects β-cell AP firing. The AP firing frequency after a 10 min exposure to 14 mM glucose was significantly reduced in islet β-cells expressing EAC3I-GFP (1.39±0.17 Hz) compared with control β-cells (RIP-rtTA-cells, 1.9±0.16 Hz) (Figure 7A, 7B, and 7E). Furthermore, inhibition of β-cell KATP channels by a 2 min exposure to tolbutamide increases the AP firing frequency in control β-cells (RIP-rtTA, 2.35±0.10 Hz), but this increase is significantly attenuated in EAC3I-GFP expressing β-cells (1.78±0.21 Hz) (Figure 7C, 7D, and 7E). Thus, CaMKII has an important role in glucose-induced and LTCC-dependent increase in AP firing frequency, which further increases Ca2+ influx. CaMKII enhances β-cell function in the presence of insulin resistance

To determine the role of CaMKII in augmenting Ca2+ influx and insulin secretion during periods of increased insulin demand, RIP-rtTA and EAC3I-GFP mice (n=5 each) were

placed on a HFD at 3 weeks of age to induce systemic insulin resistance. GTTs revealed that both groups of mice exhibited comparable glucose tolerance, as expected since the eAC3I-GFP transgene is not expressed under these conditions (Figure 8A and 8C). Both cohorts of animals were then placed on doxycycline while the HFD was continued. Reassessment of glucose tolerance after 2 weeks on doxycycline revealed a substantial impairment of glucose tolerance in animals with EAC3I-GFP expression in β-cells when compared with RIP-rtTA animals (Figure 8B and 8C, n=5 per group). Since increased Ca2+ influx augments insulin secretion during conditions of insulin resistance (32), glucose-stimulated Ca2+ influx was also monitored in islets of HFD treated animals. Glucose-stimulated Ca2+ influx was significantly diminished in EAC3I-GFP expressing islets compared to RIP-rtTA islets, which were both isolated from HFD treated mice (Figure 8D and 8E). This demonstrates an important role for CaMKII in augmenting glucose-stimulated β-cell Ca2+ influx and insulin secretion under conditions of systemic insulin resistance. DISCUSSION It is well established that Ca2+ entry into pancreatic β-cells stimulates insulin secretion and that CaMKII activity influences Ca2+ homeostasis in many cell types (20,33-35). While CaMKII has been implicated in regulating islet insulin secretion, mechanisms underlying CaMKII action in β-cells are poorly understood (2). The results presented here demonstrate that CaMKII modulates Ca2+ handling and insulin secretion in a mouse model with conditional β-cell expression of a CaMKII inhibitory peptide. The results suggest that CaMKII enhances glucose tolerance by amplifying glucose-stimulated Ca2+ entry and insulin secretion.

Ca2+-dependent amplification and/or reduction of the channels required for cytosolic Ca2+ entry dynamically modulates Ca2+ signals in many cell types. Amplification can occur through many mechanisms, including Ca2+-dependent activation of ER Ca2+ release via ryanodine receptor (RYR) channels, as well as through Ca2+ dependent facilitation of plasma membrane LTCCs (33,36,37). Although some of these mechanisms involve direct binding of Ca2+-

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binding proteins (e.g., calmodulin) to the channels, others involve additional Ca2+ activated proteins such as CaMKII (33). In cardiac cells CaMKII regulates multiple proteins involved in Ca2+ homeostasis, tightly modulating excitation-contraction and excitation-transcription coupling (33,36,37). Similarly we find that CaMKII controls Ca2+ homeostasis in pancreatic β-cell, in this case modulating excitation-secretion coupling. When activated during glucose-stimulation, β-cell CaMKII facilitates LTCCs and amplifies glucose-stimulated islet Ca2+ entry. Although LTCC facilitation by CaMKII can be induced by depolarization, it has also been shown to result from ER Ca2+ release in neurons as well as sarcoplasmic reticulum Ca2+ release in myocytes (26,38,39). Therefore, the CaV1.2 and/or CaV1.3 LTCCs in -cells may undergo CaMKII-dependent facilitation via multiple mechanisms under different physiological conditions, which influence insulin secretion. The results suggest that β-cell CaMKII provides a Ca2+ sensitive feedback loop for enhancing GSIS secretion by amplifying Ca2+ influx.

CaMKII can also indirectly regulate LTCCs by regulating the plasma membrane potential via K+ channel modulation (40). Data presented here suggest that CaMKII does not modulate -cell KV and KATP currents. We also find that CaMKII enhances AP firing frequency during KATP inhibition. Our data suggest that CaMKII regulates -cell electrical activity and Ca2+ entry independently of any effects on KATP or KV channel activity. However, CaMKII can inhibit the inward rectifier subunit of the -cell KATP channel complex (Kir6.2) (41). Moreover, CaMKII modulates H2O2 activation of sarcolemmal KATP channels and oxidative stress activates Kv2.1 channels (42,43). Thus, it is important to note that our data do not exclude potential effects of CaMKII on -cell K+ channels under specific conditions not assessed in this study such as oxidative stress.

Endoplasmic reticulum Ca2+ stores also play an important role in regulating β-cell excitability and GSIS (44-47). CaMKII modulates ER Ca2+ levels by regulating both RYR and SERCA (21-23). RYR2 is phosphorylated by CaMKII in islets and mutations of RYR2 that mimic CaMKII phosphorylation increase -cell

ER Ca2+ release (23). Therefore, diminished ER Ca2+ leak through RYRs following chronic inhibition of β-cell CaMKII may cause the reductions in both thapsigargin-induced ER Ca2+ release and basal cytoplasmic Ca2+ levels. However, carbachol induced ER Ca2+ release through IP3 receptors is also decreased in islets with β-cell CaMKII inhibition. Therefore, -cell ER Ca2+ levels appear to be reduced by CaMKII inhibition, possibly due to SERCA inhibition. A brief reduction in SERCA activity with acute inhibition of CaMKII would account for the increase of basal Ca2+ (Fig. 5C) due to the inability of ER to take up cytoplasmic Ca2+. A sustained reduction of SERCA activity with chronic CaMKII inhibition would ultimately decrease -cell ER Ca2+ stores (Fig. 5A). The exact mechanism(s) responsible for CaMKII modulation of β-cell ER Ca2+ handling are currently being investigated.

CaMKII-dependent modulation of Ca2+ influx likely causes the changes in GSIS reported here because ionomycin-stimulated insulin secretion is unaffected by CaMKII inhibition. However, other studies suggest roles for CaMKII in regulating components of insulin granule priming and or fusion. For example, one study found that CaMKII inhibition decreased insulin secretion even when islet intracellular calcium levels are clamped (48). These effects may involve the direct phosphorylation of synapsin1 and microtubule-associated protein, MAP2, by CaMKII (49,50). The studies presented here confirm that synapsin1 phosphorylation is indeed regulated by CaMKII. Synapsin1 controls the targeting of synaptic like vesicles in primary pancreatic β-cells and rat insulinoma cells (17), and is localized to insulin granules in mouse insulinoma cells (51). This indicates the possibility that β-cell synapsin1 either directly influences insulin granule release via association with the insulin granule or indirectly influences insulin secretion through release of neurotransmitters from synaptic like vesicles. MAP2 is also phosphorylated by CaMKII and may influence insulin granule priming through insulin granule trafficking to the membrane on microtubules. This would be predicted to influence 2nd phase insulin secretion and interestingly the studies presented here identify significant reductions in 2nd phase insulin secretion when CaMKII is inhibited in islet

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β-cells. Thus, MAP2 may be important in regulating trafficking of insulin granules in response to CaMKII activation during GSIS. Future studies will determine the roles of synapsin1 and MAP2 regulation of insulin secretion and islet function in response to glucose activation of CaMKII. In summary, although the changes in Ca2+ induced by -cell CaMKII inhibition are likely to impair first phase insulin secretion, our data cannot exclude additional roles for CaMKII during insulin secretion such as granule trafficking and priming during 2nd phase insulin secretion.

Although CaMKII physiologically modulates islet function, not much is known about the influence of CaMKII signaling during the pathogenesis of diabetes (2). Perturbations in CaMKII signaling are observed following palmitate treatment of insulinoma β-cells (52). Therefore, changes in CaMKII signaling may contribute to aberrant β-cell function following HFD feeding, during the progression of diabetes (52,53). Indeed, we found that the inhibition of CaMKII in mice maintained on a HFD results in a rapid exacerbation of glucose intolerance. These

data indicate that β-cell CaMKII activity is critical for the maintenance of glucose stimulated islet Ca2+ influx and thus insulin secretion in the face of chronic hyperglycemia and the initial progression of insulin resistance induced by a high fat diet, thereby protecting mice from glucose intolerance. This indicates the exciting possibility that augmenting CaMKII activity may also increase human glucose-stimulated β-cell Ca2+ entry and enhance GSIS in a diabetic setting. Future studies will determine how CaMKII signaling influences human -cell GSIS during the pathogenesis of diabetes.

In conclusion, this study suggests that CaMKII plays an important role as a β-cell Ca2+ sensor that modulates Ca2+ handling under both high energy/glucose and low energy/glucose conditions. CaMKII amplifies Ca2+ influx and insulin secretion during glucose stimulation and maintains cytoplasmic Ca2+ levels under fasting conditions. Thus, CaMKII is a key Ca2+ sensor that controls pancreatic β-cell Ca2+ homeostasis to dynamically modulate insulin secretion.

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REFERENCES 1.  Ammala, C., Eliasson, L., Bokvist, K., Berggren, P. O., Honkanen, R. E., Sjoholm, A., and Rorsman, 

P. (1994) Activation of protein kinases and inhibition of protein phosphatases play a central role in the regulation of exocytosis in mouse pancreatic beta cells. Proc Natl Acad Sci U S A 91, 4343‐4347 

2.  Easom, R. A. (1999) CaM kinase II: a protein kinase with extraordinary talents germane to insulin exocytosis. Diabetes 48, 675‐684 

3.  Ashcroft, S.  J.  (1994) Protein phosphorylation and beta‐cell  function. Diabetologia 37 Suppl 2, S21‐29 

4.  Norling, L. L., Colca, J. R., Kelly, P. T., McDaniel, M. L., and Landt, M. (1994) Activation of calcium and calmodulin dependent protein kinase II during stimulation of insulin secretion. Cell Calcium 16, 137‐150 

5.  Wenham,  R. M.,  Landt,  M.,  and  Easom,  R.  A.  (1994)  Glucose  activates  the  multifunctional Ca2+/calmodulin‐dependent protein kinase  II  in  isolated rat pancreatic  islets.  J Biol Chem 269, 4947‐4952 

6.  Ammala,  C.,  Eliasson,  L.,  Bokvist,  K.,  Larsson,  O.,  Ashcroft,  F.  M.,  and  Rorsman,  P.  (1993) Exocytosis elicited by action potentials and voltage‐clamp calcium currents  in  individual mouse pancreatic B‐cells. J Physiol 472, 665‐688 

7.  Babb,  E.  L.,  Tarpley,  J.,  Landt,  M.,  and  Easom,  R.  A.  (1996)  Muscarinic  activation  of Ca2+/calmodulin‐dependent  protein  kinase  II  in  pancreatic  islets.  Temporal  dissociation  of kinase activation and insulin secretion. Biochem J 317 ( Pt 1), 167‐172 

8.  Gromada,  J., Hoy, M., Renstrom, E., Bokvist, K., Eliasson, L., Gopel, S., and Rorsman, P.  (1999) CaM  kinase  II‐dependent mobilization  of  secretory  granules  underlies  acetylcholine‐induced stimulation of exocytosis in mouse pancreatic B‐cells. J Physiol 518 ( Pt 3), 745‐759 

9.  Milo‐Landesman, D., Surana, M., Berkovich,  I., Compagni, A., Christofori, G., Fleischer, N., and Efrat,  S.  (2001)  Correction  of  hyperglycemia  in  diabetic  mice  transplanted  with  reversibly immortalized  pancreatic  beta  cells  controlled  by  the  tet‐on  regulatory  system.  Cell Transplantation 10, 645‐650 

10.  Klug, J. R., Mathur, B. N., Kash, T. L., Wang, H.‐D., Matthews, R. T., Robison, A. J., Anderson, M. E., Deutch, A. Y., Lovinger, D. M., Colbran, R. J., and Winder, D. G. (2012) Genetic  Inhibition of CaMKII  in Dorsal  Striatal Medium  Spiny Neurons Reduces  Functional Excitatory  Synapses  and Enhances Intrinsic Excitability. PLoS One 7, e45323 

11.  Jacobson, D. A., Kuznetsov, A., Lopez,  J. P., Kash, S., Ammala, C. E., and Philipson, L. H.  (2007) Kv2.1  ablation  alters  glucose‐induced  islet  electrical  activity,  enhancing  insulin  secretion.  Cell Metab 6, 229‐235 

12.  Philipson, L. H., Rosenberg, M. P., Kuznetsov, A., Lancaster, M. E., Worley, J. F., 3rd, Roe, M. W., and Dukes, I. D. (1994) Delayed rectifier K+ channel overexpression in transgenic islets and beta‐cells associated with impaired glucose responsiveness. J Biol Chem 269, 27787‐27790 

13.  Rae,  J., Cooper, K., Gates, P.,  and Watsky, M.  (1991)  Low  access  resistance perforated patch recordings using amphotericin B. J Neurosci Methods 37, 15‐26 

14.  Schindelin,  J., Arganda‐Carreras,  I., Frise, E., Kaynig, V.,  Longair, M., Pietzsch, T., Preibisch, S., Rueden,  C.,  Saalfeld,  S.,  Schmid,  B.,  Tinevez,  J.  Y., White,  D.  J.,  Hartenstein,  V.,  Eliceiri,  K., Tomancak, P., and Cardona, A. (2012) Fiji: an open‐source platform for biological‐image analysis. Nature Methods 9, 676‐682 

15.  Nir,  T.,  Melton,  D.  A.,  and  Dor,  Y.  (2007)  Recovery  from  diabetes  in  mice  by  beta  cell regeneration. J Clin Invest 117, 2553‐2561 

9

by guest on March 22, 2020

http://ww

w.jbc.org/

Dow

nloaded from

CaMKII modulates β-cell Ca2+ homeostasis  

16.  Pullen,  T.  J.,  Sylow,  L.,  Sun,  G.,  Halestrap,  A.  P.,  Richter,  E.  A.,  and  Rutter,  G.  A.  (2012) Overexpression  of  monocarboxylate  transporter‐1  (SLC16A1)  in  mouse  pancreatic  beta‐cells leads to relative hyperinsulinism during exercise. Diabetes 61, 1719‐1725 

17.  Krueger,  K.  A.,  Ings,  E.  I.,  Brun,  A.  M.,  Landt,  M.,  and  Easom,  R.  A.  (1999)  Site‐specific phosphorylation  of  synapsin  I  by  Ca2+/calmodulin‐dependent  protein  kinase  II  in  pancreatic betaTC3 cells: synapsin I is not associated with insulin secretory granules. Diabetes 48, 499‐506 

18.  Bernal‐Mizrachi, E., Fatrai, S., Johnson, J. D., Ohsugi, M., Otani, K., Han, Z., Polonsky, K. S., and Permutt, M. A.  (2004) Defective  insulin  secretion and  increased  susceptibility  to experimental diabetes are induced by reduced Akt activity in pancreatic islet beta cells. J Clin Invest 114, 928‐936 

19.  Anthony,  D.  F.,  Beattie,  J.,  Paul,  A.,  and  Currie,  S.  (2007)  Interaction  of  calcium/calmodulin‐dependent protein kinase IIdeltaC with sorcin indirectly modulates ryanodine receptor function in cardiac myocytes. J Mol Cell Cardiol 43, 492‐503 

20.  Wehrens, X. H., Lehnart, S. E., Reiken, S. R., and Marks, A. R. (2004) Ca2+/calmodulin‐dependent protein kinase II phosphorylation regulates the cardiac ryanodine receptor. Circ Res 94, e61‐70 

21.  Currie, S., Loughrey, C. M., Craig, M. A., and Smith, G. L. (2004) Calcium/calmodulin‐dependent protein  kinase  IIdelta  associates with  the  ryanodine  receptor  complex  and  regulates  channel function in rabbit heart. Biochem J 377, 357‐366 

22.  Xu, A., Hawkins,  C.,  and Narayanan, N.  (1993)  Phosphorylation  and  activation  of  the  Ca(2+)‐pumping  ATPase  of  cardiac  sarcoplasmic  reticulum  by  Ca2+/calmodulin‐dependent  protein kinase. J Biol Chem 268, 8394‐8397 

23.  Dixit,  S.  S., Wang,  T., Manzano,  E.  J.,  Yoo,  S.,  Lee,  J.,  Chiang, D.  Y.,  Ryan, N.,  Respress,  J.  L., Yechoor,  V.  K.,  and  Wehrens,  X.  H.  (2013)  Effects  of  CaMKII‐Mediated  Phosphorylation  of Ryanodine Receptor Type 2 on Islet Calcium Handling, Insulin Secretion, and Glucose Tolerance. PLoS One 8, e58655 

24.  Grueter, C. E., Abiria, S. A., Dzhura,  I., Wu, Y., Ham, A.  J., Mohler, P.  J., Anderson, M. E., and Colbran, R.  J.  (2006) L‐type Ca2+ channel  facilitation mediated by phosphorylation of the beta subunit by CaMKII. Molecular Cell 23, 641‐650 

25.  Hudmon, A.,  Schulman, H.,  Kim,  J., Maltez,  J. M.,  Tsien, R. W.,  and  Pitt, G.  S.  (2005)  CaMKII tethers to L‐type Ca2+ channels, establishing a local and dedicated integrator of Ca2+ signals for facilitation. The J Cell Biol 171, 537‐547 

26.  Gao, L., Blair, L. A., Salinas, G. D., Needleman, L. A., and Marshall, J.  (2006)  Insulin‐like growth factor‐1 modulation  of  CaV1.3  calcium  channels  depends  on  Ca2+  release  from  IP3‐sensitive stores  and  calcium/calmodulin  kinase  II  phosphorylation  of  the  alpha1  subunit  EF  hand.  J Neurosci 26, 6259‐6268 

27.  Jenkins, M.  A.,  Christel,  C.  J.,  Jiao,  Y.,  Abiria,  S.,  Kim,  K.  Y., Usachev,  Y. M., Obermair, G.  J., Colbran, R. J., and Lee, A. (2010) Ca2+‐dependent facilitation of Cav1.3 Ca2+ channels by densin and Ca2+/calmodulin‐dependent protein kinase II. J Neurosci 30, 5125‐5135 

28.  Abiria, S. A., and Colbran, R. J. (2010) CaMKII associates with CaV1.2 L‐type calcium channels via selected beta subunits to enhance regulatory phosphorylation. J of Neurochem 112, 150‐161 

29.  Lee, T. S., Karl, R., Moosmang, S., Lenhardt, P., Klugbauer, N., Hofmann, F., Kleppisch, T., and Welling, A. (2006) Calmodulin kinase II is involved in voltage‐dependent facilitation of the L‐type Cav1.2  calcium  channel:  Identification  of  the  phosphorylation  sites.  J  Biol  Chem  281,  25560‐25567 

30.  Braun, M., Ramracheya, R., Bengtsson, M., Zhang, Q., Karanauskaite, J., Partridge, C., Johnson, P. R.,  and  Rorsman,  P.  (2008)  Voltage‐gated  ion  channels  in  human  pancreatic  beta‐cells: electrophysiological characterization and role in insulin secretion. Diabetes 57, 1618‐1628 

10

by guest on March 22, 2020

http://ww

w.jbc.org/

Dow

nloaded from

CaMKII modulates β-cell Ca2+ homeostasis  

31.  Matthews,  E.  K.,  and  Sakamoto,  Y.  (1975)  Electrical  characteristics  of  pancreatic  islet  cells.  J Physiol 246, 421‐437 

32.  Gonzalez, A., Merino, B., Marroqui,  L., Neco,  P., Alonso‐Magdalena,  P.,  Caballero‐Garrido,  E., Vieira, E., Soriano, S., Gomis, R., Nadal, A., and Quesada, I. (2013) Insulin hypersecretion in islets from  diet‐induced  hyperinsulinemic  obese  female mice  is  associated with  several  functional adaptations in individual beta‐cells. Endocrinology 154, 3515‐3524 

33.  Dzhura,  I., Wu, Y., Colbran, R.  J., Balser,  J. R.,  and Anderson, M.  E.  (2000) Calmodulin  kinase determines calcium‐dependent facilitation of L‐type calcium channels. Nat Cell Biol 2, 173‐177 

34.  Malinow, R.,  Schulman, H.,  and  Tsien, R. W.  (1989)  Inhibition  of postsynaptic  PKC or CaMKII blocks induction but not expression of LTP. Science 245, 862‐866 

35.  Kranias, E. G., Gupta, R. C., Jakab, G., Kim, H. W., Steenaart, N. A., and Rapundalo, S. T. (1988) The role of protein kinases and protein phosphatases  in the regulation of cardiac sarcoplasmic reticulum function. Mol Cell Biochem 82, 37‐44 

36.  Zhang,  R.,  Khoo,  M.  S.,  Wu,  Y.,  Yang,  Y.,  Grueter,  C.  E.,  Ni,  G.,  Price,  E.  E.,  Jr.,  Thiel,  W., Guatimosim,  S.,  Song,  L.  S., Madu,  E.  C.,  Shah,  A.  N.,  Vishnivetskaya,  T.  A.,  Atkinson,  J.  B., Gurevich, V. V., Salama, G., Lederer, W. J., Colbran, R. J., and Anderson, M. E. (2005) Calmodulin kinase II inhibition protects against structural heart disease. Nat Med 11, 409‐417 

37.  Wu, Y., Colbran, R.  J., and Anderson, M. E.  (2001) Calmodulin kinase  is a molecular switch  for cardiac excitation‐contraction coupling. Proc Natl Acad Sci U S A 98, 2877‐2881 

38.  Xiao, R.  P., Cheng, H.,  Lederer, W.  J.,  Suzuki,  T.,  and  Lakatta,  E. G.  (1994) Dual  regulation of Ca2+/calmodulin‐dependent kinase II activity by membrane voltage and by calcium  influx. Proc Natl Acad Sci U S A 91, 9659‐9663 

39.  Wu,  Y.,  Kimbrough,  J.  T.,  Colbran,  R.  J.,  and  Anderson,  M.  E.  (2004)  Calmodulin  kinase  is functionally  targeted  to  the  action  potential  plateau  for  regulation  of  L‐type  Ca2+  current  in rabbit cardiomyocytes. J Physiol 554, 145‐155 

40.  Griffith, L. C., Wang,  J., Zhong, Y., Wu, C. F., and Greenspan, R.  J.  (1994) Calcium/calmodulin‐dependent  protein  kinase  II  and  potassium  channel  subunit  eag  similarly  affect  plasticity  in Drosophila. Proc Natl Acad Sci U S A 91, 10044‐10048 

41.  Kline, C. F., Wright, P. J., Koval, O. M., Zmuda, E. J., Johnson, B. L., Anderson, M. E., Hai, T., Hund, T.  J.,  and  Mohler,  P.  J.  (2013)  betaIV‐Spectrin  and  CaMKII  facilitate  Kir6.2  regulation  in pancreatic beta cells. Proc Natl Acad Sci U S A 110, 17576‐17581 

42.  McCord, M. C., and Aizenman, E. (2013) Convergent Ca2+ and Zn2+ signaling regulates apoptotic Kv2.1 K+ currents. Proc Natl Acad Sci U S A 110, 13988‐13993 

43.  Zhang,  D.  M.,  Chai,  Y.,  Erickson,  J.  R.,  Heller  Brown,  J.,  Bers,  D.  M.,  and  Lin,  Y.  F.  (2014) Modulation  of  Sarcolemmal  ATP‐Sensitive  Potassium  Channels  by  Nitric  Oxide  via sGC/PKG/ROS/ERK1/2/CaMKII Signaling in Ventricular Cardiomyocytes. J Physiol  

44.  Worley, J. F., 3rd, McIntyre, M. S., Spencer, B., Mertz, R. J., Roe, M. W., and Dukes, I. D. (1994) Endoplasmic reticulum calcium store regulates membrane potential  in mouse  islet beta‐cells. J Biol Chem 269, 14359‐14362 

45.  Roe, M. W., Mertz, R. J., Lancaster, M. E., Worley, J. F., 3rd, and Dukes, I. D. (1994) Thapsigargin inhibits the glucose‐induced decrease of  intracellular Ca2+  in mouse  islets of Langerhans. Am J Physiol 266, E852‐862 

46.  Wollheim,  C.  B.,  Janjic, D.,  Siegel,  E. G.,  Kikuchi, M.,  and  Sharp, G. W.  (1981)  Importance  of cellular calcium stores in glucose‐stimulated insulin release. Upsala J MedSci 86, 149‐164 

47.  Prentki, M.,  and Wollheim,  C.  B.  (1984)  Cytosolic  free  Ca2+  in  insulin  secreting  cells  and  its regulation by isolated organelles. Experientia 40, 1052‐1060 

48.  Bhatt, H. S., Conner, B. P., Prasanna, G., Yorio, T., and Easom, R. A. (2000) Dependence of insulin secretion  from  permeabilized  pancreatic  beta‐cells  on  the  activation  of  Ca(2+)/calmodulin‐

11

by guest on March 22, 2020

http://ww

w.jbc.org/

Dow

nloaded from

CaMKII modulates β-cell Ca2+ homeostasis  

dependent protein kinase II. A re‐evaluation of  inhibitor studies. Biochem Pharmacol 60, 1655‐1663 

49.  Matsumoto,  K.,  Fukunaga,  K.,  Miyazaki,  J.,  Shichiri,  M.,  and  Miyamoto,  E.  (1995) Ca2+/calmodulin‐dependent protein kinase  II and  synapsin  I‐like protein  in mouse  insulinoma MIN6 cells. Endocrinology 136, 3784‐3793 

50.  Krueger, K. A., Bhatt, H., Landt, M., and Easom, R. A. (1997) Calcium‐stimulated phosphorylation of MAP‐2 in pancreatic betaTC3‐cells is mediated by Ca2+/calmodulin‐dependent kinase II. J Biol Chem 272, 27464‐27469 

51.  Yamamoto,  H.,  Matsumoto,  K.,  Araki,  E.,  and  Miyamoto,  E.  (2003)  New  aspects  of neurotransmitter  release  and  exocytosis:  involvement  of  Ca2+/calmodulin‐dependent phosphorylation of synapsin I in insulin exocytosis. J Pharmacol Sci 93, 30‐34 

52.  Watson, M. L., Macrae, K., Marley, A. E., and Hundal, H. S.  (2011) Chronic effects of palmitate overload on nutrient‐induced insulin secretion and autocrine signalling in pancreatic MIN6 beta cells. PLoS One 6, e25975 

53.  Prentki, M., and Nolan, C. J. (2006) Islet beta cell failure in type 2 diabetes. J Clin Invest 116, 1802‐1812

FOOTNOTES * This work was supported by NIH grant DK096122 (D.A.J), DK081666 (D.A.J) as well as a Pilot and Feasibility grant through the Vanderbilt University Diabetes Research Training Center (VDRTC) P60 DK20593. The Vanderbilt Islet Procurement and Analysis Core is also supported by NIH grant DK20593. 1 To whom correspondence may be addressed: Department of Molecular Physiology and Biophysics, Vanderbilt University, 7425B MRB IV, 2213 Garland Avenue, Nashville, TN, 37232-0615, Tel: (615) 875-7655, Fax: 615-343-0475, email: david.a.jacobson@vanderbilt.edu 2 The abbreviations used are: AIP2, autocamtide-2 related Inhibitory Peptide II; CaMKII, Ca2+/calmodulin-dependent protein kinase II; CCh, carbachol; ER, endoplasmic reticulum; GSIS, glucose stimulated insulin secretion; HFD, high fat diet; HRP, horseradish peroxidase; KRB, Krebs-Ringer buffer; LTCCs, L-type Ca2+ channels; RYR, ryanodine receptor; SERCA, sarco/endoplasmic reticulum ATPase; tetO, tetracycline operator; VDCCs, voltage dependent Ca2+ channels Acknowledgements - We thank Danny Winder for the EAC3I-GFP mouse model as well as helpful discussions and suggestions. FIGURE LEGENDS FIGURE 1. Doxycycline induces inhibition of β-cell CaMKII activity in a transgenic mouse. (A) Cartoon of the transgenic design of a mouse model with doxycycline inducible β-cell CaMKII inhibition. (B) Epifluorescent detection of β-cell specific GFP in a pancreas of an EAC3I-GFP + RIPrtTA mouse on doxycycline for 1.5 weeks; white bar is equivalent to 20M. (C) Islet cell cluster from an EAC3I-GFP + RIPrtTA mouse on doxycycline for 1.5 weeks stained for GFP (green) and insulin (red), white bar is equivalent to 10M. (D) Islet phospho-synapsin1 (top panels) and GAPDH (lower panels) from islets expressing rtTA or rtTA + EAC3I-GFP in β-cells. (E) Total pancreatic insulin from RIPrtTA or EAC3I-GFP + RIPrtTA mice treated with doxycycline for 1.5 weeks (n=4 pancreata/group). (F) Total islet insulin from RIPrtTA or EAC3I-GFP + RIPrtTA mice treated with doxycycline for 1.5 weeks (n=4 independent islet preparations/group). (G-H) Doxycycline treated RIPrtTA EAC3I-GFP (G) and doxycycline treated

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RIPrtTA (H) pancreatic sections stained for insulin (red), glucagon (green) and nuclei (blue); white bars are equivalent to 20M. FIGURE 2. β-cell CaMKII enhances glucose tolerance by amplifying glucose-stimulated insulin secretion in mice (A) Glucose tolerance test on mice with β-cell CaMKII inhibition (gray line) and control (black line) animals. (B) Insulin secretion from islets isolated from β-cell EAC3I-GFP expressing or (gray line) control (black line) animals, treated as indicated in above with 5.6 mM glucose, 16.7 mM glucose, and 20 mM KCl; n = 4 islet sets from independent animals per group. (C) Total islet insulin secretion (area under the curve) during 1st (15-24 minutes) and 2nd phase (24-42 minutes) insulin secretion as well as following KCl-stimulation (66-72 minutes). Error bars represent +/-SEM. (D) Doxycyline treated RIP rtTA EAC3I (grey bars) and doxycyline treated RIP rtTA (black bars) static islet insulin secretion under 7 mM and 10 mM glucose as indicated (n=3 male islet sets for each animal type, +/-SEM, *p<0.05). FIGURE 3. -cell CAMKII does not influence glucose metabolism (A) Glucose induced NAD(P)H fluorescence in GFP+ -cells of an intact islet from RIP rtTA tetO-GFP (black line, 10 islets each from 3 independent preparations) or RIP rtTA EAC3I-GFP (grey line, 10 islets each from 4 independent preparations). (B) Glucose induced NAD(P)H fluorescence from all cells in an islet plane 30 uM deep from RIP rtTA tetO-GFP (black line, 10 islets each from 3 independent preparations) or RIP rtTA EAC3I-GFP (grey line, 10 islets each from 4 independent preparations). FIGURE 4. CaMKII does not significantly modulate -cell KV or KATP channels (A) Representative KV currents from a control (RIP-rtTA) -cell in response to voltage Steps in 10 mV increments from -80 to +80 mV. (B) Representative KV currents from an EAC3I-GFP expressing -cell in response to voltage Steps in 10 mV increments from -80 to +80 mV. (C) Current density vs Voltage plots of RIP-rtTA (black line) and EAC3I-GFP -cells (grey line) recorded in steps from −80mV to the indicated voltage +/− SEM’s (n=5 for each condition). (D) KATP currents recorded in response to a voltage ramp from -120 mV to 60 mV (black line below traces) from RIP-rtTA (black trace is the average of 11 recordings with grey SEM bars) or EAC3I-GFP -cells (grey trace is the average of 22 recordings with black SEM bars). (E) Doxycyline treated RIP rtTA EAC3I-GFP (grey bars) and doxycyline treated RIP rtTA tetO-GFP (black bars) static islet insulin secretion with 5.6 mM glucose (glucose=G.) alone or in combination with 0.25, 2.5 or 25 M ionomycin (ionomycin=I.; n=3 male islet sets for each animal type). Error bars represent +/-SEM. FIGURE 5. β-cell CaMKII increases cytoplasmic and endoplasmic reticulum Ca2+ levels (A) Relative islet Ca2+ levels following glucose-stimulation (14 mM); islets (54 from RIP rtTA and 58 from RIP rtTA EAC3I) isolated from four independent animals. (B) Relative islet Ca2+ influx during 6 minutes post initiation of glucose-stimulated islet Ca2+ influx. (C) Relative islet Ca2+ levels from islet cell clusters (~20 cells per cluster) following glucose stimulation(14 mM); AIP2 (pretreated with 20 M cell permeable AIP2 for 20 minutes), Vehicle (pretreated with vehicle for 20 minutes). (D) Relative islet Ca2+ influx (AUC) during 6 minutes post initiation of glucose-stimulated islet Ca2+ influx. (E) Relative islet Ca2+ levels from GFP positive islet cells with (grey trace, RIP rtTA EAC3I-GFP) or without EAC3I (black trace, RIP rtTA tetO-GFP) in 5.6 mM glucose KRB in response to 20 mM KCl stimulation (black bar). (F) Relative KCl induced Ca2+ increase (AUC) determined from -cell Ca2+ influx during KCl treatment. (G) Relative islet Ca2+ levels during ER Ca2+ release with 2 M thapsigargin (with no extracellular Ca2+) (H) Relative ER Ca2+ levels (AUC) determined from islet Ca2+ influx during thapsigargin treatment. (I) Relative islet Ca2+ levels during CCh induced ER Ca2+ release (with no extracellular Ca2+). (J) Relative CCh induced ER Ca2+ release (AUC) determined from islet Ca2+ influx during CCh treatment. Error bars represent +/-SEM, *** p<0.001.

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CaMKII modulates β-cell Ca2+ homeostasis  

FIGURE 6. CaMKII facilitates β-cell Ca2+ channels augmenting VDCC current amplitude (A) triple voltage step protocol used to elicit Ca2+ channel facilitation (left). β-cell Ca2+ channel currents following the voltage clamp protocol shown in panel (right). The black Ca2+ current trace is during the first voltage step (P1) and the grey trace is during the third voltage step (P3). (B) Normalized change in β-cell Ca2+ current between the first voltage step and the third voltage step. (C) β-cell Ca2+ channel currents in response to a voltage step to 10 mV. (D) Current Voltage plot of β-cell Ca2+ channel currents in response

to the indicated voltage steps. Error bars represent +/-SEM, * p<0.05, ** p<0.01. (E) Activation of ・-cell

VDCCs in response to the indicated voltage steps. (F.) Fast (fast) and slow (slow) time constants of inactivation kinetics of -cell VDCCs in response to the indicated voltage steps. FIGURE 7. CaMKII activity increases β-cell action potential firing frequency (A) Control β-cell electrical activity in response to 14 mM glucose. Inset shows the APs from the segment of activity indicated by the horizontal grey bar. (B) Electrical activity from a β-cell expressing EAC3I in response to 14 mM glucose. Inset shows the APs from the segment of activity indicated by the horizontal grey bar. (C) Control β-cell electrical activity in response to glucose (14 mM) and tolbutamide (200 M). (D) Electrical activity from a β-cell expressing EAC3I in response to glucose (14 mM) and tolbutamide (200 M). (E) β-cell action potential firing frequency 10 minutes post glucose (14 mM) treatment, rtTA (n=16) and EAC3I (n=15). (F) β-cell action potential firing frequency 10 minutes post glucose (14 mM) and 2 minutes post glucose (14 mM) with tolbutamide (200 M) treatment, rtTA (n=7) and EAC3I (n=9). Error bars represent +/-SEM, * p<0.05. FIGURE 8. β-cell CaMKII protects animals from glucose intolerance following exposure to a high fat diet by enhancing glucose stimulated Ca2+ entry. (A) Glucose tolerance test on mice (RIP rtTA, black line; RIP rtTA EAC3I, grey line) following 2 months on a high fat diet. (B) Glucose tolerance test on the mice shown in panel-A two weeks post doxycycline treatment (β-cell CaMKII inhibition, gray line; controls, black line) following 2.5 months on a high fat diet. (C) Relative islet Ca2+ levels following glucose-stimulation (14 mM); islets (n>70 islets from each group, RIP rtTA and RIP rtTA EAC3I) isolated from four independent animals.

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Colbran and David A. JacobsonPrasanna K. Dadi, Nicholas C. Vierra, Alessandro Ustione, David W. Piston, Roger J.

and insulin secretion, impairing glucose toleranceInhibition of pancreatic beta-cell CaMKII reduces glucose-stimulated calcium influx

published online March 13, 2014J. Biol. Chem. 

  10.1074/jbc.M114.562587Access the most updated version of this article at doi:

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