Blocking Ca²⁺ Channel β₃ Subunit Reverses Diabetes

Summary

Voltage-gated Ca²⁺ channels (Ca_v) are essential for pancreatic beta cell function as they mediate Ca²⁺ influx, which leads to insulin exocytosis. The β3 subunit of Ca_v (Ca_vβ₃) has been suggested to regulate cytosolic Ca²⁺ ([Ca²⁺]_i) oscillation frequency and insulin secretion under physiological conditions, but its role in diabetes is unclear. Here, we report that islets from diabetic mice show Ca_vβ₃ overexpression, altered [Ca²⁺]_i dynamics, and impaired insulin secretion upon glucose stimulation. Consequently, in high-fat diet (HFD)-induced diabetes, Ca_vβ₃-deficient (Ca_vβ₃^−/−) mice showed improved islet function and enhanced glucose tolerance. Normalization of Ca_vβ₃ expression in ob/ob islets by an antisense oligonucleotide rescued the altered [Ca²⁺]_i dynamics and impaired insulin secretion. Importantly, transplantation of Ca_vβ₃^−/− islets into the anterior chamber of the eye improved glucose tolerance in HFD-fed mice. Ca_vβ₃ overexpression in human islets also impaired insulin secretion. We thus suggest that Ca_vβ₃ may serve as a druggable target for diabetes treatment.

Graphical Abstract

Highlights

• •Pancreatic islets from diabetic mice have increased level of Ca_vβ₃
• •Overexpression of Ca_vβ₃ in islets alters Ca²⁺ dynamics and impairs insulin secretion
• •Deficiency of Ca_vβ₃ prevents islet dysfunction and glucose intolerance in diabetes
• •Blocking Ca_vβ₃ improves islet function and glucose tolerance after onset of diabetes

Lee et al. report that pancreatic islets from diabetic mice overexpress Ca_vβ₃, resulting in altered [Ca²⁺]_i dynamics and impaired insulin secretion. Reducing Ca_vβ₃ expression recovers islet dysfunction and glucose homeostasis. Experiments with human islets suggest that Ca_vβ₃ may constitute a druggable target in diabetes treatment.

Introduction

Intracellular Ca²⁺ signaling in pancreatic islets is important for their endocrine function, particularly insulin secretion. Ca²⁺ signaling evoked by glucose stimulation shows the unique feature of oscillatory increases regulated by an interplay between Ca²⁺ influx through voltage-gated L-type Ca²⁺ channels and Ca²⁺ mobilization from intracellular stores (Dupont et al., 2011, Sabourin et al., 2015). Accumulating evidences suggest that both the amplitude and the frequency of cytosolic Ca²⁺ ([Ca²⁺]_i) oscillations in the islets are important and that these are altered in subjects with type 2 diabetes mellitus (T2DM). In a diabetic mouse model, the amplitude of [Ca²⁺]_i changes decreased and the response to glucose was delayed, concurrent with a disappearance of [Ca²⁺]_i oscillations (Colsoul et al., 2014, Gilon et al., 2014). Similar findings have been reported for islets from diabetic patients (Flanagan et al., 2017, Fridlyand et al., 2013, Pittas et al., 2007, Reinbothe et al., 2013, Xu et al., 2012). In this context, it is noteworthy that restoration of Ca²⁺ signaling has been shown to result in improved glucose tolerance (Ahrén et al., 1999, Ojo et al., 2015). Although a number of genes associated with β cell dysfunction in T2DM have been identified (Dyachok et al., 2008, Gandasi et al., 2017, Healy et al., 2010, Lawlor et al., 2017, Zhou et al., 2015), the molecular mechanisms underlying alterations in Ca²⁺ dynamics in T2DM have not yet been well defined.

Voltage-gated Ca²⁺ channels (Ca_v) play a critical role in intracellular Ca²⁺ signaling and insulin exocytosis (Rutter et al., 2006, Rutter et al., 2017, Yang and Berggren, 2006). They are composed of four subunits: a pore-forming α1 subunit; α2/δ; β; and γ subunits. The β subunit is anchored to the intracellular side of the membrane and modifies Ca²⁺ channel currents through binding to the pore-forming α1 subunit (Buraei and Yang, 2010, Van Petegem et al., 2004). Among the four types of β subunits, the β3 subunit (Ca_vβ₃) is mainly expressed in pancreatic islets in addition to the β2 subunit (Ca_vβ₂). In a previous study, we reported that islets from Ca_vβ₃- deficient (Ca_vβ₃^−/−) mice showed an increased frequency of [Ca²⁺]_i oscillations and better insulin secretion, and Ca_vβ₃^−/− mice were more glucose tolerant than wild-type mice (Berggren et al., 2004). However, the role of Ca_vβ₃ in diabetes remains unclear.

Here, we report that Ca_vβ₃ plays a major role in alterations of Ca²⁺ dynamics and subsequent insulin secretion in the diabetic islets. We observed that the protein level of Ca_vβ₃ in islets from diabetic mice was elevated and that [Ca²⁺]_i dynamics in response to a high glucose concentration were altered. Deficiency of Ca_vβ₃ prevented the alteration of Ca²⁺ signaling during diabetes progression. Decreased expression of Ca_vβ₃ in islets from diabetic mice showed improvement of [Ca²⁺]_i dynamics and insulin secretion compared to islets from control mice, resulting in ameliorated glucose tolerance in the mice. Therefore, targeting of Ca_vβ₃ may be a therapeutic strategy in T2DM.

Results

Pancreatic Islets from ob/ob Mice Overexpress Ca_vβ₃ and Have Altered [Ca²⁺]_i Dynamics and Insulin Secretion

We investigated the relationship between diabetes and Ca_vβ₃ using B6.Cg-Lep^ob/J (ob/ob) mice, a diabetic mouse model. First, we compared Ca_vβ₃ protein levels in islets from 8- to 12-week-old ob/ob mice with those in islets from lean mice. The protein level of Ca_vβ₃ in islets from ob/ob mice was significantly higher than that in islets from control (lean) mice (Figure 1A). Considering the phenotype of Ca_vβ₃^−/− mice with the shorter oscillation periods in Ca²⁺ signaling and improved glucose-induced insulin secretion (GIIS), we anticipated opposite changes in ob/ob mice. We measured [Ca²⁺]_i dynamics in islets from ob/ob and lean mice upon high-glucose (11 mM) stimulation. Representative Ca²⁺ traces are shown in Figures 1B and 1C. The first-peak amplitudes in glucose-induced Ca²⁺ traces were lower in islets from ob/ob mice than in those from lean mice (Figure 1D). For quantitative analysis of the oscillatory patterns, we analyzed the period and amplitude of oscillations based on the power spectral density. Islets from ob/ob mice showed oscillations of longer period and smaller amplitude than islets from lean mice (Figures 1E and 1F). To check Ca²⁺ increase dependent on Ca_v, we depolarized dissociated islet cells by 25 mM KCl. The [Ca²⁺]_i peak in islet cells from ob/ob mice was lower than that in islet cells from lean mice (Figures 1G and 1H). GIIS was lower in islets from ob/ob mice than in those from lean mice (Figure 1I). Consistent with these results, glucose intolerance was significantly higher in ob/ob mice than in control lean mice (Figure 1J). Hence, we observed overexpression of Ca_vβ₃ and alterations in [Ca²⁺]_i dynamics, including smaller amplitudes of the first peak of glucose-induced Ca²⁺ increases and longer oscillation periods and smaller oscillation amplitudes, in islets from ob/ob mice. These changes result in impaired GIIS and overall impaired in vivo glucose tolerance.

Figure 1.

Ca_vβ₃ Expression and [Ca²⁺]_i Dynamics Were Altered in Islets from ob/ob Mice

(A) Left panel shows protein levels of Ca_vβ₃ in islets from control lean mice and ob/ob mice. Right panel shows relative quantification of Ca_vβ₃ protein expression in left panel (n = 5; 40 islets in each case).

(B and C) Effects of 11 mM glucose on [Ca²⁺]_i in islets from control (B) and ob/ob mice (C). Representative traces out of 39 for lean and ob/ob islets are shown.

(D) First peak ratios of glucose-induced [Ca²⁺]_i changes in islets from control lean and ob/ob mice.

(E) Oscillation periods of glucose-induced [Ca²⁺]_i changes in islets from control lean and ob/ob mice.

(F) Oscillation amplitudes of glucose-induced [Ca²⁺]_i changes in islets from control lean mice and ob/ob mice.

(G) Effects of 25 mM KCl on [Ca²⁺]_i in dissociated islet cells from control lean (black) and ob/ob (red) mice. Representative traces in dissociated islet cells from control lean and ob/ob mice are shown.

(H) Peak ratios of [Ca²⁺]_i changes induced by 25 mM KCl in dissociated islet cells from lean (black) and ob/ob (red) mice (n = 10; each experiment included 50 single cells).

(I) Glucose-induced insulin release in islets from control lean and ob/ob mice. The islets were treated with 3 mM or 11 mM glucose for 30 min (n = 5; 10 islets in each case).

(J) Left panel shows intraperitoneal glucose tolerance test in control lean and ob/ob mice (n = 5 each). Right panel shows comparison of areas under the curves from left panel.

Data are presented as the mean ± SEM; ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001.

Pancreatic Islets from HFD-Fed Mice Overexpress Ca_vβ₃ and Show Altered [Ca²⁺]_i Dynamics and Insulin Secretion

Next, we investigated the relationship between Ca_vβ₃ and diabetes using high-fat diet (HFD)-fed mice, another model of T2DM. As in ob/ob mice, the Ca_vβ₃ protein level was higher in islets from mice fed a HFD for 8 weeks than in islets from normal chow diet (NCD)-fed mice (Figure 2A). Representative Ca²⁺ traces upon 11 mM glucose stimulation are shown in Figures 2B and 2C. First-peak amplitudes in glucose-induced Ca²⁺ traces were lower in islets from HFD-fed mice compared to those of NCD-fed mice (Figure 2D). Based on the power spectral density, we found that islets from HFD-fed mice produced oscillations of longer period (Figure 2E) and smaller amplitude (Figure 2F). However, 25 mM KCl did not induce significant differences in Ca²⁺ influx between islets from HFD-fed mice and those from NCD-fed mice (Figures 2G and 2H). Nevertheless, GIIS was lower in islets from HFD-fed mice than in islets from NCD-fed mice (Figure 2I). HFD-fed mice showed stronger glucose intolerance than NCD-fed mice (Figure 2J). Collectively, these results revealed overexpression of Ca_vβ₃, alterations in Ca²⁺ dynamics, and insulin secretion in islets from HFD-fed mice, similar to the observations made in ob/ob mice. Based on these findings, which were opposite to the results obtained in Ca_vβ₃^−/− mice, we suspected that overexpression of Ca_vβ₃ might be a causative factor underlying altered [Ca²⁺]_i dynamics and insulin secretion in diabetic islets.

Figure 2.

Ca_vβ₃ Expression and [Ca²⁺]_i Dynamics Were Altered in Islets of HFD Mice

(A) Left panel shows protein levels of Ca_vβ₃ in islets from NCD and HFD B6 mice. Right panel shows relative quantification of Ca_vβ₃ protein expression in left panel (n = 5; 40 islets in each case).

(B and C) Effects of 11 mM glucose on [Ca²⁺]_i in islets from NCD (B) and HFD (C) B6 mice. Representative traces out of 30 for NCD and HFD islets are shown.

(D) First peak ratios of glucose-induced [Ca²⁺]_i changes in islets from NCD and HFD B6 mice.

(E) Oscillation periods of glucose-induced [Ca²⁺]_i changes in islets from NCD and HFD B6 mice.

(F) Oscillation amplitudes of glucose-induced [Ca²⁺]_i changes in islets from NCD and HFD B6 mice.

(G) Effects of 25 mM KCl on [Ca²⁺]_i in dissociated islet cells from NCD (black) and HFD (red) B6 mice. Representative traces on dissociated islet cells from NCD and HFD B6 mice are shown.

(H) Peak ratios of [Ca²⁺]_i changes induced by 25 mM KCl in dissociated islet cells from NCD (black) and HFD (red) B6 mice (n = 10; each experiment involved 50 single cells).

(I) Glucose-induced insulin release in islets from NCD and HFD B6 mice. Islets were treated with 3 mM or 11 mM glucose for 30 min (n = 5; 10 islets in each case).

(J) Left panel shows intraperitoneal glucose tolerance test in NCD and HFD B6 mice (n = 5 in each case). Right panel shows comparison of areas under the curves from left panel.

Data are presented as the mean ± SEM; ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001.

Ca_vβ₃ Overexpression in Pancreatic Islets Alters [Ca²⁺]_i Dynamics and Insulin Secretion

To investigate whether the altered [Ca²⁺]_i dynamics and impaired insulin secretion in diabetic islets are directly resulting from Ca_vβ₃ overexpression, we measured these parameters in islets from C57BL/6 mice overexpressing Ca_vβ₃ (Figure 3A) and compared with islets from control mice. First-peak amplitudes in the glucose-induced Ca²⁺ traces were lower in islets overexpressing Ca_vβ₃ than in control islets expressing GFP (Figures 3B–3D). Oscillation periods were longer and amplitudes smaller in islets from Ca_vβ₃-overexpressing mice compared to those from control mice (Figures 3E and 3F). Upon depolarization of dissociated islet cells with 25 mM KCl, [Ca²⁺]_i peak amplitudes in islet cells overexpressing Ca_vβ₃ were lower than those in control islet cells (Figures 3G and 3H). In our previous study, we suggested that an increase in inositol triphosphate (IP₃)-mediated Ca²⁺ release from endoplasmic reticulum (ER) might contribute to changes in [Ca²⁺]_i oscillation frequency and insulin secretion in Ca_vβ₃ KO islets (Berggren et al., 2004). To check whether this mechanism is involved in islets overexpressing Ca_vβ₃, we stimulated islet cells overexpressing Ca_vβ₃ and control islet cells with carbamylcholine (Cch), triggering the cholinergic-mediated IP₃ signaling pathway. The resulting peak in [Ca²⁺]_i increase in islet cells overexpressing Ca_vβ₃ was lower than that in controls (Figure 3I). GIIS was also lower after stimulation with 11 mM glucose in islet cells overexpressing Ca_vβ₃ (Figure 3J). The results obtained in islets or islet cells overexpressing Ca_vβ₃ were consistent with the findings in ob/ob and HFD-fed mice. To shed some light on how Ca_vβ₃ levels can be elevated in diabetic islets, we investigated potential effects of several factors, such as glucose, insulin, and inflammatory cytokines, known to be associated with diabetes progression. Tumor necrosis factor alpha (TNF-α) or interleukin-1β (IL-1β) significantly increased protein levels of Ca_vβ₃ in isolated islets (Figure S1). This suggests that proinflammatory cytokines may induce overexpression of Ca_vβ₃ in islets and thereby cause islet cell dysfunction.

Figure 3.

Overexpression of Ca_vβ₃ Altered [Ca²⁺]_i Dynamics in Pancreatic Islets

(A) Left panel shows protein levels of Ca_vβ₃ in control and Ca_vβ₃-overexpressing adenovirus-treated islet. Right panel shows relative quantification of Ca_vβ₃ protein levels in left panel (n = 5; 40 islets in each case).

(B and C) Effects of 11 mM glucose on [Ca²⁺]_i in control (B) and Ca_vβ₃-overexpressing (C) islets. Representative traces out of 30 for both control and Ca_vβ₃-overexpressing islets are shown.

(D) First peak ratios of glucose-induced [Ca²⁺]_i changes in control and Ca_vβ₃-overexpressing islets.

(E) Oscillation periods of glucose-induced [Ca²⁺]_i changes in control and Ca_vβ₃-overexpressing islets.

(F) Oscillation amplitudes of glucose-induced [Ca²⁺]_i changes in control and Ca_vβ₃-overexpressing islets.

(G) Effects of 25 mM KCl on [Ca²⁺]_i in control islet cells (black) and Ca_vβ₃-overexpressing islet cells (red). Representative traces of each group are shown.

(H) Peak ratios of [Ca²⁺]_i changes induced by 25 mM KCl in control islet cells (black) and Ca_vβ₃-overexpressing islet cells (red; n = 10; each experiment involved 50 single cells).

(I) Peak ratios of [Ca²⁺]_i changes induced by 200 μM Cch in control islet cells and Ca_vβ₃-overexpressing islet cells (n = 5; each experiment involved 30 single cells).

(J) Glucose-induced insulin release in control and Ca_vβ₃-overexpressing islets. Islets treated with 3 mM or 11 mM glucose for 30 min (11 mM glucose/3 mM glucose) are shown (n = 5; 10 islets in each case).

The data are presented as the mean ± SEM; ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001. See also Figures S1 and S4.

Pancreatic Islets from Ca_vβ₃^−/− Mice Exposed to HFD Have Ameliorated [Ca²⁺]_i Dynamics and Insulin Secretion

We examined whether Ca_vβ₃ had a direct role in the islet dysfunction observed in the HFD model. Ca_vβ₃^−/− mice and their littermate controls were fed HFD for eight weeks, and no significant difference in body weight changes was observed between the two groups (Figure S2A). Next, we investigated [Ca²⁺]_i dynamics in the islets from control and Ca_vβ₃^−/− mice (Figures 4B and 4C). First-peak amplitudes in the glucose-induced Ca²⁺ traces were higher in islets from HFD-fed Ca_vβ₃^−/− mice than in those from control animals fed the HFD (Figure 4D). Ca_vβ₃^−/− islets showed shorter oscillation periods and larger amplitudes (Figures 4E and 4F). There was no significant difference in the [Ca²⁺]_i peak after stimulation with 25 mM KCl between Ca_vβ₃^−/− and control islet cells (Figures 4G and 4H). This was in agreement with similar KCl-stimulated responses in islets from control mice fed NCD and in islets from control mice fed HFD (Figure 2H). We also compared effects of Cch in islets from Ca_vβ₃^−/− mice and their littermate controls fed HFD. Ca_vβ₃^−/− islets showed increased peak of [Ca²⁺]_i compared to control islets (Figure 4I), which is consistent with the Cch effect in islets overexpressing Ca_vβ₃ (Figure 3I). The compromised GIIS in islets from HFD-fed mice was also improved in islets from HFD-fed Ca_vβ₃^−/− mice (Figure 4J). Consistent with the results on [Ca²⁺]_i dynamics and insulin secretion, glucose tolerance in Ca_vβ₃^−/− mice on HFD was better than that in control mice (Figure 4K), and insulin tolerance was not significantly different (Figure S2B). Plasma insulin level at 30 min after glucose injection was higher in HFD-fed Ca_vβ₃^−/− mice (Figure 4L). Although the fasting insulin level was not significantly different in HFD-fed Ca_vβ₃^−/− mice compared to lean mice (Figure S2C), non-fasting insulin levels were higher in HFD-fed Ca_vβ₃^−/− mice (Figure S2D). Therefore it is likely that an increase in in vivo GIIS explains the better glucose tolerance in HFD-fed Ca_vβ₃^−/− mice. Based on these observations, we suggest that overexpression of Ca_vβ₃ is an important factor causing islet dysfunction in diabetes development, and regulation of the Ca_vβ₃ level could lead to an improvement of islet function and glucose homeostasis.

Figure 4.

Ca_vβ₃^−/− Mice Fed a HFD Show a Less Severe Diabetic Phenotype

(A) Protein levels of Ca_vβ₃ in islets from control mice fed a HFD and Ca_vβ₃^−/− mice fed a HFD (n = 5; 40 islets in each case).

(B and C) Effects of 11 mM glucose on [Ca²⁺]_i in islets from control mice fed a HFD (B) and Ca_vβ₃^−/− mice fed a HFD (C). Representative traces out of 30 are shown.

(D) First peak ratios of glucose-induced [Ca²⁺]_i changes in islets from control mice and Ca_vβ₃^−/− mice fed a HFD.

(E) Oscillation periods of glucose-induced [Ca²⁺]_i changes in islets from control mice and Ca_vβ₃^−/− mice fed a HFD.

(F) Oscillation amplitudes of glucose-induced [Ca²⁺]_i changes in islets from control mice and Ca_vβ₃^−/− mice fed a HFD.

(G) Effects of 25 mM KCl on [Ca²⁺]_i in dissociated islet cells from control mice (black) and Ca_vβ₃^−/− mice (red) fed a HFD. Representative traces on dissociated islet cells are shown.

(H) Peak ratios of [Ca²⁺]_i changes induced by 25 mM KCl in dissociated islet cells from control mice (black) and Ca_vβ₃^−/− mice (red) fed a HFD (n = 5; each experiment involved 50 single cells).

(I) Peak ratios of [Ca²⁺]_i changes induced by 200 μM Cch in dissociated islet cells from Ca_vβ₃^−/− mice fed a HFD and control mice fed a HFD (n = 5; each experiment involved 30 single cells).

(J) Glucose-induced insulin release from islets of control and Ca_vβ₃^−/− mice fed a HFD. Islets were treated with 3 mM or 11 mM glucose for 30 min (n = 3; 10 islets in each case).

(K) Left panel shows intraperitoneal glucose tolerance test of control and Ca_vβ₃^−/− mice fed a HFD (n = 4 in each case). Right panel shows comparison of areas under the curves from left panel.

(L) Plasma insulin levels 30 min after glucose injection from fasted control mice (black) and Ca_vβ₃^−/− mice (red) fed a HFD (n = 5 in each case).

Data are presented as the mean ± SEM; ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001. See also Figure S2.

Treatment with Antisense Oligonucleotide Targeting Ca_vβ₃ Improves [Ca²⁺]_i Dynamics and Insulin Secretion in ob/ob Islets

The next question was whether decreasing expression of Ca_vβ₃ could improve Ca²⁺ dynamics and insulin secretion in the diabetes onset condition. We tested this idea in ob/ob islets from 8- to 12-week-old mice by treatment with antisense oligonucleotide targeting Ca_vβ₃ (Ca_vβ₃ ASO). Treatment with Ca_vβ₃ ASO effectively reduced Ca_vβ₃ expression in the ob/ob islets (Figure 5A). We measured glucose-induced Ca²⁺ dynamics in ob/ob islets treated with Ca_vβ₃ ASO or scramble ASO (Figures 5B and 5C). First-peak amplitudes in glucose-induced Ca²⁺ traces were significantly higher in ob/ob islets after Ca_vβ₃ ASO treatment (Figure 5D). Islets treated with Ca_vβ₃ ASO showed shorter oscillation periods (Figure 5E) and larger amplitudes (Figure 5F), as indicated by power spectral analysis. Upon stimulation with 25 mM KCl, dissociated islet cells treated with Ca_vβ₃ ASO showed higher Ca²⁺ peak ratios than islet cells treated with scramble ASO (Figures 5G and 5H). Moreover, treatment with Ca_vβ₃ ASO, compared to treatment with scramble ASO, increased Cch-induced Ca²⁺ release in islet cells from ob/ob mice (Figure 5I). Consistent with the Ca²⁺-influx results, islets treated with Ca_vβ₃ ASO showed improved GIIS (Figure 5J).

Figure 5.

Treatment with Antisense Oligonucleotide Targeting Ca_vβ₃ Improves [Ca²⁺]_i Dynamics and Insulin Secretion in ob/ob Islets

(A) Left panel shows protein levels of Ca_vβ₃ in ob/ob islets treated with scramble ASO and Ca_vβ₃ ASO. Right panel shows relative quantification of Ca_vβ₃ protein levels in left (n = 5; 40 islets in each case).

(B and C) Effects of 11 mM glucose on [Ca²⁺]_i in ob/ob islets treated with scramble ASO (B) and Ca_vβ₃ ASO (C). Representative traces out of 30 are shown.

(D) First peak ratios of glucose-induced [Ca²⁺]_i changes in ob/ob islets treated with scramble ASO and Ca_vβ₃ ASO.

(E) Oscillation periods of glucose-induced [Ca²⁺]_i changes in ob/ob islets treated with scramble ASO and Ca_vβ₃ ASO.

(F) Oscillation amplitudes of glucose-induced [Ca²⁺]_i changes in ob/ob islets treated with scramble ASO and Ca_vβ₃ ASO.

(G) Effects of 25 mM KCl on [Ca²⁺]_i in dissociated islet cells from scramble ASO (black) and Ca_vβ₃-ASO-treated (red) ob/ob mice. Representative traces on dissociated islet cells are shown.

(H) Peak ratios of [Ca²⁺]_i changes induced by 25 mM KCl in dissociated islet cells from scramble ASO (black) and Ca_vβ₃-ASO-treated (red) ob/ob mice (n = 5; each experiment involved 50 single cells).

(I) Peak ratios of [Ca²⁺]_i changes induced by 200 μM Cch in ob/ob-dissociated islet cells treated with scramble ASO and Ca_vβ₃ ASO (n = 5; each experiment involved 30 single cells).

(J) Glucose-induced insulin release in ob/ob islets treated with scramble ASO and Ca_vβ₃ ASO. The islets were treated with 3 mM or 11 mM glucose for 30 min (n = 5; 10 islets in each case).

Data are presented as the means ± SEM; ^∗p < 0.05, ^∗∗∗p < 0.001. See also Figure S5.

Transplantation of Ca_vβ₃^−/− Islets Improves Glycemic Control in HFD-Fed Mice

To assess the effect of targeting Ca_vβ₃ in pancreatic islets after diabetes onset in vivo, we transplanted islets from Ca_vβ₃^−/− mice and control littermates into the anterior chamber of the eye in diabetic mice. C57BL/6J mice were put on a HFD during the whole period of the experiment. After eight weeks of HFD feeding, the animals were treated with streptozotocin to avoid an effect of endogenous pancreatic islets. Thereafter, islet transplantations were performed (Figures 6A and 6B). Three weeks later, we imaged islet vascularization in vivo (Figure 6C). Vessel diameters inside the islets did not significantly differ between Ca_vβ₃^−/− islet-transplanted and control islet-transplanted mice (Figure S3A). The blood glucose level was decreased in both Ca_vβ₃^−/− and control islet-transplanted mice (Figure S3B). After 4 weeks, when transplanted islets are fully vascularized and innervated, we performed glucose tolerance tests. From five weeks after transplantation, glucose tolerance in mice transplanted with Ca_vβ₃^−/− islets improved as compared with mice transplanted with control islets. We presented the results obtained six weeks after transplantation, when glucose tolerance was more significantly improved. The area under the curve was significantly lower in Ca_vβ₃^−/− islet-transplanted compared to control islet-transplanted mice (Figure 6D). However, insulin tolerance was not significantly different (Figure 6E). In short, glucose clearance, but not insulin sensitivity, was improved in Ca_vβ₃^−/− islet-transplanted mice. These results suggest that suppression of Ca_vβ₃ after the time of onset of diabetes can significantly improve the diabetes phenotype in mice.

Figure 6.

Metabolic Transplantation of Ca_vβ₃^−/− Islet Improves Glucose Control under Diabetic Conditions

(A) Schematic diagram of experimental protocol.

(B) Representative photograph of islets engrafted on the iris.

(C) Representative islet vessel images; maximum projections of image stacks of an islet graft in the anterior chamber of the eye three weeks after transplantation. Islet is green, and blood vessels are red (Texas Red staining). The scale bar represents 100 μm.

(D) Left panel shows intraperitoneal glucose tolerance tests in HFD-fed mice transplanted with islets from control and Ca_vβ₃^−/− mice (n = 8 in each case). Right panel shows comparison of areas under the curves from left panel.

(E) Intraperitoneal insulin tolerance test in HFD-fed mice transplanted with islets from control and Ca_vβ₃^−/− mice (n = 8 in each case).

Data are presented as the mean ± SEM; ^∗p < 0.05 and ^∗∗p < 0.01. See also Figure S3.

Overexpression of Ca_vβ₃ Disrupts GIIS in Human Islets

To investigate whether Ca_vβ₃ functions in human islets, we overexpressed Ca_vβ₃ in islets from individual donors using adenovirus (Figure 7A) and measured GIIS. Overexpression of Ca_vβ₃ decreased insulin secretion specifically in the presence of a high-glucose concentration, whereas it didn’t affect basal insulin secretion (Figures 7B and 7C). From these results, we suggest that overexpression of Ca_vβ₃ might have a similar role in human islets as in rodent islets, namely decreasing GIIS.

Figure 7.

Ad-Ca_vβ₃-Transduced Human Islets Display Impaired Glucose-Stimulated Insulin Secretion

(A) Sample confocal images of control (left panel) and Ca_vβ₃-overexpressing human islets (right panel).

(B) Insulin secretion from control or Ca_vβ₃-overexpressing islets of individual human donors incubated with 3 mM or 11 mM glucose.

(C) Average insulin secretion from control or Ca_vβ₃-overexpressing islets of human donors incubated with 3 mM or 11 mM glucose.

Data are presented as mean ± SEM; ^#p < 0.01 versus 3 mM glucose/Ad-GFP, ^¤p < 0.05 versus 11 mM glucose/Ad-GFP, and ^∗∗p < 0.01 versus 11–3 mM glucose/Ad-GFP. The scale bars represent 20 μm. Δ Insulin, insulin released from islets incubated with 11 mM glucose minus that from islets incubated with 3 mM glucose.

Discussion

Ca²⁺ signaling in pancreatic β cell is critical for glucose-induced insulin secretion to regulate blood glucose levels. A number of studies have suggested that Ca²⁺ signaling is disturbed in T2DM through changes in Ca_v activity or intracellular Ca²⁺ release (Duchen et al., 2008, Gilon et al., 2014, Ramadan et al., 2011, Rorsman et al., 2012, Velasco et al., 2016). In this study, we showed that Ca_vβ₃ was overexpressed in diabetic mouse islets and that this was important for progression of T2DM. Based on our results obtained with ASO treatment and islet transplantation, we suggest that Ca_vβ₃ could be a target for treatment of T2DM.

Overexpression of Ca_vβ₃ in pancreatic islets led to alterations in Ca²⁺ dynamics during diabetes progression. Alterations in Ca²⁺ dynamics in diabetic islets, including decreases in the amplitude of Ca²⁺ influx induced by glucose and elongation of the period of Ca²⁺ oscillations, have been previously reported (Colsoul et al., 2010, Colsoul et al., 2014, Gilon et al., 2014, Tengholm and Gylfe, 2009). In our experiments using islets from ob/ob and HFD-fed mice, we also consistently observed that these phenotypes concur with overexpression of Ca_vβ₃ and that overexpression of Ca_vβ₃ per se induced similar changes in Ca²⁺ dynamics. In a series of experiments where we reduced Ca_vβ₃ expression in diabetic islets, the changes in Ca²⁺ dynamics were reversed. Together, these results indicate that Ca_vβ₃ is responsible for the alterations in Ca²⁺ dynamics in diabetic mouse islets.

Future studies will reveal how Ca_vβ₃ mechanistically decreases Ca²⁺ conductance (Figure S4) and increases the Ca²⁺ oscillation period. Ca_vβ₃ is mainly known as a subunit that can regulate Ca_v activity. Ca_vβ₃ binds a highly conserved sequence located in the intracellular loop joining the α interaction domain of the α1 subunit, thus tightly regulating pore opening (Buraei and Yang, 2010, Van Petegem et al., 2004). In other cell systems, Ca_vβ₃ generally increased Ca_v activity by channel localization or regulation of voltage-dependent activation or inhibition, a phenotype opposite to that observed in our study (Bernardo et al., 2009, Kharade et al., 2013, Murakami et al., 2008, Ohta et al., 2010). Decrease of Ca²⁺ conductance by overexpression of Ca_vβ₃ seems to be dependent on the cellular and/or molecular context. One study showed that overexpression of Ca_vβ₃ decreased the activity of N type and R type Ca_v channels, but not that of L-type Ca²⁺ channels (Yasuda et al., 2004). In pancreatic islets, we have previously shown that removal of Ca_vβ₃ did not change Ca_v activity under normal physiological conditions although it increased Ca²⁺ oscillation frequency. Hence, Ca_vβ₃ may have a unique mode of action in pancreatic islets that is lacking in other cells and that this feature is particularly important for the progression of diabetes. For the regulation of Ca²⁺ oscillation pattern by Ca_vβ₃, we previously suggested that IP₃-mediated Ca²⁺ release from ER might be important (Berggren et al., 2004). In the present study, we showed that overexpression of Ca_vβ₃ decreased Cch-induced Ca²⁺ release and that this effect was restored by decreasing Ca_vβ₃ levels (Figures 3I, 4I, and 5I). These results led us to hypothesize that overexpression of Ca_vβ₃ in diabetes negatively impacts ER Ca²⁺ handling and eventually leads to ER stress. ER stress has been associated with the loss of insulin secretion in T2DM (Rieusset, 2017) and is thought to be a major contributor to islet dysfunction (Marmugi et al., 2016). However, we could not observe significant changes in ER stress markers between ob/ob islets treated with Ca_vβ₃-ASO or scrambled ASO under conditions where we observed improvement of Ca²⁺ dynamics and insulin secretion (Figure S5). Although we cannot exclude that Ca_vβ₃ is involved in ER stress at the later time points of diabetes progression, ER stress may not be the primary mechanism whereby Ca_vβ₃ exerts its negative effects on β cell function.

We propose Ca_vβ₃ as a potential target for diabetes treatment. We showed that decreasing the level of Ca_vβ₃ in ob/ob islets recovered their GIIS and that transplantation of Ca_vβ₃^−/− islets into HFD-induced diabetic mice could recover glucose intolerance. Insulin secretion assays showed that manipulation of Ca_vβ₃ affected secretion only at elevated glucose concentrations (Figures 4L and S2). This means that targeting Ca_vβ₃ is not associated with risk for hypoglycemia, one of the main issues associated with the strategy of augmenting insulin secretion in patients with T2DM. Our data demonstrating that overexpression of Ca_vβ₃ decreased GIIS also in human islets (Figure 7) support the notion that Ca_vβ₃ may indeed represent a promising clinical target in the treatment of diabetes. To develop treatment strategies for diabetes targeting Ca_vβ₃, two approaches can theoretically be considered. One is to decrease the level of Ca_vβ₃ in pancreatic islets by ASO or small interfering RNA (siRNA) treatment, alternatively to transplant islets in which the endogenous expression levels of Ca_vβ₃ have been suppressed. The other is to block Ca_vβ₃ function. Although we did not pinpoint the exact mechanism of Ca_vβ₃ action in the diabetes islets, regulation of inositol trisphosphate generation or interaction with binding partners, such as α1 subunit, might be involved. Here, we particularly proved the in vivo application using Ca_vβ₃^−/− islets transplanted into HFD-fed mice. This may in the future pave the way for a gene therapeutic approach, where the expression of β cell Ca_vβ₃ is suppressed prior to islets being transplanted. Obviously, aspects such as efficacy, specific delivery, and side effects require further considerations before we can proceed with such strategies. In whole-body Ca_vβ₃-knockdown mice, we did not observe any phenotypic defects. However, there are some reports of abnormal behavioral activities in Ca_vβ₃^−/− mice, such as reduced anxiety, increased aggression, increased nighttime activity, and impaired working memory, although some forms of hippocampus-dependent learning are enhanced (Murakami et al., 2007, Jeon et al., 2008).

An important question is how Ca_vβ₃ levels mechanistically are upregulated in diabetic mouse islets. A recent study showed that Ca_vβ₃ protein is upregulated in diabetic rat hearts subsequent to streptozotocin treatment (Ferdous et al., 2016), suggesting that diabetes may be associated with altered Ca_vβ₃ protein levels in various cell types in multiple organs. Here, we found that pro-inflammatory cytokines, such as TNF-α or IL-1β, increased Ca_vβ₃ levels in the pancreatic islets (Figure S1). This is in line with a previous study demonstrating that Ca_vβ₃ expression is significantly increased in dendritic cells following treatment with TNF-α and IL-1β for 3 days (Bros et al., 2011). These findings imply that regulation of Ca_vβ₃ by proinflammatory cytokines can be a universal mechanism regulating Ca²⁺ signaling in cells. Recent studies have suggested that islet inflammation is associated with diabetes progression, and pro-inflammatory cytokines may therefore be critically involved in islet dysfunction (Donath, 2013, Eguchi and Nagai, 2017). The detailed relationship between islet inflammation and the roles of Ca_vβ₃ should be the focus of future studies and thereby give us valuable information how targeting Ca_vβ₃ may restore diabetes.

In conclusion, we have shown that Ca_vβ₃ is associated with a significant Ca²⁺ signaling dysfunction in diabetic mouse islets and that targeting of this subunit might have beneficial effects not only on Ca²⁺ handling but also on insulin release, thereby resulting in improved glucose homeostasis. With similar results obtained applying human islets, we propose that Ca_vβ₃ may serve as a druggable target in diabetes.

Experimental Procedures

Experimental Animal Care and Preparation of Islets and Cells

All experimental procedures were approved by the Pohang University of Science and Technology Institutional Animal Care and Use Committee (POSTECH-2015-0055-R1, POSTECH IACUC, Korea). C57BL/6J, B6.Cg-Lep^ob/J male mice (Jackson Laboratory, Bar Harbor, ME, USA) were used. Ca_vβ₃^−/− mice were generated and backcrossed over 18 generations as described previously (Namkung et al., 1998). Ca_vβ₃ heterozygous (Cavβ₃^+/−) mice were backcrossed into C57BL/6J. Wild-type (Ca_vβ₃^+/+) and Ca_vβ₃^−/− mice used for analyses were obtained by breeding Cavβ₃^+/− mice. The animals were maintained with a 12-hr light/dark cycle with free access to water. Mice were sacrificed by cervical dislocation under anesthesia with CO₂. Four-week-old mice were fed a 60% HFD (D12492, Research Diets, New Brunswick, NJ, USA) for 8 weeks, and body weight and food intake were monitored. Islets of Langerhans were isolated by collagenase digestion (1 mg/mL collagenase P; Roche Diagnostics, Indianapolis, IN) and subsequently handpicked under a stereomicroscope. Islets were cultured in RPMI 1640 medium (Gibco, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin at 37°C in a humidified atmosphere of 5% CO₂ in air for one day before the experiment. Single-islet cells were obtained by shaking islets in Ca²⁺-free medium with Accutase (Gibco) and seeded on poly-l-lysine-coated glasses and cultured overnight in RPMI 1640 culture medium. For treatment with proinflammatory cytokines, islets were pooled and incubated at 20 ng/mL TNF-α (R&D Systems, Minneapolis, MN) or 20 ng/mL IL-1β (R&D Systems) in RPMI 1640 medium (Gibco) supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37°C in a humidified atmosphere of 5% CO₂ for 24 hr.

[Ca²⁺]_i Measurements

[Ca²⁺]_i was measured using the Fura2-AM method. Islets or islet cells were incubated with Fura2-AM (2 μmol/L; Invitrogen, Carlsbad, CA) in HEPES buffer (125 mM NaCl, 5.9 mM KCl, 2.56 mM CaCl₂, 1.2 mM MgCl₂, 25 mM HEPES, 3 mM glucose, and 0.1% BSA [pH 7.4]) for 30 min at 37°C under 5% CO₂. After loading, glass coverslips containing islets and cells were mounted into an open perifusion chamber and maintained at 37°C, and [Ca²⁺]_i was measured as the 340/380 nm fluorescence ratio. Islets were stimulated with 11 mM glucose, 25 mM KCl, or 200 μM Cch (Sigma-Aldrich). The light source was equipped with a xenon arc lamp and an integrated shutter (Lambda DG-4; Sutter Instrument Company, Novato, CA) and coupled to the microscope (IX 71; Olympus, Tokyo, Japan) via a liquid light guide. Sixteen-bit gray-scale images with a binning of 1 × 1 were captured every second (exposure time ∼100 ms) with a cooled electron multiplying charge-coupled device (EM-CCD) camera (ImagEM X2; Hamamatsu Photonics, Hamamatsu, Japan). The camera and shutter were controlled by MetaFluor software (MDS Analytical Technologies, Sunnyvale, CA). Data were analyzed with the same software. Cells with bright [Ca²⁺]_i signal defined the regions of interest (ROIs). ROI signals were calculated by subtracting background noise signal. [Ca²⁺]_i oscillations were analyzed using power spectral analysis in MATLAB (MathWorks, Lowell, MA) with a code adapted for analysis of the oscillations in pancreatic islets (Uhlén, 2004) with modification. Oscillation amplitude values were calculated as the square root of the total power of periods from 6 to 600 s. The fast Fourier transform power spectrum was used to determine the dominating oscillation period from respective power spectra.

SDS-PAGE and Immunoblot Analysis

Islets were lysed in Laemmli sample buffer and heated at 95°C for 5 min. Proteins were separated in SDS-PAGE gels (6%–16% gradient) and transferred to nitrocellulose membranes (Whatman, Maidstone, UK). Blots were blocked for 30 min with 5% skim milk, incubated with Ca_vβ₃ antibody (1:2,000; C1978; Sigma-Aldrich), BIP (1:1,000; 3177; Cell Signaling Technology, Danvers, MA), ATF-6α (1:100; sc-166659; Santa Cruz Biotechnology, Santa Cruz, CA), or ARE1α (1:1,000; 3294; Cell Signaling Technology) at 4°C overnight and washed 3 times with washing buffer (50 mM Tris aminomethane, 150 mM NaCl, and 0.05% Tween). The membranes were incubated with secondary antibody (rabbit) at room temperature for 1 hr and washed 3 times with washing buffer. Immunoreactive bands were visualized with the ECL Plus immunoblotting detection system (Thermo Scientific, Waltham, MA).

Recombinant Adenovirus and Antisense Oligonucleotides

Adenovirus-overexpressing Ca_vβ₃ construct was generated through homologous recombination between linearized pAd-Track-CMV vector carrying either Flag2-wild-type (WT) or Flag2-Ca_vβ₃ and the adenoviral backbone vector pAd-Easy. Ad-GFP was used as a control for all experiments. Viruses were purified with an Adeno-X Maxi purification kit (Clontech Laboratories, Palo Alto, CA, USA) and titrated according to the manufacturer’s instructions. Antisense oligonucleotides, a series of chimeric 20-mer phosphorothioate oligonucleotides containing 2′-O-methoxyethyl groups at positions 1–5 and 16–20 targeted to mouse Ca_vβ₃, were synthesized and purified (Integrated DNA Technologies, Coralville, IA, USA). Adenovirus and antisense oligonucleotides were added directly to pancreatic islet in the culture medium, 4 hr and overnight, respectively.

Glucose and Insulin Tolerance Tests

For glucose tolerance tests, mice were fasted overnight and 1 g/kg of d-glucose (Sigma-Aldrich) was injected intraperitoneally. For insulin tolerance tests, mice were fasted for 4 hr and 0.2 U/kg of insulin (Eli Lilly, Indianapolis, Indiana, USA) was injected intraperitoneally. Blood samples were collected at 0, 15, 30, 60, and 120 min after injection by tail bleeding. Blood glucose levels were determined using a glucometer (Accu-Check Active, Roche Diagnostics).

Insulin Release

Ten islets isolated from corresponding mice were pooled and incubated at 3 mM or 11 mM glucose in HEPES buffer for 30 min at 37°C. The conditioned buffer was collected, and insulin concentration was measured with the Rat/Mouse Insulin ELISA kit (ALPCO Diagnostics, Salem, NH), according to manufacturer’s instruction. GSIS data are normalized by the number of islets. For experiments with human islets, groups of 10 human islets infected with Ad-GFP or Ad-Ca_vβ₃ were preincubated with 3 mM glucose in Krebs buffer at 37°C for 1 hr. Subsequently, preincubated islets were treated with 3 mM glucose and then 11 mM glucose in Krebs buffer at 37°C for 1 hr. Samples were collected for insulin secretion assay. Insulin concentrations in the collected samples were determined by using AlphaLISA assay. Krebs buffer consisted of (in mM) 119 NaCl, 20 HEPES, 4.6 KCl, 2 CaCl₂, 1 MgSO₄, 0.15 Na₂HPO₄, 0.4 KH₂PO₄, 5 NaHCO₃, and 0.1% BSA (pH 7.4). Human pancreata were obtained within the Nordic Network for Islet Transplantation from deceased donors. This study includes pancreatic islets from 3 donors. The experiments were approved by the Regional Ethical Review Boards in Uppsala and in Stockholm.

Islet Transplantation

Mice were fasted overnight and then injected with 150 mg/kg of streptozotocin (Sigma-Aldrich) via intraperitoneal injection. When the glucose level was >300 mg/dL, 0.2 U/kg/day of insulin (Eli Lilly) was injected to maintain glycemia. Islets were isolated from donor mice, and ∼100 islets were transplanted into the anterior chamber of the eye of the recipient mouse (Speier et al., 2008).

Electrophysiological Recordings

Adenovirus-infected single islet cells were subjected to conventional whole-cell patch-clamp analysis with an EPC-10 patch-clamp amplifier (HEKA Elektronik, Lambrecht/Pfalz, Germany). The cells were bathed in the external solution (138 mM NaCl, 10 mM TEACl, 10 mM CaCl₂, 5.6 mM KCl, 1.2 mM MgCl₂, 5 mM HEPES, and 3 mM glucose [pH 7.4]). Borosilicate glass electrodes (1.2 mm outside diameter; Warner Instrument, Hamden, CT) were pulled with a vertical pipette puller (PC-10; Narishige, Tokyo, Japan), and the recording pipette had tip resistances ranging between 2 and 3 MΩ when filled with pipette solution (150 mM N-methyl-D-glucamine, 2 mM CaCl2, 10 mM EGTA, 1 mM MgCl₂, 5 mM HEPES, and 20 mM ATP [pH 7.2]). All recordings were performed at room temperature. The amplitude of whole-cell Ca²⁺ currents was normalized to cell capacitance. Acquisition and analysis of data were done using Patchmaster (HEKA Elektronik).

Statistics

All results are presented as means ± SEM. An unpaired Student’s t test was used for pairwise comparisons. Statistical significance of results from glucose and insulin tolerance tests were assessed by two-way repeated- measures ANOVA followed by multiple comparison with Bonferroni’s correction. A p value < 0.05 was considered statistically significant.

Published: July 24, 2018