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. 2009 Mar 15;587(Pt 6):1169-78.
doi: 10.1113/jphysiol.2008.168005. Epub 2009 Jan 26.

Synaptotagmin-7 is a principal Ca2+ sensor for Ca2+ -induced glucagon exocytosis in pancreas

Natalia Gustavsson  1 , Shun-Hui Wei, Dong Nhut Hoang, Ye Lao, Quan Zhang, George K Radda, Patrik Rorsman, Thomas C Südhof, Weiping Han
Affiliations

Synaptotagmin-7 is a principal Ca2+ sensor for Ca2+ -induced glucagon exocytosis in pancreas

Natalia Gustavsson et al. J Physiol. .

Abstract

Hormones such as glucagon are secreted by Ca(2+)-induced exocytosis of large dense-core vesicles, but the mechanisms involved have only been partially elucidated. Studies of pancreatic beta-cells secreting insulin revealed that synaptotagmin-7 alone is not sufficient to mediate Ca(2+)-dependent insulin granule exocytosis, and studies of chromaffin cells secreting neuropeptides and catecholamines showed that synaptotagmin-1 and -7 collaborate as Ca(2+) sensors for exocytosis, and that both are equally involved. As no other peptide secretion was analysed, it remains unclear whether synaptotagmins generally act as Ca(2+) sensors in large dense-core vesicle exocytosis in endocrine cells, and if so, whether synaptotagmin-7 always functions with a partner in that role. In particular, far less is known about the mechanisms underlying Ca(2+)-triggered glucagon release from alpha-cells than insulin secretion from beta-cells, even though insulin and glucagon together regulate blood glucose levels. To address these issues, we analysed the role of synaptotagmins in Ca(2+)-triggered glucagon exocytosis. Surprisingly, we find that deletion of a single synaptotagmin isoform, synaptotagmin-7, nearly abolished Ca(2+)-triggered glucagon secretion. Moreover, single-cell capacitance measurements confirmed that pancreatic alpha-cells lacking synaptotagmin-7 exhibited little Ca(2+)-induced exocytosis, whereas all other physiological and morphological parameters of the alpha-cells were normal. Our data thus identify synaptotagmin-7 as a principal Ca(2+) sensor for glucagon secretion, and support the notion that synaptotagmins perform a universal but selective function as individually acting Ca(2+) sensors in neurotransmitter, neuropeptide, and hormone secretion.

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Figures

Figure 1
Figure 1. Synaptotagmin-7 expression in glucagon-secreting cells
Twenty micrometre pancreatic sections were stained with antibodies against synaptotagmin-7 (S757, Synaptic Systems) and glucagon, followed by fluorescence-conjugated secondary antibodies. Representative images of such stained sections, taken on a Leica TCS2 confocal microscope, are shown. Synaptotagmin-7 (Syt 7, red) was expressed in glucagon-positive cells and shown to have a high degree of overlap with glucagon signals (green). Arrows indicate selected overlapping signals of synaptotagmin-7 and glucagon. For comparison, no apparent synaptotagmin-7 signal was detected in islet sections from synaptotagmin-7 KO (Syt7−/−) mouse. Scale bars: 40, 20 and 5 μm for top, middle and bottom rows, respectively.
Figure 2
Figure 2. Reduced glucagon level and impaired glucagon secretion, but normal glucagon sensitivity in synaptotagmin-7 KO mice
A, plasma glucagon levels were measured in synaptotagmin-7 KO and control mice that were fasted for 20 h (Fasting state) or 2 h (Resting state) by using the Lincoplex kit. Synaptotagmin-7 KO mice (grey bar) exhibited lower glucagon levels than control (white bar) in both groups. n= 9 for fasting control, resting control and resting KO, and 12 for fasting KO. **P < 0.01 vs. control. B, blood glucose levels before and at 5, 10, 20 and 40 min after i.p. injection of 0.2 mg kg−1 glucagon in 6 h fasted synaptotagmin-7 KO mice (filled circle) and control (open circle) were measured by using a glucometer. Blood glucose response to glucagon injection was not different between the two groups. n= 10 for each group. C, synaptotagmin-7 KO and control mice were injected with 1 U kg−1 insulin, and their plasma glucagon levels were measured before and at 20 and 40 min after injection. Hypoglycaemia-induced glucagon secretion was greatly reduced in KO mice (filled circle) compared to control (open circle). n= 9 for each group. *P < 0.05; **P < 0.01. D, total stimulated glucagon secretion during insulin-induced hypoglycaemia was calculated as area under curve (AUC) in C after basal secretion subtraction. Hypoglycaemia-stimulated glucagon secretion in vivo was significantly reduced in synaptotagmin-7 KO (grey bar) mice compared to controls (white bar). **P < 0.01.
Figure 3
Figure 3. Impaired glucagon secretion in isolated synaptotagmin-7 KO mouse islets
A, low glucose-induced glucagon secretion from isolated islets was measured in perifusion experiments at a glucose concentration of 10 mm (basal) or 1 mm (stimulatory). Arrow indicates switching from basal to stimulatory perifusion buffer. The perfusate was collected in 2 min intervals, and glucagon levels were determined by using RIA. Synaptotagmin-7 KO islets (Syt7−/−, filled circle) showed impaired glucagon secretion when compared with control (open circle). B, low glucose-stimulated glucagon secretion for the entire stimulation period in the perifusion experiments was lower in isolated islets from synaptotagmin-7 KO (grey bar) than from control (white bar). Glucagon secretion was calculated by integrating the area under each curve in A after baseline subtraction. Data are presented as means ±s.e.m., n= 7 for KO and 9 for control, **P < 0.01.
Figure 4
Figure 4. Normal ultrastructure of pancreatic α-cells in synaptotagmin-7 KO mice
A, pancreatic α-cell ultrastructure was analysed by using transmission EM. One representative image from each genotype is shown. Ultrastructural organizations, including distribution of glucagon secretory granules, were similar in control and KO mouse α-cells. Scale bar = 2 μm. B, glucagon granule number per μm2 was similar in synaptotagmin-7 KO (grey bar) and control (white bar) mouse α-cells. n= 23 α-cells from 3 mice of each genotype. C, glucagon granule distribution was not different between synaptotagmin-7 KO (grey bar) and control (white bar) mouse α-cells. Numbers on the X-axis refer to distance (in nm) between glucagon granule membrane and plasma membrane. Refer to Methods for details. n= 10 α-cells from 3 synaptotagmin-7 KO, and 9 α-cells from 4 control mice.
Figure 5
Figure 5. Normal electrical properties and Ca2+ channel activity in synaptotagmin-7 KO α-cells
A, spontaneous action potentials were recorded in synaptotagmin-7 KO and control α-cells in the presence of 1 mm glucose. B, action potentials from panel A were displayed in expanded horizontal scales to depict spontaneous membrane depolarizations between action potentials. KO α-cells (Syt7−/−, grey bar) exhibited normal resting membrane potential (C), action potential frequency (D) and action potential amplitude (E) compared to control (Ctrl, white bar). F, Ca2+ currents were recorded in synaptotagmin-7 KO and control α-cells in the presence of 0.1 μg ml−1 TTX, 20 mm TEA, 4 mm 4-AP and equimolar substitution of Ba2+ for Ca2+ (2.6 mm). The currents were evoked by depolarizations from −70 mV to −8 mV. Ca2+ currents were indistinguishable between synaptotagmin-7 KO (Syt7−/−) and control (Ctrl) α-cells in the absence of glucose. G, current–voltage relationship of peak Ca2+ current amplitude against membrane depolarisations from −70 mV to +40 mV showed no difference between synaptotagmin-7 KO (filled circle) and control (open circle) α-cells. Data are presented as means ±s.e.m., n= 5 for each group.
Figure 6
Figure 6. Ca2+ dependence of residual glucagon secretion in synaptotagmin-7 KO α-cells
A, membrane capacitance was recorded in functionally identified α-cells in isolated synaptotagmin-7 KO and control islets. Capacitance increase elicited by 500 ms depolarizations from −70 to 0 mV in synaptotagmin-7 KO α-cells (red) was lower than in control α-cells (black). The N-type Ca2+ channel blocker ω-conotoxin (1 μm) inhibited membrane capacitance jump, and depolarization-induced capacitance change was not different in synaptotagmin-7 KO (pink) and control (grey) α-cells in the presence of the blocker. B, recordings from similar experiments as represented in A are summarized and presented as means ±s.e.m.n= 12 and 10 for KO (Syt7−/−) without and with ω-conotoxin treatment; and n= 9 and 10 for control (Ctrl) without and with ω-conotoxin treatment, respectively. Statistics (P values) are indicated in the graph. There was no difference in capacitance change between synaptotagmin-7 KO and control in the presence of 1 μmω-conotoxin (hatched bars).
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