Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2008 Mar 11;105(10):3992-7.
doi: 10.1073/pnas.0711700105. Epub 2008 Feb 28.

Impaired insulin secretion and glucose intolerance in synaptotagmin-7 null mutant mice

Natalia Gustavsson  1 , Ye Lao, Anton Maximov, Jen-Chieh Chuang, Elena Kostromina, Joyce J Repa, Cai Li, George K Radda, Thomas C Südhof, Weiping Han
Affiliations

Impaired insulin secretion and glucose intolerance in synaptotagmin-7 null mutant mice

Natalia Gustavsson et al. Proc Natl Acad Sci U S A. .

Abstract

Vertebrates express at least 15 different synaptotagmins with the same domain structure but diverse localizations and tissue distributions. Synaptotagmin-1,-2, and -9 act as calcium sensors for the fast phrase of neurotransmitter release, and synaptotagmin-12 acts as a calcium-independent modulator of release. The exact functions of the remaining 11 synaptotagmins, however, have not been established. By analogy to the role of synaptotagmin-1, -2, and -9 in neurotransmission, these other synaptotagmins may serve as Ca(2+) transducers regulating other Ca(2+)-dependent membrane processes, such as insulin secretion in pancreatic beta-cells. Of these other synaptotagmins, synaptotagmin-7 is one of the most abundant and is present in pancreatic beta-cells. To determine whether synaptotagmin-7 regulates Ca(2+)-dependent insulin secretion, we analyzed synaptotagmin-7 null mutant mice for glucose tolerance and insulin release. Here, we show that synaptotagmin-7 is required for the maintenance of systemic glucose tolerance and glucose-stimulated insulin secretion. Mutant mice have normal insulin sensitivity, insulin production, islet architecture and ultrastructural organization, and metabolic and calcium responses but exhibit impaired glucose-induced insulin secretion, indicating a calcium-sensing defect during insulin-containing secretory granule exocytosis. Taken together, our findings show that synaptotagmin-7 functions as a positive regulator of insulin secretion and may serve as a calcium sensor controlling insulin secretion in pancreatic beta cells.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Synaptotagmin-7 is present in mouse pancreatic β-cells. Twenty-micrometer pancreatic sections were first reacted with both a polyclonal rabbit antibody against synaptotagmin-7 (S757; Synaptic Systems) and a monoclonal guinea pig antibody against insulin, followed by fluorescence-conjugated secondary antibodies (Alexa Fluor 546 goat-anti-rabbit IgG and Alexa Fluor 488 Donkey-anti-Guinea Pig IgG). Representative images of such stained sections, taken on a Leica TCS2 confocal microscope, are shown. Synaptotagmin-7 (Syt7, red) was expressed in insulin-positive cells and shown to have high degree of overlap with insulin signals (green). Arrows indicate selected overlapping signals of insulin and synaptotagmin-7. For comparison, no apparent synaptotagmin-7 signal was detected in islet sections from synaptotagmin-7 mutant (Syt7−/−) mouse. (Scale bars: 40, 20, and 5 μm for Upper, Middle, and Bottom, respectively.)
Fig. 2.
Fig. 2.
Impaired glucose tolerance and insulin secretion, but normal insulin sensitivity in synaptotagmin-7 mutant mice. (A) An i.p. glucose tolerance test (IPGTT) was performed on overnight-fasted synaptotagmin-7 mutants and wild-type control mice. Blood glucose levels before and at 15, 30, 60, 90, and 120 min after glucose injection (2 mg per gram of body weight) were measured. Synaptotagmin-7 mutant (Syt7−/−) mice (filled square, n = 13) exhibited glucose intolerance as evidenced by higher glucose concentration after injection and delayed clearance of glucose. ∗, P < 0.02, ∗∗, P < 0.005 vs. control (filled circle, n = 17). (B) Plasma insulin levels in control and synaptotagmin-7 mutant mice before and at 8, 15, 30, and 60 min of IPGTT were determined. Synaptotagmin-7 mutant mice showed insulin-secretory deficiency, especially in the first 15 min, upon glucose challenge. n = 10 for control (filled circle) and 11 for mutant (filled square). ∗, P < 0.05, ∗∗, P < 0.005. (C) Blood glucose levels were measured in 2-h-fasted control and mutant mice before and at 15, 30, and 60 min after injection of 1 unit/kg insulin. Synaptotagmin-7 mutant mice appeared to have higher insulin sensitivity than their wild-type control. ∗, P < 0.03, n = 17.
Fig. 3.
Fig. 3.
Normal islet architecture, ultrastructural organizations, and insulin production in synaptotagmin-7 mutant mice. (A) Islet architecture and size were analyzed by using histological sections stained with hematoxylin and eosin (H&E). Two representative sections are shown for both mutant and control islets. Gross architecture and size of mutant mouse islets were not different from those of the control. (Scale bar, 50 μm.) (B) Ultrastructure of β-cells was analyzed by transmission EM. Two representative images from each genotype (mutant and control) are shown. Ultrastructural organizations, including distribution and number of insulin-containing secretory granules, were similar in control and mutant mouse β-cells. (Scale bars: 2 μm.) (C) Insulin contents were measured in isolated individual pancreatic islets from synaptotagmin-7 mutant (Syt7−/−) and control mice, using ELISA. Isolated islets were incubated at 3 mM glucose for 2 h before they were lysed by sonication. Data are presented as means ± SEM. n = 19 for control and 14 for mutant. (D) Insulin mRNA levels were analyzed by real time PCR from total RNA extracted from isolated islets. Insulin mRNA level was not altered based on two separate qPCR experiments from pooled islets of three to five mutant or control mice.
Fig. 4.
Fig. 4.
Glucose metabolism and Ca2+ response are unaffected in synaptotagmin-7 mutant mouse islets. (A) Representative traces of NADH autofluorescence from synaptotagmin-7 mutant (Syt7−/−, solid line) and control (dotted line) mouse islets perifused with 20 mM glucose (n = 14 for each group). Both synaptotagmin-7 mutant and control displayed similar time course and extent of autofluorescence change. Rise over basal level (%) and rate of rise are presented in Results. (B) Representative Ca2+ responses to 20 mM glucose from a control (dotted line) and a mutant (solid line) mouse islet (n = 20 control, n = 21 Syt7−/−). Cytosolic [Ca2+]i was measured by using Ca2+ indicator Fluo-4. Glucose-induced Ca2+ changes were similar in control and mutant mouse with regard to lag time, rise, initial lowering and oscillations. Refer to Results for mean values of lag time for [Ca2+]i rise, rise over basal (%), initial lowering nadir, and oscillation rate. Data are presented as fluorescence intensity in arbitrary units.
Fig. 5.
Fig. 5.
Stimulated insulin secretion is reduced in isolated synaptotagmin-7 mutant mouse islets. (A) Glucose-induced insulin secretion from isolated islets was measured in perifusion experiments at a glucose concentration of 3 mM (basal) or 20 mM (stimulatory). The perfusate was collected in 3-min intervals, and insulin levels were determined by using ELISA. Synaptotagmin-7 mutant islets (Syt7−/−, filled square) displayed impaired insulin secretion when compared with control (open square). (B) Glucose-induced insulin secretion for the entire stimulation period (Total) or the first phase (during the first 15 min after stimulation) in the perifusion experiments was lower in isolated islets from mutant (gray bar) than from control (white bar). Insulin secretion was calculated by integrating the area under each curve in A after baseline subtraction. Data are presented as mean ± SEM. n = 9 for mutant and 10 for control. ∗, P < 0.05.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Porte D., Jr Banting lecture 1990. Beta-cells in type II diabetes mellitus. Diabetes. 1991;40:166–180. - PubMed
    1. Ashcroft FM, Rorsman P. Molecular defects in insulin secretion in type-2 diabetes. Rev Endocr Metab Disord. 2004;5:135–142. - PubMed
    1. Henquin J.-C., et al. Signals and Pools Underlying Biphasic Insulin Secretion. Diabetes. 2002;51:S60–S67. - PubMed
    1. Rorsman P, Renstrom E. Insulin granule dynamics in pancreatic beta cells. Diabetologia. 2003;46:1029–1045. - PubMed
    1. Kahn SE, et al. Importance of early phase insulin secretion to intravenous glucose tolerance in subjects with type 2 diabetes mellitus. J Clin Endocrinol Metab. 2001;86:5824–5829. - PubMed

Publication types

MeSH terms