Protein Cell 2012, 3(8): 618–626
618 © Higher Education Press and Springer-Verlag Berlin Heidelberg 2012
Protein & Cell
Bulk-like endocytosis plays an important role
in the recycling of insulin granules in
pancreatic beta cells
Du Wen1,4, Yanhong Xue1,4, Kuo Liang3, Tianyi Yuan2, Jingze Lu1, Wei Zhao1,4, Tao Xu1 ?, Liangyi Chen2 ?
1 National Key Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China
2 Lab of Cell Secretion and Metabolism, Institute of Molecular Medicine, Peking University and National Center for Nanoscience
and Technology, Beijing, 100871, China
3 Department of General Surgery, Xuanwu Hospital, Capital Medical University, Beijing 100053, China
4 Graduate School of the Chinese Academy of Sciences, Beijing 100864, China
? Correspondence: email@example.com (L. Chen); firstname.lastname@example.org (T. Xu)
Received May 6, 2012 Accepted May 10, 2012
Although bulk endocytosis has been found in a number of
neuronal and endocrine cells, the molecular mechanism
and physiological function of bulk endocytosis remain
elusive. In pancreatic beta cells, we have observed
bulk-like endocytosis evoked both by flash photolysis and
trains of depolarization. Bulk-like endocytosis is a
clathrin-independent process that is facilitated by en-
hanced extracellular Ca2+ entry and suppressed by the
inhibition of dynamin function. Moreover, defects in bulk-
like endocytosis are accompanied by hyperinsulinemia in
primary beta cells dissociated from diabetic KKAy mice,
which suggests that bulk-like endocytosis plays an im-
portant role in maintaining the exo-endocytosis balance
and beta cell secretory capability.
KEYWORDS bulk-like endocytosis, clathrin-independent
endocytosis, dynamin, diabetic KKAy mice
After secretory vesicles fuse with the plasma membrane,
membrane retrieval must occur to maintain a constant cell
size and to facilitate the reuse of vesicular membrane com-
ponents. In the past three decades, at least three types of
vesicle recycling processes have been found in excitable
cells, such as neuronal and endocrine cells. These processes
include clathrin-dependent endocytosis and two types of
clathrin-independent endocytosis: kiss and run endocytosis
and bulk endocytosis (Royle and Lagnado, 2003). Among
these clathrin-independent types of membrane retrieval, bulk
endocytosis is a relatively new concept, which was first pro-
posed by Takei et al. in 1996 (Clayton and Cousin, 2009).
Using electron microscopy to observe nerve terminals, Takei
et al. observed stimulation-evoked internalization of larger
areas of plasma membrane as endosomes from which multi-
ple vesicles could subsequently bud off. Unlike clathrin-me-
diated endocytosis, bulk retrieval is often triggered by
high-frequency membrane depolarization (Clayton et al.,
2008), and its formation does not directly involve clathrin
(Royle and Lagnado, 2003). Bulk endocytosis has been re-
ported in various neuronal systems, including hippocampal
neurons (Takei et al., 1996), retinal bipolar cells (Paillart et al.,
2003), neuromuscular junctions (Teng et al., 1999; Richards
et al., 2000), and the calyx of Held (Wu and Wu, 2007). De-
spite the widespread existence of bulk endocytosis cell types,
the functional significance of bulk endocytosis in specific
physiological processes has not been demonstrated.
In pancreatic beta cells, stimulated insulin secretion has been
found to be coupled with enhanced vesicle recycling processes
(Orci et al., 1973). Recently, we have shown that elevated [Ca2+]i
triggers both clathrin-dependent slow endocytosis and clathrin-
independent fast endocytosis in pancreatic beta cells (He et al.,
2008). However, whether clathrin-independent endocytosis
plays a role in the recycling of insulin vesicles under physiologi-
cally relevant stimulation remains to be determined.
In the present study, we show that both flash photolysis
and depolarization pulses evoke bulk-like endocytosis in
primary pancreatic beta cells. The frequency but not the am-
plitude of the bulk-like retrieval events is increased by an
Bulk-like endocytosis in pancreatic beta cells
Protein & Cell
© Higher Education Press and Springer-Verlag Berlin Heidelberg 2012 619
increase in the intracellular Ca2+ concentration and inhibited
by dynasore (dynamin blocker). In addition, we found signifi-
cant inhibition of depolarization-induced bulk-like endocytosis
in hyperinsulinemic beta cells isolated from diabetic KKAy
mice compared with age-matched C57 controls, which sug-
gested a role of bulk-like endocytosis in maintaining beta cell
function during the development of diabetes.
Flash photolysis and depolarization can elicit clathrin-
independent bulk-like endocytosis
Similar to a previous report from our group (He et al., 2008),
homogenously elevating [Ca2+]i in primary beta cells via con-
trolled UV photolysis ("uncaging") of 1-(4,5-dimethoxy-2-
(DMNP-EDTA) evoked excessive membrane retrieval, which
contains a fast and a slow component (Fig. 1A). By magnify-
ing the capacitance trace in the time scale, step-like de-
creases in capacitance were frequently observed in the rapid
declining phase (Fig. 1A). These downward capacitance
shifts were not accompanied by the alteration of membrane
conductance (Gm, Fig. 1A) and may occur concomitantly with
a transient decrease in series conductance (Gs, Fig. 1A). The
electrophysiological characteristics shown in Fig. 1 have
been defined as bulk-like endocytosis (Wu and Wu, 2007).
Because similar responses were also obtained in insu-
lin-secreting INS-1 cells (Fig. 1B), we performed molecular
manipulations in INS-1 cells. An average step-like capaci-
tance decline of 19 ± 0.7 fF was recorded in cells with levels
of [Ca2+]i elevated to approximately 20 μmol/L, which corre-
sponded to the scission of a vacuole of approximately 780 nm
in diameter (assuming 1 fF/μm2 as the specific membrane
capacitance). When we knocked down clathrin in INS-1 cells
(He et al., 2008), the capacitance drops persisted with similar
amplitude (19 ± 1 fF) and frequency (clathrin shRNA, 0.54 ±
0.11 event/s, n = 15; control, 0.51 ± 0.04 event/s, n = 40),
which confirmed their association with clathrin-independent
endocytosis (Fig. 1D). This clathrin-independent endocytosis
is obviously different from the “cavicapture” model, in which
similar increases and decreases in capacitance are observed
during large fusion pore flickering (MacDonald et al., 2006).
We also tested whether bulk-like endocytosis occurred un-
der physiological-relevant stimulation. To record bulk-like en-
docytosis, we used trains of 200 ms depolarization (1 Hz) from
–70 mV to 0 mV to repetitively depolarize primary pancreatic
beta cells (using an interval of 10 s after each sweep), thereby
simulating the membrane potential oscillation of the beta cells
during blood glucose elevation. Indeed, bulk-like endocytosis
was observed in 39.3% of the beta cells (12/28 cells) under the
standard whole-cell configuration (an example is shown in Fig.
2). The time required for bulk-like endocytosis to occur was
variable, and bulk-like retrieval events can be observed during
both the depolarization sweep (synchronized) and the interval
between different sweeps (asynchronized). We also directly
tested whether physiological stimulation induced bulk-like en-
docytosis in vivo using electron microscopy. Occasionally,
large invaginations connected to the plasma membrane could
be observed after intact islets were stimulated with KCl (Fig.
2D). More frequently, fused chain of insulin granules were
observed in the primary beta cells (Fig. 2A, arrow), similar to
previous reports (Kwan and Gaisano, 2005; Hoppa et al.,
2012). They collapse into the plasma membrane and release
insulin in a multiple-granular manner (Fig. 2C) and can be
retrieved back as a whole unit (Fig. 2B, arrowhead), which may
suggest a type of bulk-like membrane retrieval.
Depolarization-induced bulk-like endocytosis is Ca2+ and
Wu et al. demonstrated that Ca2+ initiates bulk-like endocytosis
during depolarization at calyx nerve terminals (Wu et al., 2009).
Similarly, we attempted to determine whether the bulk- like en-
docytosis in primary beta cells depends on the amount of cal-
cium that enters the cells. A depolarization train evokes an ap-
proximately 20% greater capacitance in a 10 mmol/L Ca2+ bath
solution compared with a 2.6 mmol/L Ca2+ bath solution (Fig. 3A
and 3B). In contrast, depolarization in a 10 mmol/L Ca2+ bath
solution evoked an approximately 2.5-fold increase in the fre-
quency but not the average amplitude of bulk-like endocytosis
(the mean frequency was 0.010 ± 0.003 Hz in 2.6 mmol/L Ca2+
compared with 0.025 ± 0.005 Hz in 10 mmol/L Ca2+, P < 0.05)
(Fig. 3C and 3D). The increased number of bulk-like events was
primarily due to an increase in the bulk retrieval during the inter-
vals between the depolarization trains (P = 0.08, Fig. 3E), which
suggested that bulk-like endocytosis is a delayed mechanism
that is triggered by prolonged Ca2+ elevation.
Dynamin is a large GTPase that is principally involved in
the scission of newly formed vesicles from the membrane
(Cao et al., 1998). Dephosphorylation of dynamin I has been
demonstrated to participate in activity dependent bulk endo-
cytois in neurons. To verify the role of dynamin in bulk-like
endocytosis in pancreatic beta cells, we preincubated beta
cells in RPMI 1640 with 80 μmol/L dynasore for 30 min, which
potently inhibits dynamin function (Macia et al., 2006; Newton
et al., 2006). Thereafter, cells were transferred to a recording
chamber containing 10 mmol/L Ca2+ and 80 μmol/L dynasore
(to prevent a washout effect). In contrast to previous reports
(Tsai et al., 2009; Wu et al., 2009), short-term pretreatment
with dynasore did not inhibit exocytosis. Instead, depolariza-
tion evoked an approximately 2-fold increase in capacitance
in the 10 mmol/L Ca2+ solution (Fig. 3A and 3B) and signifi-
cantly reduced the frequency of the delayed bulk-like event
(Fig. 3D and 3E). Given that knocking-down of clathrin did not
affect bulk-like endocytosis (Fig. 1), dynamin may play a
clathrin -independent facilitatory role in stimulation -activated
bulk-like endocytosis in primary beta cells.
Protein & Cell
Du Wen et al.
620 © Higher Education Press and Springer-Verlag Berlin Heidelberg 2012
Figure 1. Flash photolysis can elicit clathrin-independent bulk-like endocytosis. (A and B) An example of UV
photolysis-induced fast decay in the capacitance of primary beta cells and INS-1 cells. The trace enclosed in the red box is enlarged
in the inset, which shows a single step-like decrease in capacitance. (C) The size histogram of the step-like capacitance declines in
INS-1 cells after a homogenous increase in [Ca2+]i to aproximately 20 μmol/L. (D) A summary of the average step-like capacitance
decreases in control INS-1 cells ([Ca2+]i approximately 20 μmol/L, n = 203) and in INS-1 cells where clathrin expression was
knocked down ([Ca2+]i approximately 20 μmol/L, n = 81).
Impairment of the bulk-like endocytic mechanism in beta
cells from diabetic KKAy mice
Insulin secretion from pancreatic beta cells plays a crucial
role in the regulation of blood glucose homeostasis. There-
fore, it is well accepted that type II diabetes involves a per-
turbation of the exocytotic machinery of pancreatic beta cells
(Eliasson et al., 2008). However, whether changes in the
Bulk-like endocytosis in pancreatic beta cells
Protein & Cell
© Higher Education Press and Springer-Verlag Berlin Heidelberg 2012 621
Figure 2. Depolarization elicit bulk-like endocytosis in pancreatic β cells. (A) Trains of ten 200-ms pulses (frequency 1 Hz)
induced a capacitance jump (Cm), except for abrupt bulk endocytosis (arrows). The measured values of Gm and Gs are shown
below the figure. (B) After 5 min of 20 mmol/L glucose stimulation, two insulin glanules are contacted and fused with each other
(indicated by arrow) and three fused glanules are retrieval together after the release of the contents (indicated by arrow head). (C)
Secretory granules fuse together (also partially emptied) after 70 mmol/L KCl stimulation for 5 min. (D) Large invagination
coonected to the plasma membrane after 70 mmol/L KCl stimulation. Scale bar: 500nm in all EM figures.
membrane recycling process participate in such pathological
processes has not been investigated. We used yellow-coated
KKAy mice that exhibit spontaneous hyperglycemia and glu-
cose intolerance at 8 weeks of age (Srinivasan and Ramarao,
2007). Consistent with a previous report (Srinivasan and
Ramarao, 2007), KKAy mice had greater body weight than
age-mated C57Bl/6 controls (Fig. 4A) and exhibited impaired
glucose tolerance and pronounced insulin resistance at 10
weeks (Fig, 4B and 4C). Compared with the control group,
depolarization evoked a greater than two-fold increase in
capacitance in KKAy beta cells in both 2.6 mmol/L and
10 mmol/L Ca2+ bath solutions (Fig. 4D and 4E), which is
Protein & Cell
Du Wen et al.
622 © Higher Education Press and Springer-Verlag Berlin Heidelberg 2012
Figure 3. Depolarization-induced bulk-like endocytosis is Ca2+ and dynamin dependent. (A) The average Cm traces
induced by ten 200-ms pulses (frequency 1 Hz) in 2.6 mmol/L Ca2+ (black), 10 mmol/L Ca2+ (red) or 10 mmol/L Ca2+ plus 80 μmol/L
dynasore (blue) (2.6 mmol/L Ca2+, n = 28; 10 mmol/L Ca2+, n = 31; 10 mmol/L Ca2+ plus 80 μmol/L dynasore, n = 17). (B) The
average capacitance jump (ΔCm) in the first sweep. (C and D) The average bulk amplitude and frequency under the three
conditions (*P < 0.05, ** P < 0.01). (E) The average bulk frequency during the depolarization time and the resting time (*P < 0.05).
consistent with the beta cell hyperinsulinemia reported at an
earlier stage in diabetic animals or patients (Homo-Delarche,
1997; Liang et al., 2011). Although the mean amplitude of
bulk-like endocytosis was not changed, markedly fewer
bulk-like retrieval events were observed in the diabetic beta
cells stimulated in either 2.6 mmol/L or 10 mmol/L Ca2+ bath
solutions (Fig. 4F and 4G). Taken together, these data sug-
gest that bulk-like endocytosis is severely impaired in pan-
creatic beta cells isolated from diabetic KKAy mice.
Eliasson et al. was the first author to report the induction of
endocytosis by transient elevation of cytosolic calcium in
mouse pancreatic beta cells, including large amplitude ca-
pacitance fluctuations that resemble bulk endocytosis
(Eliasson et al., 1996). Our data corroborate their findings by
demonstrating bulk-like endocytosis evoked by both flash
photolysis and whole-cell depolarization in primary beta cells.
Moreover, we show that bulk-like endocytosis is not altered
when endogenous clathrin is knocked down with shRNA,
which has been demonstrated to inhibit both transferrin in-
ternalization and clathrin-mediated slow endocytosis in a
previous study by our group (He et al., 2008). Although
clathrin-coated pits have been shown to bud from large in-
foldings on the plasma membrane (Lenzi et al., 2002), we
believe that bulk-like endocytosis in beta cells is a clathrin-
independent process. Interestingly, studies have proposed
that calcium activates bulk-like endocytosis in various types
of neurons (Clayton et al., 2009; Wu et al., 2009). Qualita-
tively, our data fit with the hypothesis that bulk-like endocyto-
sis is a Ca2+-dependent process because enhanced Ca2+
influx doubled the frequency of bulk endocytosis, whereas only
30% increase in exocytosis was observed. However, high
levels of Ca2+ in the bath solution did not enhance the mean
amplitude of bulk-like endocytic events and failed to affect the
Bulk-like endocytosis in pancreatic beta cells
Protein & Cell
© Higher Education Press and Springer-Verlag Berlin Heidelberg 2012 623
Figure 4. Impairment of the bulk-like endocytic mechanism in beta cells from diabetic KKAy mice. Comparison of (A) body
weight, (B) blood glucose during GTT (i.p. administration of 2 g/kg body weight glucose), and (C) blood glucose during ITT (i.p.
administration of 0.5 U/kg body weight insulin) between C57Bl/6 and KKAy mice. (D) The average Cm traces induced by ten
200-ms pulses (frequency 1 Hz) in C57 and KKAy beta cells (blue, 2.6 mmol/L Ca2+, C57, n = 28; green, 2.6 mmol/L Ca2+, KKAy,
n = 25; red, 10 mmol/L Ca2+, C57, n = 31; black, 10 mmol/L Ca2+, KKAy, n = 15). Beta cells from C57 and KKAy mice are of similar
size in resting condition (C57 beta cells, Cslow = 5.0 ± 0.1 pF; KKAy Cslow = 4.9 ± 0.3 pF). (E) The average capacitance jump (ΔCm) in
the first sweep. (F and G) The average bulk amplitude and frequency under 2.6 mmol/L Ca and 10 mmol/L Ca conditions (*P < 0.05,
** P < 0.01).
Protein & Cell
Du Wen et al.
624 © Higher Education Press and Springer-Verlag Berlin Heidelberg 2012
frequency of bulk retrieval during the depolarization (Fig. 3A
and 3C). This suggests that the stimulatory effect of Ca2+ on
bulk-like endocytosis is facilitatory rather than obligatory.
Although inhibition of dynamin function with dynasore in
primary beta cells failed to inhibit exocytosis, it abolished the
stimulatory effects of the high bath concentration of Ca2+ on
bulk-like endocytosis, which is consistent with the function of
Ca2+ and dynamin in the same pathway in bulk-like endocyto-
sis. Bulk-like endocytosis is activated by strong stimulation in
neurons (Clayton et al., 2008). In parallel, elevated Ca2+ acti-
vates the calcium/calmodulin -dependent protein phosphatase
calcineurin, which leads to the dephosphorylation of dynamin I
and its interaction with the F-BAR protein, syndapin I (Anggono
et al., 2006; Clayton et al., 2009). Therefore, our data appear to
agree with the specific role of dynamin I rephosphorylation at
SER-774 in bulk-like endocytosis in neurons (Clayton et al.,
2010). Interestingly, bulk-like endocytosis persisted during
depolarization in cells pretreated with dynasore, which argues
against the mandatory role of dynamin in bulk-like endocytosis.
Similarly, many more endosome-like structures have been
observed in dynamin I KO synapses after acute strong stimu-
lation compared with control synapses (Hayashi et al., 2008),
which indicates the existence of a bulk retrieval mechanism
that is independent of dynamin I. Therefore, we postulate that
neither Ca2+ nor dynamin is required for the initiation of
clathrin-independent bulk-like endocytosis; however, they fa-
cilitate bulk -like endocytosis in an activity-dependent manner.
In addition, extremely high increases in the intracellular calcium
concentration evoked by flash photolysis may temporally coor-
dinate different bulk-like endocytic events that contribute to the
rapid clathrin-independent phase of endocytosis that has been
observed in primary beta cells and corticotrophs (Eliasson et
al., 1996; Lee and Tse, 2001; He et al., 2008).
Although bulk-like endocytosis has been found in a num-
ber of neurons and endocrine cells, its mechanism and
physiological significance remain elusive. Bulk-like endocyto-
sis has been proposed to be a retrieval mechanism evoked
by conditions under which classical endocytosis cannot
match pace with exocytosis (e.g., dynamin I knockout or
(Hayashi et al., 2008; Heerssen et al., 2008). Moreover, the
intermediate organelle generated by bulk-like endocytosis is
proposed to be incapable of efficiently generating fu-
sion-competent synaptic vesicles (Hayashi et al., 2008;
Heerssen et al., 2008). However, compound exocytosis of
prefused insulin granules was reported in rat pancreatic beta
cells in 2005 (Kwan and Gaisano, 2005). Recently, com-
pound exocytosis has been further demonstrated to play a
important role under conditions associated with the global
elevation of cytosolic Ca2+ (Hoppa et al., 2012), which is the
condition that augments the bulk-like endocytosis found in the
present study. Our electron microscope data also show ex-
amples of compound fusion of several granules to the plasma
membrane (Fig. 2C) and retrieval of three inter-connected
nearly empty vacuoles (Fig. 2B, arrowhead). Therefore,
bulk-like endocytosis may be a result of compound or se-
quential exocytosis evoked by physiological stimulation in
pancreatic beta cells. Most importantly, we demonstrate the
selected reduction of bulk-like endocytosis in beta cells iso-
lated from mice at earlier stage of diabetes. This contrasts
with the enhanced insulin secretion from the beta cells com-
pared with the control cells. Because bulk-like endocytosis
may play an important role in the recycling of insulin granules,
the combination of defective recycling and enhanced secre-
tion will lead to the run-down and down-regulation of beta cell
secretory ability as diabetes progresses, which has been
observed in human diabetic patients (Ostenson et al., 2006).
Therefore, elucidating the molecular mechanism of bulk-like
endocytosis and discovering reagents that upregulate the
endocytic pathway may prolong the preservation of primary
beta cell function and postpone the onset of type II diabetes.
MATERIALS AND METHODS
Islets and single beta cell preparation
C57BL/6 male mice were purchased from the Vital River Experi-
mental Animal Company, Beijing, China, and age-mated KKAy mice
were purchased from the Institute of Laboratory Animal Science at
the Chinese Academy of Medical Sciences, Beijing, China. All of the
mice were maintained under specific pathogen-free conditions. The
handling of the mice and the experimental procedures were con-
ducted in accordance with the Committee of the Use of Live Animals
in Teaching and Research at the Institute of Biophysics at the Chi-
nese Academy of Sciences. The mice were sacrificed by cervical
dislocation, and primary islets and beta cells were isolated as previ-
ously described. The isolated cells were plated on coverslips precoated
with poly-l-lysine and maintained in a 37°C, 5% CO2 incubator for
24–48 h in modified RPMI 1640 medium. We selected cells with di-
ameters >11 μmol/L with the expectation that more than 80%–90% of
the cells would be beta cells (Rorsman and Trube, 1986) more than.
Patch capacitance measurements
Electrophysiological experiments were conducted with standard
whole-cell recordings using an EPC-10 patch-clamp amplifier (HEKA).
The bath solution contained (mmol/L) 138 NaCl, 5.6 KCl, 1.2 MgCl2,
2.6 CaCl2, 5 glucose, and 10 HEPES (pH 7.4, adjusted with NaOH).
For the flash experiments, the intracellular solution contained
(mmol/L) 105 Cs- Glutamate, 2 MgATP, 0.3 GTP, 33 HEPES, 0.4
Mag-Fura-2, 0.4 Fura-4F, 4 CaCl2, and 5 NP-EGTA (pH 7.2, adjusted
with NaOH or HCl). For the depolarization experiments, the pipette
solution contained (mmol/L) 125 Cs-Glutamate, 2 Mg-ATP, 0.3
Na2-GTP, 1 MgCl2, and 0.1 EGTA (pH 7.2, adjusted with CsOH). In
addition, 10 mmol/L TEA-Cl and 2 mmol/L forskolin were added to
the bath solution. The extracellular calcium concentration was 2.6
mmol/L unless otherwise indicated. Capacitance measurements
were performed using the ‘sine + DC’ mode of the software lock-in
extension of Pulse software. An 800-Hz, 20-mV peak-to-peak sinu-
Bulk-like endocytosis in pancreatic beta cells
Protein & Cell
© Higher Education Press and Springer-Verlag Berlin Heidelberg 2012 625
soidal voltage stimulus was applied at a DC holding potential of –70
mV. Ten trains of depolarization (from –70 mV to 0 mV for 200 ms)
were applied to single beta cells to evoke an increase in capacitance,
which was indicative of insulin secretion. Flash photolysis and in-
creases in the membrane capacitance were performed as previously
described (Duman et al., 2006).
Transmission electron microscopy
The mouse islets were grown in culture medium for one day before
being harvested. On the day of experiments, islets were stimulated
with either 70 mmol/L KCl or 20 mmol/L glucose for 5 minute before
fixed in 2.5% glutaraldehyde in PBS (pH 7.4) at room temperature for
30 min. The islets were then postfixed with 1% osmium tetroxide in
0.1 M sodium cacodylate for one hour. Following dehydration in a
graded acetone series, the samples were embedded in Embed 812
and sliced into 70-nm sections using a Leica ultramicrotome EM UC6
(Leica, Germany). After being contrasted with uranyl acetate and lead
citrate, the sections were observed under a Spirit transmission elec-
tron microscope (FEI, the Netherlands).
Intraperitoneal glucose tolerance and insulin
For the intraperitoneal glucose tolerance test (IGTT), the mice were
starved overnight and then given an i.p. glucose injection (2 g/kg
body weight). Venous blood was drawn from the tail vein, and the
blood glucose level was measured using a glucometer (ACCU-CHEK
Active, Roche). For the insulin tolerance test (ITT), the mice were
starved for 4–5 hours prior to an i.p. injection of 0.5 U/kg body weight
of human regular insulin (Sigma-Aldrich, St. Louis, MO, USA). Blood
glucose levels were measured using a glucometer.
All of the data were analyzed using Igor Pro software (Wavemetrics,
Lake Oswego, OR). The results are presented as the mean value ±
SEM. Statistical significance was evaluated using either Student’s t
test for single Gaussian distributed datasets or the Mann-Whitney
rank sum test for non-single Gaussian distributed datasets. The
symbols * and ** denote statistical significance with P values of less
than 0.05 and 0.01, respectively.
This work was supported by a grant from the National Natural Sci-
ence Foundation of China (Grant No. 30871225), a grant from Beijing
Municipal Science and Technology Commission (Grant No. 7121008),
a grant from the Ministry of Science and Technology (Grant No.
SQ2011SF11B01041) and the fund from The Key Construction Pro-
gram of the National “985” Project from the Department of Education
of China to Peking University.
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