Insulin Resistance in Tetracycline-Repressible Munc18c
Transgenic Mice
Beth A. Spurlin,1 Rhonda M. Thomas,1 Angela K. Nevins,1 Hyo-Jeong Kim,2 Yoon-Jung Kim,2
Hye-Lim Noh,2 Gerald I. Shulman,2,3 Jason K. Kim,2 and Debbie C. Thurmond1
To investigate the physiological effects of modulating
the abundance of Munc18c or syntaxin 4 (Syn4) proteins on the regulation of glucose homeostasis in vivo,
we generated tetracycline-repressible transgenic mice
that overexpress either Munc18c or Syn4 proteins in
skeletal muscle, pancreas and adipose tissue seven-,
five-, and threefold over endogenous protein, respectively. Munc18c transgenic mice displayed whole-body
insulin resistance during hyperinsulinemic-euglycemic
clamp resulting from >41% reductions in skeletal muscle and white adipose tissue glucose uptake, but without
alteration of hepatic insulin action. Munc18c transgenic
mice exhibited ⬃40% decreases in whole-body glycogen/
lipid synthesis, skeletal muscle glycogen synthesis, and
glycolysis. Glucose intolerance in Munc18c transgenic
mice was reversed by repression of transgene expression using tetracycline or by simultaneous overexpression of Syn4 protein. In addition, Munc18c transgenic
mice had depressed serum insulin levels, reflecting a
threefold reduction in insulin secretion from islets isolated therefrom, thus uncovering roles for Munc18c
and/or Syn4 in insulin granule exocytosis. Taken together, these results indicate that balance, more than
absolute abundance, of Munc18c and Syn4 proteins
directly affects whole-body glucose homeostasis through
alterations in insulin secretion and insulin action.
Diabetes 52:1910 –1917, 2003
I
nsulin resistance, in large part, results from an
inability to recruit adequate quantities of GLUT4
protein to the cell surface. Skeletal muscle and
adipose tissue clearance of circulating blood glucose
accounts for the majority of insulin-stimulated glucose
uptake, as these tissues express the insulin-responsive
glucose transporter GLUT4 (1). In the basal non–insulinFrom the 1Department of Biochemistry and Molecular Biology, Center for
Diabetes Research, Indiana University School of Medicine, Indianapolis,
Indiana; the 2Department of Internal Medicine, Yale University School of
Medicine, New Haven, Connecticut; and the 3Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut.
Address correspondence and reprint requests to Debbie C. Thurmond,
Department of Biochemistry and Molecular Biology, Center for Diabetes
Research, Indiana University School of Medicine, Indianapolis, IN 46202.
E-mail: [email protected].
Received for publication 13 December 2002 and accepted in revised form 21
April 2003.
HRP, horseradish peroxidase; IPGTT, intraperitoneal glucose tolerance test;
IR, insulin receptor; IRAP, insulin-responsive aminopeptidase; IRS, insulin
receptor substrate; NEFA, nonesterified fatty acid; PI 3-kinase, phosphatidylinositol 3-kinase; PVDF, polyvinylidene difluoride; SNARE, SNAP [soluble
NSF (N-ethylmaleimide sensitive factor) attachment protein] receptor; Syn4,
syntaxin 4; Tg, transgenic.
© 2003 by the American Diabetes Association.
1910
stimulated state, GLUT4 localizes to tubulovesicular elements and small intracellular vesicles throughout the cell
cytoplasm (2). Upon stimulation with insulin, these GLUT4containing compartments translocate to and fuse with the
plasma membrane (3–7). This ultimately results in a large
increase in the number of functional glucose transporters
on the cell surface, which accounts for nearly all of the
insulin-stimulated increase in glucose uptake.
The insulin-stimulated translocation of GLUT4-containing vesicles is a complex multistep process necessary for
normal maintenance of glucose homeostasis (4,6,7). Insulin binding to its receptor activates the intrinsic protein
kinase of the receptor ␤ subunit, resulting in its autophosphorylation and tyrosine phosphorylation of the family of
insulin receptor substrate (IRS) proteins (8) and Cbl (9).
The phosphorylation of IRS results in the association,
activation, and targeting of the phosphatidylinositol 3-kinase (PI 3-kinase) (10 –12). The active PI 3-kinase generates phosphatidylinositol-3,4,5-trisphosphate, which is
necessary for the stimulation of both protein kinase B and
atypical protein kinase C isoforms (13,14). This insulin
signaling cascade leads to vectorial movement, or “trafficking,” of the GLUT4 vesicle toward the plasma membrane. Once near the inner leaflet of the plasma membrane,
GLUT4 vesicles associate with t-SNARE (SNAP [soluble
NSF (N-ethylmaleimide sensitive factor) attachment protein] receptor) proteins syntaxin 4 (Syn4) and SNAP-23
present in the plasma membrane (15–20) via the GLUT4
vesicle cargo v-SNARE protein VAMP2 (17,21–24). VAMP2,
Syn4, and SNAP-23 proteins form the heterotrimeric
SNARE core complex essential for the eventual incorporation of GLUT4 into the plasma membrane to facilitate
glucose uptake.
SNARE core complex interactions in Saccharomyces
cerevisiae, Caenorhabditis elegans, and Drosophila melanogaster are regulated by the Sec1-type proteins, through
specific and high-affinity binding to their cognate syntaxins
(25–27). Three Sec1 homologs have been identified in
mammalian plasma membranes: Munc18a, Munc18b, and
Munc18c. Munc18a is predominantly expressed in neurons, whereas Munc18b and Munc18c are expressed ubiquitously, and only Munc18c binds Syn4 with high affinity
(28 –32). Null mutations in the genes for the SM proteins
(Sec1/Munc18) cause dramatic reductions in vesicle exocytosis, suggesting that these proteins are essential for
normal SNARE function (27,33,34). Similar to other SM
proteins, disruption of Munc18c binding to Syn4 impairs
vesicle fusion, indicating that it is required for the insulinstimulated GLUT4 translocation process (35,36).
DIABETES, VOL. 52, AUGUST 2003
B.A. SPURLIN AND ASSOCIATES
Overexpression of Munc18c results in inhibition of
insulin-stimulated vesicle fusion in vitro, but its effect on
glucose uptake in vivo is unknown (29,37–39). Munc18c
binds to Syn4 in a manner mutually exclusive of either
Syn4 binding proteins (SNAP-23 and VAMP2) in adipocytes, and competes for Syn4 when overexpressed (36).
Overexpression of Munc18c in 3T3L1 adipocytes inhibits
insulin-stimulated GLUT4 translocation (38,39). This inhibitory effect was fully reversed by increased expression of
Syn4, indicating a functional requirement for excess Syn4
over Munc18c protein (36). Consistent with this, increased
expression in skeletal muscle of GLUT4-EGFP mice by
adenoviral-Munc18c resulted in inhibition of insulin-stimulated GLUT4 translocation, specifically in the transverse
tubules and not in the sarcolemma (40). Similarly, sarcolemmal membranes contained much higher levels of
Syn4 protein than transverse tubules, suggesting that
overexpression of Munc18c was sufficient to saturate the
available endogenous Syn4 binding sites in transverse
tubules yet not in the sarcolemmal membrane (40). Evidence favoring this model stems from a growing number
of cellular systems reporting an unequal stoichiometric
abundance of Munc18 protein relative to syntaxin protein
(36,41,42).
The physiological relevance of Syn4 and Munc18c protein abundance with respect to insulin-stimulated GLUT4
translocation and glucose homeostasis has not been investigated in vivo. Therefore, we generated transgenic mice
that overexpress Munc18c and/or Syn4 proteins under the
control of a tetracycline-repressible promoter in peripheral insulin-sensitive tissues (skeletal muscle and fat) and
pancreas. In this report, we demonstrate that Munc18c but
not Syn4 protein overexpression alone results in insulin
resistance and impaired insulin secretion. Hyperinsulinemic-euglycemic clamp analyses showed defective peripheral glucose disposal, consistent with a defect in skeletal
muscle insulin-stimulated GLUT4 translocation. Oral administration of tetracycline resulted in normalization of
glucose intolerance and serum insulin levels coordinate
with the downregulation of transgene expression, indicating that the deleterious results of Munc18c overexpression
are reversible by restoration of endogenous Munc18c
protein levels. Furthermore, glucose intolerance is also
normalized by coordinate overexpression of Syn4 with
Munc18c, suggesting that Munc18c protein must be balanced by an overabundance of Syn4 to maintain normoglycemia in vivo.
RESEARCH DESIGN AND METHODS
Materials. The rabbit polyclonal GLUT4, Syn4, VAMP2, and Munc18c antibodies were obtained as described (17,39). The SNAP-23, GLUT1, transferrin
receptor, phosphotyrosine-horseradish peroxidase (PY20-HRP), and phosphoserine-specific Akt antibodies were purchased from Affinity Bioreagents
(Golden, CO), Chemicon (Temecula, CA), Zymed Laboratories (South San
Francisco, CA), Transduction Laboratories (Lexington, KY), and New England
Biolabs (Beverley, MA), respectively. Goat anti-rabbit HRP and anti– digoxigenin HRP-conjugated secondary antibodies were purchased from Bio-Rad
(Hercules, CA) and Roche (Indianapolis, IN), respectively. The GST-Syn4
⌬TM, GST-SNAP-23, and GST-Munc18cCT plasmids were gifts of Dr. Jeffrey
Pessin (SUNY Stony Brook, NY). The rat insulin radioimmunoassay kit was
acquired from Linco Research (St. Charles, MO).
Generation of pCOMBICMV-Munc18c transgenic mice. All studies involving mice followed the Guidelines for the Use and Care of Laboratory Animals.
The pUC-COMBICMV plasmid for generation of tetracycline-repressible transgenic (Tg) mice was a gift from Dr. Ulli Certa (Hoffman la Roche, Switzerland)
DIABETES, VOL. 52, AUGUST 2003
(43). Flag-tagged Munc18c (39) was inserted into the PmeI site pCombi-CMV
vector. The construct was linearized by digestion with NotI and microinjected
into the nucleus of preimplantation embryos and transferred into the oviduct
of pseudo-pregnant C57Bl6J female mice by staff at the the University of Iowa
Transgenic Animal Facility (Iowa City, IA). Pups were screened for the
presence of the transgene using PCR of genomic DNA, and the positive
animals were bred with C57Bl6J stock.
Tissue homogenization and immunoblotting. Tissues were homogenized
in a 1% Igepal detergent buffer (25 mmol/l Tris, pH 7.4, 1% Igepal, 10% glycerol,
50 mmol/l sodium fluoride, 10 mmol/l sodium pyrophosphate, 137 mmol/l
sodium chloride, 1 mmol/l sodium vanadate, 1 mmol/l phenylmethylsulfonyl
fluoride, 10 ␮g/ml aprotinin, 1 ␮g/ml pepstatin, and 5 ␮g/ml leupeptin) for 15 s
and centrifuged at 2000g for 5 min; subsequent supernatants were microcentrifuged at 13,500g for 20 min at 4°C. Proteins were separated on 10% or 12%
SDS-PAGE followed by transfer to polyvinylidene difluoride (PVDF) or
nitrocellulose membrane for immunoblotting.
Intraperitoneal glucose tolerance test (IPGTT). Male Munc18c Tg and
wild-type mice (4 – 6 months old) were fasted overnight for 18 h. Blood was
collected from the tail vein and blood glucose monitored (Hemocue). After
sample collection of fasted blood, mice were administered glucose (2 g/kg) by
intraperitoneal injection, and subsequent blood glucose readings were taken
at 30-min intervals over 120 min.
Hyperinsulemic-euglycemic clamp. Surgery was performed to chronically
cannulate the left jugular vein 5 days before the in vivo experiment. After an
overnight fast, a 120-min hyperinsulinemic-euglycemic clamp experiment in
awake mice with a primed continuous infusion of insulin (Humulin R; Eli Lilly,
Indianapolis, IN), high-performance liquid chromatography–purified [3-3H]glucose, and 2-deoxy-D-[1-14C]glucose was conducted as previously described (44).
Isolation, culture, and stimulation of insulin secretion of mouse islets.
Pancreatic mouse islets were isolated using a modification of the previously
described method (45). Briefly, pancreata from 8- to 12-week-old male mice
were digested with collagenase and purified using a Ficoll density gradient.
After isolation, islets were cultured overnight in CMRL-1066 medium. Fresh
islets were hand-picked into groups of 10, preincubated in Krebs-Ringer
bicarbonate buffer (10 mmol/l HEPES pH 7.4, 134 mmol/l NaCl, 5 mmol/l
NaHCO3, 4.8 mmol/l KCl, 1 mmol/l CaCl2, 1.2 mmol/l MgSO4, 1.2 mmol/l
KH2PO4) containing 2 mmol/l glucose and 0.1% BSA for 2 h, followed by
stimulation with 20 mmol/l glucose for 2 h. Media was collected to measure
insulin secretion, and islets were harvested in NP-40 lysis buffer to determine
cellular insulin content by radioimmunoassay.
Quantitation of SNARE proteins in tissue homogenates. GST-Munc18CT
(13 residues of the far COOH-terminus of Munc18c), GST-Syn4⌬TM, and
GST-SNAP-23 were expressed in DH5␣ strain of Escherichia coli using
isopropylthiogalactoside induction (36). Skeletal muscle homogenate proteins
were subjected to electrophoresis alongside known quantities of GSTMunc18c, GST-Syn4, or GST-SNAP-23 recombinant proteins on 10% SDSPAGE followed by transfer to PVDF membranes and immunoblotting with
anti-Munc18c, anti-Syn4, or anti-SNAP-23, respectively. Proteins were detected using enhanced chemiluminescence, using exposures well within the
linear range of the film, and quantitated using the BioRad Quantity One
software package (Hercules, CA).
RESULTS
Protein expression in Munc18c Tg mice. To determine
the importance of Munc18c protein in insulin-stimulated
GLUT4 translocation in vivo, we generated transgenic
mice with overexpression of Munc18c in skeletal muscle
and adipose tissue. Of the six founders, four lines transmitted the transgene and two lines, 14131/2 and 14140/2,
overexpressed the Munc18c protein in skeletal muscle by
threefold and sevenfold, respectively. Mice overexpressing
Munc18c protein in skeletal muscle by threefold showed
no significant glucose intolerance, whereas the line expressing sevenfold was glucose intolerant and insulin
resistant. Subsequent studies were performed using the
14140/2 line of Munc18c Tg mice.
The initial use of this particular vector described differential levels of transgenic protein expression among various tissues, with skeletal muscle exhibiting the largest
increase in expression and no changes in brain tissue (43).
The overexpression of Munc18c protein in the Tg male and
female mice was directly compared to endogenous levels
1911
Munc18c OVEREXPRESSION IN VIVO
FIG. 1. Trangenic protein expression in pancreas, skeletal muscle, and fat of Munc18c Tg and Syn4 transgenic
mice. Heart, lung, liver, kidney (Kid), spleen, pancreas
(Panc), gastrocnemius skeletal muscle (Musc), and epididymal adipose (Fat) tissues were isolated from
Munc18c Tg (Tg) and wild-type littermate (Wt) mice and
homogenized as described in RESEARCH DESIGN AND METHODS. Samples (50 ␮g per lane) were resolved by SDSPAGE and immunoblotted with antibodies for Munc18c,
Syn4 (Syn 4), SNAP-23 or VAMP2 (A) or IRAP and
GLUT4 (B) (5 ␮g per lane). C: Pancreas, gastrocnemius
skeletal muscle, and epididymal adipose tissue homogenates were prepared from Syn4 transgenic and wild-type
littermate mice and immunoblotted for the presence of
Syn4 protein as described above. Data are representative of at least three independent sets of tissues.
in a wild-type littermate expressed in heart, lung, liver,
kidney, spleen, pancreas, skeletal muscle, and epididymal
fat by immunoblot analysis. Of these tissues, only pancreas
(Fig. 1A, lanes 11–12), fat (Fig. 1A, lanes 13–14), and
skeletal muscle (Fig. 1A, lanes 15–16) showed a repeated
significant increase in Munc18c protein (five-, three-, and
sevenfold, respectively), without effecting Syn4, SNAP-23,
or VAMP2 abundance. No significant differences in overall
body weight (28 ⫾ 1 g) or tissue weight between wild-type
and Munc18c Tg mice were detected (data not shown)
with the exception of heart weight—Munc18c Tg heart was
14% heavier than that of wild-type (0.21 ⫾ 0.01 vs. 0.18 ⫾
0.01 g; P ⬍ 0.05). No changes in GLUT4 or the GLUT4
vesicle cargo protein IRAP (insulin responsive aminopeptidase) protein were detected in insulin-responsive tissues,
and characteristic tissue-specific expression was displayed
in heart, skeletal muscle, and fat but not liver (Fig. 1B),
with no differences in GLUT1 protein abundance (data not
shown). Syn4 protein was similarly overexpressed in only
pancreas, skeletal muscle, and adipose tissues of the Syn4
Tg mice compared with wild-type mice (Fig. 1C), without
alterations in VAMP2 or SNAP-23 abundance (data not
shown). Thus the transgenic mouse lines overexpressed
the Munc18c or Syn4 protein in insulin-secreting and
insulin-responsive tissues.
Munc18c Tg mice exhibit glucose intolerance and
insulin resistance. To determine the effects of Munc18c
overexpression in vivo, male Munc18c Tg and wild-type
littermate male mice were fasted overnight and subjected
to an intraperitoneal injection of glucose (2 g/kg), and
disposal was monitored over a 2-h period (Fig. 2A). Peak
blood glucose was reached by 30 min after injection in the
wild-type mice and was cleared over the remaining 90 min.
Although fasting glucose levels were similar, the blood
glucose levels of Munc18c Tg mice peaked higher at 30 min
and remained significantly higher for the duration of the
glucose challenge. In contrast to the Munc18c Tg mice, the
Syn4 transgenic mice demonstrated the ability to clear
glucose to the same level as wild-type mice throughout the
experiment (Fig. 2B). These data clearly demonstrated that
the Munc18c Tg mice had impaired ability to clear glucose
from the peripheral circulation.
1912
To investigate the mechanism of glucose intolerance,
hyperinsulinemic-euglycemic clamps were conducted in
the Munc18c Tg mice. The plasma glucose concentrations
FIG. 2. Munc18c Tg mice are glucose intolerant. Glucose tolerance
testing (GTT) of Munc18c Tg and Syn4 transgenic mice was performed
by intraperitoneal injection of D-glucose (2 g/kg) into 4- to 6-month-old
male mice fasted for 18 h. Blood glucose was monitored at 0, 30, 60, 90,
and 120 min postinjection as described in RESEARCH DESIGN AND METHODS.
Data shown are the averages ⴞ SE from 11 Munc18c Tg (Tg) and 10
wild-type (Wt) mice (A) or 8 Syn4 Tg and 8 Wt mice (B). P < 0.05 vs.
wild-type mice, unpaired Student’s t test.
DIABETES, VOL. 52, AUGUST 2003
B.A. SPURLIN AND ASSOCIATES
FIG. 3. Munc18c Tg mice exhibit impaired whole-body
glucose homeostasis as assessed by hyperinsulinemiceuglycemic clamp. A: Steady-state glucose infusion rate
obtained from averaged rates during 90 –120 min of
clamp procedure. B: Insulin-stimulated glucose uptake,
glycolysis, and glycogen/lipid synthesis. Data shown are
the averages ⴞ SE from seven wild-type (䡺) or Munc18c
Tg mice (f). P < 0.05 vs. wild-type mice, unpaired
Student’s t test.
were maintained at 7 mmol/l, and plasma insulin concentrations were raised from 16 to 77 pmol/l during the
clamps. The glucose infusion rate necessary to maintain
euglycemia under conditions of constant infusion of insulin (2.5 mU 䡠 kg⫺1 䡠 min⫺1) was reduced by 32% in Munc18c
Tg mice compared with wild-type mice (Fig. 3A). Consistent with this, Munc18c Tg mice exhibited 31, 24, and 39%
reductions in insulin-stimulated whole-body glucose turnover, glycolysis, and glycogen/lipid synthesis, respectively,
compared with the wild-type mice (Fig. 3B). Conversely,
neither basal hepatic glucose production nor insulin’s
ability to suppress hepatic glucose production was affected in the Munc18c Tg mice compared with wild-type
mice (wild-type: basal, 92 ⫾ 13; insulin, 0.0 ⫾ 6.2; Munc18c
Tg: basal, 84 ⫾ 14; insulin, 0.0 ⫾ 7.8 ␮mol 䡠 kg–1 䡠 min–1).
Taken together, these data indicate that glucose intolerance in the Munc18c Tg mice was due to reduced peripheral glucose disposal in response to insulin and not to
alteration in hepatic insulin action.
Impaired glucose uptake in skeletal muscle and adipose tissue of the Munc18c Tg mice. Because glucose
uptake into skeletal muscle and adipose tissue accounts
for the majority of peripheral glucose disposal, we examined tissue-specific glucose uptake in vivo during hyperinsulinemic-euglycemic clamps. Insulin-stimulated glucose
uptake in skeletal muscle (gastrocnemius) was decreased
by 42% in the Munc18c Tg mice compared with the
wild-type littermate mice (Fig. 4A). Similarly, skeletal
muscle glycolysis and glycogen synthesis rates were reduced, by 41 and 47%, respectively, in the Munc18c Tg
mice. In white adipose tissue, glucose uptake was significantly decreased by 43% in the Munc18c Tg mice compared with wild-type littermates (Fig. 4B). Glucose uptake
in brown adipose tissue of the Munc18c Tg was also
reduced by 25% compared with wild-type mice (Fig. 4C). In
contrast, glucose uptake in cardiac muscle of Munc18c Tg
mice did not differ from that of wild-type mice, consistent
with a lack of Munc18c protein overexpression in cardiac
muscle (Fig. 4D). Overall, whole-body insulin resistance in
the Munc18c Tg mice was accounted for by the 41%
decrease in skeletal muscle and 43% decrease in white
adipose tissue glucose uptake in these mice and was
without systemic effects in tissues that do not overexpress
Munc18c.
DIABETES, VOL. 52, AUGUST 2003
The mechanism for glucose uptake into peripheral tissues involves initiation of the insulin signaling cascade,
which leads to the translocation of GLUT4-containing
vesicles to the cell surface membranes. We have previously shown that overexpression of Munc18c by adenoviral infection of skeletal muscle of GLUT4-EGFP mice
resulted in inhibition of insulin-stimulated GLUT4 translocation (40), as assessed by fractionation of hindquarter
muscles into cell surface and intracellular membrane
compartments (46). Consistent with these data, the
Munc18c Tg mice failed to translocate GLUT4 to the cell
surface fractions in response to insulin (compared with
wild-type mice), whereas all mice showed similar quantities of GLUT4 protein in the cell surface fractions under
basal conditions (data not shown). By contrast, insulin
signaling was unaffected in the overexpression of Munc18c
in skeletal muscle tissue homogenates, since transgenic
and wild-type mice injected with insulin showed equivalently elevated levels of tyrosine phosphorylation of insulin receptor (IR) and IRS-1 proteins as well as increased
serine phosphorylation of PKB/Akt (data not shown).
Taken together, these data are consistent with the concept
that Munc18c overexpression impairs insulin-stimulated
GLUT4 translocation and is independent of effects on
proximal insulin signaling events.
Munc18c overexpression reduces insulin secretion.
To examine the metabolic characteristics of Munc18c Tg
and wild-type mice with and without tetracycline administration, fasting levels of serum glucose, insulin, triglycerides, cholesterol, and nonesterified fatty acid (NEFA)
levels were quantitated (Table 1). Munc18c Tg mice
showed no significant increase in fasting blood glucose
compared with wild-type mice, and no statistically significant differences were observed for serum triglycerides,
cholesterol or NEFAs, although Munc18c Tg mice tended
to have slightly elevated triglycerides and cholesterol
levels. In contrast, Munc18c Tg mice exhibited significantly reduced fasting insulin levels compared with wildtype, and after tetracycline treatment these levels rose to
the wild-type levels. This alteration in insulin levels might
reflect the potential effect of fivefold overexpression of
Munc18c on pancreatic functions, especially since it was
normalized by tetracycline administration.
To investigate the possibility that Munc18c overexpres1913
Munc18c OVEREXPRESSION IN VIVO
FIG. 5. Insulin secretion is impaired in islets from Munc18c Tg mice.
Islets were isolated as described in RESEARCH DESIGN AND METHODS and
placed in culture for 15 h at 37°C in CMRL medium. Islet cells were
preincubated for 2 h in low glucose Krebs-Ringer buffer (2.8 mmol/l)
followed by a 2-h incubation under basal (2.8 mmol/l glucose) or
stimulated (20 mmol/l glucose) conditions. Insulin secretion was measured by radioimmunoassay and normalized for insulin content, and
data subsequently normalized to basal to obtain fold secretion. Data
shown are the averages ⴞ SE; n ⴝ 3 replicates for one of three
independent experiments. P < 0.01 vs. basal or glucose-stimulated Tg
islets.
FIG. 4. Munc18c Tg mice show defects in skeletal muscle glucose
uptake, glycolysis, and glycogen synthesis in hyperinsulinemic-euglycemic clamp. A: Insulin-stimulated glucose uptake, glycolysis, and
glycogen synthesis in gastrocnemius muscles. B: Glucose uptake into
white adipose tissue. C: Glucose uptake into brown adipose tissue. D:
Glucose uptake into cardiac muscle. Data shown are the averages ⴞ SE
from seven wild-type (䡺) or Munc18c Tg mice (f). P < 0.05 vs.
wild-type mice, unpaired Student’s t test.
sion reduced insulin secretion, islets were isolated from
Munc18c Tg or wild-type mice. Glucose stimulation (20
mmol/l) resulted in a 27-fold increase in insulin release
compared with unstimulated islets of wild-type mice (Fig.
5). By contrast, glucose stimulation resulted in only a
sixfold increase in insulin release from Munc18c Tg islets.
Data shown are corrected for total insulin content, although no significant alterations of total insulin content of
wild-type or Munc18c Tg islets incubated with or without
glucose were detected. These data demonstrate that overexpression of Munc18c in pancreatic islet cells reduced
glucose-stimulated insulin secretion.
Restoration of glucose tolerance in Munc18c Tg mice
by tetracycline or by coexpressing Syn4 protein. The
Munc18c transgene is under the regulation of the Tet
operator, such that oral administration of tetracycline
causes downregulation of the transgene (43). To evaluate
the effectiveness of the Tet-repressible system, 4- to
6-month-old Munc18c Tg and wild-type littermate mice
were pair-fed tetracycline in drinking water (1 mg/ml) for
7 days, after which their tissues were homogenized and
immunoblotted for protein expression. Similar to the
results from non–tetracycline-fed mice, Munc18c protein
content was similar in heart, lung, liver, kidney, and spleen
(Fig. 6A, lanes 1–10). However, tetracycline administration downregulated Munc18c protein overexpression to
levels near those observed in wild-type mice in pancreas,
skeletal muscle, and fat (Fig. 6A, lanes 11–16). Moreover,
tetracycline feeding had no apparent adverse effects on
expression of Syn4, SNAP-23, or VAMP2, indicating its
TABLE 1
Metabolic characteristics of wild-type and Munc18c Tg mice
Mice
Wild-type
Munc18c Tg
Glucose
(mg/dl) (n ⫽ 10)
Con
Tet
188 ⫾ 10
177 ⫾ 9
173 ⫾ 6
168 ⫾ 3
Insulin
(ng/ml) (n ⫽ 6)
Con
Tet
0.4 ⫾ 0.05
0.2 ⫾ 0.03*
0.3 ⫾ 0.10
0.3 ⫾ 0.08
Triglycerides
(mg/dl) (n ⫽ 6)
Con
Tet
86 ⫾ 4
109 ⫾ 12
83 ⫾ 7
115 ⫾ 13
Cholesterol
(mmol/l) (n ⫽ 6)
Con
Tet
100 ⫾ 6
118 ⫾ 5
102 ⫾ 5
118 ⫾ 5
Fatty acids
(mg/dl) (n ⫽ 6)
Con
Tet
846 ⫾ 98
853 ⫾ 75
917 ⫾ 68
969 ⫾ 113
Data are averages ⫾ SE. Serum was collected from fasted wild-type or Munc18c Tg male mice (4 – 6 months of age) administered tetracycline
(Tet, 1 mg/ml) or vehicle (Con) in drinking water for 7 days for determination of the metabolic parameters shown. *P ⬍ 0.05 vs. wild-type,
as determined by unpaired Student’s t test.
1914
DIABETES, VOL. 52, AUGUST 2003
B.A. SPURLIN AND ASSOCIATES
FIG. 6. Downregulation of the Munc18c transgene or coexpression of the Syn4 transgene results in normalized glucose tolerance. A: Munc18c Tg
and wild-type (Wt) male mice 4 to 6 months old were fed tetracycline (1 mg/ml) in drinking water for 7 days. Tissue extracts from heart, lung,
liver, kidney (Kid), spleen, pancreas (Panc), skeletal muscle (Musc), and epididymal adipose (Fat) were prepared and protein (50 ␮g) separated
by 10% SDS-PAGE and immunoblotted as described in Fig. 1. Data are representative of at least three independent sets of tissues. B: Before being
killed for tissue analysis, mice were fasted for 18 h and glucose tolerance tested as described in Fig. 2 (data represent the averages ⴞ SE of seven
Tg and seven Wt mice). C: Glucose intolerance is normalized in mice overexpressing Syn4 with Munc18c. Munc18c Tg mice were crossed with Syn4
Tg mice and 4- to 6-month-old male offspring carrying both Munc18c and Syn4 transgenes (SM Tg, open stars) and compared with Syn4 Tg mice
(F) in glucose tolerance tests as described in Fig. 1. The averages ⴞ SE are shown for six SM Tg and six Syn4 Tg mice.
specific repressive effect on the tetracycline-sensitive
Munc18c transgene.
The advantage of the tetracycline-repressible transgene
system is the flexibility of using a mouse as its own control
in metabolic studies. To determine whether the profound
metabolic phenotype (i.e., peripheral insulin resistance) in
the Munc18c Tg mice was directly caused by the increased
Munc18c protein, we administered tetracycline to the
Munc18c Tg and wild-type mice used in the data set for
Fig. 2A for 1 week and performed the IPGTT. Consistent with the immunoblotting results, normalization of
Munc18c protein content in the transgenic mice resulted in
normalization of glucose tolerance to levels indistinguishable from those of wild-type mice fed tetracycline (Fig.
6B). With the exception of insulin levels, which were
normalized by tetracycline administration, tetracycline
exerted no changes in metabolic parameters, in either
wild-type or Munc18c Tg mice.
To determine if glucose intolerance in the Munc18c Tg
mice could be normalized by simultaneous overexpression
of Syn4 in the same tissues, Munc18c Tg and Syn4 transgenic mice were cross-bred to generate Munc18c/Syn4
double-Tg mice (SM Tg). The SM Tg mice were as glucose
tolerant as the Syn4 transgenic mice (Fig. 6C). In addition,
the SM Tg mice displayed insulin levels (0.3 ⫾ 0.09) not
significantly different from wild-type. Taken together,
these data demonstrate that simultaneous increase of Syn4
with Munc18c in vivo reestablished normoglycemia by
DIABETES, VOL. 52, AUGUST 2003
restoring the necessary stoichiometric balance between
these proteins.
DISCUSSION
In this report, we document the generation and characterization of transgenic mouse models in which the level of
Munc18c or Syn4 protein can be modulated by tetracycline. Our data clearly showed that Munc18c overexpression in skeletal muscle, adipose tissue, and pancreas led to
insulin resistance and impaired insulin secretion, which
were reversible upon tetracycline administration or by the
simultaneous overexpression of Syn4 in vivo. Glucose
intolerance resulted from significant 40% reductions in
whole-body glucose turnover and glycogen/lipid synthesis,
and similar decreases in skeletal muscle glucose uptake,
glycolysis, and glycogen synthesis, in addition to a 43%
decrease in white adipose glucose uptake. Importantly,
insulin resistance in the Munc18c Tg mice was independent of any effect on hepatic insulin action, indicating that
the observed whole-body insulin resistance was the result
of defects in peripheral glucose uptake. Moreover, our
Munc18c Tg mice showed no differences from littermate
wild-type mice in body weight or adipose mass, similar to
the adipose-specific GLUT4 knockout mice (47). Intriguingly, Munc18c Tg mice showed defective insulin secretion
that was reversible by tetracycline—the first report on the
effects of Munc18c on insulin secretion. In all, these
1915
Munc18c OVEREXPRESSION IN VIVO
alterations in Munc18c protein abundance culminated in
insulin resistance and impaired insulin secretion in vivo.
The glucose intolerance of the Munc18c Tg mice was
fully reversed after 1 week of oral tetracycline administration to ablate transgene expression, using the same mice
initially used to characterize the glucose intolerant phenotype. Quantitative immunoblotting using total homogenates of skeletal muscle (gastrocnemius) from male wildtype and male Munc18c Tg mice revealed that Syn4 and
SNAP-23 were present at ⬃50 nmol/l and 150 nmol/l,
respectively. By contrast, Munc18c was present in wildtype mice at 2 nmol/l, elevated to 15 nmol/l in Munc18c Tg
mice. These quantitative results were consistent with a
previously reported threefold overabundance of SNAP-23
relative to Syn4 in 3T3L1 adipocytes (42), with Syn4 and
Munc18 abundances reported in rat liver and kidney
tissues (41), and also with our previous findings using
3T3L1 adipocytes (36). Interestingly, even with a more
than sevenfold increase in Munc18c protein, there remains
a greater than threefold excess of Syn4, and yet this still
results in the insulin-resistant phenotype and dramatically
impaired skeletal muscle GLUT4 translocation. Because
the Munc18-syntaxin complexes form in a 1:1 stoichiometry (30), further studies using variable dosage and duration
of tetracycline treatment in these mice are underway to
relate this phenomenon to glucose homeostasis.
Unexpectedly, Munc18c Tg mice exhibited significant
reductions in fasting serum insulin levels, which were
reversed by tetracycline administration to downregulate
Munc18c transgene expression, overall suggesting that
Munc18c protein affected islet function. Munc18c was
overexpressed by fivefold in pancreas tissue from
Munc18c Tg mice; however, there were no apparent
differences in pancreas tissue weight or islet size compared with those of wild-type mice. Although not believed
to regulate glucose uptake in ␤-cells, Munc18 and SNARE
proteins have been shown to mediate glucose-stimulated
exocytosis of insulin-containing granules (48). Consistent
with our finding here with Munc18c overexpression, a
recent Munc18a overexpression study implicated Munc18a
(Munc18 –1/n-Sec1) as a negative regulator of the insulin
secretory machinery via a mechanism involving its binding
partner syntaxin 1 (49), and Munc18c overexpression in
islets may inhibit insulin granule exocytosis via a mechanism involving Syn4. Alternatively, Munc18c may bind and
sequester a different protein required for insulin secretion.
It has also been reported that the distribution of Munc18c
in pancreatic acinar cells can affect exocytosis (50). Thus,
more studies will be necessary to resolve the mechanism
underlying the inhibition of insulin secretion in Munc18c
Tg islets, and ultimately techniques other than overexpression will be required to determine the true function of the
Munc18 proteins in insulin secretion.
ACKNOWLEDGMENTS
Supported by a predoctoral fellowship from the Indiana
University Diabetes Graduate Training Program (B.A.S.), a
Career Development Award from the American Diabetes
Association (D.C.T.), and a research grant from the Indiana University School of Medicine Showalter Research
Trust Fund (D.C.T.). The clamp studies were conducted at
the Yale Mouse Metabolic Phenotyping Center and sup1916
ported by grants from the U.S. Public Health Service (U24
DK-59635, J.K.K. and G.I.S.) and the American Diabetes
Association (7– 01-JF-05, J.K.K.). Gerald I. Shulman is an
investigator of the Howard Hughes Medical Institute.
We are very grateful to Dr. Jeffrey E. Pessin for generating the transgenic mice at the University of Iowa Transgenic Animal Facility. We would also like to thank Drs. Ulli
Certa, Richard Scheller, and Steve Waters for the pCOMBI
vector, Syn4 cDNA, and IRAP antibody, respectively. The
Indiana University School of Medicine Analyte Core Facility was invaluable for their assistance with metabolic
measurements of serum samples.
REFERENCES
1. Olson AL, Pessin JE: Structure, function and regulation of the mammalian
facilitative glucose transporter gene family. Ann Rev Nutr 16:235–256, 1996
2. Slot JW, Geuze HJ, Gigengack S, Lienhard GE, James DE: Immunolocalization of the insulin regulatable glucose transporter in brown adipose
tissue of the rat. J Cell Biol 113:123–135, 1991
3. Czech MP: Molecular actions of insulin on glucose transport. Annu Rev
Nutr 15:441– 471, 1995
4. Kahn BB: Facilitative glucose transporters: regulatory mechanisms and
dysregulation in diabetes. J Clin Invest 89:1367–1374, 1992
5. Kandror KV, Pilch PF: Compartmentalization of protein traffic in insulinsensitive cells. Am J Physiol 271:E1–E14, 1996
6. Klip A, Tsakiridis T, Marette A, Ortiz PA: Regulation of expression of
glucose transporters by glucose: a review of studies in vivo and in cell
cultures. FASEB J 8:43–53, 1994
7. Thurmond DC, Pessin JE: Molecular machinery involved in the insulinregulated fusion of GLUT4-containing vesicles with the plasma membrane
(Review). Mol Membr Biol 18:237–245, 2001
8. White MF: The IRS-signalling system: a network of docking proteins that
mediate insulin action. Mol Cell Biochem 182:3–11, 1998
9. Saltiel AR, Pessin JE: Insulin signaling pathways in time and space. Trends
Cell Biol 12:65–71, 2002
10. Chen KS, Friel JC, Ruderman NB: Regulation of phosphatidylinositol
3-kinase by insulin in rat skeletal muscle. Am J Physiol 265:E736–E742, 1993
11. Folli F, Saad MJA, Backer JM, Kahn CR: Regulation of phosphatidylinositol
3-kinase activity in liver and muscle of animal models of insulin-resistant
and insulin-deficient diabetes-mellitus. J Clin Invest 92:1787–1794, 1993
12. Ruderman NB, Kapeller R, White MF, Cantley LC: Activation of phosphatidylinositol 3-kinase by insulin. Proc Natl Acad Sci U S A 87:1411–1415,
1990
13. Bandyopadhyay G, Standaert ML, Zhao L, Yu B, Avignon A, Galloway L,
Karnam P, Moscat J, Farese RV: Activation of protein kinase C (alpha, beta,
and zeta) by insulin in 3T3/L1 cells: transfection studies suggest a role for
PKC-zeta in glucose transport. J Biol Chem 272:2551–2558, 1997
14. Kotani K, Ogawa W, Matsumoto M, Kitamura T, Sakaue H, Hino Y, Miyake
K, Sano W, Akimoto K, Ohno S, Kasuga M: Requirement of atypical protein
kinase C␭ for insulin stimulation of glucose uptake but not for Akt
activation in 3T3–L1 adipocytes. Mol Cell Biol 18:6971– 6982, 1998
15. Araki S, Tamori Y, Kawanishi M, Shinoda H, Masugi J, Mori H, Niki T,
Okazawa H, Kubota T, Kasuga M: Inhibition of the binding of SNAP-23 to
syntaxin 4 by Munc18c. Biochem Biophys Res Commun 234:257–262, 1997
16. Kawanishi M, Tamori Y, Okazawa H, Araki S, Shinoda H, Kasuga M: Role
of SNAP23 in insulin-induced translocation of GLUT4 in 3T3–L1 adipocytes: mediation of complex formation between syntaxin4 and VAMP2.
J Biol Chem 275:8240 – 8247, 2000
17. Olson AL, Knight JB, Pessin JE: Syntaxin 4, VAMP2, and/or VAMP3/
cellubrevin are functional target membrane and vesicle SNAP receptors
for insulin-stimulated GLUT4 translocation in adipocytes. Mol Cell Biol
17:2425–2435, 1997
18. Rea S, Martin LB, McIntosh S, Macaulay SL, Ramsdale T, Baldini G, James
DE: Syndet, an adipocyte target SNARE involved in the insulin-induced
translocation of GLUT4 to the cell surface. J Biol Chem 273:18784 –18792,
1998
19. Volchuk A, Wang Q, Ewart HS, Liu Z, He L, Bennett MK, Klip A: Syntaxin
4 in 3T3–L1 adipocytes: regulation by insulin and participation in insulindependent glucose transport. Mol Biol Cell 7:1075–1082, 1996
20. Yang C, Coker KJ, Kim JK, Mora S, Thurmond DC, Davis AC, Yang B,
Williamson RA, Shulman GI, Pessin JE: Syntaxin 4 heterozygous knockout
mice develop muscle insulin resistance. J Clin Invest 107:1311–1318, 2001
21. Cain CC, Trimble WS, Lienhard GE: Members of the VAMP family of
DIABETES, VOL. 52, AUGUST 2003
B.A. SPURLIN AND ASSOCIATES
synaptic vesicle proteins are components of glucose transporter-containing vesicles from rat adipocytes. J Biol Chem 267:11681–11684, 1992
22. Cheatham B, Volchuk A, Kahn CR, Wang L, Rhodes CJ, Klip A: Insulinstimulated translocation of GLUT4 glucose transporters requires SNAREcomplex proteins. Proc Natl Acad Sci U S A 93:15169 –15173, 1996
23. Tamori Y, Hashiramoto M, Araki S, Kamata Y, Takahashi M, Kozaki S,
Kasuga M: Cleavage of vesicle-associated membrane protein (VAMP)-2 and
cellubrevin on GLUT4-containing vesicles inhibits the translocation of
GLUT4 in 3T3–L1 adipocytes. Biochem Biophys Res Commun 220:740 –
745, 1996
24. Volchuk A, Sargeant R, Sumitani S, Liu Z, He L, Klip A: Cellubrevin is a
resident protein of insulin-sensitive GLUT4 glucose transporter vesicles in
3T3–L1 adipocytes. J Biol Chem 270:8233– 8240, 1995
25. Gengyo-Ando K, Kamiya Y, Yamakawa A, Kodaira K, Nishiwaki K, Miwa J,
Hori I, Hosono R: The C. elegans unc-18 gene encodes a protein expressed
in motor neurons. Neuron 11:703–711, 1993
26. Salzberg A, Cohen N, Halachmi N, Kimchie Z, Lev Z: The Drosophila Ras2
and Rop gene pair: a dual homology with a yeast Ras-like gene and a
suppressor of its loss-of-function phenotype. Development 117:1309 –1319,
1993
27. Hosono R, Hekimi S, Kamiya Y, Sassa T, Murakami S, Nishiwaki K, Miwa
J, Taketo A, Kodaira KI: The unc-18 gene encodes a novel protein affecting
the kinetics of acetylcholine metabolism in the nematode Caenorhabditis
elegans. J Neurochem 58:1517–1525, 1992
28. Garcia EP, Gatti E, Butler M, Burton J, De Camilli P: A rat brain Sec1
homologue related to Rop and UNC18 interacts with syntaxin. Proc Natl
Acad Sci U S A 91:2003–2007, 1994
29. Pevsner J, Hsu SC, Scheller RH: n-Sec1: a neural-specific syntaxin-binding
protein. Proc Natl Acad Sci U S A 91:1445–1449, 1994
30. Hata Y, Slaughter CA, Sudhof TC: Synaptic vesicle fusion complex
contains unc-18 homologue bound to syntaxin. Nature 366:347–351, 1993
31. Tellam JT, McIntosh S, James DE: Molecular identification of two novel
Munc-18 isoforms expressed in non-neuronal tissues. J Biol Chem 270:
5857–5863, 1995
32. Tellam JT, Macaulay SL, McIntosh S, Hewish DR, Ward CW, James DE:
Characterization of Munc-18c and syntaxin-4 in 3T3–L1 adipocytes: putative role in insulin-dependent movement of GLUT-4. J Biol Chem 272:
6179 – 6186, 1997
33. Harrison SD, Broadie K, van de Goor J, Rubin GM: Mutations in the
Drosophila Rop gene suggest a function in general secretion and synaptic
transmission. Neuron 13:555–566, 1994
34. Verhage M, Maia AS, Plomp JJ, Brussaard AB, Heeroma JH, Vermeer H,
Toonen RF, Hammer RE, van den Berg TK, Missler M, Geuze HJ, Sudhof
TC, de Vries KJ, Geijtenbeek A, Brian EC, de Graan PN, Ghijsen WE:
Synaptic assembly of the brain in the absence of neurotransmitter secretion: dynamics of Munc18 –1 phosphorylation/dephosphorylation in rat
brain nerve terminals. Science 287:864 – 869, 2000
35. Thurmond DC, Pessin JE: Discrimination of GLUT4 vesicle trafficking from
fusion using a temperature-sensitive Munc18c mutant. EMBO J 19:3565–
3575, 2000
36. Thurmond DC, Kanzaki M, Khan AH, Pessin JE: Munc18c function is
DIABETES, VOL. 52, AUGUST 2003
required for insulin-stimulated plasma membrane fusion of GLUT4 and
insulin-responsive amino peptidase storage vesicles. Mol Cell Biol 20:379 –
388, 2000
37. Schulze KL, Littleton JT, Salzberg A, Halachmi N, Stern M, Lev Z, Bellen HJ:
rop, a Drosophila homolog of yeast Sec1 and vertebrate n-Sec1/Munc-18
proteins, is a negative regulator of neurotransmitter release in vivo.
Neuron 13:1099 –1108, 1994
38. Tamori Y, Kawanishi M, Niki T, Shinoda H, Araki S, Okazawa H, Kasuga M:
Inhibition of insulin-induced GLUT4 translocation by Munc18c through
interaction with syntaxin4 in 3T3–L1 adipocytes. J Biol Chem 273:19740 –
19746, 1998
39. Thurmond DC, Ceresa BP, Okada S, Elmendorf JS, Coker K, Pessin JE:
Regulation of insulin-stimulated GLUT4 translocation by munc18c in
3T3L1 adipocytes. J Biol Chem 273:33876 –33883, 1998
40. Khan AH, Thurmond DC, Yang C, Ceresa BP, Sigmund CD, Pessin JE:
Munc18c regulates insulin-stimulated GLUT4 translocation to the transverse tubules in skeletal muscle. J Biol Chem 276:4063– 4069, 2001
41. Fujita H, Tuma PL, Finnegan CM, Locco L, Hubbard AL: Endogenous
syntaxins 2, 3 and 4 exhibit distinct but overlapping patterns of expression
at the hepatocyte plasma membrane. Biochem J 329:527–538, 1998
42. Hickson GR, Chamberlain LH, Maier VH, Gould GW: Quantification of
SNARE protein levels in 3T3–L1 adipocytes: implications for insulinstimulated glucose transport. Biochem Biophys Res Commun 270:841–
845, 2000
43. Schultze N, Burki Y, Lang Y, Certa U, Bluethmann H: Efficient control of
gene expression by single step integration of the tetracycline system in
transgenic mice. Nat Biotechnol 14:499 –503, 1996
44. Kim JK, Gavrilova O, Chen Y, Reitman ML, Shulman GI: Mechanism of
insulin resistance in A-ZIP/F-1 fatless mice. J Biol Chem 275:8456–8460, 2000
45. Lacy PE, Kostianovsky M: Method for the isolation of intact islets of
Langerhans from the rat pancreas. Diabetes 16:35–39, 1967
46. Zhou M, Sevilla L, Vallega G, Chen P, Palacin M, Zorzano A, Pilch PF,
Kandror KV: Insulin-dependent protein trafficking in skeletal muscle cells.
Am J Physiol 275:E187–E196, 1998
47. Zisman A, Peroni OD, Abel ED, Michael MD, Mauvais-Jarvis F, Lowell BB,
Wojtaszewski JF, Hirshman MF, Virkamaki A, Goodyear LJ, Kahn CR,
Kahn BB: Targeted disruption of the glucose transporter 4 selectively in
muscle causes insulin resistance and glucose intolerance. Nat Med 6:924 –
928, 2000
48. Rhodes CJ: Processing of the insulin molecule. In Diabetes mellitus: a
fundamental and clinical text. LeRoith T, Olefsky E., Eds. Philadelphia,
Lippincott Williams & Wilkins, 2000, p. 20 –38
49. Zhang W, Efanov A, Yang SN, Fried G, Kolare S, Brown H, Zaitsev S,
Berggren PO, Meister B: Munc-18 associates with syntaxin and serves as a
negative regulator of exocytosis in the pancreatic beta-cell. J Biol Chem
275:41521– 41527, 2000
50. Gaisano HY, Lutz MP, Leser J, Sheu L, Lynch G, Tang L, Tamori Y, Trimble
WS, Salapatek AM: Supramaximal cholecystokinin displaces Munc18c
from the pancreatic acinar basal surface, redirecting apical exocytosis to
the basal membrane. J Clin Invest 108:1597–1611, 2001
1917