1 Departments of Medicine, Microbiology and Immunology, and Hormone Research Institute, 2 Department of Biochemistry and Biophysics and Metabolic Research Unit, 3 Department of Biochemistry and Biophysics and Hormone Research Institute, School of Medicine, University of California, San Francisco 94143; and 4 Department of Biology, University of California, Los Angeles, California 90095
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ABSTRACT |
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The functional role of glutamate
decarboxylase (GAD) and its product GABA in pancreatic islets has
remained elusive. Mouse -cells express the larger isoform GAD67,
whereas human islets express only the smaller isoform GAD65. We have
generated two lines of transgenic mice expressing human GAD65 in
pancreatic
-cells (RIP7-hGAD65, Lines 1 and 2) to study the effect
that GABA generated by this isoform has on islet cell function. The ascending order of hGAD65 expression and/or activity in
-cells was
Line 1 heterozygotes < Line 2 heterozygotes < Line 1 homozygotes. Line 1 heterozygotes have normal glucose tolerance,
whereas Line 1 homozygotes and Line 2 heterozygotes exhibit impaired
glucose tolerance and inhibition of insulin secretion in vivo in
response to glucose. In addition, fasting levels of blood glucose are
elevated and insulin is decreased in Line 1 homozygotes. Pancreas
perfusion experiments suggest that GABA generated by GAD65 may function as a negative regulator of first-phase insulin secretion in response to
glucose by affecting a step proximal to or at the KATP+ channel.
regulation of insulin secretion; neurotransmitter; pancreas perfusion; glucose intolerance
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INTRODUCTION |
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PANCREATIC
-CELLS in islets of Langerhans express the enzyme
glutamate decarboxylase (GAD) and GABA at levels comparable to those
encountered in the central nervous system (14, 44, 57).
However, the physiological function of these molecules in islets
remains unclear (reviewed in 37, 53).
GABA produced in -cells has been suggested to serve as a functional
regulator of pancreatic hormone release or as a paracrine signaling
molecule for communication between
-cells and the other endocrine
cells in islets of Langerhans (53). These hypotheses have
been tested by different investigators by use of in vitro assays and
exogenous GABA or its mimics. These investigations have yielded
conflicting data. In perfused rat and dog pancreas, GABA was shown to
inhibit arginine-stimulated insulin release (20, 27). In
contrast, perfusion of rat pancreas with GABA or GABA mimics had no
detectable effect on insulin secretion (14, 50).
Similarly, conflicting results have been reported on the effect of GABA
on glucagon and somatostatin secretion (13, 14, 27, 50,
51). There is convincing evidence to suggest that GABA may have
an inhibitory effect on glucagon release in vitro (13, 14,
51). It is not clear, however, whether GABA acts as a signal
molecule for glucose-induced inhibition of glucagon secretion
(14, 51).
There are at least two distinct GABA receptors for inhibitory
neurotransmission in the brain, GABAA and GABAB
receptors (26, 36). GABAA receptors, which
regulate chloride conductance and can cause cell inhibition by
hyperpolarization, are expressed on islet - and
-cells but not on
-cells (51, 58). It has been proposed that the
inhibitory effect of GABA on glucagon secretion may be mediated by
GABAA receptors (13, 51). Recent results suggest that GABAB receptors are expressed in pancreatic
islets (Chang and Baekkeskov, unpublished results). GABAB
receptors are coupled to G proteins and can mediate inhibition of the
closure of K+ channels or the opening of Ca2+
channels (26 and references therein). A GABAB receptor
agonist has been reported to inhibit both insulin secretion and the
rise in cytoplasmic Ca2+ of
-cells (20).
In mammals, there are two highly homologous nonallelic isoforms of GAD,
GAD65 and GAD67 (11). The two forms differ mainly in their
association with the co-enzyme pyridoxal 5'-phosphate (PLP) (22,
35) and may differ in some aspects of subcellular targeting
(6, 7, 23, 25, 48, 54). GAD65, which is less saturated
with co-enzyme (22, 35), is found both in the cytosol and
associated with the cytosolic face of the membrane of synaptic-like
microvesicles in -cells (6, 7, and unpublished results). GAD65 is
the major GAD isoform in rat islets and the only isoform in human
islets (28, 34, 45). In contrast, mouse
-cells seem to
express exclusively the cytosolic and highly PLP-saturated GAD67
isoform, even though both isoforms were detected at mRNA levels
(12, 28, 45-47). Perhaps not surprisingly, therefore, GAD65
/
mice exhibit normal glucose homeostasis (24).
Targeted disruption of the GAD67 gene in the mouse is more likely to
result in a phenotype because of loss of GABA in islets of Langerhans. Neonatal lethality of GAD67
/
mice has, however, precluded analysis of glucose homeostasis and islet cell function in mice deficient in
this isoform (2, 8). Targeted disruption of the genes encoding the GAD isoforms has therefore not provided information about
the role of GABA in the endocrine pancreas. Thus the role of GAD and
GABA in islets of Langerhans remains elusive.
The experiments reported so far on the effect of GABA on hormone
release in pancreatic islets have been limited to analyzing the effect
of exogenous GABA and GABA mimics, which may function differently from
the endogenous transmitter produced in -cells. Thus the
physiological conditions that affect GABA synthesis and release may not
be approximated by the in vitro assays. Furthermore, the dose of
exogenous GABA used in most of the experiments may exceed the level of
endogenous GABA (53).
It is not clear how GABA release from -cells is regulated in vivo.
GABA can be measured in conditioned medium from insulinoma cell lines
(13, 40, 55), but not from islets (40),
consistent with a paracrine rather than an endocrine function. There
are conflicting data as to whether the release from insulinoma cell lines is regulated by glucose (13, 40, 55).
To assess the role of GABA in pancreatic islets in an in vivo setting,
we have taken advantage of the low or absent expression of GAD65 in
mouse islets and targeted expression of this isoform to -cells in
two lines of transgenic mice. We show in this study that expression of
transgenic GAD65 and elevated levels of GABA in pancreatic
-cells
result in inhibition of glucose-induced insulin release and impaired
glucose tolerance and diabetes in transgenic mice. Based on results of
studies of the perfused pancreas of transgenic and control mice, we
propose that GABA acts as a specific inhibitor of first-phase insulin
release and exerts its effect at a step before or at
-cell depolarization.
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MATERIALS AND METHODS |
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Generation of transgenic mice.
A 9.7-kb rat insulin promoter sequence (RIP7), including the first
intron of the rat insulin II gene (41), was used to direct pancreatic -cell specific expression of human GAD65. A transgenic construct (Fig. 1A) was
generated by inserting a 1,757-bp BamH I cDNA fragment
encoding the human GAD65 into a Cla I site of RIP7 at +180.
The RIP7 vector carries polyadenylation sequences from the SV-40 large
T antigen gene. To generate cohesive ends for ligation, the
BamH I fragment was partially filled with dGTP, dATP, and
dTTP, and the Cla I site with dCTP, by use of reverse transcriptase. The transgenic construct, RIP7-hGAD65, was linearized with Sal I and tested for transient expression in a
TC3
cell line (10) in parallel with transgenic constructs
generated by use of shorter versions of the rat insulin promoter, RIP1
and RIP5 (9, 30). The RIP7-hGAD65 construct was shown to
mediate the highest expression of GAD65. The distribution of the enzyme between cytosolic and membrane fractions was similar to rat islets (6) (results not shown). Two transgenic founder mice,
RIP7-hGAD65 mouse 1 and mouse 2, were generated
by microinjection of the RIP7-hGAD65 cDNA at a concentration of ~2
ng/ml into B6D2/F2 mouse embryos, according to established procedures
(21). RIP7-hGAD65 mouse 1 and mouse
2 were backcrossed into C57Bl/6 mice. Genetic transmission of the
transgene was assessed by Southern blot and by polymerase chain
reaction (PCR) analyses with DNA isolated from tail biopsies. Physiological studies were carried out on third- and fourth-generation backcrossed heterozygous 10- to 15-wk-old Line 1 and Line 2 mice. After
nine backcrosses into C57Bl/6 mice, Line 1 was bred to homozygosity, and homozygous mice were subjected to physiological studies at 8-15 wk of age. Control mice for all experiments with heterozygote transgenic mice were the transgene-negative littermates of the mice
analyzed. Control mice for homozygous Line 1 mice were wild-type C57Bl/6 mice of the same age and sex.
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PCR analysis of transgenic expression. mRNA expression of the human GAD65 transgene in the transgenic line was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) using a 5' primer from the 5' untranslated sequences of RIP7 (AAGTGACCAGCTACAGTCGG) and a 3' primer from the coding region of the human GAD65 gene (AGCAGGTCTGTTGCATGGAG). The tubulin gene was used as a control for the RT-PCR analyses. Total RNAs were isolated from freshly removed mouse pancreases using RNAzol (Tel-Test, Friendswood, TX), followed by digestion with RNase-free DNase I (Promega, Madison, WI) to remove contaminated genomic DNA. PCR amplification conditions were 94°C (2 min), followed by 35 cycles of 94°C (1 min), 60°C (1 min), 72°C (3 min), and finally 72°C for 10 min. The amplified DNA fragments (350 bp) were resolved on a 1.5%-agarose gel.
Immunohistochemical analysis. Immunohistochemical analysis of protein expression of the transgene was performed on frozen sections of mouse pancreas, as previously described (28), using a mixture of three human monoclonal antibodies to GAD65, MICA 2, 4, and 6 (49).
For immunohistochemical analysis of GABA expression, 25 ml of fixative containing 4% paraformaldehyde (Polyscience, PA) and 0.1% glutaraldehyde (Sigma, St. Louis, MO) in PBS, pH 7.3, were perfused directly into the left ventricle of transgenic mice and negative littermates. After perfusion, the pancreas was removed and postfixed in the same fixative for 1 h at 4°C. The tissue was rinsed in 30% sucrose-PBS overnight at 4°C, embedded in Optimal Cutting Temperature (OCT) embedding medium (Tissue Tek, Miles Diagnostic Division, Elkhart, IN), and stored atIslet isolation and protein expression analysis. Mice were anesthesized with pentobarbital, and the pancreas was inflated with Hanks' balanced salt solution (HBSS; Sigma) before excision. Pancreases were subjected to two sequential incubations of 10 min each with collagenase type XI (Sigma) in a siliconized scintillation vial (2 pancreases per vial) at 37°C under vigorous shaking. The collagenase digest was washed three times in ice-cold HBSS containing 1% BSA (Sigma). Islets were mainly released in the second incubation and were handpicked several times under a stereomicroscope to reach 100% purity. Islets were subjected to a brief recovery period in RPMI 1640 and 10% FCS (GIBCO-BRL, Grand Island, NY), washed in PBS, and snap-frozen on dry ice.
For enzyme, protein, and Western blot assays, islets were extracted at a concentration of 10 islets/µl in 50 mM HEPES-NaOH, pH 7.0, 1 mM 2-aminoethylisothiouronium bromide, 1 mM phenylmethylsulfonyl fluoride, 10 mM benzamidine-HCl, 5 mM sodium fluoride, and 1% Triton X-114. After 1 h of incubation on ice, the lysate was centrifuged at 150,000 g for 1 h to remove cellular debris. For GAD activity assay, the supernatant was aliquoted and diluted in the same buffer without Triton X-114 but containing 0.1% BSA and 10 mM [1-14C]glutamate in the presence and absence of 0.1 mM PLP. Reactions were incubated for 2 h at 37°C and quenched by addition of 6 N HCl (5). Lysis buffer was used as a negative control in the presence and absence of PLP. Protein concentration in the different lysates was determined using the bicinchoninic acid protein assay reagent (Pierce, Rockford, IL) and BSA as standard. For analysis of GAD expression in islets by immunoblotting, aliquots of the islet lysates were boiled for 3 min in SDS sample buffer (62.5 mM Tris · HCl, pH 6.8, 2% SDS, 2.5%Glucose tolerance tests and measurement of insulin and glucagon secretion in vivo. Glucose tolerance tests were conducted in overnight-fasted animals, as previously described (30). Serum insulin levels were measured by radioimmunoassay with a kit (Binax, South Portland, MA) according to the manufacturer's instructions. Serum glucagon levels were measured by a double antibody radioimmunoassay with a kit from Diagnostic Products (Los Angeles, CA) according to the manufacturer's instructions.
Pancreatic perfusion. In vitro pancreas perfusion was performed as previously described for the mouse or Chinese hamster pancreas (17, 30, 32). Perfusate consisted of bicarbonate-phosphate-calcium buffer (17) containing 0.2% purified, "stabilized" bovine albumin and 3% T-40 Dextran (Aldrich Chemical, Milwaukee, WI). Perfusate was introduced into the celiac artery at 1 ml/min, and effluent was collected at 1- to 2-min intervals from the portal vein after a single passage through the pancreas. To minimize loss of soluble oxygen during the transit time from oxygenator to pancreas at these low flow rates (17), glass was employed for the influx tubing. As described (17), perfusate pressure was continuously monitored. Surgical integrity of the system was also evaluated by comparing perfusate inflow rate to efflux rate from the portal vein into the collection tubes. Experiments in which efflux rate was <80% of influx were discarded. As previously described (31), the pancreas was perfused in each experiment for 20 min without glucose to test for nonstimulated leakage or washout of insulin, a potential problem in perfusion experiments using mouse pancreas. This period was also used to establish whether the transgenic mouse pancreas released insulin in a constitutive manner (31). Agents, including glucose, IBMX (Aldrich Chemical), and KCl (Sigma), were added as indicated in the individual experiments. Insulin levels in pancreatic perfusate were measured by solid-phase radioimmunoassay (29), with rat insulin as the reference standard and antiporcine insulin antibody (Linco Research, Eureka, MO). Data analyses were carried out using Student's t-test for paired as well as unpaired values.
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RESULTS |
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Heterozygous RIP7-hGAD65 transgenic mice express elevated levels of
GAD65 and GABA, and Line 2 has the highest expression.
The RIP7 transgenic vector directs high levels of -cell specific
expression (41). An RIP7-hGAD65 construct was generated (Fig. 1A) and used for microinjection of mouse embryos.
Transgenic RIP7-hGAD65 lines were established from two founder mice
identified by Southern blot and PCR analyses. RT-PCR (Fig.
1B), immunohistochemical analysis (Fig.
2, a and b), and
immunoblot and enzyme assays (Fig. 3,
A and B) established expression of the transgene
in Lines 1 and 2. Thus immunofluorescence studies using human GAD65
specific monoclonal antibodies that recognize the mouse and human
protein equally well (28) revealed significant expression
of GAD65 in islets of both RIP7-hGAD65 Lines 1 and 2 heterozygous mice
(Fig. 2, a and b). At the heterozyygote stage,
the expression of GAD65 was highest in the RIP7-hGAD65 Line 2 islets.
As reported earlier (28), no expression of endogenous
GAD65 was detected in normal mouse pancreas by immunofluorescence
analysis (results not shown).
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Line 1 homozygotes express the highest levels of GAD65. After breeding of Line 1 to homozygosity, islets of Langerhans were isolated from Line 1 and Line 2 heterozygotes, from Line 1 homozygous mice, and from wild-type C57Bl/6 mice for analysis of GAD65 expression and activity. Immunoblot analysis was performed using the antibody 1701, which recognizes GAD65 and GAD67 equally well. In some experiments, equal amounts of protein were loaded from transgenic and wild-type islets (Fig. 3A), whereas in other experiments up to a fivefold excess of wild-type islets was loaded on the gel (results not shown). The analyses revealed a high expression of GAD65 in Line 1 and Line 2 transgenic mice, which was severalfold higher than expression of endogenous GAD67 in wild-type and transgenic mice (Fig. 3A). Overloading of protein and prolonged exposures of autoradiograms did not reveal expression of endogenous GAD65 in islets of wild-type mice (results not shown). Quantitative immunoblot analysis (a representative blot is shown in Fig. 3A) revealed that expression of GAD65 was highest in Line 1 homozygous mice, followed by Line 2 heterozygotes and then Line 1 heterozygotes. The levels of transgenic GAD65 exceeded endogenous GAD67 levels by ~8.7-fold (Line 1 homozygotes), 7.5-fold (Line 2 heterozygotes), and 4.5-fold (Line 1 heterozygotes). Thus Line 1 homozygotes, Line 2 heterozygotes, and Line 1 heterozygotes represent three distinct levels of GAD65 expression.
We next analyzed GAD enzyme activity in isolated islets from Line 1 heterozygotes, Line 2 heterozygotes, Line 1 homozygotes, and wild-type C57Bl/6 mice to confirm that the increased expression levels in transgenic mice also reflect increased enzyme activity in islets, and therefore a potential for GABA production. GAD65 provides the majority of PLP-inducible apoenzyme activity in brain but is also present as a holoenzyme (35). Enzyme analysis in the presence and absence of exogenous PLP revealed no PLP-inducible enzyme activity in wild-type C57Bl/6 mice, consistent with the absence of GAD65 in normal mouse islets. Approximately 1.6-fold and 3.4-fold increases in holoenzyme activity over wild-type mice were observed in Line 2 heterozygotes and in Line 1 homozygotes, respectively, whereas no increase was detected in Line 1 heterozygotes (Fig. 3B). Addition of PLP resulted in ~7-fold, 11-fold, and 15-fold increases in GAD activity in Line 1 heterozygotes, Line 2 heterozygotes, and Line 1 homozygotes, respectively. Thus transgenic GAD65 contributes to holoenzyme activity in the two highest expressing lines but most significantly enhances the apoenzyme pool in all three lines.Exhibition of impaired glucose tolerance and elevated blood sugars
in vivo in RIP7-hGAD65 transgenic mice.
We addressed the question of whether increased levels of GAD65 and GABA
affect glucose homeostasis. Blood glucose levels were analyzed in
transgenic mice after an overnight fast and at different time points
after glucose administration (Fig. 4).
Fasting blood glucose levels in transgenic and control mice were not
significantly different for Line 2 heterozygotes (Fig. 4B)
or in Line 1 heterozygotes (not shown). However, they were
significantly elevated in Line 1 homozygotes (Fig. 4A).
Analyses of blood glucose levels after intraperitoneal administration
of glucose revealed impaired glucose tolerance in Line 1 homozygotes
and in Line 2 heterozygotes (Fig. 4), whereas Line 1 heterozygotes
remained normal (results not shown). Blood glucose levels maximized at
20 min in control mice and approached basal values by 90 min. In
contrast, Line 1 homozygotes and Line 2 heterozygotes showed prolonged
high blood glucose levels. Whereas Line 2 mice reached normal blood
glucose levels at 150 min, glucose levels in Line 1 were still elevated
at 180 min (Fig. 4). Thus impairment of glucose tolerance is most
severe in Line 1 homozygotes, which express the highest levels of
antigen, and these mice also exhibit elevated fasting blood sugar.
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Exhibition of decreased insulin secretion in vivo in transgenic
mice.
To assess whether the hyperglycemia in homologous Line 1 mice and
heterologous Line 2 mice was associated with abnormal secretion of
islet hormones, serum insulin and glucagon levels were monitored in
separate glucose stimulation experiments. No significant differences were found in serum glucagon levels between the transgenic and control
mice (data not shown). As shown in Fig.
5A, fasting serum insulin
levels were significantly lower in Line 1 homozygotes [0.33 ± 0.02 (SD) ng/ml] compared with control C57Bl/6 mice of the same age
and sex (0.42 ± 0.02 ng/ml; P < 0.01) and
remained significantly lower after administration of glucose. The
increment over basal in Line 1 homozygous mice was also significantly
different between transgenic and control animals at both 30 min
[0.11 ± 0.07 (SD) vs. 0.55 ± 0.14; P < 0.001] and 90 min (0.12 ± 0.07 vs. 0.87 ± 0.08;
P < 0.001). Although there was a tendency for lower
fasting serum insulin levels in Line 2 heterozygotes compared with
nontransgenic littermates, the differences were not statistically significant either in the set of animals shown in Fig. 5B or
in a set of 10 transgenic and 10 control animals subsequently analyzed. The combined data for basal insulin levels obtained for 15 transgenic Line 2 mice and 15 nontransgenic littermates of the same sex and age
were 0.20 ± 0.10 (SD) ng/ml for transgenic mice vs. 0.26 ± 0.17 ng/ml for control mice (P < 0.15). Administration
of glucose, however, elicited significantly lower blood insulin levels
in Line 2 mice at both 30 and 90 min (Fig. 5B). The
increment above basal in Line 2 heterozygotes was also significantly
different between transgenic and control animals, at both 30 min:
0.15 ± 0.15 (transgenic) vs. 0.47 ± 0.18 ng/ml (controls)
(P < 0.025) and at 90 min: 0.31 ± 0.22 (transgenic) vs. 0.84 ± 0.19 ng/ml (controls) (P < 0.001). These results suggest that the fasting hyperglycemia
observed in Line 1 homozygotes and the hyperglycemia measured during a
glucose tolerance test in both lines are caused by a decrease in
glucose-induced insulin secretion.
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RIP7-hGAD65 Line 2 mice exhibit a selective inhibition of
first-phase insulin secretion in vitro.
To identify the nature of the inhibition of insulin secretion in
transgenic mice, the kinetics of insulin secretion during stimulation
with glucose and IBMX were examined using in vitro pancreas perfusion
of Line 2 heterozygotes. An initial perfusion without glucose
(31) showed no evidence of leakage or constitutive release
of insulin. Glucose at 7 mM was selected for the first stimulatory
step, because it approximates the basal glucose levels in those mice
(Fig. 6). It also is the level at which a
submaximal first-phase insulin release is consistently detected
(15) and can be used to assess either enhancement or
impairment of insulin secretion. This concentration of glucose elicited
a sharp first-phase insulin secretion from control pancreases (Fig. 6),
as previously reported (32). In contrast, the first-phase
insulin response of transgenic pancreases was significantly lowered
(17.38 ± 4.22 vs. 45.6 ± 5.07 ng/ml in nontransgenic
littermates; Fig. 6). Only a single time point at 38 min was
significantly suppressed during second-phase release in transgenic
mice. The perfused pancreas of both transgenic and control mice
responded to subsequent ascending levels of 11 and 22 mM glucose. The
declining insulin responses to higher glucose steps observed for both
transgenic and control mice (Fig. 6) have been previously shown
(15, 30) and were ascribed to a packet storage of insulin
with differing sensitivities to glucose, or alternatively to feedback
inhibition (43), also termed time-dependent inhibition
(42). In the presence of 22 mM glucose, IBMX, an inhibitor
of cAMP phosphodiesterase, potentiated the response to glucose with a
large and rapid increase in insulin secretion that was identical in
transgenic and control pancreases (Fig. 6). Hence, inhibition of
glucose-induced insulin secretion in RIP7-hGAD65 Line 2 heterozygous
mice primarily affects the first phase of insulin secretion in response
to physiological levels of glucose. The single time point that was
significantly suppressed during second-phase release could indicate an
additional minor effect on this phase.
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A defect before membrane depolarization of the pancreatic -cell.
Insulin secretion in response to glucose involves coordinated steps,
including 1) elevation of intracellular ATP levels as a
result of glucose metabolism; 2) closure of
sulfonylurea-sensitive KATP+ channels, and
-cell
depolarization; 3) activation of Ca2+ channels,
and oscillation of cytoplasmic Ca2+ levels; and
4) induction of exocytosis (1, 39). To assess whether the defect in first-phase insulin secretion in transgenic mice
is at a step before or after depolarization of the
-cell, K+ was used to artificially depolarize the
-cell and
bypass the closure of KATP+ channels in Line 2 heterozygotes. It has been shown that stimulation of the pancreas with
depolarizing concentrations of K+ alone primarily results
in first-phase insulin secretion (16). In such
experiments, glucose has an additive potentiating effect on insulin
release (18). Because we had found the response to glucose
to be atypical (Fig. 4), glucose was excluded in the experiments with
K+ to eliminate it as a variable and to allow a direct
measurement of the effect of depolarization on insulin release in the
transgenic and the normal pancreas. K+ at 20 mM stimulated
a sharp first-phase insulin response and a low prolonged insulin
secretion that were identical in transgenic and control mice (Fig.
7). A subsequent stimulation with
physiological glucose (7 mM) at 50 min again revealed a similar
defective first-phase insulin response in transgenic mice, much as when
glucose was used as the initial stimulus (compare Fig. 7 with Fig. 6).
Thus, in the same pancreas, both the normal response to K+
and the impaired response to glucose are demonstrable.
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DISCUSSION |
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In this study, we show that expression of the smaller isoform of
the GABA synthesizing enzyme GAD in pancreatic -cells of RIP7-hGAD65
transgenic mice results in elevated GAD enzyme activity and increased
GABA levels in
-cells. Line 1 heterozygotes, which have the lowest
expression of transgenic GAD65, do not display a phenotype. However,
homozygous mice of this line, which express almost twice the levels of
the enzyme, have the most severe phenotype and exhibit elevated fasting
blood glucose, impaired glucose tolerance, and inhibition of fasting as
well as glucose-induced insulin secretion. Line 2 heterozygotes, which
express ~1.6-fold the levels of Line 1 heterozygotes, have a more
subtle phenotype, which includes impaired glucose tolerance, inhibition
of insulin secretion in response to glucose, but normal fasting blood
glucose and insulin levels. Thus increasing levels of GAD65 expression
in the transgenic lines correlate with severity of the phenotype. Line
2 heterozygotes do not develop chronic hyperglycemia, diabetes, or
obesity over a lifespan of 2.5 yr (not shown). Line 1 homozygous mice
are currently being monitored over a prolonged period to assess these parameters.
Our in vitro studies, using the perfused pancreas in Line 2 heterozygotes, suggest that an inhibition of insulin secretion occurs
primarily at the first phase of insulin release stimulated at
physiological glucose levels. A single time point, however, was
significantly suppressed during second-phase release. Thus the
inhibitory effect may not be strictly limited to the first phase of
insulin secretion. These results are clearly distinct from a more
general inhibition of the insulin secretory pathway seen in perfusion
studies of some transgenic models, which overexpress membrane proteins
in pancreatic -cells (19). The normal potentiating effect of IBMX in RIP7-hGAD65 mice suggests that the inhibition of
first-phase insulin secretion does not involve cAMP-mediated signaling pathways.
Whereas the in vivo analysis of RIP7-hGAD65 Line 2 mice showed a consistently impaired insulin release at all glucose levels during glucose challenge, the in vitro pancreas perfusion analysis detected impairment only at the first step of glucose stimulation (7 mM) but not at subsequent steps of 11 mM and 22 mM glucose. It is possible that, in vitro, the inhibitory effect of the elevated GABA levels is obscured by the nature of stepwise increments in glucose concentration and the lack of feedback mechanisms by circulating inhibitors and potentiators (15, 42, 43).
It was shown previously that stimulation of the pancreas with
depolarizing concentrations of K+ in the absence of glucose
causes mainly first-phase insulin secretion (16). In the
present study, we found that the response to K+ in the
transgenic pancreas is normal. These results indicate that the -cell
machinery distal to
-cell depolarization, including Ca2+
channels and exocytosis of secretory vesicles, is intact in RIP7-hGAD65 transgenic mice. Thus inhibition of first-phase insulin secretion probably results from events occurring before membrane depolarization, in the glucose metabolic cascade, or at the K+ ATP channel
(1, 39).
How does GAD65/GABA expressed in -cells exert an inhibitory effect
on first-phase insulin secretion? At least two possible mechanisms can
be proposed. First, recent evidence suggests that glutamate may act as
a messenger that enters secretory vesicles and induces their priming to
become part of a readily releasable pool of granules (33).
The levels of GAD65 in RIP7-hGAD65 Line 2 islets are similar to the
endogenous levels of the protein in rat and human islets, in which the
protein is still relatively rare (4). It is possible,
however, that transgenic GAD65 significantly affects levels of
glutamate (substrate), resulting in an inhibition of priming. Such
inhibition would, however, be predicted to affect mainly second-phase
insulin release, which is inconsistent with the observations in
RIP7-hGAD65 mice. The second possibility is that the phenotype of
RIP7-hGAD65 mice is mediated by the elevated levels of GABA synthesized
by GAD65. Metabolism of GABA in the tricarboxylic acid cycle via the
GABA shunt (38) seems to be excluded, because this process
is likely to generate ATP and stimulate insulin secretion.
Alternatively, GABA accumulated in vesicles and secreted from the
-cell may mediate a paracrine or autocrine inhibitory effect on
insulin secretion via GABA receptors. GABAA receptors are
expressed on
- or
-cells (51), and GABAB
receptors are expressed on
-cells (Chang and Baekkeskov, unpublished
results). One hypothesis is that GABA secreted by
-cells inhibits
first-phase insulin secretion in an autocrine fashion via G
protein-linked GABAB receptors.
Earlier studies, using exogenous GABA, have reported a wide variety of
effects on -cell function, including a general inhibition of both
phases of insulin secretion, a stimulation of insulin secretion, or no
effect at all (37, 53 for review). The inherent contradiction of the
studies using exogenous GABA is not understood, but it may involve
differences in GABA uptake and/or transport in the different
experimental systems. Our results, with a transgenic model, which
increases GAD65 and GABA levels in
-cells, suggest that the action
of the endogenous transmitter synthesized in these cells is primarily,
although perhaps not exclusively, to regulate the first phase of
insulin secretion elicited by glucose at step(s) proximal to or at the
KATP+ channel.
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ACKNOWLEDGEMENTS |
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We thank John Kim, Ann Neill, Raquel Nagal, and Mary Ann Jones for excellent technical assistance, and Patti Keefe for help with references.
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FOOTNOTES |
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This study was supported by National Institutes of Health Grants R01 DK-47043 (to S. Baekkeskov) and P01 DK-41822 (to S. Baekkeskov and D. Hanahan), the Nora Eccles Treadwell Foundation (to S. Baekkeskov), an American Diabetes Association Mentor-Based Fellowship (to S. Baekkeskov), and a Fellowship Award from the Juvenile Diabetes Foundation International (to Y. Shi).
Present address of Y. Shi: Diabetes Research, Endocrine Division, Lilly Research Laboratories, Eli Lilly and Co., Indianapolis, IN 46285.
Address for reprint requests and other correspondence: S. Baekkeskov, Hormone Research Institute, Univ. of California San Francisco, 513 Parnassus Ave., Rm. HSW 1090, San Francisco, CA 94143-0534 (E-mail: s_baekkeskov{at}biochem.ucsf.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Received 5 August 1999; accepted in final form 25 April 2000.
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