Inhibition of glucose- and calcium-induced insulin secretion from βTC3 cells by novel inhibitors of protein isoprenylation

Article abstract of DOI:10.1124/jpet.102.036160

The majority of low molecular weight G proteins undergoes a series of post-translational modification steps, e.g., isoprenylation, at their C-terminal cysteine, which seem to be critical for the transport of the modified proteins to the membrane sites for interaction with their respective effector proteins. Using lovastatin, an inhibitor of mevalonic acid, and hence, isoprenoid biosynthesis, we demonstrated previously that protein isoprenylation is critical for physiological insulin secretion from normal rat islets. Herein, we used more selective synthetic inhibitors of protein prenylation to examine their effects on glucose- and calcium-mediated insulin secretion from βTC3 cells. Both 3-allyl- and vinylfarnesols, which inhibit and/or modulate protein farnesyl transferases, significantly (80-95%) inhibited glucose-and KCI-stimulated insulin secretion from these cells. In a similar manner, the allyl and vinyl forms of geranylgeraniol, reagents targeted toward protein geranylation, attenuated insulin secretion elicited by glucose and KCI. Furthermore, manumycin A, a natural inhibitor of protein farnesylation, and geranylgeranyl transferase inhibitor-2147 (GGTI-2147), a peptidomimetic inhibitor of protein geranylgeranylation, also inhibited glucose-and KCI-induced insulin secretion to comparable degrees. Treatment of βTC3 cells with either 3-vinylfarnesol or 3-vinyl geranylgeraniol resulted in accumulation of unprenylated proteins in the cytosolic fraction. These data further support our original formulation that inhibition of isoprenylation of small molecular weight G proteins might impede their interaction with their putative effectors, which may be required for physiological insulin secretion.

Full text of DOI:10.1124/jpet.102.036160

0022-3565/02/3031-82–88
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
U.S. Government work not protected by U.S. copyright
JPET 303:82–88, 2002
Vol. 303, No. 1
36160/1006171
Printed in U.S.A.
Inhibition of Glucose- and Calcium-Induced Insulin Secretion
from ␤TC3 Cells by Novel Inhibitors of Protein Isoprenylation
RAJESH AMIN, HAI-QING CHEN, MARIE TANNOUS, RICHARD GIBBS, and ANJANEYULU KOWLURU
Department of Pharmaceutical Sciences, Wayne State University, and ␤ Cell Biochemistry Research Laboratory, John D. Dingell VA Medical
Center, Detroit, Michigan (R.A., H.-Q.C., M.T., A.K.); and Department of Medicinal Chemistry and Molecular Pharmacology, School of
Pharmacy and Pharmacal Sciences, Purdue University, West Lafayette, Indiana (R.G.)
Received March 13, 2002; accepted May 28, 2002
ABSTRACT
The majority of low molecular weight G proteins undergoes a ilar manner, the allyl and vinyl forms of geranylgeraniol, re-
series of post-translational modification steps, e.g., isopreny- agents targeted toward protein geranylation, attenuated insulin
lation, at their C-terminal cysteine, which seem to be critical for secretion elicited by glucose and KCl. Furthermore, manumycin
the transport of the modified proteins to the membrane sites for A, a natural inhibitor of protein farnesylation, and geranylgera-
interaction with their respective effector proteins. Using lova- nyl transferase inhibitor-2147 (GGTI-2147), a peptidomimetic
statin, an inhibitor of mevalonic acid, and hence, isoprenoid inhibitor of protein geranylgeranylation, also inhibited glucose-
biosynthesis, we demonstrated previously that protein isopre- and KCl-induced insulin secretion to comparable degrees.
nylation is critical for physiological insulin secretion from normal Treatment of ␤TC3 cells with either 3-vinylfarnesol or 3-vinyl
rat islets. Herein, we used more selective synthetic inhibitors of geranylgeraniol resulted in accumulation of unprenylated pro-
protein prenylation to examine their effects on glucose- and teins in the cytosolic fraction. These data further support our
calcium-mediated insulin secretion from ␤TC3 cells. Both 3-al- original formulation that inhibition of isoprenylation of small
lyl- and vinylfarnesols, which inhibit and/or modulate protein molecular weight G proteins might impede their interaction with
farnesyl transferases, significantly (80–95%) inhibited glucose- their putative effectors, which may be required for physiological
and KCl-stimulated insulin secretion from these cells. In a sim- insulin secretion.
Most low molecular weight G proteins and the ␥ subunits nylated cysteine by a protease of microsomal origin, resulting
of trimeric G proteins possess a specific C-terminal sequence
(referred to as the CAAX motif), which makes them suitable
candidates for a series of post-translational modifications
(Clarke, 1992; Takai et al., 1992; Casey, 1995; Wedegaertner
et al., 1995; Kowluru et al., 2000). The first of these modifi-
cations involves attachment of either a 15-carbon (referred to
as farnesylation) or a 20-carbon (referred to as geranylgera-
nylation) derivative of MVA to the cysteine via a thioether
linkage. These reactions are catalyzed by protein farnesyl- or
geranylgeranyl-transferases, respectively (Casey et al., 1989;
Kato et al., 1992; James et al., 1993; Kohl et al., 1993;
Kowluru et al., 2000). Subsequent modifications include,
cleavage of the three terminal amino acids after the isopre-
in the exposure of the carboxylate anion of the prenylated
cysteine residue (Hancock et al., 1991; Clarke 1992; Kowluru
et al., 1996a). This site is further subjected to methylation by
a prenyl cysteine methyltransferase, which neutralizes the
carboxylate anion, thus making the candidate G protein more
hydrophobic, resulting in its translocation to the membrane
sites for interaction with their respective effector proteins
(Clarke, 1992). Besides the aforementioned modifications,
certain G proteins (e.g., H- and N-Ras and the ␣ subunits of
trimeric G proteins) undergo acylation (typically, incorpora-
tion of palmitate) at a cysteine residue, which is frequently
upstream to the prenylated cysteine (Hancock et al., 1989;
Wedegaertner et al., 1995).
By using inhibitors of each of these modifications, previous
studies from our laboratory have demonstrated involvement
of these proteins in physiological insulin secretion (Li et al.,
1993; Metz et al., 1993). For example, using lovastatin
(LOVA), an inhibitor of MVA biosynthesis from 3-hydroxy-3-
methylglutaryl coenzyme A, which is a precursor of farnesyl
and geranylgeranyl pyrophosphates, we have reported inhi-
These studies are supported by grants from the Department of Veterans
Affairs (Merit Review grant and the Research Enhancement Award Program
grant; both to A.K.), the National Institutes of Health (1 R01-DK-56005-01 to
A.K. and 1 R01-CA 78819-01 to R.A.G.). A.K. is the recipient of a Career
Research Scientist Award from the Department of Veterans Affairs.
Article, publication date, and citation information can be found at
http://jpet.aspetjournals.org.
DOI: 10.1124/jpet.102.036160.
ABBREVIATIONS: MVA, mevalonic acid; LOVA, lovastatin; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium; ELISA, enzyme-linked
immunosorbent assay; GGTI-2147, geranylgeranyl transferase inhibitor-2147; 3vFOH, 3-vinylfarnesol; 3aFOH, 3-allylfarnesol; 3-vGGOH, 3-vinyl
geranylgeraniol; FTase, farnesyltransferase; GGTase, geranylgeranyl transferase I.
82

Role of Protein Prenylation in Insulin Secretion
83
bition of glucose-induced insulin secretion from normal rat
islets (Metz et al., 1993). Studies by Li et al. (1993) have
demonstrated similar inhibitory effects by LOVA on bomb-
esin- and vasopressin-induced insulin secretion from HIT-
T15 cells. In each case, inhibition of isoprenylation of ␤-cell G
proteins also resulted in altered subcellular distribution of
these proteins in these cells as evidenced by accumulation of
nonprenylated proteins in the cytosolic fraction (Li et al.,
1993; Metz et al., 1993; Kowluru and Metz, 1996). Further-
more, using acetyl farnesyl cysteine, a specific inhibitor of
Synthesis of 3-Allyl- and 3-Vinylfarnesols and Geranylge-
raniols. These compounds were synthesized (Figs. 1 and 2) via our
previously described vinyltriflate to isoprenoid analogs (Gibbs et al.,
1999). Details of the specificity of these inhibitors, in terms of their
ability to inhibit prenyl transferases, were described in detail in our
previous publications (Gibbs et al., 1999; see Discussion).
Insulin Release Studies. ␤TC3 cells were cultured overnight in
Dulbecco’s modified Eagle’s medium containing 5 mM glucose and
10% fetal calf serum in the presence of diluent alone or various
concentrations of inhibitors as indicated in the text. After preincu-
bation in the presence of 3.3 mM glucose, cells were then incubated
prenyl cysteine methyltransferases and/or cerulenin, a spe- in the presence of either 3 or 20 mM glucose for 45 min at 37°C. The
supernatant was then removed, centrifuged at 300g for 10 min, and
assayed for insulin. To assess insulin content, cells were extracted
overnight in acid/ethanol mixture as described previously (Metz et
al., 1993). The amount of insulin was quantitated by ELISA (Amer-
ican Laboratory Products Company; Kowluru et al., 2001).
Cell Viability Assay. ␤TC3 cells were seeded at a density of 1 ϫ
106 cells/ml in round-bottomed 96-well plates and then treated with
inhibitors (as described above). Cell viability was determined by a
colorimetric assay (at 550–690 nm), using MTT, which measures the
reduction of 3-(4,5-dimethylthiazolyl-2) 2,5-diphenyltetrazolium bro-
cific inhibitor of fatty acylation, we (Metz et al., 1993;
Kowluru et al., 2000) have provided evidence for the regula-
tory roles of these post-translational modification steps in
physiological insulin secretion; these findings have been sub-
sequently confirmed by other investigators (Deeney et al.,
2000; Yajima et al., 2000; Straub et al., 2002).
The current study further explores the roles of protein
isoprenylation steps in physiological insulin secretion from
insulin-secreting clonal ␤ (␤TC3) cells. We felt a critical need
for these studies because LOVA, which was used in previous mide into the blue formazan product by metabolically active cells.
Subcellular Distribution of G Proteins: GTP Overlay Assay.
To identify the effects of our inhibitors on the hydrophobicity of G
proteins, we used the [␣-32P]GTP overlay assay. Experimental de-
tails for this method, which determine the relative abundance of the
G proteins in total membrane and soluble fractions, have been de-
scribed in Li et al. (1993), Kowluru et al. (1994), and Kowluru and
Metz (1996). Briefly, homogenates of control and inhibitor-treated
cells were centrifuged at 105,000g for 90 min in a TL-100 ultracen-
trifuge (Beckman Coutler, Inc., Fullerton, CA). The supernatant and
pellets were designated as the cytosol and membrane fractions, re-
studies, also inhibits both sterol and nonsterol limbs of the
cholesterol pathways; affecting many compounds that could
have specific functional roles in the ␤ cell (Li et al., 1993;
Metz et al., 1993; Kowluru and Metz, 1996). Specifically,
LOVA inhibits the biosynthesis of both farnesyl and gera-
nylgeranyl pyrophosphates and, thus, it is difficult to deter-
mine precisely which of the two signaling pathways (e.g.,
farnesylation or geranylgeranylation) is critical for the reg-
ulation of insulin secretion from the islet ␤ cell. With these
facts in mind, we undertook the present study to evaluate spectively. They were separated by SDS-PAGE (12.5%) and trans-
ferred to a nitrocellulose membrane using a transblot apparatus
(Bio-Rad, Hercules, CA). The blots were then washed in a medium
containing 50 mM Tris-HCl pH 7.4, 5 mM MgCl2, 1 mM EGTA, and
0.3% Tween 20 for 10 min. The membranes were incubated in the
above-described medium containing [␣-32P]GTP (1 ␮Ci/ml) for 1 h at
room temperature followed by extensive washing in the same buffer
to remove unbound label from the membranes. Labeling of proteins
was determined by autoradiography using an X-OMAT film (East-
specific inhibitors of farnesyl and geranylgeranyl trans-
ferases and examine their effects on glucose- and calcium-
mediated insulin secretion from ␤TC3 cells to further exam-
ine the roles of these modification steps in glucose- and
calcium-induced insulin secretion. To provide corroboration
for these results, we compared the effects of other commer-
cially available inhibitors of protein farnesylation and gera-
nylgeranylation on glucose- and KCl-induced secretion from
these cells. Our data clearly support the original formulation
that both farnesylation and geranylgeranylation of proteins
are necessary for glucose- and calcium-mediated exocytotic
secretion of insulin.
Methods and Materials
Materials. Rat Insulin ELISA kit was purchased from American
Laboratory Products Company (Windham, NH). [␣-32P]GTP was
purchased from Amersham Biosciences (Piscataway, NJ). Enhanced
chemiluminescence reagents and Hyper film were from Amersham
Biosciences. GGTI-2147 was purchased from Calbiochem (San Diego,
CA). Manumycin A was obtained from Sigma-Aldrich (St. Louis,
MO). MTT assay reagents were purchased from Roche Applied Sci-
ence (Indianapolis, IN). All other reagents used in the present study
were of analytical grade and were of highest purity available.
Cell Culture. ␤TC3 cells were kindly provided by Dr. Shimon
Efrat (Sackler School of Medicine, Tel Aviv University, Tel Aviv,
Israel). Cells were cultured in Dulbecco’s modified Eagle’s medium
containing 25 mM glucose supplemented with 10% fetal bovine se-
rum, 100 IU/ml penicillin, 100 IU/ml streptomycin, and 2 mM L-
glutamine under 95% O2/5% CO2 (Efrat et al., 1990) The medium
was changed twice weekly and cells were trypsinized and subcloned
weekly. Cells were used between passages 20 and 65.
Fig. 1. Scheme for the synthesis of 3vFOH and 3alFOH. a, nBuLi; THF,
0°C; geranyl bromide, 0°C to room temperature. b, (Me3Si)2NK, THF,
Ϫ78°C; AvN (SO2CF3)2, Ϫ78°C and brought to room temperature. c,
vinylSnBu3, Pd (AsPh3)2, CuI, NMP, room temperature. d, allylSnBu3,
Pd(AsPh3)2, CuI, NMP, 80°C. e, DIBAL-H, PhMe, Ϫ78°C.

84
Amin et al.
Fig. 2. Scheme for the synthesis of 3VGGOH and 3alGGOH. a, nBuLi;
THF, 0°C; farnesyl bromide, 0°C to room temperature. b, (Me3Si)2NK,
THF, Ϫ78°C; AvN (SO2CF3)2, Ϫ78°C and brought to room temperature. c,
vinylSnBu3, Pd (AsPh3)2, CuI, NMP, room temperature. d, allylSnBu3,
Pd(AsPh3)2, CuI, NMP, 80°C. e, DIBAL-H, PhMe, Ϫ78°C.
Fig. 3. Inhibition by 3vFOH of glucose- or KCl-induced insulin secretion
from ␤TC3 cells. Cells were incubated (for 18 h) in the presence of varying
concentrations (0–20 ␮M) of 3vFOH as indicated in the figure. After
incubation in low glucose (3 mM for 45 min), cells were then incubated in
Krebs-Ringer medium for 45 min in the presence of either 20 mM glucose
or 40 mM KCl in the continuous presence of either the inhibitor or the
diluent as indicated in the figure. Insulin was quantitated by ELISA (see
Materials and Methods). Data are expressed as mean Ϯ S.E.M. from
three different experiments carried out in triplicate. ૺ, p Ͻ 0.05 versus
the control value in the absence of inhibitor.
man Kodak, Rochester, NY). Molecular weights of labeled proteins
were determined using prestained, authentic molecular weight stan-
dards (Bio-Rad). Gel scanning was performed using UN-SCAN-IT
software and Adobe Photoshop (Adobe Systems, Mountain View,
CA).
Results
Insulin release elicited in the presence of either a stimu-
latory concentration (20 mM) of glucose or a depolarizing
concentration (40 mM) of KCl was measured in ␤TC3 cells in
the absence or presence (0–20 ␮M) of 3-vinylfarnesol
(3vFOH). Data in Fig. 3 indicate minimal effects of this
compound on glucose-stimulated insulin secretion up to 10
␮M. However, at 20 ␮M, glucose-induced insulin secretion
was completely abolished by 3vFOH. Potassium-induced in-
sulin release was inhibited by 80% in the presence of 10 ␮M
3vFOH, and a comparable degree of inhibition was demon-
strable even at 20 ␮M of this compound. Data in Fig. 4
demonstrate inhibition by 3-allylfarnesol (3aFOH) of glucose-
or KCl-induced insulin secretion from ␤TC3 cells. At 10 ␮M
3aFOH, the glucose- and KCl-induced insulin secretion was
inhibited by 83 and 94%, respectively. These data indicate
potential regulation by protein farnesylation of glucose- and
calcium-induced insulin secretion from isolated ␤ cells.
We next examined the effects of inhibitors of protein gera-
nylgeranylation on glucose- and KCl-mediated insulin secre-
tion. Data in Fig. 5 demonstrate significant attenuation in
glucose- and calcium-induced insulin secretion by 3-vinyl-
geranylgeraniol (3-vGGOH). We observed complete inhibi-
tion by 3-vGGOH (20 ␮M) on glucose-induced insulin secre-
tion, whereas KCl-induced secretion was inhibited by Ͼ90%
under similar experimental conditions. Comparable degrees
of inhibition of KCl-induced insulin secretion were demon-
strable in the presence of 20 ␮M 3-allyl geranylgeraniol (ad-
ditional data not shown). These data indicate a requirement
Fig. 4. Inhibition by 3aFOH of glucose- or KCl-induced insulin secretion
from ␤TC3 cells. Cells were incubated (for 18 h) in the presence of varying
concentrations (0–10 ␮M) of 3aFOH as indicated in the figure. After
incubation in low glucose (3 mM for 45 min), cells were then incubated in
Krebs-Ringer medium for 45 min in the presence of either 20 mM glucose
or 40 mM KCl in the continuous presence of inhibitor or diluents as
indicated in the figure. Insulin was quantitated by ELISA (see Materials
and Methods). Data are expressed as mean Ϯ S.E.M. from three different
experiments carried out in triplicate. ૺ, p Ͻ 0.05 versus the control value
in the absence of inhibitor.
for protein farnesylation and geranylgeranylation modifica- next studied the effects of two commercially available inhib-
tion steps in glucose- as well as calcium-induced insulin itors of protein farnesylation and geranylgeranylation on
secretion from the ␤TC3 cells.
glucose or KCl-induced insulin secretion. Manumycin, a nat-
To further solidify our observations that protein farnesy- ural substance isolated from Streptomyces parvulus, is a
lation and geranylation are critical to insulin secretion, we selective farnesyl pyrophosphate competitive inhibitor of far-

Role of Protein Prenylation in Insulin Secretion
85
suggest that both farnesylation and geranylation of proteins
are critical to glucose as well as calcium-induced insulin
secretion ␤TC3 cells.
To assess whether the effects of these inhibitors on insulin
secretion are not due to their effects on insulin synthesis, we
measured the total insulin content in control and inhibitor-
treated (18 h; 10 ␮M) cells. Data from these studies indicated
no major differences in insulin content between the control
and inhibitor-treated ␤TC3 cells. For example, these values
represented 96 Ϯ 5 and 109 Ϯ 3% of control cells when they
were exposed to 3aFOH and 3vFOH, respectively (mean Ϯ
variance from two experiments). However, a modest reduc-
tion (78 Ϯ 3% of control; mean Ϯ variance from two experi-
ments) in insulin content was demonstrable in cells treated
with 3vGGOH. These data indicate a possible modulatory
role for geranylgeranylated proteins in insulin biosynthesis
as well.
We also examined the effects of our inhibitors on the met-
abolic viability of ␤ TC3 cells under the conditions they
inhibited glucose- and KCl-stimulated insulin secretion.
Data in Fig. 8 indicated no significant effects of either the
allyl- or the vinylfarnesyl transferase inhibitors (0–20 ␮M)
on the viability of these cells. A modest (Ϫ16%), but insignif-
icant (p ϭ 0.33) loss in the viability of cells was demonstrable
at 20 ␮M GGTI-2147. These data suggest that the inhibition
of glucose- and KCl-induced insulin secretion by these inhib-
itors is largely due to their ability to inhibit protein preny-
lation, and not due to their nonspecific cytotoxic effects.
Previous studies (Li et al., 1993; Metz et al., 1993; Kowluru
and Metz, 1996) have demonstrated that inhibition of protein
isoprenylation by LOVA, an inhibitor of 3-hydroxy-3-methyl-
glutaryl coenzyme A reductase, resulted in accumulation of
unprenylated G proteins in the cytosolic fraction in normal
rat islets and clonal ␤ cells. Based on those findings, we
Fig. 5. Inhibition by 3vGGOH of glucose- or KCl-induced insulin secre-
tion from ␤TC3 cells. Cells were incubated (for 18 h) in the presence of
varying concentrations (0–20 ␮M) of 3vGGOH as indicated in the figure.
After incubation in low glucose (3 mM for 45 min), cells were then
incubated in Krebs-Ringer medium for 45 min in the presence of either 20
mM glucose or 40 mM KCl in the continuous presence of either the
inhibitor or diluent as indicated in the figure. Insulin was quantitated by
ELISA (see Materials and Methods). Data are expressed as mean Ϯ
S.E.M. from three different experiments carried out in triplicate. ء, p Ͻ
0.05 versus the control value in the absence of inhibitor.
nesyl transferase (Tannous et al., 2001). At 10 ␮M concen-
tration, manumycin A markedly reduced both glucose- and
KCl-induced insulin release (Fig. 6), further confirming data
described in Figs. 3 and 4. Likewise, GGTI-2147, a peptido-
mimetic inhibitor of geranylgeranyl transferases, at 20 ␮M
inhibited glucose- and KCl-induced secretion by 95 and 65%,
respectively (Fig. 7). Together, data in Figs. 3 to 7 clearly
Fig. 6. Inhibition by manumycin A of glucose- or KCl-induced insulin
secretion from ␤TC3 cells. Cells were incubated (for 18 h) in the absence
or presence of manumycin (10 ␮M) as indicated in the figure. After
incubation in low glucose (3 mM for 45 min), cells were then exposed in
Krebs-Ringer medium for 45 min in the presence of either 20 mM glucose
or 40 mM KCl in the continuous presence of either manumycin or diluent
as indicated in the figure. Insulin was quantitated by ELISA (see Mate-
rials and Methods). Data are expressed as incremental response to either
20 mM glucose or 40 mM KCl and are expressed as mean Ϯ S.E.M. from
three different experiments carried out in triplicate. ૺ, p Ͻ 0.05 versus
the control value in the absence of inhibitor.
Fig. 7. Inhibition by GGTI-2147 of glucose- and KCl-induced secretion
from ␤TC3 cells. Cells were incubated (for 18 h) in the presence of varying
concentrations (0–20 ␮M) of GGTI-2147 as indicated in the figure. After
incubation in low glucose (3 mM for 45 min), cells were then incubated in
Krebs-Ringer medium for 45 min in the presence of either 20 mM glucose
or 40 mM KCl in the continuous presence of either GGTI-2147 or diluent
as indicated in the figure. Insulin was quantitated by ELISA (see Mate-
rials and Methods). Data are expressed as mean Ϯ S.E.M. from three
different experiments carried out in triplicate. ૺ, p Ͻ 0.05 versus the
control value in the absence of inhibitor.

86
Amin et al.
Fig. 9. Altered subcellular distribution of GTP-binding proteins in ␤TC3
cells after exposure to 3vFOH or 3vGGOH; GTP overlay assay. Cytosolic
and membrane proteins (30 ␮g/lane) from control or inhibitor-treated
cells (as indicated in figure) were separated by SDS-PAGE and trans-
ferred to a nitrocellulose membrane. After transfer, the membrane was
incubated in a binding buffer containing [␣-32P]GTP. After incubation,
membranes were washed extensively and labeled bands were identified
by autoradiography (see Materials and Methods for additional details).
Data are representative of two experiments with similar results. CON,
control; FTI, 3vFOH; GGTI, 3vGGOH; C, cytosol; and M, membrane.
Discussion
One of the main objectives of this study is to further ex-
amine the contributory roles of protein farnesylation and
geranylgeranylation in glucose- and calcium-induced insulin
secretion from isolated ␤ cells. Our data clearly support orig-
inal observations from our laboratory (Metz et al., 1993) and
those of others (Li et al., 1993) suggesting that isoprenylation
steps are critical to physiological insulin secretion. To the
best of our knowledge, there have been only two studies that
examined the contributory roles of protein isoprenylation in
Fig. 8. Lack of effects of farnesyl- and geranylgeranyl transferase inhib-
itors on metabolic cell viability of ␤TC3 cells. Metabolic cell viability was
determined using an MTT assay (see Materials and Methods for addi-
tional details) in ␤TC3 cultured in the presence of various inhibitors for
18 h as indicated in the figure. Data are mean Ϯ variance from two
different experiments carried out in triplicate.
proposed that inhibition of glucose-induced insulin secretion insulin secretion. Using LOVA, Li et al. (1993) first reported
by LOVA might, in part, be due to the accumulation of critical that inhibition of isoprenylation resulted in significant atten-
G proteins in the cytosolic fraction, thereby impeding the uation in bombesin and vasopressin potentiation of nutrient-
interaction of the candidate G proteins with their membrane- induced insulin secretion from HIT-T15 cells. These investi-
associated effector proteins. To further confirm our hypoth- gators have also provided evidence to indicate abnormalities
esis and to determine the putative modes of action of these in subcellular distribution (i.e., significant accumulation of
inhibitors, we examined the subcellular distribution (i.e., unprenylated proteins in the cytosolic fraction) of small G
membrane versus cytosolic) of the ␤-cell G proteins in control proteins in LOVA-treated cells. Based on these data, they
and inhibitor-treated cells. To address this, isolated ␤TC3 proposed that protein isoprenylation plays a critical regula-
cells were exposed to these inhibitors overnight, and the tory role in bombesin and vasopressin-induced insulin secre-
relative abundance of GTP-binding proteins in the total sol- tion from HIT-T15 cells. At the same time, independent stud-
uble and membranous fractions was determined by the ies from our laboratory have provided evidence to suggest
[␣-32P]GTP overlay assay (see Materials and Methods for that protein isoprenylation, carboxyl methylation, and fatty
additional details). Data in Fig. 9 indicate that exposure of acylation steps play important regulatory roles in physiolog-
␤TC3 cells either to farnesols or geranylgeraniols results in ical insulin secretion from isolated rat islets (Metz et al.,
significant increase in the abundance of soluble G proteins, 1993). We have shown that pretreatment of isolated rat islets
as evidenced by increase in the labeling in the soluble frac- with LOVA resulted in significant inhibition of glucose-in-
tion. A corresponding decrease in the abundance of G pro- duced insulin secretion; such an inhibition was reversed by
teins in the membrane fraction was also demonstrable (Fig. exogenous provision of MVA in the culture medium indicat-
9). These data clearly indicate that treatment of isolated ␤ ing that LOVA-induced inhibition of insulin secretion is due
cells with either the farnesols or geranylgeraniols results in to its inhibition of MVA (and subsequent isoprenoid) biosyn-
abnormal subcellular distribution of the candidate G pro- thesis. Under conditions in which LOVA inhibited insulin
teins, presumably interfering with interaction of these sig- secretion, we have also demonstrated significant accumula-
naling proteins with their effector proteins in the membrane tion of unprenylated G proteins in the cytosolic fraction.
fraction. These data also imply that the abundance of isopre- Together, these studies provided initial evidence for the con-
nylated proteins within the ␤ cell is significantly decreased in tributory roles of isoprenylation in insulin secretion.
cells treated with inhibitors of protein prenylation as evi-
The current studies form a logical extension to our original
denced by the accumulation of nonprenylated proteins in the studies in the sense that they use more specific inhibitors of
cytosolic fraction (Fig. 9). We have provided evidence in our these pathways (i.e., farnesylation and geranylgeranylation)
earlier studies that such an interaction of specific proteins to study their individual roles in both glucose- and KCl-
(e.g., Cdc42) with their effector proteins (e.g., phospholipase mediated secretion. We have been able to further establish
C) is critical for physiological insulin secretion (Kowluru et the roles of these modification steps in insulin secretion by
al., 1996a).
using commercially available inhibitors as well. It may also

Role of Protein Prenylation in Insulin Secretion
87
be mentioned that the purpose of the current studies was not alogs have similar effects in the present study on insulin
to identify the ␤-cell endogenous farnesylated and gera- secretion. This may seem surprising in view of the fact that
nylgeranylated G proteins that are required for physiological the vinyl analogs are alternative substrates. These differ-
insulin secretion. Several previous studies have identified ences may, in part, be due to differences in the cell types used
the candidate G proteins that may be required for insulin (i.e., NIH3T3 cells versus the pancreatic ␤ cells used in this
secretion (Kowluru and Metz, 1994; Kowluru et al., 1994, study). However, recent preliminary unpublished data in our
1996a,b,c, 1997a,b, 2000; Robertson et al., 1991; Regazzi et laboratory have suggested that 3-vinyl-FPP may be an alter-
al., 1992; Leiser et al., 1995; Sharp, 1996; Lang, 1999; native substrate with some proteins (such as H-Ras), but an
Kowluru and Amin, 2002). Instead, the purpose of the inhibitor with other proteins (such as RhoB). These in vitro
present studies was to synthesize more specific inhibitors to data suggest that the in vivo mechanism of action of the vinyl
probe for the roles of protein farnesylation and geranylgera- analogs is complex. However, the combined use of the allyl
nylation in insulin secretion. In addition to their utility in and vinyl analogs may provide more information on the sig-
studies that examined putative contributory roles of these nificance of various prenylated proteins in signaling path-
modification steps in physiological insulin secretion (this ways.
study), we recently used these inhibitors to demonstrate the
Interestingly, in our previous studies, we have observed
roles of farnesylated proteins (e.g., H-Ras) in the signaling only a modest, but significant (Ϫ30%) inhibition by LOVA
cascade leading to interleukin-induced nitric oxide release elicited by depolarizing concentrations of potassium (Metz et
from normal rat islets and clonal ␤ (HIT-T15, RIN5F, and al., 1993). In the present study, we observed a significant
INS-1) cells (Tannous et al., 2001; Kowluru and Amin, 2002; inhibition in KCl-induced insulin secretion by both FTI and
Kowluru and Morgan, 2002). Therefore, these inhibitors GGTIs in ␤TC3 cells. Even though these findings (i.e., inhi-
might subserve the roles as valuable tools to study the reg- bition of KCl-induced insulin secretion) are directionally sim-
ulatory function(s) of specific G proteins in various aspects of ilar, reasons underlying such pronounced differences (i.e.,
␤-cell function. It should be noted that the 3-allyl- and about 30% inhibition by LOVA in contrast to Ͼ80–90% inhi-
3-vinylfarnesols and geranylgeraniol analogs have similar bition by farnesols and geranylgeraniols) remain unclear at
effects in the present study on insulin secretion in ␤TC3 cells. this time. It may be due to the specificity of the inhibitors
The key issue of the selectivity of the farnesylation and used in the present study as well as differences in cell types
geranylgeranylation inhibitors has been addressed in some used in the two studies (i.e., rat islets in LOVA experiments
detail. Previous studies have demonstrated that 3-allyl-FPP, and ␤TC3 cells in this study). Our data indicate relatively a
the active form of the prodrug 3-allylfarnesol, exhibits greater degree of inhibition of KCl-induced insulin secretion
ϳ1600-fold selectivity for the inhibition of farnesyltrans- by these inhibitors compared with their effects on glucose-
ferase (FTase) versus geranylgeranyl transferase I (GGTase induced insulin secretion. Based on these data, we speculate
I) (Gibbs et al., 1999). In a similar manner, 3-vinyl-FPP, the that isoprenylated proteins might exert differential contrib-
active form of 3-vinylfarnesol, is an effective and tight-bind- utory roles in insulin secretory responses elicited by glucose
ing substrate for FTase, and exhibits no productive interac- or KCl.
tion with GGTase I (selectivity for FTase/GGTase I, ϳ600-
Several previous studies have demonstrated regulatory
fold). The in vitro selectivity of 3-allyl-FPP is reflected in the roles for both trimeric and small molecular weight G proteins
in vivo selectivity of 3-allylfarnesol, which blocks the growth in insulin secretion (Robertson et al., 1991; Sharp, 1996;
of NIH3T3 cells transformed with the oncogenic variant of Kowluru et al., 2000). Most of these studies used pharmaco-
farnesylated H-Ras, but not the growth of NIH3T3 cells logical inhibitors or select bacterial toxins to identify these
transformed with the oncogenic variant of geranylgerany- two classes of G proteins. For example, using acetyl farne-
lated H-Ras, consistent with a lack of intracellular effect on sylcysteine, an inhibitor of prenyl cysteine methyl trans-
protein geranylgeranylation. Evaluation of 3-vinylfarnesol ferases, it has been shown that carboxyl methylation of ␥
versus these two NIH3T3 cell lines demonstrated only mod- subunits of trimeric G proteins (Kowluru et al., 1997a) as
est selectivity of this compound for the farnesylated H-Ras well as small G proteins, such as Cdc42, Rac, and Rap, may
cell line, which may be consistent with a potentially different be involved in glucose- and calcium-mediated insulin secre-
mechanism of action for this compound (vide infra). A similar tion (Leiser et al., 1995; Kowluru et al., 1996a; Kowluru et
in vivo pattern was observed with the geranylgeraniol ana- al., 1997b). Likewise, using cerulenin, a specific inhibitor of
logs and the two NIH3T3 cell lines; 3-allyl-geranylgeraniol protein palmitoylation, several previous studies have dem-
exhibits excellent selectivity for the geranylgeranylated H- onstrated that acylation of proteins is relevant to insulin
Ras cell line versus the farnesylated variant, whereas 3-vi- secretion (Metz et al., 1993; Yajima et al., 2000; Straub et al.,
nylgeranylgeraniol exhibits only modest selectivity between 2002). Some of the candidate proteins for palmitoylation in-
the two cell lines. However, 3-allyl-GGPP and 3-vinyl-GGPP clude, but are not limited to, the Ras subfamily of small G
exhibit no selectivity for GGTase I in vitro. We have previ- proteins as well as the ␣ subunits of trimeric G proteins.
ously suggested that the observed cellular selectivity for the Furthermore, bacterial toxins have been used in previous
geranylgeraniol analogs is due to a low intracellular concen- studies to assign roles for these proteins in modulation of
tration of the natural GGTase I substrate GGPP, relative to insulin secretion. For example, using clostridial toxins that
the FTase substrate FPP. However, this has not been con- specifically monoglucosylate and inhibit functions of Rho
clusively demonstrated, and thus we have also used the al- subfamily of small G proteins (Kowluru and Metz, 1994;
ternative GGTase I inhibitor GGTI-2147, which exhibits a Kowluru et al., 1997b), we demonstrated inhibition of both
Ͼ60-fold in vivo selectivity for the inhibition of protein gera- glucose- and KCl-induced insulin secretion from normal rat
nylgeranylation (Vasudevan et al., 1999). It should be noted islets and clonal ␤ cells. Some of the farnesylated proteins
that the 3-allyl- and 3-vinylfarnesol and geranylgeraniol an- identified in the ␤ cell include Ras G proteins (Kowluru et al.,

88
Amin et al.
gamma subunits of trimeric GTP-binding proteins in pancreatic ␤ cells. Modula-
tion in vivo by calcium, GTP and pertussis toxin. J Clin Invest 15:1596–1610.
Kowluru A, Li G, Rabaglia ME, Segu VB, Hofmann F, Aktories K, and Metz SA
(1997b) Evidence for differential roles of the Rho subfamily of GTP-binding pro-
teins in glucose- and calcium-induced insulin secretion from pancreatic ␤ cells.
Biochem Pharmacol 54:1097–1108.
Kowluru A and Metz SA (1994) Stimulation by prostaglandin E2 of a high-affinity
GTPase in the secretory granules of normal rat and human pancreatic islets.
Biochem J 297:399–406.
Kowluru A and Metz SA (1996) Subcellular distribution and posttranslational mod-
ifications of GTP-binding proteins in insulin-secreting cells. Methods Neurosci
29:298–318.
Kowluru A and Morgan NG (2002) GTP-binding proteins in cell survival and demise:
the emerging picture in the pancreatic ␤ cell. Biochem Pharmacol 63:1027–1035.
Kowluru A, Rabaglia ME, Muse KE, and Metz SA (1994) Subcellular localization and
kinetic characterization of guanine nucleotide binding proteins in normal rat and
human pancreatic islets and transformed ␤ cells. Biochim Biophys Acta 1222:348–
359.
Kowluru A, Robertson RP, and Metz SA (2000) GTP-binding proteins in the regula-
tion of pancreatic ␤ cell function, in Diabetes Mellitus: A Fundamental and Clinical
Text (LeRoith D, Taylor SI, and Olefsky JM eds) pp 78–94, Lippincott Williams &
Wilkins, Philadelphia.
Kowluru A, Seavey SE, Rhodes CJ, and Metz SA (1996b) A novel regulatory mech-
anism for trimeric GTP-binding proteins in the membrane and secretory granule
fractions of human and rodent ␤ cells. Biochem J 313:97–107.
Kowluru A, Seavey SE, Li G, Sorenson RL, Weinhaus A, Nesher R, Rabaglia ME,
Vadakekalam J, and Metz SA (1996c) Glucose-and GTP-dependent stimulation of
the carboxyl methylation of Cdc42 in rodent and human pancreatic islets and pure
␤ cells. Evidence for an essential role of GTP-binding proteins in nutrient-induced
insulin secretion. J Clin Invest 98:540–555.
2000; Tannous et al., 2001; Kowluru and Amin, 2002), ␥
subunits of trimeric G proteins (Kowluru et al., 1996b), and
the nuclear lamin B (Kowluru, 2000). Examples of geranyl
geranylated proteins include Cdc42, Rac, and Rap (Regazzi et
al., 1992; Leiser et al., 1995; Kowluru et al., 1997b, 2000).
In conclusion, our current studies further confirm and re-
inforce the postulation that both farnesylation and gera-
nylgeranylation of proteins play important regulatory roles
in physiological insulin secretion. These data, together with
previous findings, suggest that inhibition of post-transla-
tional prenylation impedes the maturation, membrane asso-
ciation, and biological effects of at least a subgroup of these
proteins, leading to inhibition of physiological insulin secre-
tion.
Acknowledgments
We thank Prof. Shimon Efrat for providing the ␤TC3 cell line.
Portions of this work have been presented at the Annual Meetings of
the Endocrine Society in Denver, CO, 2001 and accepted for presen-
tation at the Annual Meetings of the American Diabetes Association
in San Francisco in June 2002.
Lang J (1999) Molecular mechanisms and regulation of insulin exocytosis as a
paradigm of endocrine secretion. Eur J Biochem 259:3–17.
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Address correspondence to: Dr. Anjan Kowluru, Department of Pharma-
ceutical Sciences, Applebaum College of Pharmacy and Health Sciences,
Wayne State University, 259 Mack Ave., Detroit, MI 48201. E-mail:
akowluru@wizard.pharm.wayne.edu
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