Download Diabetes Research and Clinical Practice 28 Suppl. (1995) S115

Diabetes Research and Clinical Practice 28 Suppl. (1995) S115-S137
DIABETES
RESEARCH
AND CLINICAL
PRACTICE
Extrapancreatic effects of sulfonylureas — a comparison
between glimepiride and conventional sulfonylureas
Gunter Muller*a, Yusuke Satohb, Karl Geisena
a
Hoechst AG Frankfurt, Pharmaceutical Research Divison, SBU Metabolic Diseases, H 825, 65926 Frankfurt, Germany
b
Hoechst Japan Limited, Pharma Research and Development Divison, Kawagoe, Saitama 350-11 Japan
Abstract
The contribution of extrapancreatic effects of sulfonylureas to the blood glucose-decreasing activity was reevaluated in vivo and in vitro with several conventional sulfonylureas and with the new one glimepiride. In vivo, in
dogs, after single approximately equipotent blood glucose-decreasing doses, the sulfonylureas were tested for a
ranking in the ratios of mean plasma insulin-increasing and blood glucose-decreasing activity. Studies were also
performed in hyperglycemic hyperinsulinemic KK-Ay mice under once daily treatment for 8 weeks. In vitro,
glimepiride and glibenclamide were tested for the ranking of their extrapancreatic activity with respect to the
stimulation of glucose transport and glucose metabolizing processes in normal and insulin-resistant fat cells as well as
in the isolated diaphragm. Furthermore, in vitro studies were performed, especially with glimepiride, in order to
characterize the molecular mechanism for the extrapancreatic activity. The dog studies revealed a marked ranking in
the ratios of plasma insulin-increasing and blood glucose-decreasing activity between the different sulfonyiureas
(glimepiride < glipizide < gliclazide < glibenclamide). In the hyperglycemic hyperinsulinemic KK -Ay mice,
glimepiride reduced blood glucose by 40%, plasma insulin by 50% and HBAlc by 33%, whereas glibenclamide and
gliclazide had no effect on these parameters. In vitro, glimepiride and glibenclamide had extrapancreatic effects
within the lower uM range, with glimepiride exhibiting 2-3-fold lower ED50 values than glibenclamide. In the
absence of insulin, both stimulated glucose transport — up to 60% of the maximum insulin response in the rat
diaphragm and up to 35% in 3T3 adipocytes. Glycogenesis was stimulated in the rat diaphragm — up to 55% of the
maximum insulin effect; lipogenesis in 3T3 adipocytes — up to 40%. The studies on the molecular mechanism of
extrapancreatic activity with rat adipocytes and diaphragm suggest that these direct insulin -mimetic effects rely on
the induction of GLUT4 translocation from internal stores to the plasma membrane and on the activation of the key
metabolic enzymes, glycogen synthase and glycerol-3-phosphate acyltransferase. These processes occur within the
same drug concentration range and with the same ranking between glimepiride and glibenclamide as observed for
glucose utilization and transport. The direct effects of sulfonylureas may ultimately be regulated by a glycosyl-phosphatidylinositol-specific phospholipase C, shown to be activated by glimepiride in rat adipocytes. Lipolytic cleavage
products thereby generated from glycolipidic structures may in turn stimulate specific protein phosphatases which
activate key regulatory proteins/enzymes of glucose and lipid metabolism. Conclusion: From the ranking of the
sulfonylureas in the ratio of mean insulin-releasing and mean blood glucose decreasing-activity at single doses in
vivo, it is deduced that sulfonylureas have a variable degree of blood glucose decreasing-acivity which is independent
of their insulin secretion-stimulating activity. The different degree of this extrapancreatic activity in vivo is confirmed
* Corresponding author, Tel.: +49 069 3054271; Fax: +49 069 311454.
0168-8227/95/$09.50 © 1995 Elsevier Science Ireland Ltd. All rights reserved.
SSDI 0168-8227(95)01089-U
G. Müller et al. /Diabetes Research and Clinical Practice 28 Suppl. (1995) S115-S137
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by a corresponding ranking in stimulation of glucose utilization in vitro. Dephosphorylation and activation of key
enzymes of glucose transport and metabolism induced by a glycosyl phosphatidylinositol-specific phospholipase C
might be the molecular basis for the extrapancreatic activity of sulfonylureas.
Keywords:
cAMP
Glibenclamide; Glimepiride; Insulin action; Adipocytes; Diaphragm; Glucose transport; Metabolism;
1. Introduction
Since the introduction of sulfonylureas into the
treatment of non-insulin-dependent diabetes mellitus (NIDDM) about four decades ago, there has
been some debate as to whether their blood glucose-decreasing activity is based solely on the
stimulation of insulin release—or—is-also -due to
extrapancreatic activity (for reviews see Refs. 1
and 2). The observation that, after long-term
treatment of NIDDM patients with sulfonylurea
compounds, plasma insulin levels often return to
pre-treatment levels without loss of improved
blood glucose control, raised suspicion of extrapancreatic effects [3-5], especially since, in in
vitro studies with isolated or cultured muscle and
fat cells, direct (as assayed in the absence of
insulin) stimulation of glucose transport and
metabolism was demonstrated [6-14]. The interpretation of this long-term effect of sulfonylureas
in patients as extrapancreatic activity [15-17]
has, however, been questioned. Blood glucose
control without adequately elevated mean plasma
insulin levels has rather been explained as a
drug-induced postprandial kick of insulin release
resulting in lower mean blood glucose values and
thereby also reduced basal insulin levels [18-20].
Furthermore, long-term treatment with sulfonylureas has been suspected of reducing insulin
resistance by normalizing blood glucose and/or
basal insulin levels since permanently increased
blood glucose and/or basal plasma insulin levels
are said to induce insulin resistance in peripheral
tissues of Type II diabetics [21-23] and of normal
[24] and diabetic rats [25]. The molecular mechanism for desensitization due to increased mean
glucose or basal insulin concentrations has been
considered to reside within receptor and post-receptor steps of the insulin signaling cascade
[26-28] (e.g. phosphorylation and inactivation of
IRS-129).
With different sulfonylureas, we have tried in
vivo and in vitro to re-evaluate the extrapancreatic activity. In addition, we have attempted to
elucidate the underlying molecular mechanism.
We hypothetised that the ratio of mean plasma
insulin increase and mean blood glucose decrease
should be the same for all sulfonylureas if time
response curves for blood glucose decrease and
plasma insulin increase had a similar shape and
blood glucose were solely decreased by insulin
release. Ranking of plasma insulin/blood glucose
ratios, however, would be an indication of extrapancreatic blood glucose decreasing activity which
does not rely on additional insulin release. Furthermore, we postulated that in vivo ranking in
plasma insulin/blood glucose ratios should also
be reflected in extrapancreatic activity in vitro of
the sulfonylureas tested. Identical ranking in vivo
and in vitro would indicate that the cellular sites
of action of sulfonylureas, as identified in the in
vitro studies, are responsible for the extrapancreatic activity in vivo.
In vivo, to exclude the postulated indirect effect of sulfonylureas via metabolic improvement
during long-term treatment, we performed the
studies at single approximately equipotent blood
glucose-decreasing doses. Study animals were
normal fasted dogs. Study parameters were the
mean plasma insulin-increasing and mean blood
glucose-decreasing activity during the whole study
period. The sulfonylureas tested were glibenclamide, gliclazide, glipizide and glimepiride.
Glimepiride is a new sulfonylurea. Its pharmacological characterization is presented elsewhere
[30]. Glimepiride, glibenclamide and gliclazide
were additionally studied subchronically in diabetic hyperinsulinemic KK-Ay-mice for their ef-
G. Müller et al /Diabetes Research and Clinical Practice 28 Supp. (1995)S115-S137
feet on plasma insulin, blood glucose and HBAlc.
In vitro, glimepiride and glibenclamide were examined in normal and insulin-resistant fat cells
and in the isolated diaphragm. Potency ratios and
EC50 values of dose response curves were used as
ranking parameters for the extrapancreatic activity in vitro. The in vitro studies on the molecular
mechanism of the extrapancreatic activity were
performed with isolated and cultured fat cells
predominantly using glimepiride.
2. Experimental procedures
2. 1 Biological
Methods which have already been published
were used for the determination of blood glucose, plasma insulin and glycated hemoglobin
[30,31]; for preparation of rat diaphragms and
adipocytes [32]; for differentiation of 3T3
adipocytes [33]; for primary culture [34], metabolic
labeling with 32Pi or myo-[14C]inositol and cellular
fractination of rat adipocytes [35,36]; for immunoblotting and immunoprecipitation of
GLUT4 [35]; for assays of 2-deoxyglucose or 3-omethylglucose transport [35], lipogenesis,
glycogenesis, glycogen synthase, glycerol-3-phosphate acyltransferase, protein kinase A, low Km
cAMP-specific phosphodiesterase [32] and glycosyl-phosphatidylinositol-specific phospholipase C
[33,3.6].
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with confidence limits of two drugs were determined as the ratio of equieffective concentrations of each drug from the respective graded
dose-response relations [37]. The level of significance for all test procedures was 5%.
3. Results
3.1 Extrapancreatic activity in vivo
After intravenous administration of 60 ug/kg
to normal fasted dogs, glimepiride lowered the
blood glucose markedly less than glibenclamide
up to 16 h, and thereafter up to 51 h, twice as
much as glibenclamide (Fig. 1). Over the whole
study period, glimepiride had a total blood glucose-decreasing activity of 18,4 ±1,8% (mean S.E.M.), that of glibenclamide being 11.7 + 2.6%.
Both compounds showed a plasma insulin peak
after 30 min, the peak value after glibenclamide
being twice as high as that after glimepiride.
After glibenclamide, insulin gradually declined
up to the end of the study, whereas, 2 h after
2.2. Statistical
Raw data were used for the evaluation of
plasma insulin, and raw data or raw data expressed as a percent of the initial values were
used to evaluate the blood glucose. Mean plasma
insulin and mean blood glucose changes over the
whole study period were calculated by the trapezoidal rule. In case of dose-response curves for
plasma insulin and blood glucose, the equipotent
mean blood glucose decrease and corresponding
mean plasma insulin change over the whole study
period were each taken from the respective regression lines. For comparison of means of several groups, analysis of variance was performed
and subsequently Duncan's multiple range test
[37]. EC50 values were determined by a fourparameter logistic function fit [38]. Potency ratios
Fig. 1. Time course for blood glucose and plasma insulin
levels in fasted male beagle dogs after a single intravenous
dose of glimepiride or glibenclamide as clear aqueous sodium
salt solutions (mean values), cf. Fig. 2, Exp. 1.
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Fig. 2. Ratios of mean plasma insulin increase/mean percentage blood glucose decrease (PI/BG ratio) over the whole study period
in fasted male beagle dogs after a single oral dose of different sulfonylureas at approximately equipotent blood glucose-decreasing
doses.
administration of glimepiride, insulin did not differ markedly from the control. The mean plasma
insulin concentration after glimepiride was decreased by about 1.06 ± 1.84 U/ml, and after
glibenclamide it was increased by about 1.34 ±
1.62 U/ml. The ratio for plasma insulin change and
blood glucose decrease after glimepiride was < 0,
and after glibenclamide 0.12 (Fig. 2, Exp. 1).
After oral administration of 90 g/kg to normal
fasted dogs, the blood glucose decrease
induced by glimepiride was initially faster than
that induced by glibenclamide; subsequently, up
to 16 h it was lower, and thereafter up to 48 h, it
was two times greater than that induced by
glibenclamide (Fig. 3). Over the whole study period, glimepiride had a total blood glucose-decreasing activity of 15.3 ± 1.2%, that of glibenclamide being 15.7 ± 2.4%. Both compounds
showed a plasma insulin peak after 60 min, this
being 37.7 ± 7.4 U/ml for glibenclamide and
G. Muller et al. / Diabetes Research and Clinical Practice 28 Suppl. (1995) S115S137
Fig. 3. Time course for blood glucose and plasma insulin
levels in fasted male beagle dogs after a single oral dose of
glimepiride or glibenclamide as clear aqueous sodium salt
solutions (mean values), cf. Fig. 2, Exp. 2.
31.1 ± 4.3 U/ml for glimepiride. After
glimepiride, the plasma insulin values declined
within 3 h to those of the controls; after glibenclamide they gradually declined up to the end of
study. The mean total plasma insulin increase
after glimepiride was about 0.41 ± 0.47 U/ml, that
after glibenclamide being about 2.70 ± 1.44. The
ratio for plasma insulin change and blood glucose
decrease after glimepiride was 0.03 and after
glibenclamide 0.17 (Fig. 2, Exp. 2).
After oral administration to dogs, glimepiride,
at single doses of 0.01, 0.03 or 0.1 mg/kg, and
gliclazide, at single doses of 0.4, 1.2 or 4.0 mg/kg,
both decreased the blood glucose dose-dependently almost to the same degree (Fig. 4). However, the corresponding plasma insulin levels with
glimepiride at all doses were significantly lower
than those with gliclazide. The regression lines
through the mean percentage blood glucose decrease over 8 h yielded an ED 25 of 0.056 mg/kg
for glimepiride, and one of 1.5 mg/kg for gliclazide. The corresponding mean plasma insulin increase was 0.88 U/ml for glimepiride and 3.70
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U/ml for gliclazide, resulting in a total plasma
insulin increase/total blood glucose decrease ratio of 0.04 for glimepiride and 0.15 for gliclazide
(Fig. 2, Exp. 4).
After oral administration of glimepiride or
glibenclamide at doses of 90 g/kg, or of gliclazide
or glipizide at doses of 1.8 and 180 g/kg,
respectively, to dogs, glimepiride had the highest
total blood glucose-lowering activity and lowest
insulin-releasing activity; glipizide had the lowest
total blood glucose-lowering activity and highest
insulin-releasing activity (Fig. 5). The ranking in
maximal blood glucose decrease was: glibenclamide > gliclazide = glipizide > glimepiride,
the ranking in mean total blood glucose decrease,
however, being: glimepiride (-20.4 ± 1.5%) >
gibenclamide (-19.0 ± 1.0%) > gliclazide (16.8 ± 1.2%) > glipizide (-13.0 ± 2.2%).
The ranking in plasma insulin peak height was:
glipizide > gliclazide > glibenclamide >
glimepiride, the ranking in mean total plasma
insulin increase being gliclazide (12.2 ± 0.7
U/ml) < glimepiride (12.4 ± 1.0 U/ml) <
glibenclamide (13.6 ± 1.0 U/ml) < glipizide
(14.7 ± 0.7 U/ml). The total plasma insulin
increase/total blood glucose decrease ratio for
glimepiride was 0.03, for gliclazide 0.07, for glipizide 0.11 and for glibenclamide 0.16 (Fig. 2, Exp.
3).
After a single oral dose of 90 g/kg glibenclamide
or glimepiride to fasted dogs which, 30 min after
administration, had been repeatedly loaded orally
with glucose (2 g/kg) at 5-h intervals up to 50 h,
the plasma insulin peaks and the peaks and nadirs
of blood glucose were lower than those in the
control animals (Fig. 6). The mean blood
glucose concentration over 52 h in the
glimepiride animals was 5.7 ± 0.13 mmol/l and
in the glibenclamide animals 5.6 ± 0.10 mmol/l.
Both means were statistically significantly lower
than that in the controls (6.2 ± 0.14 mmol/l),
however, they did not differ from each other. The
mean plasma insulin concentration over 52 h in
the glimepiride animals was 46.9 ± 4.1 U/ml and
in the glibenclamide treated animals 53.5 ± 4.4
U/ml. Both means were also statistically
significantly lower than that in the
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Time in hrs after administration
Fig. 4. Time course for blood glucose and plasma insulin levels in fasted male beagle dogs after a single oral dose of glimepiride or
gliclazide (mean values), cf. Fig. 2, Exp. 4.
Fig. 5. Time course for blood glucose and plasma insulin levels in fasted male beagle dogs after a single oral dose of glimepiride,
glibenclamide, gliclazide or glipizide as clear aqueous sodium salt solutions (mean ± S.E.M.). cf. Fig. 2, Exp. 3.
G. Muller et al. /Diabetes Research and Clinical Practice 28 Suppl. (1995) S115-S137
controls (60.6 ± U/ml).However, in contrast to the mean blood glucose concentrations,
they also differed statistically from each other.
After 8 weeks' oral treatment of hyperglycemic
hyperinsulinemic KK-Ay mice once daily with 0.5
mg/kg glimepiride, 1.25 mg/kg glibenclamide or
20 mg/kg gliclazide (approximately equipotent
blood glucose-decreasing doses in rabbits),
glimepiride reduced blood glucose by 40%, plasma
insulin levels by 50% and HBAlc by 33% (Fig:7).
Glimepiride treatment had provoked changes in
these parameters already after 3 weeks of treatment. In contrast, even after 8 weeks of treatment, glibenclamide and gliclazide had no effect
on blood glucose, plasma insulin and HBA lc levels.
Thepresented animal studies suggest that sulfonylureas also manage to decrease blood glucose independently of their capacity to stimulate
insulin release and that sulfonylureas of different
structure exhibit this extrapancreatic activity to a
variable degree. Since short-term studies were
performed; a secondary sulfonylurea effect, i.e. an
Blood glucose in
mmol/I
Plasma insulin in
U/ml
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Fig. 7. Time course for blood glucose, plasma insulin and HBAlc levels in insulin-resistant diabetic KK-Ay mice treated once daily
for 8 weeks with glimepiride, glibenclamide or gliclazide (mean
values). *Significant from the control values with P <
0.05;
** significant from the control values with P < 0.01.
tivity of sulfonylureas might be direct stimulation
of peripheral glucose disposal. Evidence of direct
so-called insulin-mimetic activity — the stimulation of glucose utilization — of sulfonylureas, in
particular of glibenclamide, has accumulated during the past two decades in the literature
stemming from in vitro studies with isolated and
cultured muscle [6-10] and fat cells [11-14].
Effect of sulfonylureas on glucose transport and
metabolism. The two major pathways for nonoxidative glucose metabolism in muscle and fat
tissue are glycogen and lipid synthesis which are
mainly involved in the overall peripheral glucose
Fig. 8. Time course for blood glucose levels in fasted male
beagle dogs after a single oral dose of glimepiride or glibenclamide as clear aqueous sodium salt solutions (mean values).
utilization. We therefore studied glimepiride and
glibenclamide for their effects on lipogenesis and
glycogenesis in vitro. For lipogenesis, incorporation of [3H]glucose into toluene-extractable acylglycerides in cultured 3T3 adipocytes and, for
glycogenesis, incorporation of [14C]glucose into
ethanol-precipitable glycogen in the isolated rat
diaphragm were determined (Fig. 9). Both drugs
stimulated glycogenesis (Panel A) to up to 2.5
times the basal state (corresponding to 65-70%
of the maximal insulin effect) and lipogenesis
(Panel B) to up to four times the basal state
(corresponding to 40-45% of the maximal insulin
effect), the stimulation being concentration dependent. There was no difference in the maximal
response between the two drugs. However, both
dose-response curves of glimepiride were statistically significantly shifted to the left of those of
glibenclamide, resulting in a glimepiride/
glibenclamide potency ratio of about two. The
EG50 values for lipogenesis were 1.7 M for
glimepiride and 2.9 M for glibenclamide; the
EC50 values for glycogenesis were 0.4 M for
glimepiride and 0.8 M for glibenciamide.
The rate-limiting step of lipogenesis and glycogenesis under physiological conditions is glucose
transport [39]. To compare glimepiride and
glibenclamide for their effect on glucose transport,
the uptake of the non-metabolizable glucose ana-
G. Muller et al/Diabetes Research and Clinical Practice 28 Suppl. (1995) S115-S137
Panel A
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Panel B
Fig. 9. (according to Ref. 32). Panel A: Effect of sulfonylureas on glycogenesis in isolated rat diaphragms was assayed after
incubation with the indicated concentrations of glimepiride and glibenclamide for 4 h or insulin for 30 min. The 14C-incorporation
into ethanol-precipitated glycogen measured for the-basal state was set at 100%(mean ± sem, n = 6). Panel B: Effect of
sulfonylureas on lipogenesis in 3T3 adipocytes was tested after incubation with differentiated and confluent cells with the indicated
concentrations of glimepiride and glibenclamide for 20 h or insulin for 15 min. The 3H-incorporation into toluene-extractable lipids
measured_ for the basal state and corrected for a blank value (incubation mixture lacking cells) was set at at 100% <mean ± sem,
n = 6).
logue, 2-deoxyglucose, in the isolated rat diaphragm and in 3T3 adipocytes was determined
(Fig. 10). Both drugs increased the 2-deoxyglucose uptake in the diaphragm (Panel A) by up to
1.8-fold (corresponding to 70-75% of the maximal insulin effect) and in 3T3 cells (Panel B) by
up to 7-fold (corresponding to 40% of the maxi-
Panel A
mal insulin effect), the increase being concentration dependent, The maximal transport velocities
for glimepiride and glibenclamide did not differ.
Again, the dose-response curves for glimepiride
were statistically significantly shifted to the left of
those for glibenclamide. The EC50 values in 3T3
cells were 3.2 M for glimepiride and 5.2 M for
Panel B
Fig. 10. (according to Ref. 32). Panel A: Effect of sulfonylureas on 2-deoxyglucose transport in isolated rat diaphragms was assayed
after incubation with the indicated concentrations of glimepiride and glibenclamide or 300 nM insulin for 4 h. The diaphragm-associated 3H-radioactivity measured for the basal state and corrected for non-specific trapping (presence of 25 M cytochalasin B) was
set at 100% (mean ± S.E.M., n =4). Panel B: Effect of sulfonylureas on 2-deoxyglucose transport in 3T3 adipocytes was assayed
after incubation for 30 min with the indicated drug concentrations or 30 nM insulin. The cell-associated 3H-radioactivity measured for
the basal state and corrected for non-specific trapping (presence of 25 M cytochalasin B) was set at 100% (mean ± S.E.M., n = 8).
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G. Muller et al. /Diabetes Research and Clinical Practice 28 Suppl. (1995) S115-S137
glibenclamide. The EC50 values in rat diaphragm
were 0.8 M for glimepiride and 1.4 M for
glibenclamide.
NIDDM is associated with desensitization of
the glucose transport system towards stimulation by
insulin in muscle and fat tissue, resulting in lower
glucose disposal rates in diabetic patients as
revealed by euglycemic hyperinsuimemic clamp
studies [40,41]. A primary culture of isolated rat
adipocytes desensitized in vitro for insulin-stimulated glucose transport by long-term incubation
with high concentrations of insulin, glucose and
glutamine is regarded as a corresponding in vitro—
model [42,43]. Compared to non-desensitized cells,
the maximal insulin response of 3-o-methylglucose transport in these adipocytes was reduced to
about one third, and the insulin dose response
curve (data not shown) was shifted to the right
(EC50 = 1.9 vs. 0.5 nM). With glimepiride, however,
there was only a 15% reduction in the maximal
response, and only a marginal shift to the right in
the dose response curve was observed (EC50 = 22.5
vs. 16.5 M), this shift not being significant
statistically. This indicates that the ex-trapancreatic
activity of glimepiride is hardly affected in the
insulin-resistant state.
Effect of sulfonylureas on GLUT4 translocation /
dephosphorylation. The molecular mechanism of
glucose transport stimulation by insulin in muscle
and fat cells is based primarily on the translocation of the glucose transporter isoform GLUT4
[44] from intracellular stores — the low density
microsomes — to the plasma membrane [45,46].
The increase in the number of functional glucose
transporter molecules in the plasma membrane in
response to insulin is dramatically impaired in
adipocytes isolated from insulin-resistant hyperinsulinemic Zucker fatty rats, providing a molecular
explanation of the defective insulin-stimulated
glucose transport in these cells and reduced glucose clearance in these animals [47,48]. The effect
of glimepiride (10 M) on the cell surface expression
of GLUT4 in normal and desensitized rat
adipocytes was studied in comparison to insulin
(10 nM). GLUT4 was determined in total membranes, low density microsomes and plasma membranes using SDS-PAGE of the proteins with
subsequent immunoblotting with anti-GLUT4
antibodies and [125I]protein A, followed by autoradiography (Fig. 11). Densitometric quantita-tion
of the GLUT4 band in the autoradiogram revealed
that, in normal cells (upper panel), glimepiride
increased the amount of immunore-active GLUT4
in the plasma membrane 3-3.5-fold, while insulin
increased it 7-8-fold. This increase was correlated
with a decrease in GLUT4 in the low-density
microsomes. The observed GLUT4 translocation
induced by glimepiride and insulin was in
reasonable agreement with the glucose transport
stimulation in 3T3 adipocytes (Fig. 10, Panel B). In
the insulin-resistant cells (middle-panel), insulinstimulated GLUT4 trans-location was reduced to
about 2-fold. The glimepiride-induced translocation
was about 3-fold, and thus hardly diminished
compared to normal cells, as was the case for
glucose transport activation by glimepiride.
Obviously, glimepiride causes a redistribution of
GLUT4 from low-density microsomes to plasma
membranes in rat adipocytes, which is not
significantly impaired in insulin-resistant cells
compared to normal cells and is presumably
responsible for the stimulation of glucose transport
by glimepiride.
The distribution of GLUT4 between low-density microsomes and the plasma membrane is
believed to be regulated by its phosphorylation
state. Serine/threonine phosphorylation at the
carboxyl terminus may function as an internalization signal for GLUT4 [49,50]. In comparison to
insulin, the effect of glimepiride on the phosphorylation state of GLUT4 was determined in normal and desensitized rat adipocytes in vitro. Rat
adipocytes were metabolically labeled with 32Pi
until equilibrium, prior to short-term incubation
with 10 nM insulin or 10 M glimepiride. Subsequently, total membranes were isolated and
GLUT4 immunoprecipitated with anti-GLUT4
antibodies and analyzed by SDS-PAGE and fluorography (Fig. 12). In normal cells, in the absence
of insulin or glimepiride, there was considerable
phosphate incorporation into GLUT4. Densitometric quantitation of the GLUT4 band in the
fluorogram revealed that both insulin and
glimepiride reduced the phosphorylation state of
GLUT4 in total membranes by about 25-35%. In
desensitized adipocytes, in the absence of insulin
G. Muller et al. /Diabetes Research and Clinical Practice 28 Suppl. (1995) S115-S137
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Fig 11 (according to ref. 35). Effect of insulin and glimepiride on GLUT4 translocation was assayed in cultured normal (upper
panel) and desensitized rat adipocytes (middle panel) after full deactivation of the glucose transport system and incubation of the
cells in the absence (CON) or presence of 10 nM insulin (INS) or 10 M glimepiride (GLI) for 20 min. The amount of GLUT4 was
determined in isolated total membranes (T), low-density microsomes (L) and plasma membranes (P) after SDS-PAGE of equivalent
amounts of protein by immunoblotting with anti-GLUT4 antibodies and [ I25I]protein A and subsequent autoradiography. A
representative autoradiogram of a typical experiment repeated three times with similar results is shown.
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Fig. 12. (according to Ref. 35). Effect of insulin and glimepiride on the phosphorylation of GLUT4 was assayed in cultured normal
and desensitized rat adipocytes after metabolic labeling with 32 P1 for 2 h. After full deactivation of the glucose transport system, the
washed cells were incubated in the absence or presence of 10 nM insulin (INS) and 10 M glimepiride (GLI) for 20 min. again
washed and then homogenized in the presence of 1 mM sodium phosphate. GLUT4 was immunoprecipitated from equivalent
amounts of isolated total membranes with anti-GLUT4 antibodies and protein A-Sepharose and then analyzed by SDS-PAGE and
autoradiography. A representative autoradiogram of a typical experiment repeated twice with similar results is shown.
or glimepiride, the GLUT4 phosphorylation was
increased about 5-fold compared to that in normal adipocytes. Insulin caused only a 20-25%
reduction in the amount of phosphorylated
GLUT4. In contrast, the phosphorylation state of
GLUT4 after incubation with glimepiride was de-
G. Muller et al. /Diabetes Research and Clinical Practice 28 Suppl. (1995) S115-S137
creased to 40-35%, approaching a2-fold level of
GLUT4 phosphoprotein as observed in normal
cells (Fig. 12). Obviously, glimepiride causes dephosphorylation of GLUT4, this desphosphorylation being more efficient in desensitized
adipocytes than in normal cells. The dephpsphorylation of GLUT4 in response to sulfonylureas
may provide-a molecular mechanism for the stimulation of GLUT4 translocation and glucose
transport by glimepiride in normal and especially
in insulin-resistant adipocytes.
Effect of sulfonylureas on glucose-metabolizing
enzymes. The key metabolic enzymes of lipogenesis, glycerol-3-phosphate acyltransferase, and of
glycogenesis, glycogen synthase, are both activated_by_ insulin via dephosphorylation (for reviews see Refs. 51 and 32). Since glimepiride—
like insulin, as already shown for GLUT4 —
seems to regulate certain dephosphorylation
processes in adipocytes, the effect of
glimepiride
and glibenclamide on the activity of glycogen
synthase and glycerol-3-phosphate acyltransferase
was studied. Glycogen synthase activity was measured in the homogenate from drug-incubated rat
diaphragms and expressed as the ratio of activity
in the presence of 0.1 mM and 10 mM glucose-6phosphate. At 0.1 mM glucose-6-phosphate, only
the dephospho-form of glycogen synthase is active; at 10 mM, the total enzyme activity is measured. Both glibenclamide and glimepiride increased the activity ratio of glycogen synthase up
to 45-50% of the maximal insulin effect (Fig. 13),
this increase being concentration dependent.
Fig. 13. (according to Ref. 32). Effect of sulfonylureas on
glycogen synthase activity ratio (in the presence of 0.1 mM
and 10 mM glucose-6-phosphate, respectively) in the homogenate of isolated rat diaphragm was assayed after 4 h incubation with the indicated concentrations of glimepiride and
glibenclamide or 300 nM insulin (mean ± S.E.M., n = 3).
S127
Thus, sulfonylureas lead to a considerable
increase in the dephospho-form of glycogen synthase in adipocytes, which may indicate a sulfonylurea-induced dephosphorylation of this enzyme.
Glycerol-3-phosphate acyltransferase activity was
determined in the homogenate from drug-incubated rat adipoeytes. The activity of glycerol-3phosphate acyltransferase rose up to 35-40% of
the maximal insulin effect in response to both
drugs (Fig. 14), this rise being concentration dependent. For both enzymes, the glimepiride dose
response curves showed a statistically significant
shift to the left compared to those of glibenclamide. For glycogen synthase, the EC50 values
were 0.8 M for glimepiride and 2.0 M for
glibenclamide; for glycerol-3-phosphate acyltransferase the EC50 values were 19 M and 3.7 M,
respectively. The sulfonylurea-induced simultaneous stimulation of glucose transport (via dephosphorylation of GLUT4) and of enzymes of the
non-oxidative glucose metabolism (via dephosphorylation of key metabolic enzymes) suggests
that the activation of specific protein phosphatases in muscle and fat cells is the molecular
mechanism for the increase of glucose utilization
in peripheral tissues provoked by sulfonylureas.
Effect of sulfonylureas on the glycosyl-phosphatidylinositol-specific phospholipase C. The apparent coordinated activation of the multitude of
protein phosphatases involved in the regulation
Fig. 14. (according to Ref. 32). Effect of sulfonylureas on
glycerol-3-phosphate acyltransferase activity in the homogenate from isolated rat adipocytes was assayed after incubation with the indicated concentrations of glimepiride and
glibenclamide for 4 h or 20 h, or with 30 nM insulin for 30
min. The 3H-incorporation into butanol-extractable lipids
measured for the basal state and corrected for a blank value
(incubation mixture lacking protein) was set at 100% (mean ±
S.E.M., n = 5).
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G. Muller et al. /Diabetes Research and Clinical Practice 28 Suppl (1995) S115-S137
of—glucose transport and metabolism by glimepiride
raises the question about putative upstream
regulatory steps involved in glimepiride signaling.
Cleavage products of a certain subtype of free
glycolipids and glycolipid membrane anchors,
glycosyl-phbsphatidylinositol, generated by a
specific phospholipase C can alter the total
phosphorylation patterns in rat adipocytea in an
insulin-like manner [53-55]. These: putative protein phosphatase-modulating effector molecules,
phosphoinositol-glycans of yet unknown structure,
stimulate glucose transport [56], lipogenesis [57],
glycogenesis [58], glycogen synthase [59] and
glyc-erol-3-phosphate acyltransferase [60] in
intact adipocytes and muscle cells in vitro. In
isolated normal and desensitized rat adipocytes,
the effect
of glimepiride on the activity of the glycosyl phosphatidylinositol-specific phospholipase C was
tested. The assay is based on the cleavage of the
covalently attached glycosyl-phosphatidylinositol
plasma membrane anchor (for a review see Cross
[61]) of an isoform of lipoprotein lipase in rat
adipocytes [62]. After metabolic labeling of the
cells with myo-[14C]inositol and subsequent incubation with insulin or glimepiride, plasma
membranes were isolated. For detection of anchor cleavage, the membrane proteins were partitioned between a Triton X-114 and an aqueous
phase [63] to separate the hydrophilic form of
lipoprotein lipase (with the degraded anchor
structure) from the amphiphilic version (linked to
the intact anchor structure). Subsequently, hydrophilic lipoprotein lipase was immunoprecipitated from the aqueous phase and analyzed by
SDS-PAGE and fluorography for l4 C-radioactivity (Fig. 15), The retention of the radiolabeled
myo-inositol within the hydrophilic lipoprotein lipase indicates cleavage of the glycosyl-phosphatidylinositol membrane anchor by a phospholipase. Densitometric quantitation of the lipoprotein lipase band in the fluorogram revealed that,
in both normal and desensitized cells, insulin and
glimepiride caused a concentration-dependent increase in hydrophilic lipoprotein lipase. In normal
cells, the maximal effect of glimepiride (10 M)
approached 25-30% of the maximal insulin effect
(30 nM), the EC50 value for glimepiride being 3.1
M. In desensitized adipocytes, the maximal sti-
mulation of the-phospholipase by glimepiride was
slightly reduced compared to that in normal cells
(5.5-fold vs. 6.3-fold), but markedly exceeded the
maximal insulin effect (2.7-fold in desensitized vs.
12.5-fold in normal cells). The EC50 value for
insulin increased from 1.9 nM in normal cells to
5.1 nM in desensitized cells, whereas the EC50
value for glimepiride remained constant (3.1 vs.
3.5 M). Obviously, the desensitization toward
insulin and the maintenance of sensitivity toward
glimepiride for stimulation of glucose transport
and GLUT4 translocation/dephosphorylation is
also reflected in the stimulation of the phospholipase C. Furthermore, the time response curves of
activation of the phospholipase and of glucose
transport by glimepiride in desensitized as well as
in normal adipocytes were similar. These data
suggest a mechanistic coupling between the sulfonylurea-mducible phospholipase activity and
GLUT4 translocation/dephosphorylation. The
maintenance of extrapancreatic activity of
glimepiride in the desensitized adipocytes might
be responsible for its extrapancreatic activity in
the insulin-resistant KK-Ay mice. Taken together
with the demonstration of a defect in releasing
functional phosphoinositol-glycan structures in
insulin-resistant Type II diabetic Goto-Kakizaki
rats [64], the data provide evidence that sulfonylureas bypass the site of insulin resistance at the
level of the generation of phosphoinositol-glycan
structures via direct activation of the glycosylphosphatidylinositol-specific phospholipase C.
Effect of sulfonylureas on the cAMP regulatory
cascade. Sulfonylureas inhibit concentration-dependently, isoproterenol-stimulated lipolysis in rat
adipocytes in vitro [65], presumably via interference with the cAMP-dependent activation or direct inhibition of protein kinase A [66,67]. To test
for these possibilities and for differences between
glimepiride and glibenclamide, the activity of protein kinase A was assayed in the cytosol from drugtreated rat adipocytes which had been preincubated with isoproterenol to increase cytosolic
cAMP levels. Protein kinase A was tested in the
absence (for determination of the amount of active enzyme) and presence (for determination of
the total amount of enzyme) of saturating concentrations of exogenous cAMP. Glimepiride and
G. Muller et al /Diabetes Research and Clinical Practice 28 Suppl. (1995) S115-S137
S129
Fig. 15. (according to Ref. 36). Effect of insulin and glimepiride on the glycosyl-phosphatidylinositol-specific phospholipase C was
assayed in isolated rat adipocytes which had been incubated in primary culture in the presence of 0.5 mM glucose (normal) or 20
mM glucose, 16 mM glutamine, 10 nM insulin (desensitized) for 20 h. The washed cells were metabolically labeled with
myo-[14 C]inositol and then incubated for 30 min with the indicated concentrations of insulin or glimepiride. Isolated plasma
membranes were subjected to TX-114 partitioning. Hydrophilic lipoprotein lipase was purified from the aqueous phase by
immunoprecipitation and analyzed by SDS-PAGE and fluorography. A representative fluorogram of a typical experiment repeated
three times with similar results is shown.
glibenclamide reduced the protein kinase A activity ratio, i.e. the portion of active enzyme (Fig.
16), this reduction being concentration dependent. There was no difference in the maximal
inhibition between glimepiride and glibenclamide.
However, the dose response curve for glimepiride
was shifted to the left, this shift being statistically
significant. The EC 50-value was 0.7 M for
glimepiride and 1.1 M. for glibenclamide. The
protein kinase A activity ratio is assumed mainly
to reflect the intracellular cAMP concentration
at the time of homogenization. Thus, sulfonylureas seem to lower protein kinase A activity and
consequently lipolysis via causing a decrease in
cytosolic cAMP levels. One potential mechanism
for the cAMP-lowering capacity of sulfonylureas
is the stimulation of degradation of cAMP. Consequently, with glimepiride and glibenclamide, the
effect on the activity of the particulate low Km
cAMP-specific phosphodiesterase in microsomes
from drug-incubated rat adipocytes was tested.
Both drugs increased the phosphodiesterase activity up to 60% of the maximal insulin effect (Fig.
17). The dose response curve for glimepiride
showed a shift to the left of that of glibenclamide
(EC50 = 0.8 vs. 2.2 M), this shift being statisti-
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G. Muller et al. /Diabetes Research and Clinical Practice 28 Suppl. (1995) S115-S137
cAMP-specific phosphodiesterase [72]. These glycolipids were isolated from insulin-sensitive tissues and are structurally related to the lipolytic
cleavage product from the lipoprotein lipase glycosyl-phosphatidylinositol membrane anchor.
4. Discussion
Fig. 16. (according to Ref. 32). Effect of sulfonylureas on
protein kinase A activity ratio (in the absence or presence of 1 M
cAMP) in the cytosol of isolated rat adipocytes was assayed
after incubation of adipocytes with 1 M isopro-terenol for 15
min and then with the indicated concentrations of glimepiride
and glibenclamide or 5 nM insulin for 4 h (mean ± S.E.M.,
n = 4).
cally significant.
These data confirm previous reports on the
activation of phosphodiesterase by sulfonylureas
[68,69]. The consistently lower EC50 values of
glimepiride for inhibition of protein kinase A, for
activation of cAMP-specific phosphodiesterase,
and for activation of glycosyl-phosphatidylinositol
specific phospholipase C compared to those of
glibenciamide suggest a mechanistic linkage
between the cAMP regulatory cascade and the
generation of phosphoinositol-glycan molecules
in fat cells. In agreement with this putative causal
relationship, phosphoinositol-glycans prepared
from free glycolipids have been shown to inhibit
catecholamine-induced lipolysis [70] and protein
kinase A activation [71] as well as to stimulate
Fig. 17. (according to Ref. 32). Effect of sulfonylureas on low
Km cAMP-specific phosphodiesterase activity in microsomes
from isolated rat adipocytes was assayed after 2 h incubation
of adipocytes with the drug concentrations indicated or 5 nM
insulin (mean S.E.M., n = 5).
The ranking in the plasma insulin/blood glucose ratio in vivo suggests that sulfonylureas
have extrapancreatic activity and that they differ
in their extrapancreatic activity. The dogs treated
once with glimepiride or glibenciamide and then
repeatedly glucose-loaded showed a smaller blood
glucose increase despite a smaller plasma insulin
increase than the controls. Although both drugs
inhibited blood glucose increase to the same degree, the plasma insulin increase with glimepiride
was smaller than that with glibenciamide. The
dogs treated with glimepiride, glibenclamide, gliclazide or glipizide showed at a similar total blood
glucose decrease a ranking in insulin release,
glibenclamide having the highest insulin-releasing
activity and glimepiride the lowest. Of the dogs
treated with glimepiride or glibenciamide, those
given glimepiride showed, 24-48 h after treatment, lower plasma insulin values than the
glibenclamide-treated animals, despite their blood
glucose values also being significantly lower. In
the diabetic hyperinsulinemic KK-Ay mice treated
for 8 weeks with glimepiride, glibenclamide or
gliclazide, only glimepiride induced a significant
blood glucose decrease, this being accompanied
by a decrease in plasma insulin. Of the sulfonylureas tested, glimepiride obviously has the most
pronounced extrapancreatic activity in vivo.
The demonstration of extrapancreatic activity
of sulfonylureas and of a similar ranking between
glimepiride and glibenclamide in vitro supports
the in vivo findings, in the isolated or cultured fat
cells and the muscle diaphragm, the EC50 values
for the stimulation of lipogenesis, glycogenesis,
glucose transport, cAMP-specific phosphodiesterase and glycerol-3-phosphate acyltransferase,
as well as for the inhibition of protein kinase A
were two to three times lower for glimepiride
than for glibenclamide. This argues in favour of
G. Muller et al / Diabetes Research and Clinical Practice-2S-Suppl (1995) S115-S137
The physiological relevance of the direct stimulation of glucose utilization in peripheral cells
observed in vitro for sulfonylureas in the present
and also in previous studies [6-14]. It may result
in an increase of glucose disposal in muscle and
fat tissues in vivo, which does not depend on
insulin release.
The present studies on the molecular mechanism of sulfonylurea action in vitro provide an
explanation as to how sulfonylureas manage to
affect the multiple steps required for increased
glucose utilization, i.e. (i) activation of glucose
transport and glucose-metabolizing enzymes leading to stimulation of glycogen and lipid synthesis;
and (ii) downregulation of the cAMP regulatory
cascade, leading to inhibition of lipolysis. The
simultaneous and coordinated regulation of these
activities resembles the action of insulin. According to our data, glimepiride seems to exert these
pleiotropic effects via independent mecha nisms: (a) the stimulation of specific protein phosphatases responsible for regulation of GLUT4
translocation and of glycogen synthase/glycerol3-phosphate acyltransferase activity; and (b) the
stimulation of the cAMP-specific phosphodiesterase. At the ultimate hierarchy of regulation,
glimepiride, however, also causes rapid and efficient activation of the
glycosyl-phosphatidylinositol-specific phospholipase C, as shown in
rat adipocytes. The soluble phosphoinositol-glycan
molecules thereby generated are known to regulate phospho-/dephosphorylation processes, and
the cAMP regulatory cascade (for reviews see
Refs. 73 and 74). It is therefore tempting to
speculate about the glycosyl-phosphatidylinositol-specific phospholipase C as the primary target
for sulfonylurea action on protein phosphorylation and cAMP metabolism in peripheral cells.
Insulin has also recently been shown to increase
the activity of this enzyme in adipocytes [33,36,75]
and myocytes [76]. Thus, sulfonylureas may partly
mimic the insulin action by interfering with the
metabolic branch of the insulin signaling cascade
at the level of the glycosyl-phosphatidylinositolspecific phospholipase.
Besides the argument that the effect of sulfonylureas under chronic treatment might be an indirect effect due to an improved metabolic control
S131
resulting in increased insulin sensitivity, there are,
however, four more concerns about the physiological relevance of extrapancreatic effects of sulfonylureas: i) potential differences between peripheral and portal vein insulin concentrations ii)
discrepancy between protein binding in vivo and
in vitro, iii) discrepancy between total in vivo and
in vitro concentrations, iv) missing effect on blood
glucose in totally insulin-deficient animals and
diabetic patients.
(i) At the first pass, liver removes up to 50%
insulin front the portal blood [77, 78]. One
might therefore suspect that sulfonylureas
may have induced a small plasma insulin
increase in the portal vein not detectable in
the peripheral plasma, which would have
resulted in greater inhibition of the hepatic
glucose output by glimepiride. This agrument, however, cannot be sustained. Although the blood glucose in the dogs 24 h
after the administration of glimepiride was
lower than that after glibenclamide, the insulin was lower too.
(ii) The free drug concentration of glimepiride
and glibenclamide in medium containing
bovine serum albumin is, according to Panten [79, 80], 10-15% in human serum, according to Lehr [81] it is less than 1%. Since
only the drug not bound to protein is supposed to be effective [82], the higher protein
binding in vivo raises doubts as to whether,
even under the assumption of similar total
drug concentrations in serum and incubation medium, the effects observed in vitro
might become effective in vivo [83]. However, according to Hornke and Jantz (personal communication), the protein binding
of glimepiride in vitro is more than 99.5%
with bovine serum albumin.
(iii) The total drug concentrations required in
vitro for half-maximal effects (1-5 /xM) are
higher than even the cmax plasma concentrations under the therapeutical oral doses
administered to the dogs. The plasma halflife of glimepiride in dogs is 12 h [84]. After
administration of 100 g/kg glimepiride
orally to dogs, the c max plasma concentra-
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G. Muller et al. /Diabetes Research and Clinical Practice 28 Suppl. (1995) S115-S137
tion was 2.2 M (at 2 h); after 24 h, the_
plasma concentralion was 6.4 M [85]. It
seems unlikely that the short-lasting, early
cmax phase of glimepiride would have been
sufficient to induce the long-lasting blood
glucose decrease without increased plasma
insulin in our animals. Activation of the
glucose transport by insulin in rat adipocytes
in vitro persists only for 10-20 min after
removal of insulin [86]. Duration of extrapancreatic activity of sulfonylureas in vitro
after drug removal is not known. This is
difficult to determine due to the undefined
efficiency of a washout procedure.
Alternatively, the confict between total drug
concentrations effective in vivo and in vitro
might be overcome by accepting that the
total amount of molecules which interact
with the target cell in a functional manner
will be dependent on the product of the
concentration and exposure time. In the
present in vitro studies, the extrapancreatic
effects were determined immediately after
incubation, with drug, of fat cells for 30 min
to 20 h or of diaphragm for 4 h. The short
incubation time might have been compensated by a higher drug concentration. The
importance of the exposure time is underlined by preliminary experiments which indicate that, in vitro, the maximal cytochalasin B-inhibitable glucose transport velocity
in 3T3 adipocytes increases with the
incubation time in the presence of 20 M
glimepiride from about 6.8-fold over the
basal state at 30 min to 9.3-fold at 20 h. At
submaximal concentrations, glimepiride was
also more efficient with longer exposure
times. Furthermore, our studies on the activation of glycerol-3-phosphate acyltransferase by glimepiride and glibenclamide in
rat adipocytes revealed an even more striking
dependency on the incubation time rising
from an approximate 35% increase at 4 h to
90% at 20 h with 100 M drug (Fig. 14).
The maximal enzyme activity after the longterm exposure was 2.5-3 times higher
throughout the concentration range tested
and the do dose response curve was shifted to
the left.
Time-dependent penetration of the sulfony-lurea molecules into the plasma membrane
of the target cell might be the mechanism
for the increasing activity. There is strong
evidence for a high lipophilicity of sulfonylureas [87], especially of glimepiride and
glibenclamide, based on their distributionbetween an octanol and a water phase. The
partition coefficients of the unionized forms
at pH 7.4 are 2.8 for glipizide, 4.1 for gliclazide, 94 for glibenclamide and 50 for
glimepiride [80, 88, 89]. The amount of protein-bound drug may represent the pool for
molecules which time-dependently insert
into the lipid-bilayer of the membrane either via passage across the aqueous environment or via close physical contact of
serum proteins loaded with sulfonylurea
with the surface of the target cell. The
altered physical state of the lipid bilayer
induced by the intercalated sulfonylurea
may affect protein components of the signal
transduction cascade located in the plasma
membrane of fat and muscle cells. Possible
candidates for those components are the
glycosylphosphatidylinositol-specific phospholipase C in the fat cell and the membrane-associated activated protein kinase C
isoforms in the muscle cell Activation of
protein kinase C by sulfonylureas has been
demonstrated for BC3H1 myocytes [9, 10]
and correlated with the increase of glucose
transport induced by these drugs. The
suggested accumulation of sulfonylurea
molecules in the membrane of the target
cell and its functional relevance is in conflict with a typical receptor-ligand interaction which solely depends on the affinity of
the receptor and the free ligand concentration. It is, however, compatible with the
findings that in ventricular myocytes isolated
from guinea pig hearts sulfonylurea-induced
inhibition of ATP-dependent K+ channels
depends on the rates of drug absorption
into and release from the membrane lipids
[90] and on the presence of the unionized
G. Muller et al. / Diabetea Research and Clinical Practice 28 Suppl. (1995) S115-S137
form of the drug which is-charaeterized by-high
lipophilicity [91]. Furthermore no high affinity
binding sites for sulfonylureas have been
described so far in plasma membranes of
isolated and cultured adipocytes [92]. Low
affinity binding sites recently identified in
membranes of isolated rat adipocytes [93]
are presumably not involved in mediating the
extrapancreatic effects. Their high Kd values
exceed the EC 50 values for extrapancreatic
effects. With the proposed "partitioning"
mechanism, the specificity of extra-pancreatic
sulfonylurea action would be defined by the
presence of protein components of the
signaling cascade regulating glucose
transport and metabolism in the membrane
of the target cell instead of by a receptorligand interaction. The lower partitioning
coefficient of glimepiride compared to that
of glibenclamide suggests that lipophilicity
is not the main determinant for the strength of
the extrapancreatic activity iv) There are
several explanations of the missing effect
on blood glucose [30] and rate of glucose
disposal in totally insulin-deficient animals
[94] and type I diabetic patients [95, 96]:
a) Insulin may control the activity or
protein/gene expression of certain
components of the signal transduction
cascade mediating the direct insulin-mimetic
effects of sulfonylureas in fat and muscle
cells (e.g. glycosyl-phosphatidylinositolspecific phospholipase C, protein kinase C).
Consequently, sulfonylurea signaling will require a certain basal insulin level, b) The
drastically altered overall metabolic state of
completely insulin-deficient animals may
abolish the direct insulin-mimetic action of
sulfonylureas. c) When insulin is deficient,
glucose is not only less utilized from the
periphery but also excessively produced by
the liver due to absence of inhibition of
gluconeogenesis. This results in hyperglycemia which exceeds the renal glucose
threshold and leads to glucosuria. In vitro,
sulfonylureas have been shown to inhibit
gluconeogenesis in hepatocytes by stimulation
of fructose-2,6-bisphosphate formation
S133
[97, 98, 99,l00,l01] and possibly via
inhibi-tion of protein kinase A [66, 67]. The
sulfonylurea concentrations, however, required
for inhibition of hepatic gluconeogenesis in
vitro are 10-100 times higher than those for
the extrapancreatic effects in peripheral
cells in vitro. This discrepancy in the effective concentrations might be due to the
mechanism of protein kinase A inhibition in
hepatic and adipocyte tissue being fundamentally different. Protein kinase A inhibition
was demonstrated with hepatocytes as well
as with the isolated rat liver enzyme [102].
However, the sulfonylurea concentration
required for inhibition of protein kinase A
was also higher for the isolated liver enzyme
compared to that of the adipocyte enzyme for
incubation of intact cells in agreement
with the observed discrepancy between the
EC50 for inhibition of hepatic
gluconeogenesis and of lipolysis in fat cells.
Phosphoinositol-glycans may mediate the
inhibition of protein kinase A in fat cells,
whereas, in the liver, direct inhibition by
sulfonylureas seems to be effective [67]. It is
thus possible that, in the totally insulin-deficient animals and diabetic patients, the
peripheral extrapancreatic effect was
balanced by insufficient inhibition of the
hepatic glucose output. The increased
glimepiride-induced glucose flux into the
peripheral tissues might have led to reduced
glucosuria but did not lower blood glucose.
Consequently, for manifestation of direct
stimulation of glucose disposal in peripheral
tissues by sulfonylureas, a certain amount of
insulin may be necessary for inhibition of
hepatic glucose output.
The direct stimulation of glucose utilization in
peripheral tissues might be one explanation tor
the extrapancreatic activity of sulfonylureas as
observed in the above animal studies. Particular
attention has been paid to this mode of action in
the present and previous studies over the past 2
decades. However, other mechanisms, such as
modulation of glucagon release from the pancreas [30, 103], alteration of insulin clearance by
S134
G. Muller et al. /Diabetes Research and Clinical Practice 28 Suppl. (1995) SI15-S137
the fiver [104], facilitation of the passage of
insulin from plasma to tissues or increase of the
sensitivity of peripheral tissues towards insulin
action [105-109] may equally well result in a
blood glucose decrease without the need for an
increased insulin release.
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