Institute of Physiological Sciences, Departments of 1 Pharmacology and 2 Surgery, University of Lund, S-221 84 Lund; and 3 Department of Clinical Chemistry, Helsingborg Hospital, S-25187 Helsingborg, Sweden
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ABSTRACT |
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We examined the relation
between nutrient-stimulated insulin secretion and the islet lysosome
acid glucan-1,4--glucosidase system in rats undergoing total
parenteral nutrition (TPN). During TPN treatment, serum glucose was
normal, but free fatty acids, triglycerides, and cholesterol were
elevated. Islets from TPN-infused rats showed increased basal insulin
release, a normal insulin response to cholinergic stimulation but a
greatly impaired response when stimulated by glucose or
-ketoisocaproic acid. This impairment of glucose-stimulated insulin
release was only slightly ameliorated by the carnitine
palmitoyltransferase 1 inhibitor etomoxir. However, in parallel with
the impaired insulin response to glucose, islets from TPN-infused
animals displayed reduced activities of islet lysosomal enzymes
including the acid glucan-1,4-
-glucosidase, a putative key enzyme in
nutrient-stimulated insulin release. By comparison, the same lysosomal
enzymes were increased in liver tissue. Furthermore, in intact control
islets, the pseudotetrasaccharide acarbose, a selective inhibitor of
acid
-glucosidehydrolases, dose dependently suppressed islet acid
glucan-1,4-
-glucosidase and acid
-glucosidase activities in
parallel with an inhibitory action on glucose-stimulated insulin
secretion. By contrast, when incubated with intact TPN islets, acarbose
had no effect on either enzyme activity or glucose-induced insulin
release. Moreover, when acarbose was added directly to TPN islet
homogenates, the dose-response effect on the catalytic activity of the
acid
-glucosidehydrolases was shifted to the right compared with
control homogenates. We suggest that a general dysfunction of the islet
lysosomal/vacuolar system and reduced catalytic activities of acid
glucan-1,4-
-glucosidase and acid
-glucosidase may be important
defects behind the impairment of the transduction mechanisms for
nutrient-stimulated insulin release in islets from TPN-infused rats.
total parenteral nutrition; insulin secretion; islet acid
glucan-1,4--glucosidase activity; lysosomal/vacuolar system; plasma
lipids; etomoxir; acarbose
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INTRODUCTION |
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DESPITE INTENSIVE
RESEARCH, the mechanisms behind pancreatic -cell dysfunction
in patients with non-insulin-dependent diabetes mellitus (NIDDM) remain
largely unclear. There is general agreement that multiple deficiencies
are probably involved, among which is a particular desensitization of
the
-cell to glucose-stimulated insulin secretion (9,
40). Glucose-stimulated insulin release itself is composed of a
complex cascade of events (23, 42), the details of which
are far from elucidated. We (14-20, 31-36) have
proposed that one of these multiple signals, which regulates insulin
release stimulated by glucose and other nutrient secretagogues such as
leucine and
-ketoisocaproic acid (KIC), is transduced through the
vacuolar system involving the activation of the lysosomal acid
glucan-1,4-
-glucosidase. This
-glucosidehydrolase type of enzyme
produces nonphosphorylated free glucose and preferentially cleaves
-1,4-linked glucose polymers such as glycogen (15, 17,
26). It has been known for a long time that the islets of
Langerhans contain glycogen (8, 22). This glycogen level is fairly constant at a wide range of blood glucose concentrations (22), suggesting that the major part of it is not
integrated into the metabolic pool of glucose phosphorylation processes
in the cytoplasm but rather is restricted to a vacuolar pool of signal glycogen. In this context, it should be noted that the phosphorolytic breakdown of glycogen in vitro in islet tissue is known to be very slow
(22). Hence, we have hypothesized that the acid
glucan-1,4-
-glucosidase might attack certain pools of islet vacuolar
glycogen to produce high compartmentalized concentrations of glucose,
which in turn could act as a further transducer, e.g., cybernetic,
metabolic, or osmotic, in the multifactorial process of insulin release
(14-20, 31-36). In fact, recent data emphasize
the role of compartmentalization and acidification in the final stages
of exocytosis (1, 21, 41). In addition, the activated
enzyme might have the ability to modify membrane glycoproteins with
-1,4-linked glucose residues of importance for the exocytotic
process. In accordance with this idea, a series of recent in vitro
studies revealed a close relationship between islet acid
glucan-1,4-
-glucosidase activity and nutrient-stimulated insulin
release at different Ca2+ concentrations as well as in the
presence and absence of various selective
-glucosidehydrolase
inhibitors such as the pseudotetrasaccharide acarbose, the
deoxynojirimycin derivatives miglitol and emiglitate, and the
indolizine alkaloid castanospermine, whereas receptor-activated insulin
secretion induced, for example, by a cholinergic stimulus was
independent of this enzyme activity (19, 20, 30-36).
Furthermore, previous in vivo experiments disclosed a surprisingly good
correlation between glucose-stimulated insulin release and islet acid
glucan-1,4-
-glucosidase activity both in normal mice and in the
insulin-hypersecreting ob/ob mouse, an animal model with
certain similarities to the obese type of human NIDDM
(15-19).
Because patients with NIDDM often display abnormalities not only
in glucose metabolism but also in lipid metabolism, the most prominent
being hypertriglyceridemia with elevated free fatty acid (FFA) levels
(2, 24, 29), and because such abnormalities have been
suggested to greatly impair -cell function (2, 24, 37,
43), the question arose as to whether elevated levels of
triglycerides and FFA would have any impact on the insulin-secretory signal transduced through the lysosome acid glucan-1,4-
-glucosidase system in the
-cell. It has been known for a long time that, similar
to other nutrients such as glucose and certain amino acids, FFA can
acutely stimulate insulin release both in vitro and in vivo (3,
29, 39). In contrast, recent data have shown that long-term
elevation of FFA either infused into rats for 48 h or added to
isolated islets during long-term culture, greatly impair
-cell
function (2, 24, 29, 39, 43, 44). Such an impairment was
reportedly further exaggerated in the obese, prediabetic Zucker (ZDF)
rat, where islet triglyceride content was markedly increased, probably
as a consequence of increased plasma FFA levels (39).
The aim of the present investigation was to characterize, in a rat
model of total parenteral nutrition (TPN), where the TPN solution was
infused for 12 days in healthy, normal Sprague-Dawley rats, the effects
of long-term elevation of plasma levels of FFA and triglycerides
on insulin release induced by two nutrient insulin secretagogues,
glucose and KIC, and one receptor-activating secretagogue, the
cholinergic muscarinic agonist carbachol, in relation to the activity
of the islet lysosome acid glucan-1,4--glucosidase system. For
comparison, measurements of different lysosomal enzyme activities in
liver tissue after TPN infusion were also performed. Finally, to
directly test the function of the islet lysosomal/vacuolar system, we
investigated, in islets isolated from both TPN-infused and freely fed
control rats, the ability of the pseudotetrasaccharide acarbose, a
potent and selective inhibitor of islet acid glucan-1,4-
-glucosidase and glucose-stimulated insulin release (36), to enter into
the lysosomal system and modulate enzyme activity and glucose-induced insulin secretion.
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RESEARCH DESIGN AND METHODS |
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Animals. Male Sprague-Dawley rats (B&K Universal, Sollentuna, Sweden) weighing 225-235 g at the start of the infusion experiments were used. All animals were housed in metabolic cages. The temperature was maintained at a constant level, and a 12:12-h light-dark cycle was provided.
Drugs and chemicals.
Collagenase (CLS 4) was purchased from Worthington Biochemical
(Freehold, NJ). Methylumbelliferyl-coupled substrates, KIC, and
carbachol were obtained from Sigma Chemical (St. Louis, MO). Etomoxir
2-[b-(4-chlorophenoxy)-hexyl]oxirane-2-carboxylate was purchased as
the sodium salt (RBJ Research Biochemicals International, Natick, MA).
Bovine serum albumin (BSA) was from ICN Biomedicals (High Wycombe, UK).
The pseudotetrasaccharide acarbose was generously supplied by Bayer
(Leverkusen, Germany). All other drugs and chemicals were from British
Drug Houses (Poole, UK) or Merck (Darmstadt, Germany). The
radioimmunoassay (RIA) kits for insulin determination were obtained
from Novo Nordisk (Bagsvrd, Denmark) or Diagnostika (Falkenberg, Sweden).
Experimental procedures: TPN.
Rats were anesthetized intraperitoneally with chloral hydrate and
operated on under sterile conditions. The neck of the rat was gently
washed with an iodine solution. A silicon-rubber catheter, 0.037 in. OD
(Silastic, Dow Corning, Midland, MI), was inserted into the right
jugular vein. The catheter was transferred to the skull subcutaneously
and connected to a swivel via a protective coil attached to the skin of
the skull. Immediately after surgery, all rats in the TPN group were
infused with a 5% glucose solution at 2.0 ml/h for 12 h, followed
by TPN at 200 ml · kg1 · day
1. Every 2nd
day, the catheters were flushed with 100 U/kg of low molecular weight
heparin (Fragmin; Pharmacia, Uppsala, Sweden). No oral intake,
including water, was allowed during the infusion period. Control
animals underwent the same operative procedure, including insertion of
a catheter, but no TPN infusion was performed. The catheters were
similarly flushed with 100 U/kg of Fragmin every 2nd day. The control
animals were allowed free access to a standard pellet diet (B&K
Universal) and tap water ad libitum. Details of the methodology as well
as the composition of the TPN solution were recently described
(28). The TPN infusion experiments lasted 12 days. All
animals were housed in metabolic cages with a constant temperature. A
12:12-h light-dark cycle was provided. No significant differences in
body weights were detected between the TPN and the control groups at
the end of the experiments.
Determination of serum lipids.
Concentrations of FFA, triglycerides without free glycerol (TG), and
cholesterol in serum were determined enzymatically with kits from Wako
Chemicals (Neuss, Germany) and Boehringer Mannheim (Indianapolis, IN).
High-density lipoprotein (HDL) cholesterol was determined as
cholesterol in the supernatant after precipitation with polyethylene
glycol 6000. Sera were stored at 20°C until analyzed.
Insulin secretion from isolated islets.
Insulin secretion studies were performed with freshly isolated islets.
The islets were isolated directly after the TPN infusion device was
disconnected, with the exception of one series of experiments (illustrated in Fig. 1, D-F),
where the TPN rats (and the controls) were fasted for 12 h after
disconnection. After decapitation, preparation of isolated pancreatic
islets from TPN-infused and control rats was performed by retrograde
injection of a collagenase solution via the bile-pancreatic duct
(4). The islets were then preincubated for 30 min at
37°C in Krebs-Ringer bicarbonate (KRB) buffer, pH 7.4, supplemented
with 10 mmol/l HEPES, 0.1% BSA, and 1 mmol/l glucose. Each incubation
vial was gassed with 95% O2-5% CO2 (vol/vol)
to obtain constant pH and oxygenation. After preincubation, the buffer
was changed to a medium containing different glucose concentrations and
test agents, and the islets were incubated for 60 min. All incubations
were performed at 37°C in an incubation box. Immediately after
incubation, aliquots of the medium were removed and frozen for
subsequent RIA of insulin (7). In the experiments with
acarbose, the islets were first preincubated (with or without acarbose)
for 60 min (to allow for acarbose uptake) at 1 mmol/l glucose
(36). After this preincubation, the buffer was changed to
a medium containing 1 or 16.7 mmol/l glucose with or without acarbose,
and then the islets were incubated for 120 min (36).
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Determination of lysosomal enzyme activities and neutral
-glucosidase in isolated islets and liver.
For determination of lysosomal enzyme activities, 150-200 isolated
islets (35 islets in the acarbose incubation experiments) were
thoroughly washed in a glucose-free Hanks' solution and collected and
stored in acetate-EDTA buffer (1.1 mmol/l EDTA and 5 mmol/l acetate, pH
5.0) at
20°C. After thawing in an ice bath and subsequent sonication, the islet homogenates were analyzed for enzyme activities as previously described in detail (13, 15, 17, 30).
Similarly, the determination of enzyme activities in liver tissue has
previously been described (15, 30). Protein was analyzed
according to Lowry et al. (11).
In vivo experiment.
Before the start of the in vivo experiment, the TPN infusion was
stopped, and 100 U/kg of Fragmin were infused, whereafter the rats had
no access to food for 12 h. Control rats were handled similarly.
The rats were then anesthetized with 1% lidocaine (Pharmacia) at the
tail root, and glucose (4.4 mmol/kg) was rapidly injected via the
jugular catheter. Blood samples were taken from the tip of the tail at
0, 2, 6, 15, and 30 min. Plasma was then frozen at 20°C until
analysis of insulin (7). During the experiments, the
animals were kept conscious in Bollman cages.
Statistics. Levels of significance between sets of data were assessed using Student's t-test for unpaired data or, where applicable, analysis of variance followed by Tukey-Kramer's multiple comparisons test.
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RESULTS |
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Basal metabolic characteristics of TPN-infused rats and their
controls.
Table 1 shows the basal metabolic status
as reflected in serum of the TPN-infused rats and their freely fed
controls on days 4, 8, and 12 after start of the
treatment. It was seen that the TPN rats displayed greatly elevated
serum levels of FFA, TG, and cholesterol, whereas HDL-cholesterol was
modestly decreased. There was no difference in the serum glucose levels
between the two groups (Table 1). Furthermore, in ancillary
experiments, we found no difference in the basal plasma levels of
either insulin or glucose recorded on days 7, 10, and
12 (data not shown).
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Effect of TPN on the insulin secretory response stimulated by glucose, KIC, or carbachol. Figure 1A shows the effect of low (1 mmol/l) and high (16.7 mmol/l) glucose on insulin release from islets isolated either from freely fed controls or from TPN rats directly after the infusion of TPN was stopped. It is seen that insulin secretion from TPN islets was increased at low glucose. At high glucose, however, the increase in insulin release from control islets was 14-fold above basal, whereas the increase from TPN islets was only approximately fourfold. Figure 1B illustrates the effect of another nutrient secretagogue, KIC (10 mmol/l), on insulin release from isolated islets after TPN treatment. This series of experiments was performed in a glucose-free KRB medium. It is seen that insulin secretion from TPN islets, in the absence of glucose, was increased twofold above the release observed in control islets. Addition of KIC had practically no effect on insulin release in TPN islets but induced a fivefold increase in control islets. Figure 1C shows that insulin secretion at a more physiological glucose concentration (4 mmol/l) was unaffected in islets isolated from TPN rats. Moreover, cholinergic receptor-activated stimulation of insulin release by the cholinergic muscarinic agonist carbachol (20 µmol/l) was of the same magnitude in TPN and control islets (Fig. 1C).
Effect of a 12-h normalization period after TPN treatment cessation. To elucidate whether the TPN-induced impairment of glucose-stimulated insulin release was rapidly and readily reversible, we performed a series of experiments with islets isolated at 12 h after the TPN infusion was stopped. During this 12-h period, all animals were allowed drinking water but no food. Figure 1, D and E, shows that the impairment of glucose-induced insulin release from isolated islets of the TPN rats compared with the control group was still very obvious. Insulin secretion at 1 or 4 mmol/l glucose, as well as insulin release stimulated by carbachol, was similar to that of the controls. To test whether the poor insulin release in response to glucose was also present in the in vivo situation an intravenous glucose load (4.4 mmol/kg) was given to both groups of animals. Figure 1F shows that the glucose-induced in vivo insulin response in the TPN rats was also reduced. The glucose tolerance curve was slightly impaired at 15 and 30 min in the TPN group, but the area under the curve was not significantly different from that of the controls (data not shown).
Influence of etomoxir on glucose-induced insulin release in
TPN-treated islets.
We next investigated whether the profound impairment of glucose-induced
insulin release after TPN treatment could be explained by a direct
influence of the elevated serum FFA on islet glucose metabolism,
because long-term exposure to FFA has been reported to inhibit
glucose-stimulated insulin secretion through a glucose-fatty acid cycle
(29, 43). To this purpose, etomoxir, a mitochondrial carnitine palmitoyltransferase 1 inhibitor, was added to the test tubes. Figure 2 shows that
glucose-induced insulin release was suppressed by ~55% in
TPN-treated islets. Only a minor fraction of this inhibition was
reversed by etomoxir treatment. Etomoxir did not influence basal (1 mmol/l) or glucose-stimulated (16.7 mmol/l) insulin release in control
islets but slightly increased basal insulin secretion in TPN-treated
islets (Fig. 2).
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Lysosomal enzyme activities in isolated islets and liver tissue.
To test whether the elevated serum lipids might influence the lysosomal
system, we performed an analysis of islet and liver lysosomal enzyme
activities after TPN. Directly after the TPN infusion was stopped,
pancreatic islets were isolated, and liver specimens were removed for
analysis of the activity of the acid glucan-1,4--glucosidase as well
as analysis of other lysosomal enzymes and the neutral
-glucosidase
(an enzyme attributed to the endoplasmic reticulum). Figure
3A shows the activities of the
different lysosomal enzymes recorded in islet tissue. It is seen that
the activities of the glycogen-hydrolyzing enzyme acid glucan-1,4-
-glucosidase (
45%) and acid
-glucosidase (
30%) were significantly reduced. Likewise, the activities of other types of
lysosomal enzymes i.e., acid phosphatase (
50%),
N-acetyl-
-D-glucosaminidase (
50%) and
-glucuronidase (
20%) were depressed. The activity of the
proteolytic lysosomal enzyme cathepsin D, however, was unaffected, as
was the nonlysosomal neutral
-glucosidase.
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Effects of acarbose.
To elucidate whether the TPN islets might suffer from a general
dysfunction of the lysosomal/vacuolar system, we performed a series of
experiments with the the selective acid glucan-1,4--glucosidase inhibitor acarbose, a pseudotetrasaccharide, which is known to enter
the cell through endocytosis (12, 19, 30, 31, 36). Figure
4 shows the dose-response relationship
for the effect of acarbose on lysosomal enzyme activities after direct
addition to islet homogenates as well as the effect of acarbose on
glucose-induced insulin release in isolated islets. Acid
glucan-1,4-
-glucosidase (Fig. 4A) and acid
-glucosidase (Fig. 4B) activities were dose dependently
inhibited by acarbose in islet homogenates from both controls and TPN
rats. However, compared with controls, the ED50 for the
inhibitory effect of acarbose on acid glucan-1,4-
-glucosidase activity in TPN islet homogenates was clearly shifted to the right by
more than one order of magnitude. The acarbose inhibition curve for
acid
-glucosidase was also shifted to the right, although it was
less pronounced. Other lysosomal enzyme activities in freely fed
control and TPN islets were not affected by acarbose (Fig. 4,
E and F). Neutral
-glucosidase activity was
reduced only at very high concentrations of acarbose and equally for
both control and TPN islets (Fig. 4D). In parallel with the
effects of acarbose on islet acid
-glucosidehydrolase activities,
the glucose-stimulated insulin release from islets of control rats was
dose dependently and markedly suppressed by the pseudotetrasaccharide
(Fig. 4C). In contrast, acarbose displayed no effect at all
on glucose-stimulated insulin release from islets isolated from
TPN-rats (Fig. 4C).
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DISCUSSION |
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Acute stimulation of insulin release by FFA at basal glucose has
been well established for a long time (3, 27). However, the mechanisms of action of FFA in stimulating the -cell secretory machinery are still open to debate and have been suggested to include,
for example, increased mitochondrial oxidation of FFA, increased
Ca2+ influx, and increased long-chain acyl-CoA esters,
which in turn activate protein kinase C (39). In contrast,
direct demonstration of long-term effects of elevated serum FFA on
-cell function, thus in a way mimicking the situation in obese
NIDDM, has received less attention. However, very recently, Shimabukuro
et al. (37) and Unger (39) demonstrated, by
investigating the ZDF (fa/fa) rat (having a mutated leptin
receptor), that the
-cell failure in this type of obesity-associated
diabetes with high plasma FFA was correlated with excessive
accumulation of fat within the islet tissue due to an increased
capacity to esterify, and a decreased capacity to oxidize, FFA.
The present study was initially encouraged by an early article by
Greenberg et al. (5), showing that TPN treatment in human subjects for 25 days greatly reduced the insulin response to a standardized meal without any change in gut hormone release, and by
more recent studies by Sako and Grill (29) and Zhou and
Grill (43, 44) in the normal rat. They observed that
48 h of fat infusion or islet culture with elevated FFA in the
medium greatly impaired the insulin secretory capacity in response to
glucose and that the decreased insulin response in their experiments
could be explained largely by an FFA-induced decrease in islet glucose oxidation and decreased pyruvate dehydrogenase activity. However, in
contrast to these studies (29, 43) we found in our
long-term experiments that the TPN-induced suppression (~55%) of
glucose-stimulated insulin release was only slightly restored by
exposure to the carnitine palmitoyltransferase 1 inhibitor etomoxir.
Indeed, when the elevated release of basal insulin from TPN islets is
taken into account, the inhibitory effect of glucose-stimulated release exerted by TPN, when calculated as IRI (i.e., increase of insulin above basal), was found to be 66%, of which only 11% was reversed by
etomoxir. Thus only a minor part of the inhibition could be explained
by an inhibitory influence of FFA on glucose oxidation. Moreover, in
contrast to Roth et al. (28) and Zhou and Grill, we found
that the insulin-releasing action of KIC, a keto acid directly
metabolized in the mitochondria, was totally abolished by TPN
treatment. Because this nutrient is oxidized directly in the citric
acid cycle without involvement of pyruvate dehydrogenase, it appears
that the major inhibitory effect on insulin release stimulated by
glucose and KIC in TPN islets probably is exerted distally to glucose
oxidation and pyruvate dehydrogenase activity. However, in this
context, it should be noted that it cannot be excluded that part of the
impairing effect of our long-term infusion of TPN on
nutrient-stimulated insulin release could be elicited not only by FFA
but also by other (lipid?) components of the TPN solution.
The pattern of stimulated insulin release from the islets of
TPN-infused rats was strikingly similar to that previously seen after
selective blockade of islet acid glucan-1,4--glucosidase. That is,
nutrient-stimulated insulin release was markedly suppressed, whereas
receptor-mediated insulin secretion induced by phospholipase C-protein
kinase C activating agents was unaffected (20,
31-36). In accord with this, we observed a marked
inhibition of acid glucan-1,4-
-glucosidase activity in islets
isolated from TPN-treated rats assayed directly ex vivo.
Moreover, the activities of other lysosomal enzymes were also greatly
reduced. In contrast, liver tissue taken from the same animals
displayed a marked increase in the various lysosomal enzyme activities.
These results thus reveal a surprisingly great variability among
different lysosomal enzyme activities in islets and liver,
respectively, after TPN treatment. It has been known for a long time
that a normal physiological variability in lysosomal enzyme activities
may exist among different tissues as well as within a given tissue
(25). The total pattern of the lysosomal enzyme activies
in islets and liver of TPN-treated rats is also suggestive of enzyme
heterogeneity among the lysosomes and/or that lysosomal enzyme
activities in these tissues can be modulated relatively independently
of one another. Furthermore, it has previously been reported that
catabolic, destructive, and degenerative processes in a given tissue
are accompanied by increased levels in the activities of classical
lysosomal enzymes (38). Such a reaction pattern was
clearly evident in liver tissue and was most likely due to an excessive
stimulation of the lysosomal/vacuolar system by fat overloading during
the long-standing TPN infusion. In contrast, the lysosomal enzyme
activities in the islets of Langerhans were all highly suppressed by
the same treatment. It may be that the liver, by virtue of its
important physiological role in lipid metabolism, is liable to take up
comparatively more fat and, when being heavily "overloaded," its
lysosomal/vacuolar system is activated. The islets, on the other hand,
are not primarily a fat-storing organ and thus are affected differently
by the abnormal fat loading, resulting in a state of suppressed
activity of the whole lysosomal/vacuolar system. It is also possible
that specialized subpopulations of lysosomal organelles with differing
physiological functions are reacting differently in the islets and the
liver, respectively. Indeed, our present data on islet lysosomal enzyme
activities after in vitro incubation of intact islets (Fig. 5) suggest
a different action of the TPN infusion on different organelles of the
islet lysosomal/vacuolar system, because the acid
-glucosidehydrolase activities were strongly suppressed, as was
glucose-stimulated insulin release, whereas the activities of other
lysosomal enzymes were markedly increased. It is known from previous
data recorded in other tissues that fat overloading may affect
lysosomal enzyme activities differently. Thus aortic cells taken from
cholesterol-fed rabbits displayed markedly increased activities of acid
cholesteryl esterase,
N-acetyl-
-D-glucosaminidase, and
-galactosidase (6). On the other hand, addition of
oxidized low-density lipoproteins to cultured J-774 cells (murine
macrophage cell line) resulted in a significant decrease of the total
activities of N-acetyl-
-D-glucosaminidase and
cathepsin L (10).
Our results from the present acarbose experiments provide the evidence
to assume that, in the TPN islets, it is both the acid glucan-1,4--glucosidase activity itself and the whole
lysosomal/vacuolar system that were malfunctioning. In previous
experiments, we had observed that the pseudotetrasaccharide acarbose, a
potent inhibitor of islet acid
-glucosidehydrolases
(36), when incubated together with mouse islets, was taken
up and exerted a marked inhibitory effect on both acid
glucan-1,4-
-glucosidase activity and glucose-stimulated insulin
release. In contrast, a close acarbose analog, the tetrasaccharide maltotetraose, which is devoid of enzyme-inhibitory properties, did not
affect either enzyme activity or insulin release (36). It
should be emphasized that, because acarbose is a selective
-glucosidehydrolase inhibitor, our data strongly suggest a direct cause-effect relationship between inhibition of islet acid
glucan-1,4-
-glucosidase activity on the one hand and suppression of
glucose-stimulated insulin release on the other. Indeed, the present
results revealed that, in the presence of acarbose in the control
islets, the inhibition curve for glucose-stimulated insulin release was
dose dependent and similar in shape to the inhibition curve for acid
glucan-1,4-
-glucosidase activity. In contrast, no inhibitory effect
by acarbose was observed in intact, incubated islets from TPN-infused
rats, although a slight tendency to inhibition was found at a very high
concentration of acarbose (10 mM). These data suggested that, in the
TPN islets, relevant amounts of acarbose were not given access to its
target, the acidic lysosomal/vacuolar compartment in the
-cells.
This is in accord with very recent data (30) showing that
dysfunction of the islet lysosomal/vacuolar system in the spontaneously
diabetic Goto-Kakizaki rat may be an important factor involved in the
impaired glucose-stimulated insulin release of this animal NIDDM model.
In conclusion, the data of the present study suggest, but do not
directly prove, that a TPN-induced generalized suppression of the islet
lysosomal/vacuolar system and its acid glucan-1,4--glucosidase activity is associated with an impairment of glucose-stimulated insulin
secretion. Our in vitro observations further suggest that this
defective insulin response to nutrient secretagogues in TPN islets may
be referred to both as a selective suppression of the acid
glucan-1,4-
-glucosidase activity and as a dysfunction of the whole
lysosomal/vacuolar system possibly conveyed, for example, by a hitherto
unrecognized lipid-induced impairment of the membrane function of the
acidic organelles involved and/or other regulatory factors modulating
the acid glucan-1,4-
-glucosidase activity.
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ACKNOWLEDGEMENTS |
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The skillful technical assistance of Elsy Ling and Britt-Marie Nilsson and the secretarial help of Eva Björkbom are gratefully acknowledged.
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FOOTNOTES |
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This study was supported by the Stig and Ragna Gorthon Foundation, the Swedish Medical Research Council (14X-4286 and 17X-11616), the Crafoord Foundation, the Swedish Diabetes Association, the Åke Wiberg Foundation, and the Albert Påhlsson Foundation. All TPN constituents were kindly supplied by Pharmacia, Uppsala, Sweden.
Address for reprint requests and other correspondence: I. Lundquist, Dept. of Pharmacology, Univ. of Lund, Sölvegatan 10, S-221 84 Lund, Sweden
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 12 August 2000; accepted in final form 28 February 2001.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Bokvist, K,
Eliasson L,
Ämmälä C,
Renström E,
and
Rorsman P.
Co-localization of L-type Ca2+ channels and insulin-containing secretory granules and its significance for the initiation of exocytosis in mouse pancreatic -cells.
EMBO J
14:
50-57,
1995[Abstract].
2.
Elks, ML.
Chronic perifusion of rat islets with palmitate suppresses glucose-stimulated insulin release.
Endocrinology
133:
208-214,
1993[Abstract].
3.
Felber, JP,
and
Vanotti A.
Effects of fat infusions on glucose tolerance and insulin plasma levels.
Med Exp
10:
153-156,
1964[ISI].
4.
Gotoh, M,
Maki T,
Kiyoizumi T,
Satomi S,
and
Monaco AP.
An improved method for isolation of mouse pancreatic islets.
Transplantation
40:
437-438,
1985[ISI][Medline].
5.
Greenberg, GR,
Wolman SL,
Christofides ND,
Bloom SR,
and
Jeejeebhoy KN.
Effect of total parenteral nutrition on gut hormone release in humans.
Gastroenterology
80:
988-993,
1981[ISI][Medline].
6.
Haley, NJ,
Fowler S,
and
de Duve C.
Lysosomal acid cholesteryl esterase activity in normal and lipid-laden aortic cells.
J Lipid Res
21:
961-969,
1980[ISI][Medline].
7.
Heding, L.
A simplified insulin radioimmunoassay method.
In: Labelled Proteins in Tracer Studies, edited by Donato L,
Milhaud G,
and Sirchis J.. Brussels: Euratom, 1966, p. 345-350.
8.
Hellman, B,
and
Idahl L-Å.
On the functional significance of the pancreatic -cell glycogen.
In: The Structure and Metabolism of the Pancreatic Islets, edited by Falkmer S,
Hellman B,
and Täljedal I-B.. Oxford, UK: Pergamon, 1970, p. 253-262.
9.
Leahy, JL,
Bonner Weir S,
and
Weir GC.
Beta-cell dysfunction induced by chronic hyperglycemia. Current ideas on mechanism of impaired glucose-induced insulin secretion.
Diabetes Care
15:
442-455,
1992[Abstract].
10.
Li, W,
Yuan M,
Olsson AG,
and
Brunk UT.
Uptake of oxidized LDL by macrophages results in partial lysosomal enzyme inactivation and relocation.
Arterioscler Thromb Vasc Biol
18:
177-184,
1998
11.
Lowry, OH,
Rosebrough NJ,
Farr AL,
and
Randall RJ.
Protein measurement with the Folin phenol reagent.
J Biol Chem
193:
265-275,
1951
12.
Lüllman-Rauch, R.
Lysosomal glycogen storage mimicking the injection of an -glucosidase inhibitor.
Virchows Arch
38:
89-100,
1981.
13.
Lundquist, I.
Method for determination of acid amyloglucosidase in isolated islets of the pancreas.
Enzyme (Basel)
12:
647-657,
1971[ISI][Medline].
14.
Lundquist, I.
Carbohydrate content and regulation following injection of different glycogenolytic enzymes.
Enzyme (Basel)
20:
234-247,
1975[ISI][Medline].
15.
Lundquist, I.
Lysosomal enzyme activities in pancreatic islets from normal and obese hyperglycemic mice.
Metabolism
34:
1-9,
1985[ISI][Medline].
16.
Lundquist, I.
Differential changes in islet lysosomal enzyme activities in aging obese hyperglycemic mice.
Diabetes Res
3:
25-30,
1986[ISI][Medline].
17.
Lundquist, I.
Islet amyloglucosidase activity: some characteristics, and its relation to insulin secretion stimulated by various secretagogues.
Diabetes Res
3:
31-41,
1986[ISI][Medline].
18.
Lundquist, I,
and
Lövdahl R.
Pattern of islet lysosomal enzyme activities and insulin secretory response.
Enzyme (Basel)
22:
385-390,
1977[ISI][Medline].
19.
Lundquist, I,
and
Panagiotidis G.
The relationship of islet amyloglucosidase activity and glucose-induced insulin secretion.
Pancreas
7:
352-357,
1992[ISI][Medline].
20.
Lundquist, I,
Panagiotidis G,
and
Salehi A.
Islet acid glucan-1,4--glucosidase: a putative key enzyme in nutrient-stimulated insulin secretion.
Endocrinology
137:
1219-1225,
1996[Abstract].
21.
Maechler, P,
and
Wollheim CB.
Mitochondrial glutamate acts as a messenger in glucose-induced insulin exocytosis.
Nature
402:
685-689,
1999[ISI][Medline].
22.
Matschinsky, FM,
and
Ellerman JE.
Metabolism of glucose in the islets of Langerhans.
J Biol Chem
243:
2730-2736,
1967
23.
Metz, SA.
The pancreatic islet as Rubik's cube. Is phospholipid hydrolysis a piece of the puzzle?
Diabetes
40:
1565-1573,
1991[Abstract].
24.
Milburn, JL, Jr,
Hirose H,
Lee YH,
Nagasawa Y,
Ogawa A,
Ohneda M,
BeltrandelRio H,
Newgard CB,
Johnson JH,
and
Unger RH.
Pancreatic beta-cells in obesity. Evidence for induction of functional, morphologic, and metabolic abnormalities by increased long chain fatty acids.
J Biol Chem
270:
1295-1299,
1995
25.
Novikoff, AB.
Lysosomes: a personal account.
In: Lysosomes and Storage Diseases, edited by Hers HG,
and van Hoof F.. New York: Academic, 1973, p. 1-41.
26.
Pazur, JH,
and
Kleppe K.
The hydrolysis of -D-glucosides by amyloglucosidase from Aspergillus niger.
J Biol Chem
237:
1002-1006,
1962
27.
Pelkonen, R,
Miettinen TA,
Taskinen MR,
and
Nikkila EA.
Effect of acute elevation of plasma glycerol, triglyceride and FFA levels on glucose utilization and plasma insulin.
Diabetes
17:
76-82,
1968[ISI][Medline].
28.
Roth, B,
Ekelund M,
Fan B-G,
Hägerstrand I,
Salehi A,
Lundquist I,
and
Nilsson-Ehle P.
Biochemical and ultra-structural reactions to parenteral nutrition with two different fat emulsions in rats.
Intensive Care Med
24:
716-724,
1998[ISI][Medline].
29.
Sako, Y,
and
Grill VE.
A 48-hour lipid infusion in the rat time-dependently inhibits glucose-induced insulin secretion and cell oxidation through a process likely coupled to fatty acid oxidation.
Endocrinology
127:
1580-1589,
1990[Abstract].
30.
Salehi, A,
Henningsson R,
Mosén H,
Östenson C-G,
Efendic S,
and
Lundquist I.
Dysfunction of the islet lysosomal system conveys impairment of glucose-induced insulin release in the diabetic GK rat.
Endocrinology
140:
3045-3053,
1999
31.
Salehi, A,
and
Lundquist I.
Changes in islet glucan-1,4--glucosidase activity modulate sulphonylurea-induced but not cholinergic insulin secretion.
Eur J Pharmacol
243:
185-191,
1993[ISI][Medline].
32.
Salehi, A,
and
Lundquist I.
Islet glucan-1,4--glucosidase: differential influence on insulin secretion induced by glucose and isobutylmethylxanthine in mice.
J Endocrinol
138:
391-400,
1993[Abstract].
33.
Salehi, A,
and
Lundquist I.
Ca2+ deficiency, selective -glucosidehydrolase inhibition, and insulin secretion.
Am J Physiol Endocrinol Metab
265:
E1-E9,
1993
34.
Salehi, A,
Mosén H,
Linell M,
and
Lundquist I.
Castanospermine inhibits islet lysosomal acid glucan-1,4--glucosidase activity and glucose stimulated insulin release in parallel.
Pharmacol Rev Comm
10:
165-173,
1998.
35.
Salehi, A,
Mosén H,
and
Lundquist I.
Insulin release transduction mechanism through acid glucan 1,4--glucosidase activation is Ca2+ regulated.
Am J Physiol Endocrinol Metab
274:
E459-E468,
1998
36.
Salehi, A,
Panagiotidis G,
Borg LA,
and
Lundquist I.
The pseudotetrasaccharide acarbose inhibits pancreatic islet glucan-1,4--glucosidase activity in parallel with a suppressive action on glucose-induced insulin release.
Diabetes
44:
830-836,
1995[Abstract].
37.
Shimabukuro, M,
Ohneda M,
Lee Y,
and
Unger RH.
Role of nitric oxide in obesity-induced cell disease.
J Clin Invest
100:
290-295,
1997
38.
Thesleff, S,
Libelius R,
and
Lundquist I.
Endocytosis as inducer of degenerative changes in skeletal muscle.
In: Muscle, Nerve and Brain Degeneration, edited by Kidman AD,
and Tomkins JK.. Amsterdam and Oxford: Excerpta Medica, 1979, p. 119-138.
39.
Unger, RH.
Lipotoxicity in the pathogenesis of obesity-dependent NIDDM. Genetic and clinical implications.
Diabetes
44:
863-870,
1995[Abstract].
40.
Unger, RH,
and
Grundy S.
Hyperglycaemia as an inducer as well as a consequence of impaired islet cell function and insulin resistance: implications for the management of diabetes.
Diabetologia
28:
119-121,
1985[ISI][Medline].
41.
Ungermann, C,
Wickner W,
and
Xu Z.
Vacuole acidification is required for trans-SNARE pairing, LMA1 release, and homotypic fusion.
Proc Natl Acad Sci USA
96:
11194-11199,
1999
42.
Zawalich, WS,
and
Rasmussen H.
Control of insulin secretion: a model involving Ca2+, cAMP and diacylglycerol.
Mol Cell Endocrinol
70:
119-137,
1990[ISI][Medline].
43.
Zhou, YP,
and
Grill VE.
Long-term exposure of rat pancreatic islets to fatty acids inhibits glucose-induced insulin secretion and biosynthesis through a glucose fatty acid cycle.
J Clin Invest
93:
870-876,
1994[ISI][Medline].
44.
Zhou, YP,
and
Grill VE.
Palmitate-induced beta-cell insensitivity to glucose is coupled to decreased pyruvate dehydrogenase activity and enhanced kinase activity in rat pancreatic islets.
Diabetes
44:
394-399,
1995[Abstract].
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