1 Molecular Nutrition Unit, Department of Nutrition, University of Montreal, and the Centre de Recherche du Centre Hospitalier de l'Université de Montréal and Institut du Cancer, Montreal, Quebec, Canada H2L 4M1; 2 Department of Medical Biochemistry, Centre Médical Universitaire, University of Geneva, Geneva, Switzerland 1211; and 3 Center for Obesity and Metabolism at Boston University Medical Center, Boston University Medical School, Boston, Massachussets 02118
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ABSTRACT |
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A comprehensive
metabolic study was carried out to understand how chronic exposure of
pancreatic -cells to fatty acids causes high basal secretion and
impairs glucose-induced insulin release. INS-1
-cells were exposed
to 0.4 mM oleate for 3 days and subsequently incubated at 5 or 25 mM
glucose, after which various parameters were measured. Chronic oleate
promoted triglyceride deposition, increased fatty acid oxidation and
esterification, and reduced malonyl-CoA at low glucose in association
with elevated basal O2 consumption
and redox state. Oleate caused a modest (25%) reduction in glucose
oxidation but did not affect glucose usage, the glucose 6-phosphate and
citrate contents, and the activity of pyruvate dehydrogenase of INS-1
cells. Thus changes in glucose metabolism and a Randle-glucose/fatty
acid cycle do not explain the altered secretory properties of
-cells
exposed to fatty acids. The main response of INS-1 cells to chronic
oleate, which is to increase the oxidation and esterification of fatty
acids, may contribute to cause high basal insulin secretion via
increased production of reducing equivalents and/or the generation of
complex lipid messenger molecule(s).
fatty acids; insulin secretion; obesity; type 2 diabetes
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INTRODUCTION |
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OBESE PATIENTS with type 2 diabetes show altered
insulin release with respect to prevailing blood glucose characterized
by elevated circulating insulin in the fasting state and relatively low
insulin levels postprandially when glucose is high (29, 30). The
biochemical basis of these alterations is not known, but the emerging
evidence suggests that elevated circulating free fatty acids (FFA) as
well as long chain acyl-CoA esters (Lc-CoA) and triglyceride deposition
in the -cell may be causally implicated (25, 32).
FFA are important nutrients for -cell function because they are used
as fuels (23) and play a permissive role for glucose-induced insulin
release, as evidenced with a pharmacological approach (44) and leptin
gene transfer experiments (13). In addition, their Lc-CoA or complex
lipid derivatives may act as coupling factors in nutrient-stimulated
insulin secretion (8, 31).
Acute exposure of -cells to fatty acids potentiates glucose-induced
insulin secretion by mechanisms that apparently do not implicate
KATP channels but may involve
C-kinase activation by Lc-CoA and
Ca2+ channels (33, 45). By
contrast, over longer time ranges (>24 h) fatty acids such as
palmitate and oleate cause both in vitro and in vivo high basal insulin
secretion characterized by a lowered glucose set point and markedly
reduced secretion in response to a further elevation of the sugar (4,
12, 17, 38, 48, 49). At relatively high concentrations, fatty acids
also activate the nitric oxide transduction system of islet tissue, a
process that may induce cell apoptosis and be implicated in final
-cell decompensation (42, 43).
Regarding secretion, several studies have suggested that the long-term
action of fatty acids is caused by changes in lipid and glucose
metabolism, possibly as a consequence of variations in the expression
of genes encoding key enzymes implicated in fuel signaling. In this
respect, a complex picture with conflicting results has emerged. Thus
palmitate and oleate not only increase the total activity of hexokinase
in rat islets (12) and HC9 -cell (17) but also decrease the
expression of glucokinase and GLUT-2, possibly via reduced expression
of the transcription factor IDX-1 (11). In addition, fatty acids induce
the carnitine palmitoyltransferase I gene (CPT-I; Ref. 3)
and reduce acetyl-CoA carboxylase expression (4) in INS-1
-cells,
thus causing enhanced fat oxidation (4). An enhanced rate of oxidation
of fatty acids might reduce glucose metabolism and consequently insulin
secretion if a so-called Randle cycle (34) were operative in the
-cell after fat exposure. In this respect, FFA were shown to cause a modest (~20%) reduction of glucose oxidation in islets (49) and HC9
-cells (17) or to increase glucose oxidation in another islet study
(12). A reduced islet pyruvate dehydrogenase (PDH) activity predicted
by the Randle cycle has been observed (50), but the associated
decreased glucose oxidation caused by fatty acid reported by the same
group was noted only at very high (27 mM) glucose and not at
intermediate (11 mM) or low glucose concentrations (38, 49). Thus it is
presently uncertain whether alterations in glucose metabolism provide
an explanation for the long-term action of fatty acids on insulin secretion.
As reviewed previously (34), the following sequence of events
associated with predicted alterations in metabolite levels characterize
a Randle cycle. Accelerated fat oxidation results in exaggerated NADH
and acetyl-CoA production, thus causing PDH inhibition and reduced
glucose oxidation. Citrate also rises via a mass effect in the
mitochondrion and, after its export to the cytosolic compartment,
allosterically inhibits 6-phosphofructo-1-kinase, thus
causing reduced glycolytic flux and a rise in glucose 6-phosphate. To
better understand how fatty acids increase insulin secretion at low
glucose and to determine whether altered glucose metabolism via a
Randle glucose-fatty acid cycle may account for the lack of glucose
response in cells chronically exposed to FFA, we have carried out a
comprehensive study of glucose and fatty acid metabolism in INS-1
-cells after long-term exposure to oleate.
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METHODS |
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Cell culture and incubation
conditions. The experiments have been carried out with
the -cell line INS-1, which responds to glucose at physiological
concentrations of the sugar to an extent similar to that of pancreatic
islets in culture (2, 6, 39). All experiments have been carried out
with cell passages below 85 because we noticed that at higher passage
numbers INS-1 cells loose their response to glucose (>5 mM). INS-1
cells were seeded in 21-cm2 petri
dishes (1.4 × 106 cells/dish) or in 24-well
plates (105 cells/well) and grown
as described previously (2) in RPMI 1640 medium at 11 mM glucose
supplemented with 10 mM HEPES, 10% heat-inactivated fetal calf serum,
2 mM L-glutamine, 1 mM sodium pyruvate, 50 µM
-mercaptoethanol, 100 IU/ml penicillin, and 100 µg/ml streptomycin in a humidified atmosphere (5%
CO2-95% air). When cells reached
80% confluence after ~7 days, they were washed twice with PBS and
preincubated at 37°C for 2 days in culture medium containing 5 mM
glucose. Cells were then washed with PBS and incubated for 3 days in
culture medium at 5 mM glucose in the absence or presence of oleate
(0.4 mM). Albumin-bound oleate was prepared by stirring the fatty acid
Na salt at 45°C with defatted BSA (fraction V, Sigma). After
adjustment of the pH to 7.4, the solution was filtered through a
0.22-µm filter and the fatty acid concentration was measured with a
NEFA C kit (Wako Chemicals). For incubations longer than 24 h, the
medium was changed daily to maintain a constant concentration of fatty
acid. The final concentration of BSA in the culture medium was 0.5%.
Metabolites and insulin secretion
measurements. Citrate and malate were determined as
follows. After a culture period of 3 days at 5 mM glucose with or
without oleate, cells were washed with PBS and preincubated at 37°C
for 30 min in Krebs-Ringer bicarbonate buffer (KRBB) with 25 mM HEPES,
pH 7.4 (35), at 5 mM glucose, containing 0.07% BSA (fraction V). Cells
were then incubated for 30 min in fresh KRBB-HEPES at 5 or 25 mM
glucose. Incubation media were discarded or collected for insulin
measurement, and 0.5 ml of 13% perchloric acid was added to the cells.
Precipitated proteins were removed by centrifugation, and the
supernatants were neutralized by adding 2 N
KH2CO3.
The precipitated potassium perchlorate salt was eliminated by
centrifugation, and the resulting supernatants were used for metabolite
measurements. Total cellular proteins were measured by the Bradford
(Bio-Rad) assay after dissolution of the protein pellets in 0.5 N NaOH.
Malate was determined with the malate dehydrogenase (Boehringer)
reaction (46). Citrate was measured by coupling the citrate
lyase-malate dehydrogenase (Boehringer) reactions according to
Williamson and Corkey (46). Malonyl-CoA and glucose 6-phosphate were
extracted from cells with 10% trichloroacetic acid. After
centrifugation of precipitated proteins, cell extracts were brought to
pH 5-6 by successive ether extractions. Samples were lyophilized
and stored at 70°C. Malonyl-CoA was assayed with a
radioactive method with fatty acid synthase (27). Glucose 6-phosphate
was measured by a fluorometric method with glucose 6-phosphate
dehydrogenase (6). ATP and ADP were measured by a bioluminescence assay
(40). For insulin secretion determinations, INS-1 cells were plated
(105 cells/well) into 24-well plates. They were cultured
and incubated as described previously for metabolite measurements. The
insulin concentration in the medium was determined by RIA with rat
insulin as standard (2). Total cellular insulin content was measured after acid-ethanol (1.5% HCl, 75% ethanol) extraction. DNA was measured according to Labarca and Paigen (14).
Fatty acid metabolism and triglyceride measurements. Fatty acid oxidation was measured in INS-1 cells cultured in 21-cm2 petri dishes. After a preexposure for 3 days at 5 mM glucose in the absence or presence of oleate (0.4 mM), cells were washed with PBS and preincubated at 37°C for 30 min in KRBB-HEPES (pH 7.4) medium, at 5 mM glucose, containing 0.07% BSA. Cells were then incubated for 1 h at 37°C in 5 ml of fresh KRBB-HEPES at 5 or 25 mM glucose in the presence of 0.1 mM palmitate, 0.5% defatted BSA, 1 mM carnitine, and 0.11 µCi of [1-14C]palmitate (55 mCi/mmol; Amersham). At the end of the incubations, media were collected and transferred to 25-ml Erlenmeyer flasks covered with septa caps. Media were acidified by injecting perchloric acid (6% final concentration) with a syringe. The liberated CO2 was trapped in a plastic well, suspended from the septa caps, containing 0.4 ml of methanolic benzethonium hydroxide. After 1 h of incubation at 37°C, the wells were removed and the trapped 14CO2 was measured by liquid scintillation counting. Cells were scraped in cold PBS, pelleted by centrifugation, and resuspended in 4 ml of Folch reagent (10). Total lipids were extracted and separated by thin-layer chromatography to measure the incorporation of labeled palmitate into triglycerides and phospholipids (36). An aliquot of the chloroform phase was dried, and total cell triglycerides were measured with a commercial kit (PAP 150, bioMérieux, Marcy l'Etoile, France). Triglyceride recovery assessed with triolein was 94 ± 5% (n = 4).
Glucose metabolism, cell respiration, pyridine nucleotide fluorescence, and enzymatic activity measurements. Glucose usage was determined radiometrically as the production of 3H2O from [5-3H]glucose (16). Cells were cultured in 24-well plates with or without oleate as described in Cell culture and incubation conditions and subsequently incubated for 1 h at 5 or 25 mM glucose in KRBB-HEPES medium containing 0.15 µCi/µmol of [5-3H]glucose (Amersham). Glucose oxidation was measured as 14CO2 production from [U-14C]glucose (0.1 µCi/µmol) with the same experimental design as for the measurement of palmitate oxidation, except that cells were incubated for 1 h with [U-14C]glucose in the absence of fatty acids (3). In some experiments, enzymatic activity measurements were carried out at the end of the 1-h incubation period. Lactate dehydrogenase activity was determined according to Schuit et al. (39). PDH activity measurements were made according to a published procedure (47). Total PDH activity was assayed after conversion of inactive PDH complex with PDH phosphatase extracted from minipig heart (47, 50). For cell respiration and pyridine nucleotide measurements, cells were cultured as described Cell culture and incubation conditions for 3 days in 21-cm2 petri dishes in the absence or presence of oleate. Cells were detached from culture flasks by incubation in the presence of EDTA without trypsin for 5 min at 37°C (7). They were subsequently washed twice with KRBB-HEPES medium and incubated as a suspension at 37°C in acrylic cuvettes as described before (7). After 10 min of incubation at 5 mM glucose to observe basal O2 consumption, glucose was added to the cuvette to reach 25 mM. O2 consumption was measured with a Clark-type electrode in a stirred thermostatized chamber designed by the Bio-Instrumentation Group of the University of Pennsylvania, which allows simultaneous measurements of O2 and fluorescence. The pyridine nucleotide fluorescence was measured with an MB-2 air turbine fluorescence spectrophotometer set at wavelengths of 340 nm (excitation) and 460 nm (emission) (7).
Statistical analysis. All results are expressed per milligrams of protein or micrograms of DNA as means ± SE of the indicated number of experiments. Statistical significance was calculated with the Student's t-test.
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RESULTS |
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Insulin secretion, glucose metabolism, and anaplerosis
in INS-1 -cells after long-term exposure to oleate.
Figure 1 shows that the acute addition of
oleate stimulated insulin release at low glucose and amplified the
action of elevated glucose. By contrast, a 3-day preexposure of INS-1
cells to oleate caused elevated secretion at low glucose and suppressed
the action of elevated glucose on insulin release. Similar effects were
noted either in the absence or in the presence of oleate during the
30-min incubation period when insulin measurements were made. These
short- and long-term actions of oleate reproduce previous observations
made with pancreatic islets (12, 38, 48, 49) and HC9
-cells (17) and
therefore indicate that INS-1 cells are an appropriate cell model for
studying the biochemical basis of the action of FFA on
-cell
metabolic signaling.
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To better understand the mechanism of the chronic action of fatty
acids, we endeavored to carry on a detailed metabolic study of INS-1
cells after oleate exposure in which many metabolic pathways, metabolites, and enzymatic activities are measured. For feasibility reasons, we studied INS-1 cells after a 3-day exposure to the fatty
acid, a time that is long enough to establish the secretory defect
described previously, and after incubation at 5 and 25 mM glucose only.
The results indicated that oleate did not alter glucose usage either at
low or high glucose (Fig. 2). Chronic exposure of INS-1 cells to oleate caused a modest (~28%)
nonsignificant (P < 0.075) decrease
in glucose oxidation at low (5 mM) glucose and reduced by 25%
(P < 0.01) the oxidation of the
sugar at 25 mM glucose. However, the differences in the rate of
oxidation of glucose between 5 and 25 mM were similar in oleate and
control cells.
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The aforementioned results of glucose usage and oxidation do not favor
the view that fatty acids inhibit the action of glucose through a
Randle cycle. This is further supported by the data shown in Fig.
3 indicating that oleate did not affect
basal and glucose-stimulated levels of citrate and glucose 6-phosphate
in INS-1 -cells. The apparent slight decrease in glucose 6-phosphate at high glucose in oleate-treated cells was not significantly different
[24.2 ± 3.0 (n = 9) vs. 17.1 ± 1.2 (n = 7);
P < 0.075]. Glucose oxidation
and glucose 6-phosphate measurements were similar at either low or high
glucose whether oleate was present or absent during the preincubation
and incubation periods (not shown). The acute addition of oleate did
not affect glucose oxidation and the glucose 6-phosphate content of
INS-1 cells at low or high glucose (not shown). In addition, chronic
oleate treatment of INS-1 cells did not alter total PDH activity of
INS-1 cells (measured after activation by PDH phosphatase) nor did it
change its active form (measured in the absence PDH phosphatase
treatment). The following values were obtained for total PDH activity
in control and oleate-treated cells: 0.40 ± 0.01 and 0.38 ± 0.01 mU/mg protein, respectively (means ± SE of 6 experiments). The
proportion of active PDH of control and oleate-treated cells was 64 ± 3 and 58 ± 4%, respectively (not significantly different).
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Low lactate dehydrogenase is a characteristic feature of normal
-cells and INS-1 cells (39, 41). Dedifferentiated cell lines showing
a left shift in the dose dependence of glucose-stimulated insulin
secretion express high amounts of the enzyme (41). To determine whether
oleate alters insulin secretion by changing the expression of LDH, we
measured the activity of the enzyme in control and oleate-treated
cells. The LDH activity of cells chronically exposed to oleate was 40.8 ± 3.8 mU/mg protein, a value not different from that of control
cells, which was 40.5 ± 4.8 mU/mg protein (means ± SE of 3 experiments). This is consistent with the lack of action
of fatty acids on the glucose usage of INS-1 cells.
Increased anaplerotic input into the citric acid cycle is thought to be
implicated in the mechanism whereby nutrients activate the metabolic
stimulus secretion coupling of the -cell (5, 39). To assess whether
this parameter of
-cell activation is altered by oleate, malate
measurements were carried out. A rise in glucose from 5 to 25 mM caused
an ~10-fold increase in the malate content of INS-1 cells (0.25 ± 0.05 vs. 3.26 ± 0.13 nmol/mg protein). Oleate treatment barely
affected the malate content of INS-1 cells either at low or high
glucose (0.35 ± 0.05 vs. 2.77 ± 0.17 nmol/mg protein, means ± SE of 6 experiments). In view of the fact that citrate levels
also remained unaffected by oleate (Fig. 3), the data indicate that
chronic exposure of INS-1 cells to FFA does not affect anaplerosis in
the
-cell.
Lipid metabolism in INS-1 -cells chronically
exposed to oleate. Malonyl-CoA is known to rise in the
-cell in response to glucose stimulation and may act as a signaling
molecule for the short- and/or long-term control of insulin secretion
(8). This intracellular signal of glucose abundance is a key regulator
of fuel partitioning in various tissues (19, 26, 32, 37). Chronic
exposure of INS-1
-cells to oleate decreased the malonyl-CoA content
at low glucose by 30% (P < 0.001)
without affecting the glucose-induced rise of this metabolic switch
molecule (Fig. 4). Because malonyl-CoA
controls fat oxidation and esterification, an associated rise in the
rate of oxidation of fatty acids measured at low glucose occurred as
expected in oleate-treated cells (P < 0.05; Fig. 4). Likewise, an increased incorporation of FFA into total lipids was also noted at low glucose
(P < 0.05; Fig.
5). Consistent with the malonyl-CoA
measurements made at high glucose, oleate did not affect the action of
glucose to reduce fat oxidation (Fig. 4) or to promote the
esterification of FFA into total lipids (Fig. 5). Similar observations
were made with respect to the incorporation of FFA into phospholipids
(data not shown). It should be mentioned that due to tracer palmitate
dilution in the large intracellular pool of fatty acids in cells
chronically treated with oleate, the increase in palmitate oxidation
and esterification is likely to be much underestimated. Figure 5 also
indicates that oleate caused a pronounced deposition of triglycerides
in INS-1 cells (P < 0.001). The
results are consistent with the view that the reduced malonyl-CoA
content at low glucose associated with an increased fat oxidation may
contribute to cause high basal secretion. By contrast, the lack of
glucose-stimulated insulin release cannot be ascribed to altered levels
of malonyl-CoA or changes in fat oxidation and esterification
processes.
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Energy metabolism in INS-1 -cells chronically
exposed to oleate. The rates of reducing equivalent and
ATP production in response to fuel stimuli are believed to be key
components of the
-cell metabolic signal transduction cascade (9,
20, 21, 24, 31). We therefore wished to determine whether the altered
secretion caused by oleate correlates with changes in the INS-1
-cell NAD(P)H-to-NAD(P) ratio and in oxygen consumption, which
reflect the overall cellular fuel oxidation and ATP production.
Simultaneous measurements of the pyridine nucleotide oxidation state,
with native fluorescence at 340 nm (-excitation) and 540 nm
(
-emission), and of medium O2
showed a rise in the
-cell redox state and an increase in
O2 consumption in response to
elevated glucose in control cells. When cells were cultured for 3 days in the presence of oleate, basal redox state (Fig.
6) and respiration (Fig.
7) were higher, achieving levels reached by
glucose-stimulated control cells. Furthermore, the responses to glucose
in both respiration and redox were severely dampened. Interestingly,
Fig. 7 also shows that oleate increased by ~2.5-fold the maximal rate
of respiration measured at saturating substrate concentrations (25 mM
exogenous glucose + 0.4 mM oleate) in the presence of the uncoupler
carbonyl cyanide p-trifluoromethoxyphenylhydrazone. The
precise biochemical nature of this phenomenon is uncertain. It shows
that oleate enhances the total oxidative capacity of INS-1
-cells.
This observation is possibly explained by an induction of limiting
enzyme(s) of the respiratory chain.
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Finally, the cellular content of ATP, ADP as well as the ATP-to-ADP ratio at low glucose, was found not to be affected by oleate. The values for ATP at 5 mM glucose were for control and chronic oleate 29.8 ± 1.5 and 33.3 ± 1.2 nmol/mg protein, respectively (n = 6). The ATP-to-ADP ratios of control and oleate-treated cells at low glucose were 11.3 ± 0.3 and 10.3 ± 1.1, respectively. This indicates that changes in the ATP level or the ATP-to-ADP ratio do not account for high basal secretion at low glucose in oleate-treated cells.
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DISCUSSION |
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The results indicate that the cellular levels of glucose 6-phosphate,
malate, citrate, as well as glucose usage, remain unaltered at either
high or low glucose after a 3-day preexposure of INS-1 cells to oleate.
A modest (~25%) inhibition of glucose oxidation, which is
quantitatively similar to previous observations made in rat islets and
the -cell line HC9, occurs in oleate-treated cells. However, the
difference in the rate of glucose oxidation between 5 and 25 mM glucose
remains unchanged by the FFA. In addition, PDH activity is similar in
both oleate-treated and control cells. By contrast, long-term exposure
of INS-1 cells to oleate causes a massive triglyceride (TG) deposition
that is associated at low glucose with the following changes: a
decrease in the cellular content of malonyl-CoA, an increase in the
rate of fat oxidation, and a promotion of fatty acid esterification
processes, all of which finally result in considerable increases in the
cellular redox state and the rate of oxygen consumption.
Together these observations allow the following conclusions.
1) Long-term exposure of INS-1
-cells to FFA does not induce a Randle cycle in the
-cell because
the predictions that characterize this inverse relationship between fat
and glucose oxidation in some tissues (34) have not been verified.
2) The rate of fatty acid oxidation,
either in control cells or cells chronically exposed to oleate, barely
affects glucose metabolism. 3) The
lack of response to elevated glucose in oleate-treated cells cannot be
explained by the metabolic hypothesis, which predicts that reduced
glucose metabolism and PDH activity account for this effect.
4) It is, however, attractive to
believe that enhanced fat oxidation caused by chronic oleate in
association with increased intracellular FFA availability due to TG
deposition may contribute to cause high basal insulin secretion. In
accordance with this possibility, the modifications of insulin
secretion caused by oleate were found to correlate with changes in
NAD(P)H fluorescence and
O2-consumption measurements. In
this respect, FFA induction of the CPT-I gene (3) and repression of the
acetyl-CoA carboxylase gene (4) may be instrumental because CPT-I
catalyzes the limiting step of fat oxidation and acetyl-CoA
carboxylase catalyzes the formation of the CPT-I inhibitor
malonyl-CoA. Increased esterification processes may also contribute to
high basal secretion by providing complex lipid messenger molecules
such as diacylglycerol and phosphatidic acid (28, 31).
Our comprehensive metabolic study by assessing in addition to glucose
metabolism, lipid metabolism, and anaplerosis, the redox state, PDH
activity, and the Randle cycle, extends the results of a recent
publication concerning HC9 -cells (17) showing that ATP production
at different glucose concentrations (calculated on the basis of the
rates of glucose usage, lactate production, and glucose oxidation) is
not modified by chronic cell exposure to oleate. In
accordance with our results, these authors concluded that altered
glucose metabolism is unlikely to explain the lack of glucose response
of FFA-treated cells (17). They are, however, at variance with other
reports carried out in human and rat islets (38, 48, 49), which
proposed that a Randle cycle accounts for the lack of glucose
responsiveness of
-cells after long-term exposure to FFA. It should
be stated that fatty acid inhibition of glucose metabolism was modest
in these studies (~20%) and only observed at 27 mM glucose and not
at 11 or 3 mM. In addition, the fatty acids were added to cells in
ethanol solutions and not bound to BSA, which might have resulted in
very high nonphysiologically free concentrations of the fatty
acids. It should be underlined that when one considers
the relative capacities of
-cells to metabolize fatty acids vs.
glucose it appears unlikely that accelerated fat oxidation may more
than marginally influence glucose metabolism. Accordingly, the ratio of
glucose oxidation (at ~16-20 mM) vs. that of palmitate (at
~0.3 mM) has been found to be 80 (1) and 60 (22) in two rat islets
studies and >85 in INS-1 cells (3). Thus a twofold rise in fat
oxidation rate caused by chronic oleate should not affect in a major
way glucose metabolism in the
-cell. Irrespective of whether Randle
effects have or have not been previously demonstrated in islets (38,
48, 49), those studies do not prove a causative relationship between
the glucose-fatty acid cycle and the accompanying secretory alterations of fatty acid-treated cells; in contrast, the current study shows that
an active Randle cycle is not necessary for observed alterations in
secretory response in lipid-treated cells. Furthermore, some previous
islets studies have also shown metabolic changes incompatible with a
Randle cycle, i.e., increased glucose oxidation in palmitate-treated islets (12) and decreased glucose 6-phosphate content after treatment
with oleate (18).
The dissociation between the action of chronic oleate on
glucose-induced insulin release on the one hand and on glucose
metabolism, anaplerosis, and malonyl-CoA formation on the other
indicates that other factors account for the inhibitory action of FFA
on insulin secretion promoted by the sugar. Several possibilities may
be considered. 1) If the redox state
acts as a metabolic coupling factor in secretion not only via ATP
production, for example by controlling sulfhydryl groups
of transducing proteins in the -cell, it may be that a rise in
glucose from 5 to 25 mM cannot further enhance secretion because the
reduction state of pyridine nucleotides is already maximal at low (5 mM) glucose and consequently secretion cannot be further enhanced.
2) The elevated TG content of the
-cell is expected to result, after lipolysis, in a rise in cytosolic FFA and Lc-CoA. FFA and Lc-CoA stimulate C-kinase enzymes (1, 28),
which might be downregulated after long-term exposure to FFA with a
resulting loss of a possible important component implicated in
secretion. 3) Lc-CoA directly
stimulate the exocytotic release of insulin in permeabilized HIT
-cells (J. Deeney, B. Corkey, C. Rhodes, M. Prentki, and P.O.
Berggren, unpublished observations). Thus glucose might not be active
because secretion is already maximally promoted by this candidate
metabolic coupling factor. 4)
Lc-CoA, which are known to be elevated in FFA-treated
-cells, are
very effective openers of KATP
channels (15). Thus the
-cell might resist depolarization by glucose
after chronic oleate treatment.
In addition to these direct effects, pretreatment with FFA may alter the expression of various proteins, including acetyl-CoA carboxylase, CPT-I, uncoupling protein 2, and enzymes of fat oxidation. Our data support a major alteration in fat oxidation, most likely due to changes in enzyme expression, in particular elevated CPT-I activity (3). This is supported by the increase in basal respiration, the more than doubling in maximum respiratory capacity, and the elevated basal redox state.
In conclusion, chronic FFA treatment markedly alters the energy
metabolism of the -cell. The main response of INS-1
-cells to
long-term FFA treatment is to increase the mitochondrial capacity to
oxidize and esterify FFA rather than altering glucose metabolism.
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ACKNOWLEDGEMENTS |
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This work was supported by grants from the Canadian Diabetes Association, the Juvenile Diabetes Foundation (197047), the Medical Research Council of Canada (to M. Prentki); National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-35914 and DK-50662 (to B. E. Corkey); Grant 32-45957.95/1 from the Swiss National Science Foundation (to F. Assimacopoulos-Jeannet). S. Thumelin was supported by a postdoctoral fellowship from the Juvenile Diabetes Foundation International. M. Prentki is a Medical Research Council of Canada Scientist.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: M. Prentki, CR-CHUM, Pavillon de Sève, 4e, 1560 Sherbrooke Est, Montreal, PQ, Canada H2L 4M1 (E-mail: prentkim{at}ere.umontreal.ca).
Received 8 March 1999; accepted in final form 4 May 1999.
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