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Laura Segall, Nathalie Lameloise, Franoise Assimacopoulos-Jeannet, Enrique

Roche, Pamela Corkey, Stphane Thumelin, Barbara E. Corkey and Marc Prentki
Am J Physiol Endocrinol Metab 277:521-528, 1999.

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on the following topics:
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Lipid rather than glucose metabolism is implicated in
altered insulin secretion caused by oleate in INS-1 cells
LAURA SEGALL,1 NATHALIE LAMELOISE,2 FRANC OISE ASSIMACOPOULOS-JEANNET,2
ENRIQUE ROCHE,1 PAMELA CORKEY,3 STE PHANE THUMELIN,1 BARBARA E. CORKEY,3
AND MARC PRENTKI1
1Molecular Nutrition Unit, Department of Nutrition, University of Montreal, and the Centre de

Recherche du Centre Hospitalier de lUniversite de Montreal and Institut du Cancer, Montreal,


Quebec, Canada H2L 4M1; 2Department of Medical Biochemistry, Centre Medical Universitaire,
University of Geneva, Geneva, Switzerland 1211; and 3Center for Obesity and Metabolism at Boston
University Medical Center, Boston University Medical School, Boston, Massachussets 02118

Segall, Laura, Nathalie Lameloise, Francoise Assima- transfer experiments (13). In addition, their Lc-CoA or
copoulos-Jeannet, Enrique Roche, Pamela Corkey, Ste- complex lipid derivatives may act as coupling factors in
phane Thumelin, Barbara E. Corkey, and Marc Prentki. nutrient-stimulated insulin secretion (8, 31).
Lipid rather than glucose metabolism is implicated in altered Acute exposure of -cells to fatty acids potentiates
insulin secretion caused by oleate in INS-1 cells. Am. J.
Physiol. 277 (Endocrinol. Metab. 40): E521E528, 1999.A
glucose-induced insulin secretion by mechanisms that
apparently do not implicate KATP channels but may

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comprehensive metabolic study was carried out to under-
stand how chronic exposure of pancreatic -cells to fatty acids involve C-kinase activation by Lc-CoA and Ca2 chan-
causes high basal secretion and impairs glucose-induced nels (33, 45). By contrast, over longer time ranges (24
insulin release. INS-1 -cells were exposed to 0.4 mM oleate h) fatty acids such as palmitate and oleate cause both in
for 3 days and subsequently incubated at 5 or 25 mM glucose, vitro and in vivo high basal insulin secretion character-
after which various parameters were measured. Chronic ized by a lowered glucose set point and markedly
oleate promoted triglyceride deposition, increased fatty acid reduced secretion in response to a further elevation of
oxidation and esterification, and reduced malonyl-CoA at low
the sugar (4, 12, 17, 38, 48, 49). At relatively high
glucose in association with elevated basal O2 consumption
and redox state. Oleate caused a modest (25%) reduction in concentrations, fatty acids also activate the nitric oxide
glucose oxidation but did not affect glucose usage, the glucose transduction system of islet tissue, a process that may
6-phosphate and citrate contents, and the activity of pyruvate induce cell apoptosis and be implicated in final -cell
dehydrogenase of INS-1 cells. Thus changes in glucose metabo- decompensation (42, 43).
lism and a Randle-glucose/fatty acid cycle do not explain the Regarding secretion, several studies have suggested
altered secretory properties of -cells exposed to fatty acids. that the long-term action of fatty acids is caused by
The main response of INS-1 cells to chronic oleate, which is to changes in lipid and glucose metabolism, possibly as a
increase the oxidation and esterification of fatty acids, may consequence of variations in the expression of genes
contribute to cause high basal insulin secretion via increased
production of reducing equivalents and/or the generation of encoding key enzymes implicated in fuel signaling. In
complex lipid messenger molecule(s). this respect, a complex picture with conflicting results
has emerged. Thus palmitate and oleate not only
fatty acids; insulin secretion; obesity; type 2 diabetes increase the total activity of hexokinase in rat islets
(12) and HC9 -cell (17) but also decrease the expres-
sion of glucokinase and GLUT-2, possibly via reduced
OBESE PATIENTS with type 2 diabetes show altered expression of the transcription factor IDX-1 (11). In
insulin release with respect to prevailing blood glucose addition, fatty acids induce the carnitine palmitoyltrans-
characterized by elevated circulating insulin in the ferase I gene (CPT-I; Ref. 3) and reduce acetyl-CoA
fasting state and relatively low insulin levels postpran- carboxylase expression (4) in INS-1 -cells, thus caus-
dially when glucose is high (29, 30). The biochemical ing enhanced fat oxidation (4). An enhanced rate of
basis of these alterations is not known, but the emerg- oxidation of fatty acids might reduce glucose metabo-
ing evidence suggests that elevated circulating free lism and consequently insulin secretion if a so-called
fatty acids (FFA) as well as long chain acyl-CoA esters Randle cycle (34) were operative in the -cell after fat
(Lc-CoA) and triglyceride deposition in the -cell may exposure. In this respect, FFA were shown to cause a
be causally implicated (25, 32). modest (20%) reduction of glucose oxidation in islets
FFA are important nutrients for -cell function be- (49) and HC9 -cells (17) or to increase glucose oxida-
cause they are used as fuels (23) and play a permissive tion in another islet study (12). A reduced islet pyruvate
role for glucose-induced insulin release, as evidenced dehydrogenase (PDH) activity predicted by the Randle
with a pharmacological approach (44) and leptin gene cycle has been observed (50), but the associated de-
creased glucose oxidation caused by fatty acid reported
by the same group was noted only at very high (27 mM)
The costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby
glucose and not at intermediate (11 mM) or low glucose
marked advertisement in accordance with 18 U.S.C. Section 1734 concentrations (38, 49). Thus it is presently uncertain
solely to indicate this fact. whether alterations in glucose metabolism provide an
0193-1849/99 $5.00 Copyright r 1999 the American Physiological Society E521
E522 ADAPTATION OF -CELLS TO FATTY ACIDS

explanation for the long-term action of fatty acids on protein pellets in 0.5 N NaOH. Malate was determined with
insulin secretion. the malate dehydrogenase (Boehringer) reaction (46). Citrate
As reviewed previously (34), the following sequence was measured by coupling the citrate lyase-malate dehydroge-
of events associated with predicted alterations in me- nase (Boehringer) reactions according to Williamson and
tabolite levels characterize a Randle cycle. Accelerated Corkey (46). Malonyl-CoA and glucose 6-phosphate were
extracted from cells with 10% trichloroacetic acid. After
fat oxidation results in exaggerated NADH and acetyl- centrifugation of precipitated proteins, cell extracts were
CoA production, thus causing PDH inhibition and brought to pH 56 by successive ether extractions. Samples
reduced glucose oxidation. Citrate also rises via a mass were lyophilized and stored at 70C. Malonyl-CoA was
effect in the mitochondrion and, after its export to the assayed with a radioactive method with fatty acid synthase
cytosolic compartment, allosterically inhibits 6-phospho- (27). Glucose 6-phosphate was measured by a fluorometric
fructo-1-kinase, thus causing reduced glycolytic flux method with glucose 6-phosphate dehydrogenase (6). ATP
and a rise in glucose 6-phosphate. To better understand and ADP were measured by a bioluminescence assay (40). For
how fatty acids increase insulin secretion at low glucose insulin secretion determinations, INS-1 cells were plated (105
and to determine whether altered glucose metabolism cells/well) into 24-well plates. They were cultured and incu-
via a Randle glucose-fatty acid cycle may account for bated as described previously for metabolite measurements.
the lack of glucose response in cells chronically exposed The insulin concentration in the medium was determined by
RIA with rat insulin as standard (2). Total cellular insulin
to FFA, we have carried out a comprehensive study of
content was measured after acid-ethanol (1.5% HCl, 75%
glucose and fatty acid metabolism in INS-1 -cells after ethanol) extraction. DNA was measured according to Labarca
long-term exposure to oleate. and Paigen (14).

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Fatty acid metabolism and triglyceride measurements.
METHODS
Fatty acid oxidation was measured in INS-1 cells cultured in
Cell culture and incubation conditions. The experiments 21-cm2 petri dishes. After a preexposure for 3 days at 5 mM
have been carried out with the -cell line INS-1, which glucose in the absence or presence of oleate (0.4 mM), cells
responds to glucose at physiological concentrations of the were washed with PBS and preincubated at 37C for 30 min
sugar to an extent similar to that of pancreatic islets in in KRBB-HEPES (pH 7.4) medium, at 5 mM glucose, contain-
culture (2, 6, 39). All experiments have been carried out with ing 0.07% BSA. Cells were then incubated for 1 h at 37C in 5
cell passages below 85 because we noticed that at higher ml of fresh KRBB-HEPES at 5 or 25 mM glucose in the
passage numbers INS-1 cells loose their response to glucose presence of 0.1 mM palmitate, 0.5% defatted BSA, 1 mM
(5 mM). INS-1 cells were seeded in 21-cm2 petri dishes carnitine, and 0.11 Ci of [1-14C]palmitate (55 mCi/mmol;
(1.4 106 cells/dish) or in 24-well plates (105 cells/well) and Amersham). At the end of the incubations, media were
grown as described previously (2) in RPMI 1640 medium at 11 collected and transferred to 25-ml Erlenmeyer flasks covered
mM glucose supplemented with 10 mM HEPES, 10% heat- with septa caps. Media were acidified by injecting perchloric
inactivated fetal calf serum, 2 mM L-glutamine, 1 mM sodium acid (6% final concentration) with a syringe. The liberated
pyruvate, 50 M -mercaptoethanol, 100 IU/ml penicillin, CO2 was trapped in a plastic well, suspended from the septa
and 100 g/ml streptomycin in a humidified atmosphere (5% caps, containing 0.4 ml of methanolic benzethonium hydrox-
CO2-95% air). When cells reached 80% confluence after 7 ide. After 1 h of incubation at 37C, the wells were removed
days, they were washed twice with PBS and preincubated at and the trapped 14CO2 was measured by liquid scintillation
37C for 2 days in culture medium containing 5 mM glucose. counting. Cells were scraped in cold PBS, pelleted by centrifu-
Cells were then washed with PBS and incubated for 3 days in gation, and resuspended in 4 ml of Folch reagent (10). Total
culture medium at 5 mM glucose in the absence or presence of lipids were extracted and separated by thin-layer chromatog-
oleate (0.4 mM). Albumin-bound oleate was prepared by raphy to measure the incorporation of labeled palmitate into
stirring the fatty acid Na salt at 45C with defatted BSA triglycerides and phospholipids (36). An aliquot of the chloro-
(fraction V, Sigma). After adjustment of the pH to 7.4, the form phase was dried, and total cell triglycerides were
solution was filtered through a 0.22-m filter and the fatty measured with a commercial kit (PAP 150, bioMerieux,
acid concentration was measured with a NEFA C kit (Wako Marcy lEtoile, France). Triglyceride recovery assessed with
Chemicals). For incubations longer than 24 h, the medium triolein was 94 5% (n 4).
was changed daily to maintain a constant concentration of Glucose metabolism, cell respiration, pyridine nucleotide
fatty acid. The final concentration of BSA in the culture fluorescence, and enzymatic activity measurements. Glucose
medium was 0.5%. usage was determined radiometrically as the production of
Metabolites and insulin secretion measurements. Citrate 3H O from [5-3H]glucose (16). Cells were cultured in 24-well
2
and malate were determined as follows. After a culture period plates with or without oleate as described in Cell culture and
of 3 days at 5 mM glucose with or without oleate, cells were incubation conditions and subsequently incubated for 1 h at 5
washed with PBS and preincubated at 37C for 30 min in or 25 mM glucose in KRBB-HEPES medium containing 0.15
Krebs-Ringer bicarbonate buffer (KRBB) with 25 mM HEPES, Ci/mol of [5-3H]glucose (Amersham). Glucose oxidation
pH 7.4 (35), at 5 mM glucose, containing 0.07% BSA (fraction was measured as 14CO2 production from [U-14C]glucose (0.1
V). Cells were then incubated for 30 min in fresh KRBB- Ci/mol) with the same experimental design as for the
HEPES at 5 or 25 mM glucose. Incubation media were measurement of palmitate oxidation, except that cells were
discarded or collected for insulin measurement, and 0.5 ml of incubated for 1 h with [U-14C]glucose in the absence of fatty
13% perchloric acid was added to the cells. Precipitated acids (3). In some experiments, enzymatic activity measure-
proteins were removed by centrifugation, and the superna- ments were carried out at the end of the 1-h incubation
tants were neutralized by adding 2 N KH2CO3. The precipi- period. Lactate dehydrogenase activity was determined ac-
tated potassium perchlorate salt was eliminated by centrifu- cording to Schuit et al. (39). PDH activity measurements were
gation, and the resulting supernatants were used for made according to a published procedure (47). Total PDH
metabolite measurements. Total cellular proteins were mea- activity was assayed after conversion of inactive PDH com-
sured by the Bradford (Bio-Rad) assay after dissolution of the plex with PDH phosphatase extracted from minipig heart (47,
ADAPTATION OF -CELLS TO FATTY ACIDS E523

50). For cell respiration and pyridine nucleotide measure-


ments, 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 37C (7). They were subsequently washed
twice with KRBB-HEPES medium and incubated as a suspen-
sion at 37C 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 elec-
trode in a stirred thermostatized chamber designed by the
Bio-Instrumentation Group of the University of Pennsylva-
nia, which allows simultaneous measurements of O2 and
fluorescence. The pyridine nucleotide fluorescence was mea-
sured with an MB-2 air turbine fluorescence spectrophotom-
eter set at wavelengths of 340 nm (excitation) and 460 nm
(emission) (7).
Statistical analysis. All results are expressed per milli-
grams of protein or micrograms of DNA as means SE of the Fig. 2. Effect of a long-term exposure of INS-1 cells to oleate on
indicated number of experiments. Statistical significance was glucose utilization and oxidation (oxid.). Data are means SE of 45
experiments. Cells were cultured for 3 days in absence or presence of

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calculated with the Students t-test.
oleate (0.4 mM), as described in Fig. 1, and subsequently incubated for
1 h at 5 mM (open bars) or 25 mM (solid bars) glucose in absence of
RESULTS
oleate. Glucose oxidation at 5 mM glucose vs. glucose 5 mM oleate,
not significant (P 0.075). Glucose oxidation at 25 mM glucose vs.
Insulin secretion, glucose metabolism, and anaplero- glucose 25 mM oleate (* P 0.01).
sis in INS-1 -cells after long-term exposure to oleate.
Figure 1 shows that the acute addition of oleate stimu-
lated insulin release at low glucose and amplified the ments were made. These short- and long-term actions
action of elevated glucose. By contrast, a 3-day preexpo- of oleate reproduce previous observations made with
sure of INS-1 cells to oleate caused elevated secretion at pancreatic islets (12, 38, 48, 49) and HC9 -cells (17)
low glucose and suppressed the action of elevated and therefore indicate that INS-1 cells are an appropri-
glucose on insulin release. Similar effects were noted ate cell model for studying the biochemical basis of the
either in the absence or in the presence of oleate during action of FFA on -cell metabolic signaling.
the 30-min incubation period when insulin measure- 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, metabo-
lites, and enzymatic activities are measured. For feasi-
bility 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%) nonsig-
nificant (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.
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 indicat-
Fig. 1. Effect of short- and long-term exposure of INS-1 cells to oleate ing that oleate did not affect basal and glucose-
on insulin secretion. Data are means SE of 12 experiments. INS-1 stimulated levels of citrate and glucose 6-phosphate in
cells were cultured in RPMI medium containing 10% serum with
0.5% BSA for 3 days at 5 mM glucose (G) in absence or presence of INS-1 -cells. The apparent slight decrease in glucose
oleate (Ol.; 0.4 mM). Cells were subsequently incubated in Krebs- 6-phosphate at high glucose in oleate-treated cells was
Ringer bicarbonate buffer-HEPES medium at 5 or 25 mM glucose in not significantly different [24.2 3.0 (n 9) vs. 17.1
absence (open bars) or presence (solid bars) of oleate (0.4 mM). 1.2 (n 7); P 0.075]. Glucose oxidation and glucose
Insulin release during 30-min incubation period is expressed as
percentage of total cellular insulin content. Insulin content of cells
6-phosphate measurements were similar at either low
cultured in absence or presence of oleate was 1,862 12 and 1,725 or high glucose whether oleate was present or absent
5 ng/mg protein, respectively. IRI, immunoreactive insulin. during the preincubation and incubation periods (not
E524 ADAPTATION OF -CELLS TO FATTY ACIDS

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 ex-
posed 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 partition-
ing 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
Fig. 3. Lack of effect of chronic oleate treatment on citrate and the rate of oxidation of fatty acids measured at low
glucose 6-phosphate (G-6-P ) content of INS-1 cells. Data are means glucose occurred as expected in oleate-treated cells (P
SE of 69 experiments. Cells were cultured with or without oleate for

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3 days and incubated for 30 min at 5 or 25 mM glucose as described in
0.05; Fig. 4). Likewise, an increased incorporation of
Fig. 2. Glucose 6-phosphate at 25 mM glucose vs. glucose 25 mM FFA into total lipids was also noted at low glucose (P
oleate, not significant (P 0.075). 0.05; Fig. 5). Consistent with the malonyl-CoA measure-
ments made at high glucose, oleate did not affect the
shown). The acute addition of oleate did not affect action of glucose to reduce fat oxidation (Fig. 4) or to
glucose oxidation and the glucose 6-phosphate content promote the esterification of FFA into total lipids (Fig.
of INS-1 cells at low or high glucose (not shown). In 5). Similar observations were made with respect to the
addition, chronic oleate treatment of INS-1 cells did not incorporation of FFA into phospholipids (data not
alter total PDH activity of INS-1 cells (measured after shown). It should be mentioned that due to tracer
activation by PDH phosphatase) nor did it change its palmitate dilution in the large intracellular pool of
active form (measured in the absence PDH phospha- fatty acids in cells chronically treated with oleate, the
tase treatment). The following values were obtained for increase in palmitate oxidation and esterification is
total PDH activity in control and oleate-treated cells: likely to be much underestimated. Figure 5 also indi-
0.40 0.01 and 0.38 0.01 mU/mg protein, respec- cates that oleate caused a pronounced deposition of
tively (means SE of 6 experiments). The proportion of triglycerides in INS-1 cells (P 0.001). The results are
active PDH of control and oleate-treated cells was 64 consistent with the view that the reduced malonyl-CoA
3 and 58 4%, respectively (not significantly different). content at low glucose associated with an increased fat
Low lactate dehydrogenase is a characteristic feature oxidation may contribute to cause high basal secretion.
of normal -cells and INS-1 cells (39, 41). Dedifferenti- By contrast, the lack of glucose-stimulated insulin
ated cell lines showing a left shift in the dose depen-
dence of glucose-stimulated insulin secretion express
high amounts of the enzyme (41). To determine whether
oleate alters insulin secretion by changing the expres-
sion 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 Fig. 4. Effect of chronic oleate treatment on fatty acid oxidation (ox.)
the malate content of INS-1 cells (0.25 0.05 vs. 3.26 and malonyl-CoA content of INS-1 cells. Data are means SE of 46
experiments. Cells were cultured with or without oleate for 3 days
0.13 nmol/mg protein). Oleate treatment barely af- and incubated for 1 h (palmitate oxidation measurements) or 30 min
fected the malate content of INS-1 cells either at low or (malonyl-CoA determinations) at 5 or 25 mM glucose as described in
high glucose (0.35 0.05 vs. 2.77 0.17 nmol/mg Fig. 2.
ADAPTATION OF -CELLS TO FATTY ACIDS E525

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.
DISCUSSION

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 quanti-
tatively similar to previous observations made in rat
islets and the -cell line HC9, occurs in oleate-treated
Fig. 5. Effect of chronic oleate treatment on esterification of palmi-
cells. However, the difference in the rate of glucose
tate and triglyceride (TG) content of INS-1 cells. Means SE of 3 oxidation between 5 and 25 mM glucose remains un-

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experiments. Cont., control. Cells were cultured with or without changed by the FFA. In addition, PDH activity is
oleate (Ol.) for 3 days and incubated for 1 h at 5 or 25 mM glucose as similar in both oleate-treated and control cells. By
described in Fig. 2. Left panel, measurements of palmitate incorpora- contrast, long-term exposure of INS-1 cells to oleate
tion into total lipids. Right panel, triglyceride content of INS-1 cells at
5 mM glucose after culture with or without 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
release cannot be ascribed to altered levels of malonyl- increase in the rate of fat oxidation, and a promotion of
CoA or changes in fat oxidation and esterification fatty acid esterification processes, all of which finally
processes. result in considerable increases in the cellular redox
Energy metabolism in INS-1 -cells chronically ex- state and the rate of oxygen consumption.
posed to oleate. The rates of reducing equivalent and Together these observations allow the following con-
ATP production in response to fuel stimuli are believed clusions. 1) Long-term exposure of INS-1 -cells to FFA
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 con-
sumption, which reflect the overall cellular fuel oxida-
tion and ATP production.
Simultaneous measurements of the pyridine nucleo-
tide 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. Further-
more, 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 car-
bonyl cyanide p-trifluoromethoxyphenylhydrazone. The
precise biochemical nature of this phenomenon is uncer-
tain. It shows that oleate enhances the total oxidative
capacity of INS-1 -cells. This observation is possibly Fig. 6. Chronic oleate treatment alters oxidation state of pyridine
explained by an induction of limiting enzyme(s) of the nucleotides in INS-1 cells. Data are means SE of 6 (control) and 4
(oleate) experiments. Fluorescence measurements of cellular
respiratory chain. NAD(P)H were carried out at 5 or 25 mM glucose after a 3-day culture
Finally, the cellular content of ATP, ADP as well as period with or without oleate (0.4 mM). Percentage of reduced
the ATP-to-ADP ratio at low glucose, was found not to NAD(P)H at 5 mM glucose vs. 5 mM glucose plus oleate (P 0.001).
E526 ADAPTATION OF -CELLS TO FATTY ACIDS

Fig. 7. Effect of chronic oleate treatment on O2 consump-


tion of INS-1 cells. Data are means SE of 6 (control)
and 4 (oleate) experiments. Left panel, respiration mea-
surements were carried out at 5 or 25 mM glucose after
a 3-day culture period with or without oleate (0.4 mM).
Right panel, maximal rate of oxygen consumption was
measured in presence of 25 mM glucose and 0.4 mM
oleate in presence of uncoupler carbonyl cyanide p-
trifluoromethoxyphenylhydrazone (FCCP; 1 M).

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does not induce a Randle cycle in the -cell because the islets (38, 48, 49), which proposed that a Randle cycle
predictions that characterize this inverse relationship accounts for the lack of glucose responsiveness of
between fat and glucose oxidation in some tissues (34) -cells after long-term exposure to FFA. It should be
have not been verified. 2) The rate of fatty acid oxida- stated that fatty acid inhibition of glucose metabolism
tion, either in control cells or cells chronically exposed was modest in these studies (20%) and only observed
to oleate, barely affects glucose metabolism. 3) The lack at 27 mM glucose and not at 11 or 3 mM. In addition,
of response to elevated glucose in oleate-treated cells the fatty acids were added to cells in ethanol solutions
cannot be explained by the metabolic hypothesis, which and not bound to BSA, which might have resulted in
predicts that reduced glucose metabolism and PDH very high nonphysiologically free concentrations of the
activity account for this effect. 4) It is, however, attrac- fatty acids. It should be underlined that when one
tive to believe that enhanced fat oxidation caused by considers the relative capacities of -cells to metabolize
chronic oleate in association with increased intracellu- fatty acids vs. glucose it appears unlikely that acceler-
lar FFA availability due to TG deposition may contrib- ated fat oxidation may more than marginally influence
ute to cause high basal insulin secretion. In accordance glucose metabolism. Accordingly, the ratio of glucose
with this possibility, the modifications of insulin secre- oxidation (at 1620 mM) vs. that of palmitate (at
tion caused by oleate were found to correlate with 0.3 mM) has been found to be 80 (1) and 60 (22) in two
changes in NAD(P)H fluorescence and O2-consumption rat islets studies and 85 in INS-1 cells (3). Thus a
measurements. In this respect, FFA induction of the twofold rise in fat oxidation rate caused by chronic
CPT-I gene (3) and repression of the acetyl-CoA carbox- oleate should not affect in a major way glucose metabo-
ylase gene (4) may be instrumental because CPT-I lism in the -cell. Irrespective of whether Randle effects
catalyzes the limiting step of fat oxidation and acetyl- have or have not been previously demonstrated in islets
CoA carboxylase catalyzes the formation of the CPT-I (38, 48, 49), those studies do not prove a causative
inhibitor malonyl-CoA. Increased esterification pro- relationship between the glucose-fatty acid cycle and
cesses may also contribute to high basal secretion by the accompanying secretory alterations of fatty acid-
providing complex lipid messenger molecules such as treated cells; in contrast, the current study shows that
diacylglycerol and phosphatidic acid (28, 31). an active Randle cycle is not necessary for observed
Our comprehensive metabolic study by assessing in alterations in secretory response in lipid-treated cells.
addition to glucose metabolism, lipid metabolism, and Furthermore, some previous islets studies have also
anaplerosis, the redox state, PDH activity, and the shown metabolic changes incompatible with a Randle
Randle cycle, extends the results of a recent publication cycle, i.e., increased glucose oxidation in palmitate-
concerning HC9 -cells (17) showing that ATP produc- treated islets (12) and decreased glucose 6-phosphate
tion at different glucose concentrations (calculated on content after treatment with oleate (18).
the basis of the rates of glucose usage, lactate produc- The dissociation between the action of chronic oleate
tion, and glucose oxidation) is not modified by chronic on glucose-induced insulin release on the one hand and
cell exposure to oleate. In accordance with our results, on glucose metabolism, anaplerosis, and malonyl-CoA
these authors concluded that altered glucose metabo- formation on the other indicates that other factors
lism is unlikely to explain the lack of glucose response account for the inhibitory action of FFA on insulin
of FFA-treated cells (17). They are, however, at vari- secretion promoted by the sugar. Several possibilities
ance with other reports carried out in human and rat may be considered. 1) If the redox state acts as a
ADAPTATION OF -CELLS TO FATTY ACIDS E527

metabolic coupling factor in secretion not only via ATP 4. Brun, T., F. Assimacopoulos-Jeannet, B. E. Corkey, and M.
production, for example by controlling sulfhydryl groups Prentki. Long chain fatty acids inhibit acetyl-CoA carboxylase
gene expression in the pancreatic cell line INS-1. Diabetes 46:
of transducing proteins in the -cell, it may be that a 393400, 1997.
rise in glucose from 5 to 25 mM cannot further enhance 5. Brun, T., E. Roche, F. Assimacopoulos-Jeannet, B. E. Cor-
secretion because the reduction state of pyridine nucle- key, K. H. Kim, and M. Prentki. Evidence for an anaplerotic
otides is already maximal at low (5 mM) glucose and malonyl-CoA pathway in pancreatic beta-cell nutrient signaling.
consequently secretion cannot be further enhanced. 2) Diabetes 45: 190198, 1996.
6. Brun, T., E. Roche, K. H. Kim, and M. Prentki. Glucose
The elevated TG content of the -cell is expected to
regulates acetyl-CoA carboxylase gene expression in a pancreatic
result, after lipolysis, in a rise in cytosolic FFA and beta-cell line (INS-1). J. Biol. Chem. 268: 1890518911, 1993.
Lc-CoA. FFA and Lc-CoA stimulate C-kinase enzymes 7. Civelek, V. N., J. T. Deeney, N. J. Shalosky, K. Tornheim,
(1, 28), which might be downregulated after long-term R. G. Hansford, M. Prentki, and B. E. Corkey. Regulation of
exposure to FFA with a resulting loss of a possible pancreatic beta-cell mitochondrial metabolism: influence of Ca2,
important component implicated in secretion. 3) Lc- substrate and ADP. Biochem. J. 318: 615621, 1996.
8. Corkey, B. E., M. C. Glennon, K. S. Chen, J. T. Deeney, F. M.
CoA directly stimulate the exocytotic release of insulin Matschinsky, and M. Prentki. A role for malonyl-CoA in
in permeabilized HIT -cells (J. Deeney, B. Corkey, C. glucose-stimulated insulin secretion from clonal pancreatic beta-
Rhodes, M. Prentki, and P.O. Berggren, unpublished cells. J. Biol. Chem. 264: 2160821612, 1989.
observations). Thus glucose might not be active be- 9. Dukes, I. D., M. S. McIntyre, R. J. Mertz, L. H. Philipson,
cause secretion is already maximally promoted by this M. W. Roe, B. Spencer, and J. F. R. Worley. Dependence on
NADH produced during glycolysis for beta-cell glucose signaling.
candidate metabolic coupling factor. 4) Lc-CoA, which
J. Biol. Chem. 269: 1097910982, 1994.
are known to be elevated in FFA-treated -cells, are

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10. Folch, J., M. Lees, and G. H. A. Sloane-Stanley. A simple
very effective openers of KATP channels (15). Thus the method for the isolation and purification of total lipids from
-cell might resist depolarization by glucose after animal tissues. J. Biol. Chem. 226: 497509, 1957.
chronic oleate treatment. 11. Gremlich, S., C. Bonny, G. Waeber, and B. Thorens. Fatty
In addition to these direct effects, pretreatment with acids decrease IDX-1 expression in rat pancreatic islets and
reduce GLUT2, glucokinase, insulin, and somatostatin levels. J.
FFA may alter the expression of various proteins, Biol. Chem. 272: 3026130269, 1997.
including acetyl-CoA carboxylase, CPT-I, uncoupling 12. Hosokawa, H., B. E. Corkey, and J. L. Leahy. Beta-cell
protein 2, and enzymes of fat oxidation. Our data hypersensitivity to glucose following a 24-h exposure of rat islets
support a major alteration in fat oxidation, most likely to fatty acids. Diabetologia 40: 392397, 1997.
due to changes in enzyme expression, in particular 13. Koyama, K., G. Chen, M. Y. Wang, M. Shimabukuro, C. B.
Newgard, and R. H. Unger. -Cell function in normal rats
elevated CPT-I activity (3). This is supported by the made chronically hyperleptinemic by adenovirus-leptin gene
increase in basal respiration, the more than doubling in therapy. Diabetes 46: 12761280, 1997.
maximum respiratory capacity, and the elevated basal 14. Labarca, C., and K. Paigen. A simple, rapid and sensitive DNA
redox state. assay procedure. Anal. Biochem. 102: 344352, 1980.
In conclusion, chronic FFA treatment markedly al- 15. Larsson, O., J. T. Deeney, R. Branstrom, P. O. Berggren,
ters the energy metabolism of the -cell. The main and B. E. Corkey. Activation of the ATP-sensitive K channel by
long chain acyl-CoA. A role in modulation of pancreatic beta-cell
response of INS-1 -cells to long-term FFA treatment is glucose sensitivity. J. Biol. Chem. 271: 1062310626, 1996.
to increase the mitochondrial capacity to oxidize and 16. Liang, Y., G. Bai, N. Doliba, C. Buettger, L. Wang, D. K.
esterify FFA rather than altering glucose metabolism. Berner, and F. M. Matschinsky. Glucose metabolism and
insulin release in mouse -HC9 cells, as model for wild-type
This work was supported by grants from the Canadian Diabetes pancreatic -cells. Am. J. Physiol. 270 (Endocrinol. Metab. 33):
Association, the Juvenile Diabetes Foundation (197047), the Medical E846E857, 1996.
Research Council of Canada (to M. Prentki); National Institute of 17. Liang, Y., C. Buettger, D. K. Berner, and F. M. Matschinsky.
Diabetes and Digestive and Kidney Diseases Grants DK-35914 and Chronic effect of fatty acids on insulin release is not through the
DK-50662 (to B. E. Corkey); Grant 3245957.95/1 from the Swiss alteration of glucose metabolism in a pancreatic beta-cell line
National Science Foundation (to F. Assimacopoulos-Jeannet). S. (HC9). Diabetologia 40: 10181027, 1997.
Thumelin was supported by a postdoctoral fellowship from the
18. Liu, Y. Q., K. Tornheim, and J. L. Leahy. Fatty acid-induced
Juvenile Diabetes Foundation International. M. Prentki is a Medical
-cell hypersensitivity to glucose: increased phosphofructokinase
Research Council of Canada Scientist.
activity and lowered glucose-6-phosphate content. J. Clin. Invest.
Address for reprint requests and other correspondence: M. Prentki,
101: 18701875, 1998.
CR-CHUM, Pavillon de Se`ve, 4e, 1560 Sherbrooke Est, Montreal, PQ,
19. Lopaschuk, G. D., D. D. Belke, J. Gamble, T. Itoi, and B. O.
Canada H2L 4M1 (E-mail: prentkim@ere.umontreal.ca).
Schonekess. Regulation of fatty acid oxidation in the mamma-
Received 8 March 1999; accepted in final form 4 May 1999. lian heart in health and disease. Biochim. Biophys. Acta 1213:
263276, 1994.
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