Pancreatic Islet Adaptation to Fasting Is Dependent on Peroxisome Proliferator-Activated Receptor Transcriptional Up-Regulation of Fatty Ac
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内分泌学杂志 2005年第1期
Center for Integrative Genomics (S.G., B.D., L.M., W.W.), University of Lausanne, CH-1015 Lausanne, Switzerland; the Molecular Nutrition Unit and the Montreal Diabetes Research Center (C.N., R.R., M.-L.P., V.D.-A., M.P.), University of Montreal, Montreal, Quebec, Canada H2L 4M1; Hopital Rangueil (R.B.), 31403 Toulouse, France; and the Division of Medical Genetics (R.R.), Centre Hospitalier Universitaire Vaudois, CH-1011 Lausanne, Switzerland
Address all correspondence and requests for reprints to: Dr. Marc Prentki, Centre de Recherche, Centre Hospitalier de l’Université de Montréal, Pavillon de Sève, Y4603, 1560 Sherbrooke East, Montreal, Quebec, Canada H2L 4M1. E-mail: marc.prentki@umontreal.ca.
Abstract
The cellular response to fasting and starvation in tissues such as heart, skeletal muscle, and liver requires peroxisome proliferator-activated receptor- (PPAR)-dependent up-regulation of energy metabolism toward fatty acid oxidation (FAO). PPAR null (PPARKO) mice develop hyperinsulinemic hypoglycemia in the fasting state, and we previously showed that PPAR expression is increased in islets at low glucose. On this basis, we hypothesized that enhanced PPAR expression and FAO, via depletion of lipid-signaling molecule(s) for insulin exocytosis, are also involved in the normal adaptive response of the islet to fasting. Fasted PPARKO mice compared with wild-type mice had supranormal ip glucose tolerance due to increased plasma insulin levels. Isolated islets from the PPAR null mice had a 44% reduction in FAO, normal glucose use and oxidation, and enhanced glucose-induced insulin secretion. In normal rats, fasting for 24 h increased islet PPAR, carnitine palmitoyltransferase 1, and uncoupling protein-2 mRNA expression by 60%, 62%, and 82%, respectively. The data are consistent with the view that PPAR, via transcriptionally up-regulating islet FAO, can reduce insulin secretion, and that this mechanism is involved in the normal physiological response of the pancreatic islet to fasting such that hypoglycemia is avoided.
Introduction
THE NORMAL PANCREATIC islet response to starvation is reduced insulin and increased glucagon release (1). The resultant reduction in the circulating insulin/glucagon atio, together with other neurohormonal changes of starvation, leads to increased adipose tissue lipolysis, increased fatty acid oxidation (FAO) in both peripheral tissues and the liver, increased hepatic ketogenesis and increased hepatic and renal gluconeogenesis (2, 3). The net effect is the conversion of energy requirements in many tissues from glucose to fat oxidation with sparing of the limited endogenously produced glucose for obligatory requirements, such that starvation-induced hypoglycemia is avoided (2). The critical role for up-regulated FAO in this starvation response has particularly been learned from subjects with impaired mitochondrial FAO due to a variety of FAO enzyme deficiencies (4, 5, 6, 7, 8), in addition to studies of the long-chain acyl-coenzyme A (CoA) dehydrogenase-deficient mouse (9) and the peroxisome proliferator-activated receptor- (PPAR) null mouse (PPARKO) (10, 11). In each of these situations, impaired FAO is associated with starvation-induced severe hypoglycemia (4, 5, 6, 7, 8, 9, 10). Interestingly, hyperinsulinemic hypoglycemia has been observed in subjects with short-chain L-3-hydroxyacyl-CoA dehydrogenase deficiency (SCHAD) (5, 6), which is one of the FAO enzyme deficiency disorders. This suggests that enhanced FAO may also be important in the adaptive response of the islet to fasting.
PPARs are a family of nuclear receptors (PPAR, ?/, and ) that are activated by fatty acid ligands. They play key roles as lipid/nutritional state sensors and transcriptional regulators of lipid metabolism (for review see Refs. 12, 13, 14). Activated PPAR induces the transcription of genes of the peroxisomal and mitochondrial ?-oxidation pathways, and the microsomal -oxidation pathway (12) in addition to genes for fatty acid transporter proteins (15). Accordingly, PPAR is principally expressed in organs with high capacity for FAO (heart, skeletal muscle, liver, and kidney) (12). PPAR is also implicated in cholesterol metabolism and inflammation processes (for review see Refs. 12 and 16). Natural PPAR ligands comprise unsaturated and polyunsaturated fatty acids and some eicosanoids such as leukotriene B4, whereas synthetic ligands include the fibric acid derivatives (12). Of importance to the current work, PPAR is also expressed in the pancreatic islet (17) and purified rat ?-cells (18).
Many studies in the PPARKO mouse have led to the conclusion that PPAR transcriptional regulation of FAO plays a major role in the cellular metabolic response to fasting/starvation at least within cardiac muscle, liver, and kidney (11, 16, 19, 20, 21, 22). Although the mouse has a normal phenotype in the fed state, when fasted it develops severe hypoglycemia in association with elevated plasma free fatty acids (FFAs), hepatic and cardiac steatosis, and hypoketonemia, consistent with a failure of fasting to induce fatty acid catabolism by ?-oxidation and ketogenesis (11, 21). Also reduced FAO is associated with failed stimulation of gluconeogenesis in the liver (19) and kidney (23). The role of PPAR in the fasting response of the islet, however, has not been examined.
We have previously shown that PPAR expression is related to nutritional status in rat islets and rat insulinoma ?-cell lines (INS) in that it is down-regulated by elevated glucose within 6 h (17). This glucose-induced reduction in PPAR in INS(832/13) cells is associated with reduced expression of uncoupling protein 2 (UCP2) and acyl-CoA oxidase (ACO), and in INS1 cells, with reduced FAO, elevated malonyl-CoA and insulin secretion at low glucose, and impaired insulin secretion to high glucose (17). This led us to hypothesize that the normal islet response to the reverse nutritional status of fasting and starvation involves up-regulation of PPAR with induction of FAO that, via depletion of lipid signaling molecules important for insulin exocytosis (24, 25), decreases insulin secretion. In support of this hypothesis, here we demonstrate that the severe hypoglycemia of fasting in the PPARKO mouse is associated with inappropriately high insulin levels, an indication of failed islet ?-cell adaptation to fasting. This hypothesis has been further investigated in isolated islets from PPARKO and wild-type (WT) mice and in fed and fasted normal rat islets.
Materials and Methods
Animals
WT SV129 mice from The Jackson Laboratory (Bar Harbor, ME) and PPARKO mice on an SV129 background were obtained from the laboratory of FJ Gonzalez (Bethesda, MD) (10). In all experiments, 8-wk-old male mice were used. Wistar rats (200–220 g) were obtained from Charles River (St. Constant, Quebec, Canada). Mice and rats were housed in a temperature-controlled room (23 C) with an artificial 12-h light, 12-h dark cycle and were allowed unrestricted access to standard mouse chow and water. All procedures were approved by the Institutional Committee for the Protection of Animals at the Centre de Recherche du Centre Hospitalier de l’Université de Montréal and the Commission sur l’Expérimentation Animale of the Canton of Vaud.
Measurement of plasma parameters
Blood was collected 4 h after the beginning of the dark cycle for fed samples and 5 h after removal of food for fasted samples. Retro-orbital blood was collected into heparinized tubes from awake mice. Blood was immediately centrifuged and used for plasma glucose, lactate, insulin, glucagon, and FFA determinations. Plasma glucose and lactate concentrations were determined with Sigma kits (Sigma, St. Louis, MO). Plasma insulin was measured with a RIA kit (Linco Research Inc., St. Charles, MO), glucagon with a RIA kit (Linco Research Inc.) and FFA using the NEFA C kit (Wako Chemicals, Neuss, Germany).
Intraperitoneal glucose tolerance test (IPGTT)
IPGTTs were performed in conscious mice after a 5-h fast. A baseline blood sample (60 μl) was collected from the tail vein into a heparinized tube at time 0 min, after which glucose (3 g/kg body weight) was administered ip. Additional tail blood samples (60 μl) were collected at 15, 30, 60, and 120 min. Blood glucose concentrations were immediately determined by the Accutrend glucometer (Basel, Switzerland) after which the samples were centrifuged and the plasma rapidly frozen for later measurement of plasma insulin with an ELISA kit from Macromedia (Uppsala, Sweden).
Pancreatic islet isolation and culture
Pancreatic islets were isolated by collagenase digestion of pancreas according to the method of Gotoh et al. (26). After digestion and washing, mouse islets were hand-picked under a stereomicroscope. Rat islets were separated from digested exocrine tissue by histopaque gradient, after which they were hand-picked for immediate extraction of RNA. Mouse islets were kept in culture at 37 C in a humidified atmosphere containing 5% CO2. Culture medium was regular RPMI 1640 (11 mM glucose) supplemented with 10% fetal calf serum, 10 mM HEPES (pH 7.4) and 1 mM sodium pyruvate (RPMI complete). All mouse islet experiments were performed after 18 h of culture post isolation to allow recovery from the collagenase digestion and optimize secretion in response to glucose.
Measurement of islet DNA and hormonal content
Islet DNA content was measured following the technique of Hopcroft (27). Briefly, 10 islets were sonicated in 100 μl of DNA assay buffer [2 M NaCl, 0.05 M Na2HPO4, 2 mM EDTA (pH 7.4)] and then centrifuged for 5 min at 5000 rpm. The supernatant (45 μl sample or standard) was mixed with 180 μl of fluorochrome bisbenzimide H-33258 (Sigma, St. Louis, MO), after which fluorescence was measured in a spectro-fluorometer with excitation and emission wavelengths of 356 and 448 nm, respectively. For islet hormonal content measurements, islets were put in water, sonicated, and the content of insulin, glucagon and somatostatin were assayed by insulin RIA, glucagon RIA, and somatostatin RIA (Incstar Corp., Stillwater, MN) kits.
Islet immunofluorescence
Pancreases from a WT and a PPARKO mice were isolated and fixed overnight in a paraformaldehyde/lysine/periodate solution (2% paraformaldehyde, 60 mM L-lysine, 10 mM NaIO4). Pancreases were then transferred overnight in PBS-20% sucrose and again overnight in PBS-30% sucrose, before being finally embedded in Tissue-Tek O.C.T. compound (Sakura Finetek Ltd., Zoeterwoude, The Netherlands) and frozen. Pancreas sections (6 μm) were immunostained using antibodies to insulin, glucagon, and somatostatin, and the appropriate fluorescein-conjugated secondary antibodies. Immunofluorescence was examined using a fluorescence microscope.
Insulin secretion from isolated islets and INS cells
The day after isolation, 10 islets of homogeneous size from WT and PPARKO mice were picked by hand and put in perifusion chambers connected to a fraction collector. Buffer flow was set at 1 ml/min. The perifusion solution was KRBH buffer [120 mM NaCl, 4 mM KH2PO4, 20 mM HEPES, 1 mM MgCl2, 1 mM CaCl2, 5 mM NaHCO3 (pH 7.4)] containing 0.5% BSA (fraction V, RIA grade), 0.2 mM 3-isobutyl-1-methylxanthine and glucose at increasing concentrations. After a 20-min equilibration period at 2.8 mM glucose, the islets were submitted to a discontinuous glucose gradient made of 12 min steps at 2.8, 3.3, 4.2, 5.6, 8.4, 11.1, and 16.7 glucose. Effluent was recovered every min into separate tubes and insulin was measured in every fraction via RIA.
Islet metabolism determinations
For glucose usage measurements, batches of 50 islets were washed in KRBH buffer and then incubated for 2 h in a humidified incubator at 37 C, in 50 μl KRBH containing 6 or 20 mM glucose with 6 or 20 μCi/ml, respectively, of D-[5-3H]glucose. The reaction was stopped with 10 μl 1 N HCl and supernatants were passed through a DOWEX 1 x 2 column (Dow, Horgen, Switzerland). The 3H2O was eluted with 2 x 1.5 ml water. To determine column recovery efficiency, a sample of 3H2O was also passed through the column. Liquid scintillator was added to the eluates and radioactivity was measured in a ?-scintillation counter.
The experimental system for islet glucose and FAO assays consisted of a round-bottom polystyrene 15-ml Falcon tube (Becton Dickinson, Basel, Switzerland) sealed closed with a rubber stopper from which a center well was suspended. Batches of 50 islets were washed in KRBH and resuspended in 50 μl of KRBH/0.5% BSA (RIA grade) containing 6 or 20 mM glucose with 10 or 20 μCi/ml, respectively, of [U-14C]glucose (glucose oxidation) or 50 μl KRBH/1.25% BSA (fatty acid free) buffer containing 3 mM glucose and 0.8 mM L-carnitine with 1 mM palmitic acid with 4 μCi/ml of [1-14C]palmitic acid. Islets in the glucose or FAO medium were then placed at the bottom of the Falcon tubes and were sealed with the stoppers. After 3.5 h incubation at 37 C in a cell incubator, the reaction was stopped with an injection of 0.1 ml perchloric acid (10% vol/vol) (glucose oxidation) or 0.2 ml 10% trichloroacetic acid (FAO). Benzethonium hydroxide (0.3 ml) was injected into the center well to trap the 14CO2 produced. After an overnight incubation at room temperature, the benzethonium hydroxide was recovered to which scintillator fluid was added. Radioactivity was measured in a ?-scintillation counter.
Islet RNA extraction and RT-PCR analysis
Total RNA was extracted from 250 islets from fed, 12-, and 24-h fasted Wistar rats by the guanidinium thiocyanate/phenol/chloroform extraction method (28). First-strand cDNA was generated from 2 μg of RNA in a 50 μl (final volume) of a buffer containing the oligonucleotides Pd(N)6 (29). PCR amplification of PPAR (30), carnitine palmitolytransferase-1 (CPT-1) (31), UCP2 (32), ACO (31), and ?-actin (17) were performed using the primers and experimental conditions as indicated in the cited references. All PCRs had been tested to give products within the linear part of the amplification curve. Ten microliters of each of the PCR products was subjected to electrophoresis on 1.0% agarose gels followed by Southern blotting on a probe membrane (Bio-Rad, Hercules, CA). The specific radiolabeled probes used for each of the genes were according to the above cited references. The hybridization signals were quantified by scanning the autoradiograms, and the results were normalized to the ?-actin signal.
Statistical analysis
All results are expressed as means ± SEM. Statistical significance was calculated with the Student’s t test or, for multiple comparisons, one-way ANOVA with Bonferroni post hoc testing and, where indicated, two-way ANOVA.
Results
Plasma parameters in fed and fasted PPARKO and WT mice
Fasting and fed metabolic indices are shown in Table 1. Consistent with previous studies (11, 21), fed blood glucose levels were not different, but fasting blood glucose levels were 41% lower in PPARKO compared with WT mice. Plasma lactate levels were not altered by PPAR deficiency. Importantly, in association with this moderately severe fasting hypoglycemia, plasma insulin levels were 55% higher in the PPAR-deficient mice. Thus, the suppression of insulin secretion in response to hypoglycemia is impaired in PPARKO mice. In contrast, fed plasma insulin levels were significantly lower in the PPARKO compared with WT mice. Plasma glucagon levels in the PPARKO mice were appropriately elevated in response to hypoglycemia during fasting (2-fold higher than in fasted normoglycemic WT mice) and were also elevated in the fed state (35% higher than in fed WT mice). Plasma FFA levels were higher in the PPARKO mice in comparison to the WT mice in both the fed and fasted states, consistent with previous reports (11, 21).
TABLE 1. Fed and fasted plasma glucose, lactate, insulin, glucagon, and free fatty acid levels in WT (+/+) and PPARKO (–/–) mice
Elevated plasma insulin enhances ip glucose tolerance in 5-h fasted PPARKO mice
Intraperitoneal glucose tolerance was enhanced in the 5-h fasted PPARKO compared with WT mice (Fig. 1A). Blood glucose was 34% lower in the PPARKO compared with WT mice at time 0 and remained lower at every time point after the ip glucose load. Plasma insulin levels were 39% higher after 5 h of fasting (time 0) and remained higher after the glucose load in the PPARKO mice (Fig. 1B). The increment in plasma insulin from fasting levels, however, was not different between genotypes. The ratio of the insulin and glucose area under the curves (Fig. 1E) clearly show that insulin secretion for a given glucose concentration is markedly enhanced in the PPARKO mice. The glucose and insulin results of the IPGTT study, therefore, confirm the presence of abnormally high insulin levels that are inappropriate for the associated hypoglycemia in 5-h fasted PPARKO mice. Furthermore, the results suggest that hyperinsulinemia is the most likely factor contributing to the enhanced glucose tolerance in the PPAR-null mice (see also Fig. 1, C and D, representing the area under the curves of panels A and B).
FIG. 1. Blood glucose and plasma insulin levels during an IPGTT. The IPGTT test (3 g glucose/kg body weight) was carried out in 5-h fasted 8-wk-old male WT and PPARKO mice. Area under the curve (AUC). Means ± SEM of six animals. *, P < 0.05; **, P < 0.001 vs. WT using Student’s t test.
Cellular and hormonal parameters of islets from PPARKO and WT mice
To determine whether the changes in plasma insulin and glucagon levels were related to altered proportions of the three major islet cell types (, ?, and cells) or total hormonal contents, isolated islets from PPARKO and WT mice were examined for these parameters. Total islet DNA was not different between the 2 genotypes (67.2 ± 2.3 vs. 63.8 ± 1.6 ng/islet, WT vs. PPARKO, respectively), and islet size distribution was identical (data not shown). Immunofluorescent staining of whole pancreas sections using anti-glucagon, -insulin, and -somatostatin antibodies showed no obvious genotype differences in the distribution or proportion of , ?, and cells (Fig. 2). Proinsulin mRNA and glucagon mRNA corrected for ?-actin mRNA in PPARKO islets were 129 ± 25% (not significant) and 81 ± 7% (P = 0.03) of WT, respectively. The total insulin content of PPARKO and WT islets were not different (33.6 ± 4.2 and 34.1 ± 1.1 ng/islet, respectively). The total islet contents of glucagon and somatostatin, however, were 40% (291 ± 65 and 486 ± 55 pg/islet, P = 0.06) and 49% (56.4 ± 0.3 and 110.0 ± 7.2 pg/islet, P = 0.02) lower in PPARKO in comparison to WT islets, respectively. Glucagon release in static incubation was 1.7-fold higher at 2.8 mM glucose (6.0 ± 0.6 and 3.5 ± 0.6 pg/islet·h, P < 0.05) and 1.4-fold higher at 16.7 mM glucose (3.6 ± 0.5 and 2.6 ± 0.4 pg/islet·h, not significant) in islets from PPARKO compared with WT mice.
FIG. 2. Immunofluorescent staining of (A, C, and E) WT and (B, D, and F) PPARKO islets using antibodies to insulin (A and B), glucagon (C and D) and somatostatin (E and F).
Glucose-induced insulin secretion is increased in isolated islets from PPAR-deficient mice
To determine whether the hyperinsulinemia after fasting and during the IPGTT in PPARKO mice is a result of altered islet function per se causing increased insulin release, we assessed glucose-induced insulin secretion (GIIS) from isolated perifused islets from PPARKO and WT fasted mice using a step-wise glucose gradient protocol. As shown in Fig. 3, at intermediate and high glucose concentrations, PPARKO islets secreted more insulin than WT islets. At 8.4 mM and 16.7 mM glucose, the insulin responses were 65% and 36% higher from the PPARKO islets, respectively. The DNA content and size distribution of the islets chosen for the perifusion experiments for the two genotypes were identical (not shown). Thus, the results are consistent with the proposition that the hyperinsulinemia of the PPARKO mice is due to islet hypersecretion to glucose.
FIG. 3. Glucose-induced insulin secretion is enhanced in isolated islets from PPARKO mice. Ten islets of each genotype were placed into parallel perifusion chambers. After a 20-min equilibration period of perifusion at 1 ml/min with KRBH buffer containing 2.8 mM glucose and 0.5% BSA, minutely sampling of the effluent perifusate was commenced. The glucose concentration of the perifusate was increased every 12 min in seven steps from 2.8 to 16.7 mM. A–C, Results of separate experiments; D, means ± SEM from the three separate experiments.
FAO is reduced, and glucose oxidation is unaltered in islets from PPARKO mice
To provide support for the hypothesis that PPAR up-regulation of FAO is important in islets for the reduction of insulin secretion during fasting, we measured palmitate oxidation at low (6 mM) glucose in isolated PPARKO and WT islets (Fig. 4A). FAO was 44% lower in PPARKO compared with WT islets, thus confirming that PPAR is involved in the maintenance of FAO in islets. Because glucose and FAO are reciprocally regulated in several tissues (33, 34) and glucose metabolism is essential for GIIS, we also measured islet glucose usage and oxidation. Figure 4, B and C, shows that glucose usage and glucose oxidation rates at 6 and 16 mM glucose were not significantly modified by the absence of PPAR, but there was a trend for a small increase, particularly with respect to glucose usage.
FIG. 4. FAO is reduced but glucose oxidation is unchanged in isolated islets from PPARKO mice. Batches of 50 islets were washed in KRBH buffer and incubated in the presence of radioactive tracers for 2 h (glucose use) or 3.5 h (glucose and FAO). A, Palmitate oxidation; B, glucose use; and C, glucose oxidation were determined from measurements of 14CO2 or 3H2O as described in Materials and Methods. Palmitate oxidation at 100% of WT was 3.8 ± 1.4 nmol/mg prot·h. Means ± SEM of three (glucose use and oxidation) or four (FAO) experiments. *, P < 0.05 vs. WT using Student’s t test.
Fasting increases the expression of PPAR and genes under its transcriptional control in rat islets
According to the hypothesis proposing a role for PPAR in the normal adaptation of the ?-cell to fasting, the expression of PPAR and genes under its transcriptional control involved in FAO should be increased in islets with prolonged fasting. To confirm this, PPAR, CPT1, ACO, and UCP2 mRNA levels were measured by RT-PCR and Southern blotting in islets isolated from fed, 12-h fasted, and 24-h fasted Wistar rats. Islet PPAR mRNA levels were increased by 46% with 12 h and by 60% with 24 h of fasting (Fig. 5). The mitochondrial proteins under its transcriptional control, CPT1 and UCP2, increased by 62% and 82% by 24 h, respectively (Fig. 4). Expression of the peroxisomal FAO enzyme ACO, however, was not significantly altered by 24 h of fasting, although there was a trend toward an increase (Fig. 5).
FIG. 5. Fasting increases the expression of PPAR, CPT1, and UCP2 in rat islets. Islets were isolated from fed, 12-h fasted, and 24-h fasted rats. PPAR, CPT1, ACO, and UCP2 mRNA levels were measured by RT-PCR analysis as described in Materials and Methods. Visualization of PCR products by ethidium bromide staining after electrophoresis in 1% agarose gel, and expression quantified by scanning Southern blot autoradiograms with normalization to ?-actin. Means ± SEM of islets from three separate rats for each nutritional state. Statistical analysis by one-way ANOVA with Bonferroni post hoc testing. *, P < 0.05; **, P < 0.01 vs. fed state.
Discussion
The importance of PPAR and FAO in the adaptation of cardiac muscle (21), liver (11, 19, 21), and kidney (23) to fasting is well established. In this paper, we show that PPAR is also important in the adaptation of pancreatic islets to fasting, and the data support the hypothesis that the mechanism is, at least in part, via up-regulation of islet FAO. PPARKO mice fasted for 5 h developed moderately severe hyperinsulinemic hypoglycemia, consistent with failed fasting-induced suppression of insulin secretion. Isolated islets from these mice had both reduced capacity for FAO and enhanced GIIS, consistent with a role for PPAR in the maintenance of FAO in islets and a link between impaired FAO and insulin hypersecretion. The finding that glucose use and oxidation were not significantly changed in PPAR-deficient islets is also in favor of the PPAR effect on GIIS being via control of FAO. Finally, the rat islet studies confirmed that the expression of PPAR and mitochondrial FAO genes under its transcriptional control increase with fasting. Of note, both PPAR overexpression and activation of PPAR by clofibrate in INS1 cells have previously been shown to cause impaired GIIS in association with enhanced FAO (35). Thus, increased PPAR expression, as occurs in islets during conditions of low glucose (17, 36) and fasting, as shown in this study, inhibits GIIS and the absence of PPAR, as shown in the PPARKO mice, is associated with failure of the normal fasting-induced suppression of insulin secretion.
The evidence that implicates enhancement of islet FAO by PPAR as the most likely mechanism by which PPAR suppresses insulin secretion is now considerable. First, the major impact of PPAR activity in most other tissues is the promotion of FAO, such that its action in the islet is also likely to be related to this effect. Second, in this study we show that PPAR absence is associated with a 44% reduction in islet FAO, and previously it was shown that its overexpression in INS1 ?-cells causes increased FAO (35). Activation of PPAR by clofibrate in INS1 cells (35) and by clofibrate, together with 9-cis-retinoic acid, in islets (30) has also been shown to increase FAO. Third, most studies in which ?-cell FAO has been enhanced or reduced by pharmacological or molecular tools directed at sites other than PPAR are consistent with an effect of enhanced ?-cell FAO to suppress insulin secretion (25, 37, 38, 39). Fourth, islet depletion of fatty acid by adenovirus-leptin gene therapy (40) or fasting plus nicotinic acid (41) causes loss of GIIS, consistent with the hypothesis that insulin secretion can be suppressed by depletion (e.g. after enhanced FAO) of a critical lipid signaling molecule for exocytosis. Fifth, we have recently shown that enhanced FAO resulting from malonyl-CoA decarboxylase overexpression in INS cell lines and islets causes loss of fatty acid augmentation of GIIS in association with reduced ?-cell production of esterified lipid moieties diacylglycerol (25). Finally, isolated islets from 48-h fasted rats show enhanced FAO and impaired GIIS (42). 2-Bromostearate, which inhibits FAO by reducing CPT-1 activity, acutely restored GIIS in these islets (42).
The higher plasma glucagon level in PPARKO mice during fasting may simply be a counterregulatory response to the hypoglycemia, but it is inadequate to prevent the hypoglycemia. The higher glucagon level in PPARKO fed mice and the higher glucagon secretion in isolated islets, however, is more difficult to explain, but it may reflect compensatory changes in the -cell to a chronic tendency toward hypoglycemia. Alternatively, a more direct effect of PPARKO in the -cells causing glucagon hypersecretion could be possible. The lower glucagon content of PPARKO islets, in association with elevated plasma glucagon levels and mildly reduced islet glucagon mRNA expression, is consistent with the occurrence of some degranulation of the -cells due to glucagon hypersecretion without compensation by increased gene expression. Glucagon is not normally insulinotropic in situations of low glucose (43), such that it is unlikely that the elevated glucagon levels cause the fasting hyperinsulinemia of the PPARKO mice via a paracrine effect. However, it is always possible that altered lipid partitioning in the PPARKO islets has lowered the glucose set point by which glucagon is insulinotropic.
Fasting-induced hypoglycemia has often been reported in PPARKO mice, but hyperinsulinemic hypoglycemia has not been consistently reported. However, consistent with our finding, a higher ratio of insulin to glucose was reported in 24-h fasted PPARKO compared with WT mice (19), although fasting-induced hypoglycemia was not observed probably due to blood sampling during anesthesia. A nonsignificant trend for higher insulin levels, despite lower glucose levels, was found in 17-h fasted PPARKO mice (22). In other studies in which hypoglycemia has been documented in PPARKO mice (11, 20, 21), it is unfortunate that limited (20) or no (11, 21) corresponding insulin levels were reported.
It is apparent that the islet contribution to the nonketotic hypoglycemia in subjects with the various in-born errors of metabolism in FAO and in animal models of impaired FAO might differ depending on the site of the defect in FAO. Hyperinsulinemic hypoglycemia occurs in SCHAD disorders (5, 6) and in PPARKO mice (present study). It is not reported to occur when the FAO defect is secondary to defects in medium-, long-, and very-long-chain acyl-CoA dehydrogenase deficiencies (6) or deficiencies in the liver isoform of CPT-1 (8) or malonyl-CoA decarboxylase (7). However, circulating insulin levels were, in most instances, not reported in these studies. Thus, it is difficult to ascertain from the literature the extent to which inadequate suppression of insulin secretion has been excluded in the subjects with several of these rare conditions. A possible explanation for lack of islet phenotypes in some of these conditions could relate to secondary compensatory changes. For example, in very-long-chain acyl-CoA dehydrogenase-deficient mice (44), expression of acyl-CoA synthase as measured by Western blot is markedly reduced in 2-month-old mice. If acyl-CoA synthase expression is similarly reduced in islets of subjects with these more severe FAO disorders, abnormal ?-cell accumulation of LC-CoA with the stimulation of inappropriate insulin secretion would be prevented.
In conclusion, this work supports a role for increased FAO in the normal pancreatic islet response to fasting and starvation, and that PPAR is an essential transcription factor involved in this process. The increased islet FAO during fasting reduces insulin release, most probably by depletion of a lipid-signaling molecule involved in exocytosis, such that fasting hypoglycemia is avoided.
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Address all correspondence and requests for reprints to: Dr. Marc Prentki, Centre de Recherche, Centre Hospitalier de l’Université de Montréal, Pavillon de Sève, Y4603, 1560 Sherbrooke East, Montreal, Quebec, Canada H2L 4M1. E-mail: marc.prentki@umontreal.ca.
Abstract
The cellular response to fasting and starvation in tissues such as heart, skeletal muscle, and liver requires peroxisome proliferator-activated receptor- (PPAR)-dependent up-regulation of energy metabolism toward fatty acid oxidation (FAO). PPAR null (PPARKO) mice develop hyperinsulinemic hypoglycemia in the fasting state, and we previously showed that PPAR expression is increased in islets at low glucose. On this basis, we hypothesized that enhanced PPAR expression and FAO, via depletion of lipid-signaling molecule(s) for insulin exocytosis, are also involved in the normal adaptive response of the islet to fasting. Fasted PPARKO mice compared with wild-type mice had supranormal ip glucose tolerance due to increased plasma insulin levels. Isolated islets from the PPAR null mice had a 44% reduction in FAO, normal glucose use and oxidation, and enhanced glucose-induced insulin secretion. In normal rats, fasting for 24 h increased islet PPAR, carnitine palmitoyltransferase 1, and uncoupling protein-2 mRNA expression by 60%, 62%, and 82%, respectively. The data are consistent with the view that PPAR, via transcriptionally up-regulating islet FAO, can reduce insulin secretion, and that this mechanism is involved in the normal physiological response of the pancreatic islet to fasting such that hypoglycemia is avoided.
Introduction
THE NORMAL PANCREATIC islet response to starvation is reduced insulin and increased glucagon release (1). The resultant reduction in the circulating insulin/glucagon atio, together with other neurohormonal changes of starvation, leads to increased adipose tissue lipolysis, increased fatty acid oxidation (FAO) in both peripheral tissues and the liver, increased hepatic ketogenesis and increased hepatic and renal gluconeogenesis (2, 3). The net effect is the conversion of energy requirements in many tissues from glucose to fat oxidation with sparing of the limited endogenously produced glucose for obligatory requirements, such that starvation-induced hypoglycemia is avoided (2). The critical role for up-regulated FAO in this starvation response has particularly been learned from subjects with impaired mitochondrial FAO due to a variety of FAO enzyme deficiencies (4, 5, 6, 7, 8), in addition to studies of the long-chain acyl-coenzyme A (CoA) dehydrogenase-deficient mouse (9) and the peroxisome proliferator-activated receptor- (PPAR) null mouse (PPARKO) (10, 11). In each of these situations, impaired FAO is associated with starvation-induced severe hypoglycemia (4, 5, 6, 7, 8, 9, 10). Interestingly, hyperinsulinemic hypoglycemia has been observed in subjects with short-chain L-3-hydroxyacyl-CoA dehydrogenase deficiency (SCHAD) (5, 6), which is one of the FAO enzyme deficiency disorders. This suggests that enhanced FAO may also be important in the adaptive response of the islet to fasting.
PPARs are a family of nuclear receptors (PPAR, ?/, and ) that are activated by fatty acid ligands. They play key roles as lipid/nutritional state sensors and transcriptional regulators of lipid metabolism (for review see Refs. 12, 13, 14). Activated PPAR induces the transcription of genes of the peroxisomal and mitochondrial ?-oxidation pathways, and the microsomal -oxidation pathway (12) in addition to genes for fatty acid transporter proteins (15). Accordingly, PPAR is principally expressed in organs with high capacity for FAO (heart, skeletal muscle, liver, and kidney) (12). PPAR is also implicated in cholesterol metabolism and inflammation processes (for review see Refs. 12 and 16). Natural PPAR ligands comprise unsaturated and polyunsaturated fatty acids and some eicosanoids such as leukotriene B4, whereas synthetic ligands include the fibric acid derivatives (12). Of importance to the current work, PPAR is also expressed in the pancreatic islet (17) and purified rat ?-cells (18).
Many studies in the PPARKO mouse have led to the conclusion that PPAR transcriptional regulation of FAO plays a major role in the cellular metabolic response to fasting/starvation at least within cardiac muscle, liver, and kidney (11, 16, 19, 20, 21, 22). Although the mouse has a normal phenotype in the fed state, when fasted it develops severe hypoglycemia in association with elevated plasma free fatty acids (FFAs), hepatic and cardiac steatosis, and hypoketonemia, consistent with a failure of fasting to induce fatty acid catabolism by ?-oxidation and ketogenesis (11, 21). Also reduced FAO is associated with failed stimulation of gluconeogenesis in the liver (19) and kidney (23). The role of PPAR in the fasting response of the islet, however, has not been examined.
We have previously shown that PPAR expression is related to nutritional status in rat islets and rat insulinoma ?-cell lines (INS) in that it is down-regulated by elevated glucose within 6 h (17). This glucose-induced reduction in PPAR in INS(832/13) cells is associated with reduced expression of uncoupling protein 2 (UCP2) and acyl-CoA oxidase (ACO), and in INS1 cells, with reduced FAO, elevated malonyl-CoA and insulin secretion at low glucose, and impaired insulin secretion to high glucose (17). This led us to hypothesize that the normal islet response to the reverse nutritional status of fasting and starvation involves up-regulation of PPAR with induction of FAO that, via depletion of lipid signaling molecules important for insulin exocytosis (24, 25), decreases insulin secretion. In support of this hypothesis, here we demonstrate that the severe hypoglycemia of fasting in the PPARKO mouse is associated with inappropriately high insulin levels, an indication of failed islet ?-cell adaptation to fasting. This hypothesis has been further investigated in isolated islets from PPARKO and wild-type (WT) mice and in fed and fasted normal rat islets.
Materials and Methods
Animals
WT SV129 mice from The Jackson Laboratory (Bar Harbor, ME) and PPARKO mice on an SV129 background were obtained from the laboratory of FJ Gonzalez (Bethesda, MD) (10). In all experiments, 8-wk-old male mice were used. Wistar rats (200–220 g) were obtained from Charles River (St. Constant, Quebec, Canada). Mice and rats were housed in a temperature-controlled room (23 C) with an artificial 12-h light, 12-h dark cycle and were allowed unrestricted access to standard mouse chow and water. All procedures were approved by the Institutional Committee for the Protection of Animals at the Centre de Recherche du Centre Hospitalier de l’Université de Montréal and the Commission sur l’Expérimentation Animale of the Canton of Vaud.
Measurement of plasma parameters
Blood was collected 4 h after the beginning of the dark cycle for fed samples and 5 h after removal of food for fasted samples. Retro-orbital blood was collected into heparinized tubes from awake mice. Blood was immediately centrifuged and used for plasma glucose, lactate, insulin, glucagon, and FFA determinations. Plasma glucose and lactate concentrations were determined with Sigma kits (Sigma, St. Louis, MO). Plasma insulin was measured with a RIA kit (Linco Research Inc., St. Charles, MO), glucagon with a RIA kit (Linco Research Inc.) and FFA using the NEFA C kit (Wako Chemicals, Neuss, Germany).
Intraperitoneal glucose tolerance test (IPGTT)
IPGTTs were performed in conscious mice after a 5-h fast. A baseline blood sample (60 μl) was collected from the tail vein into a heparinized tube at time 0 min, after which glucose (3 g/kg body weight) was administered ip. Additional tail blood samples (60 μl) were collected at 15, 30, 60, and 120 min. Blood glucose concentrations were immediately determined by the Accutrend glucometer (Basel, Switzerland) after which the samples were centrifuged and the plasma rapidly frozen for later measurement of plasma insulin with an ELISA kit from Macromedia (Uppsala, Sweden).
Pancreatic islet isolation and culture
Pancreatic islets were isolated by collagenase digestion of pancreas according to the method of Gotoh et al. (26). After digestion and washing, mouse islets were hand-picked under a stereomicroscope. Rat islets were separated from digested exocrine tissue by histopaque gradient, after which they were hand-picked for immediate extraction of RNA. Mouse islets were kept in culture at 37 C in a humidified atmosphere containing 5% CO2. Culture medium was regular RPMI 1640 (11 mM glucose) supplemented with 10% fetal calf serum, 10 mM HEPES (pH 7.4) and 1 mM sodium pyruvate (RPMI complete). All mouse islet experiments were performed after 18 h of culture post isolation to allow recovery from the collagenase digestion and optimize secretion in response to glucose.
Measurement of islet DNA and hormonal content
Islet DNA content was measured following the technique of Hopcroft (27). Briefly, 10 islets were sonicated in 100 μl of DNA assay buffer [2 M NaCl, 0.05 M Na2HPO4, 2 mM EDTA (pH 7.4)] and then centrifuged for 5 min at 5000 rpm. The supernatant (45 μl sample or standard) was mixed with 180 μl of fluorochrome bisbenzimide H-33258 (Sigma, St. Louis, MO), after which fluorescence was measured in a spectro-fluorometer with excitation and emission wavelengths of 356 and 448 nm, respectively. For islet hormonal content measurements, islets were put in water, sonicated, and the content of insulin, glucagon and somatostatin were assayed by insulin RIA, glucagon RIA, and somatostatin RIA (Incstar Corp., Stillwater, MN) kits.
Islet immunofluorescence
Pancreases from a WT and a PPARKO mice were isolated and fixed overnight in a paraformaldehyde/lysine/periodate solution (2% paraformaldehyde, 60 mM L-lysine, 10 mM NaIO4). Pancreases were then transferred overnight in PBS-20% sucrose and again overnight in PBS-30% sucrose, before being finally embedded in Tissue-Tek O.C.T. compound (Sakura Finetek Ltd., Zoeterwoude, The Netherlands) and frozen. Pancreas sections (6 μm) were immunostained using antibodies to insulin, glucagon, and somatostatin, and the appropriate fluorescein-conjugated secondary antibodies. Immunofluorescence was examined using a fluorescence microscope.
Insulin secretion from isolated islets and INS cells
The day after isolation, 10 islets of homogeneous size from WT and PPARKO mice were picked by hand and put in perifusion chambers connected to a fraction collector. Buffer flow was set at 1 ml/min. The perifusion solution was KRBH buffer [120 mM NaCl, 4 mM KH2PO4, 20 mM HEPES, 1 mM MgCl2, 1 mM CaCl2, 5 mM NaHCO3 (pH 7.4)] containing 0.5% BSA (fraction V, RIA grade), 0.2 mM 3-isobutyl-1-methylxanthine and glucose at increasing concentrations. After a 20-min equilibration period at 2.8 mM glucose, the islets were submitted to a discontinuous glucose gradient made of 12 min steps at 2.8, 3.3, 4.2, 5.6, 8.4, 11.1, and 16.7 glucose. Effluent was recovered every min into separate tubes and insulin was measured in every fraction via RIA.
Islet metabolism determinations
For glucose usage measurements, batches of 50 islets were washed in KRBH buffer and then incubated for 2 h in a humidified incubator at 37 C, in 50 μl KRBH containing 6 or 20 mM glucose with 6 or 20 μCi/ml, respectively, of D-[5-3H]glucose. The reaction was stopped with 10 μl 1 N HCl and supernatants were passed through a DOWEX 1 x 2 column (Dow, Horgen, Switzerland). The 3H2O was eluted with 2 x 1.5 ml water. To determine column recovery efficiency, a sample of 3H2O was also passed through the column. Liquid scintillator was added to the eluates and radioactivity was measured in a ?-scintillation counter.
The experimental system for islet glucose and FAO assays consisted of a round-bottom polystyrene 15-ml Falcon tube (Becton Dickinson, Basel, Switzerland) sealed closed with a rubber stopper from which a center well was suspended. Batches of 50 islets were washed in KRBH and resuspended in 50 μl of KRBH/0.5% BSA (RIA grade) containing 6 or 20 mM glucose with 10 or 20 μCi/ml, respectively, of [U-14C]glucose (glucose oxidation) or 50 μl KRBH/1.25% BSA (fatty acid free) buffer containing 3 mM glucose and 0.8 mM L-carnitine with 1 mM palmitic acid with 4 μCi/ml of [1-14C]palmitic acid. Islets in the glucose or FAO medium were then placed at the bottom of the Falcon tubes and were sealed with the stoppers. After 3.5 h incubation at 37 C in a cell incubator, the reaction was stopped with an injection of 0.1 ml perchloric acid (10% vol/vol) (glucose oxidation) or 0.2 ml 10% trichloroacetic acid (FAO). Benzethonium hydroxide (0.3 ml) was injected into the center well to trap the 14CO2 produced. After an overnight incubation at room temperature, the benzethonium hydroxide was recovered to which scintillator fluid was added. Radioactivity was measured in a ?-scintillation counter.
Islet RNA extraction and RT-PCR analysis
Total RNA was extracted from 250 islets from fed, 12-, and 24-h fasted Wistar rats by the guanidinium thiocyanate/phenol/chloroform extraction method (28). First-strand cDNA was generated from 2 μg of RNA in a 50 μl (final volume) of a buffer containing the oligonucleotides Pd(N)6 (29). PCR amplification of PPAR (30), carnitine palmitolytransferase-1 (CPT-1) (31), UCP2 (32), ACO (31), and ?-actin (17) were performed using the primers and experimental conditions as indicated in the cited references. All PCRs had been tested to give products within the linear part of the amplification curve. Ten microliters of each of the PCR products was subjected to electrophoresis on 1.0% agarose gels followed by Southern blotting on a probe membrane (Bio-Rad, Hercules, CA). The specific radiolabeled probes used for each of the genes were according to the above cited references. The hybridization signals were quantified by scanning the autoradiograms, and the results were normalized to the ?-actin signal.
Statistical analysis
All results are expressed as means ± SEM. Statistical significance was calculated with the Student’s t test or, for multiple comparisons, one-way ANOVA with Bonferroni post hoc testing and, where indicated, two-way ANOVA.
Results
Plasma parameters in fed and fasted PPARKO and WT mice
Fasting and fed metabolic indices are shown in Table 1. Consistent with previous studies (11, 21), fed blood glucose levels were not different, but fasting blood glucose levels were 41% lower in PPARKO compared with WT mice. Plasma lactate levels were not altered by PPAR deficiency. Importantly, in association with this moderately severe fasting hypoglycemia, plasma insulin levels were 55% higher in the PPAR-deficient mice. Thus, the suppression of insulin secretion in response to hypoglycemia is impaired in PPARKO mice. In contrast, fed plasma insulin levels were significantly lower in the PPARKO compared with WT mice. Plasma glucagon levels in the PPARKO mice were appropriately elevated in response to hypoglycemia during fasting (2-fold higher than in fasted normoglycemic WT mice) and were also elevated in the fed state (35% higher than in fed WT mice). Plasma FFA levels were higher in the PPARKO mice in comparison to the WT mice in both the fed and fasted states, consistent with previous reports (11, 21).
TABLE 1. Fed and fasted plasma glucose, lactate, insulin, glucagon, and free fatty acid levels in WT (+/+) and PPARKO (–/–) mice
Elevated plasma insulin enhances ip glucose tolerance in 5-h fasted PPARKO mice
Intraperitoneal glucose tolerance was enhanced in the 5-h fasted PPARKO compared with WT mice (Fig. 1A). Blood glucose was 34% lower in the PPARKO compared with WT mice at time 0 and remained lower at every time point after the ip glucose load. Plasma insulin levels were 39% higher after 5 h of fasting (time 0) and remained higher after the glucose load in the PPARKO mice (Fig. 1B). The increment in plasma insulin from fasting levels, however, was not different between genotypes. The ratio of the insulin and glucose area under the curves (Fig. 1E) clearly show that insulin secretion for a given glucose concentration is markedly enhanced in the PPARKO mice. The glucose and insulin results of the IPGTT study, therefore, confirm the presence of abnormally high insulin levels that are inappropriate for the associated hypoglycemia in 5-h fasted PPARKO mice. Furthermore, the results suggest that hyperinsulinemia is the most likely factor contributing to the enhanced glucose tolerance in the PPAR-null mice (see also Fig. 1, C and D, representing the area under the curves of panels A and B).
FIG. 1. Blood glucose and plasma insulin levels during an IPGTT. The IPGTT test (3 g glucose/kg body weight) was carried out in 5-h fasted 8-wk-old male WT and PPARKO mice. Area under the curve (AUC). Means ± SEM of six animals. *, P < 0.05; **, P < 0.001 vs. WT using Student’s t test.
Cellular and hormonal parameters of islets from PPARKO and WT mice
To determine whether the changes in plasma insulin and glucagon levels were related to altered proportions of the three major islet cell types (, ?, and cells) or total hormonal contents, isolated islets from PPARKO and WT mice were examined for these parameters. Total islet DNA was not different between the 2 genotypes (67.2 ± 2.3 vs. 63.8 ± 1.6 ng/islet, WT vs. PPARKO, respectively), and islet size distribution was identical (data not shown). Immunofluorescent staining of whole pancreas sections using anti-glucagon, -insulin, and -somatostatin antibodies showed no obvious genotype differences in the distribution or proportion of , ?, and cells (Fig. 2). Proinsulin mRNA and glucagon mRNA corrected for ?-actin mRNA in PPARKO islets were 129 ± 25% (not significant) and 81 ± 7% (P = 0.03) of WT, respectively. The total insulin content of PPARKO and WT islets were not different (33.6 ± 4.2 and 34.1 ± 1.1 ng/islet, respectively). The total islet contents of glucagon and somatostatin, however, were 40% (291 ± 65 and 486 ± 55 pg/islet, P = 0.06) and 49% (56.4 ± 0.3 and 110.0 ± 7.2 pg/islet, P = 0.02) lower in PPARKO in comparison to WT islets, respectively. Glucagon release in static incubation was 1.7-fold higher at 2.8 mM glucose (6.0 ± 0.6 and 3.5 ± 0.6 pg/islet·h, P < 0.05) and 1.4-fold higher at 16.7 mM glucose (3.6 ± 0.5 and 2.6 ± 0.4 pg/islet·h, not significant) in islets from PPARKO compared with WT mice.
FIG. 2. Immunofluorescent staining of (A, C, and E) WT and (B, D, and F) PPARKO islets using antibodies to insulin (A and B), glucagon (C and D) and somatostatin (E and F).
Glucose-induced insulin secretion is increased in isolated islets from PPAR-deficient mice
To determine whether the hyperinsulinemia after fasting and during the IPGTT in PPARKO mice is a result of altered islet function per se causing increased insulin release, we assessed glucose-induced insulin secretion (GIIS) from isolated perifused islets from PPARKO and WT fasted mice using a step-wise glucose gradient protocol. As shown in Fig. 3, at intermediate and high glucose concentrations, PPARKO islets secreted more insulin than WT islets. At 8.4 mM and 16.7 mM glucose, the insulin responses were 65% and 36% higher from the PPARKO islets, respectively. The DNA content and size distribution of the islets chosen for the perifusion experiments for the two genotypes were identical (not shown). Thus, the results are consistent with the proposition that the hyperinsulinemia of the PPARKO mice is due to islet hypersecretion to glucose.
FIG. 3. Glucose-induced insulin secretion is enhanced in isolated islets from PPARKO mice. Ten islets of each genotype were placed into parallel perifusion chambers. After a 20-min equilibration period of perifusion at 1 ml/min with KRBH buffer containing 2.8 mM glucose and 0.5% BSA, minutely sampling of the effluent perifusate was commenced. The glucose concentration of the perifusate was increased every 12 min in seven steps from 2.8 to 16.7 mM. A–C, Results of separate experiments; D, means ± SEM from the three separate experiments.
FAO is reduced, and glucose oxidation is unaltered in islets from PPARKO mice
To provide support for the hypothesis that PPAR up-regulation of FAO is important in islets for the reduction of insulin secretion during fasting, we measured palmitate oxidation at low (6 mM) glucose in isolated PPARKO and WT islets (Fig. 4A). FAO was 44% lower in PPARKO compared with WT islets, thus confirming that PPAR is involved in the maintenance of FAO in islets. Because glucose and FAO are reciprocally regulated in several tissues (33, 34) and glucose metabolism is essential for GIIS, we also measured islet glucose usage and oxidation. Figure 4, B and C, shows that glucose usage and glucose oxidation rates at 6 and 16 mM glucose were not significantly modified by the absence of PPAR, but there was a trend for a small increase, particularly with respect to glucose usage.
FIG. 4. FAO is reduced but glucose oxidation is unchanged in isolated islets from PPARKO mice. Batches of 50 islets were washed in KRBH buffer and incubated in the presence of radioactive tracers for 2 h (glucose use) or 3.5 h (glucose and FAO). A, Palmitate oxidation; B, glucose use; and C, glucose oxidation were determined from measurements of 14CO2 or 3H2O as described in Materials and Methods. Palmitate oxidation at 100% of WT was 3.8 ± 1.4 nmol/mg prot·h. Means ± SEM of three (glucose use and oxidation) or four (FAO) experiments. *, P < 0.05 vs. WT using Student’s t test.
Fasting increases the expression of PPAR and genes under its transcriptional control in rat islets
According to the hypothesis proposing a role for PPAR in the normal adaptation of the ?-cell to fasting, the expression of PPAR and genes under its transcriptional control involved in FAO should be increased in islets with prolonged fasting. To confirm this, PPAR, CPT1, ACO, and UCP2 mRNA levels were measured by RT-PCR and Southern blotting in islets isolated from fed, 12-h fasted, and 24-h fasted Wistar rats. Islet PPAR mRNA levels were increased by 46% with 12 h and by 60% with 24 h of fasting (Fig. 5). The mitochondrial proteins under its transcriptional control, CPT1 and UCP2, increased by 62% and 82% by 24 h, respectively (Fig. 4). Expression of the peroxisomal FAO enzyme ACO, however, was not significantly altered by 24 h of fasting, although there was a trend toward an increase (Fig. 5).
FIG. 5. Fasting increases the expression of PPAR, CPT1, and UCP2 in rat islets. Islets were isolated from fed, 12-h fasted, and 24-h fasted rats. PPAR, CPT1, ACO, and UCP2 mRNA levels were measured by RT-PCR analysis as described in Materials and Methods. Visualization of PCR products by ethidium bromide staining after electrophoresis in 1% agarose gel, and expression quantified by scanning Southern blot autoradiograms with normalization to ?-actin. Means ± SEM of islets from three separate rats for each nutritional state. Statistical analysis by one-way ANOVA with Bonferroni post hoc testing. *, P < 0.05; **, P < 0.01 vs. fed state.
Discussion
The importance of PPAR and FAO in the adaptation of cardiac muscle (21), liver (11, 19, 21), and kidney (23) to fasting is well established. In this paper, we show that PPAR is also important in the adaptation of pancreatic islets to fasting, and the data support the hypothesis that the mechanism is, at least in part, via up-regulation of islet FAO. PPARKO mice fasted for 5 h developed moderately severe hyperinsulinemic hypoglycemia, consistent with failed fasting-induced suppression of insulin secretion. Isolated islets from these mice had both reduced capacity for FAO and enhanced GIIS, consistent with a role for PPAR in the maintenance of FAO in islets and a link between impaired FAO and insulin hypersecretion. The finding that glucose use and oxidation were not significantly changed in PPAR-deficient islets is also in favor of the PPAR effect on GIIS being via control of FAO. Finally, the rat islet studies confirmed that the expression of PPAR and mitochondrial FAO genes under its transcriptional control increase with fasting. Of note, both PPAR overexpression and activation of PPAR by clofibrate in INS1 cells have previously been shown to cause impaired GIIS in association with enhanced FAO (35). Thus, increased PPAR expression, as occurs in islets during conditions of low glucose (17, 36) and fasting, as shown in this study, inhibits GIIS and the absence of PPAR, as shown in the PPARKO mice, is associated with failure of the normal fasting-induced suppression of insulin secretion.
The evidence that implicates enhancement of islet FAO by PPAR as the most likely mechanism by which PPAR suppresses insulin secretion is now considerable. First, the major impact of PPAR activity in most other tissues is the promotion of FAO, such that its action in the islet is also likely to be related to this effect. Second, in this study we show that PPAR absence is associated with a 44% reduction in islet FAO, and previously it was shown that its overexpression in INS1 ?-cells causes increased FAO (35). Activation of PPAR by clofibrate in INS1 cells (35) and by clofibrate, together with 9-cis-retinoic acid, in islets (30) has also been shown to increase FAO. Third, most studies in which ?-cell FAO has been enhanced or reduced by pharmacological or molecular tools directed at sites other than PPAR are consistent with an effect of enhanced ?-cell FAO to suppress insulin secretion (25, 37, 38, 39). Fourth, islet depletion of fatty acid by adenovirus-leptin gene therapy (40) or fasting plus nicotinic acid (41) causes loss of GIIS, consistent with the hypothesis that insulin secretion can be suppressed by depletion (e.g. after enhanced FAO) of a critical lipid signaling molecule for exocytosis. Fifth, we have recently shown that enhanced FAO resulting from malonyl-CoA decarboxylase overexpression in INS cell lines and islets causes loss of fatty acid augmentation of GIIS in association with reduced ?-cell production of esterified lipid moieties diacylglycerol (25). Finally, isolated islets from 48-h fasted rats show enhanced FAO and impaired GIIS (42). 2-Bromostearate, which inhibits FAO by reducing CPT-1 activity, acutely restored GIIS in these islets (42).
The higher plasma glucagon level in PPARKO mice during fasting may simply be a counterregulatory response to the hypoglycemia, but it is inadequate to prevent the hypoglycemia. The higher glucagon level in PPARKO fed mice and the higher glucagon secretion in isolated islets, however, is more difficult to explain, but it may reflect compensatory changes in the -cell to a chronic tendency toward hypoglycemia. Alternatively, a more direct effect of PPARKO in the -cells causing glucagon hypersecretion could be possible. The lower glucagon content of PPARKO islets, in association with elevated plasma glucagon levels and mildly reduced islet glucagon mRNA expression, is consistent with the occurrence of some degranulation of the -cells due to glucagon hypersecretion without compensation by increased gene expression. Glucagon is not normally insulinotropic in situations of low glucose (43), such that it is unlikely that the elevated glucagon levels cause the fasting hyperinsulinemia of the PPARKO mice via a paracrine effect. However, it is always possible that altered lipid partitioning in the PPARKO islets has lowered the glucose set point by which glucagon is insulinotropic.
Fasting-induced hypoglycemia has often been reported in PPARKO mice, but hyperinsulinemic hypoglycemia has not been consistently reported. However, consistent with our finding, a higher ratio of insulin to glucose was reported in 24-h fasted PPARKO compared with WT mice (19), although fasting-induced hypoglycemia was not observed probably due to blood sampling during anesthesia. A nonsignificant trend for higher insulin levels, despite lower glucose levels, was found in 17-h fasted PPARKO mice (22). In other studies in which hypoglycemia has been documented in PPARKO mice (11, 20, 21), it is unfortunate that limited (20) or no (11, 21) corresponding insulin levels were reported.
It is apparent that the islet contribution to the nonketotic hypoglycemia in subjects with the various in-born errors of metabolism in FAO and in animal models of impaired FAO might differ depending on the site of the defect in FAO. Hyperinsulinemic hypoglycemia occurs in SCHAD disorders (5, 6) and in PPARKO mice (present study). It is not reported to occur when the FAO defect is secondary to defects in medium-, long-, and very-long-chain acyl-CoA dehydrogenase deficiencies (6) or deficiencies in the liver isoform of CPT-1 (8) or malonyl-CoA decarboxylase (7). However, circulating insulin levels were, in most instances, not reported in these studies. Thus, it is difficult to ascertain from the literature the extent to which inadequate suppression of insulin secretion has been excluded in the subjects with several of these rare conditions. A possible explanation for lack of islet phenotypes in some of these conditions could relate to secondary compensatory changes. For example, in very-long-chain acyl-CoA dehydrogenase-deficient mice (44), expression of acyl-CoA synthase as measured by Western blot is markedly reduced in 2-month-old mice. If acyl-CoA synthase expression is similarly reduced in islets of subjects with these more severe FAO disorders, abnormal ?-cell accumulation of LC-CoA with the stimulation of inappropriate insulin secretion would be prevented.
In conclusion, this work supports a role for increased FAO in the normal pancreatic islet response to fasting and starvation, and that PPAR is an essential transcription factor involved in this process. The increased islet FAO during fasting reduces insulin release, most probably by depletion of a lipid-signaling molecule involved in exocytosis, such that fasting hypoglycemia is avoided.
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