1 Garvan Institute of Medical Research, St. Vincents Hospital, Sydney, Australia
2 Department of Biochemistry, University of Sydney, Sydney, Australia
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ABSTRACT |
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INTRODUCTION |
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Although ongoing hyperglycemia is undoubtedly a major contributor to ß-cell decompensation, it is apparent that chronic elevations in circulating fatty acids (FAs) also accompany the progression to type 2 diabetes (3,4,5). Since FAs themselves stimulate insulin secretion, a key role in secretory compensation has been ascribed to their elevation (4,6). Carried to extremes, however, or in association with an underlying genetic predisposition, prolonged exposure to FAs could lead to "overstimulation" or "ß-cell exhaustion" (5). Moreover, chronic exposure of ß-cells to elevated FAs, both in vivo and in vitro, leads to an elevated basal secretion and a blunted response to glucose, both of which defects are reminiscent of the diabetic state (3,5,7). Furthermore, basal hypersecretion in some animal models of diabetes is ablated by reversal of the alterations in ß-cell lipid metabolism that also characterize those models (3,8). Finally, elevated FAs might also contribute to the reduction in ß-cell mass that accompanies diabetes (9). Although initial studies of ß-cell dysfunction due to chronic lipid exposure focused on metabolic alterations (5,7), more recent emphasis has been on changes in ß-cell gene expression. Around a dozen genes have now been identified as being lipid-regulated in ß-cells (1014).
At present, however, there is no comprehensive documentation of the global alterations in gene expression that would occur in lipid-treated ß-cells. In contrast, a host of glucose-regulated genes have recently been identified by transcript profiling of ß-cells using high-density oligonucleotide arrays (15). That study made use of the highly differentiated and glucose-responsive cell line MIN6 as a source of homogenous ß-cells (16). Our present aim was to undertake a comparable analysis of lipid-regulated genes. Transcript profiling of expressed genes revealed novel and multiple effects of the major circulating FAs oleate and palmitate, including induction of pro-inflammatory genes and, at least with palmitate, a partial ß-cell de-differentiation. In addition, a palmitate-induced upregulation of genes encoding a number of exocytotic and signaling enzymes suggested a previously unappreciated sensitization to noncarbohydrate stimuli, which was confirmed functionally. Most importantly, palmitate exposure was sufficient to reproduce many changes in ß-cell gene expression that have been previously shown to accompany the progression of diabetes.
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RESEARCH DESIGN AND METHODS |
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Cell culture and treatment.
MIN6 cells were passaged in 75-cm2 flasks with 20 ml Dulbeccos modified Eagles medium (DMEM) containing 25 mmol/l glucose, 24 mmol/l NaHCO3, 10 mmol/l HEPES, 10% (vol/vol) FCS, 50 IU/ml penicillin, and 50 µg/ml streptomycin. Cells were seeded at 3 x 105 per well in 0.5 ml DMEM in a 24-well dish for secretory experiments, and they were seeded at 5 x 106 per 25-cm flask for transcript profiling or RT-PCR. At 48 h before the experiment (24 h after seeding), the medium was replaced with DMEM (as above but with 6 mmol/l glucose) and supplemented with either BSA alone or BSA coupled to palmitate or oleate. In some experiments, diazoxide (0.1 mmol/l) was added to maintain cellular insulin contents during culture. For FA coupling, 18.4% BSA was dissolved in DMEM (25 mmol/l glucose) by gentle agitation at room temperature for 3 h. Palmitate or oleate (8 mmol/l) was then added as Na+ salts, and the mixture was agitated overnight at 37°C. The pH was then adjusted to 7.4, and then, after sterile filtering, FA concentrations were verified using a commercial kit, and aliquots were stored at -20°C. Similar couplings were made using glucose-free modified Krebs-Ringer bicarbonate (KRB) buffer containing 5 mmol/l NaHCO3, 1 mmol/l CaCl2, 0.5% (wt/vol) BSA, and 10 mmol/l HEPES (pH 7.4) instead of DMEM. This procedure generated BSA-coupled FA in a molar ratio of 3:1 (generally, 0.4 mmol/l to 0.92% BSA, final).
Secretory assays.
Cultured cells were washed once (twice for diazoxide-treated cells) in modified KRB buffer (see above) containing 2.8 mmol/l glucose, and then they were preincubated for a further 30 min in 0.5 ml of the same medium at 37°C. This buffer was then replaced with 0.5 ml of prewarmed KRB containing other additions as indicated, and it was incubated for a further 60 min at 37°C. An aliquot was then removed for analysis of insulin content by radioimmunoassay. The cell monolayers were washed twice in PBS and then extracted for measurement of total insulin content by lysis in 0.5 ml H2O per well, followed by sonication.
Transcript profiling.
RNA (2540 µg) was isolated using RNeasy columns, and total and double-stranded cDNA was synthesized using the Superscript Choice system with an high-performance liquid chromatographypurified T7-(dT)24 primer. This was converted to biotin-labeled, double-stranded cRNA using a high yield RNA transcript labeling kit according to the manufacturers instructions. After purification on RNeasy columns, the cRNA was fragmented at 94°C for 35 min in 100 mmol/l potassium acetate, 30 mmol/l magnesium acetate, and 40 mmol/l Tris-acetate, pH 8.1. After quality verification using Test2 arrays, the fragmented cRNA was hybridized to oligonucleotide microarrays, according to the Affymetrix instructions, in 1 mol/l NaCl, 20 mmol/l EDTA, 0.01% Tween, 100 mmol/l 2-[N-morphalino]ethanesulfonic acid (MES), pH 6.6, for 16 h at 45°C. Mouse genome U74A version 2 arrays were used, except for the first palmitiate experiment, which was conducted with the original version U74A arrays, now known to contain some faulty probe sets. The arrays were then washed on an Affymetrix fluidics station and stained with streptavidin-phycoerythrin according to the manufacturers instructions. Probe sets were visualized on a Hewlett-Packard GeneArray scanner and then quantified for intensity and comparison between experimental groups using Affymetrix Genechip software. This raw data were further analyzed using Phenzomix, a data management tool developed in-house for use with these microarrays. Based on FileMaker Pro 5.3, this tool facilitated cluster formation based on both inputs from the arrays (mRNA intensity, fold change, and present/absent calls) and known functionality. For the current study, we assembled clusters of palmitate- or oleate-regulated genes satisfying the following criteria: 1) genes were called as "changed" by the Genechip software and showed alterations of at least 1.9-fold by lipid treatment, 2) those called "increased" or "marginally increased" were also described as "present" in the experimental group, and 3) those called "decreased" or "marginally decreased" were also "present" in the control group. Gene clusters satisfying these conditions in two independent experiments for each lipid were saved and combined in multiple-comparison statements, using Phenzomix to define the smaller dataset of genes coregulated in both experiments. These genes were classified using Swiss Prot and TrEMBL databases.
Real-time RT-PCR.
Total RNA was prepared using RNeasy or Trizol, and 0.2 µg per reaction was converted to cDNA using a Superscript cDNA kit according to the manufacturers instructions. Real-time PCR was undertaken on a LightCycler (Roche) with a commercial kit containing 10 mmol/l MgCl2 and 1 µl cDNA in a PCR of 4555 cycles (annealing temperature of 55°C for all primers). Standards for each transcript were prepared using appropriate primers in a conventional PCR and purified using Wizard PCR preps. For experimental analyses, PCR products were quantified fluorometrically using SYBR-Green II. Concentrations were then calculated from standard curves as copies per microliter. These PCR products of known concentration for each primer pair were then used in the corresponding LightCycler PCR to provide standards for these reactions. ß-Actin expression was calculated in parallel as a control (this was not altered by lipid pretreatment). The following primers were used (forward and reverse): AAT CCT GTG GCA TCC ATG AAA C and CGC AGC TCA GTA ACA GTC CG (ß-actin); TGG CAA GGA AGG TGA CAA GCA C and CGG TCC CAT TTT ATT TCA GAG C (calcyclin); AAC CTG TAT GGG ATT TCG GGG and TCC AGA GGG TCA AAG CAA ACC (fructose 1,6-bisphosphatase [FBPase]); TCC TTT GTC GGC AAT CCC TAC and CCC TCC TTG ACT TTC TCT TCA TTC C (phosphatidic acid phosphohydrolase 2 [PAP2]); and TGG ATG AAC GGG AGC AGA TG and GGT TTT GTT GGA GTC AGC CTT CTC (SNAP25).
Data analysis.
Unless otherwise indicated, results are expressed as the means ± SE, with the number of observations in brackets. Statistical significance was determined with Students t test.
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RESULTS |
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There were also alterations in 15 genes controlling transcription; 4 of these genes were decreased by palmitate and are probable transcription factors. One of these, Nkx6.2, is closely related Nkx6.1, a transcription factor specific to differentiated ß-cells (24). Conversely, of the 11 transcriptional genes showing increased expression due to palmitate, as many as 7 potentially act as repressors, including ATF3 (25), Id1 (26), mTGIF (27), and EGR-binding protein 1 (28). CCAAT/enhancer binding protein-ß (C/EBPß) can either inhibit or stimulate transcription (29,30). Finally, the upregulated CREM family member is probably the inducible cAMP early repressor (ICER), the sole member documented in ß-cells (31). Of these potential repressors, only Id-1 was co-induced by oleate. Both lipids upregulated the transcriptional factors C/EBP and c-fos. They also increased expression of two genes that would be expected to stimulate cell growth: the IGF-1 receptor and amphiregulin, a close relative of ß-cellulin, which binds to the EGF family of receptors. Oleate and palmitate exerted conflicting effects on genes controlling the cell cycle, with upregulation of p21 and p15 by palmitate expected to favor growth arrest, as opposed to downregulation of p57 by oleate, which would facilitate cell cycle progression.
An unexpected category of regulated genes was that mediating inflammatory responses. Most of these genes were absent in control cells and markedly upregulated by both lipids. Three genes (MCP-1, stromal cell-derived factor 1, and GRO1 oncogene) encode chemokines, whereas pentraxins are cell surface proteins potentially implicated in proinflammatory responses. Lipocalin-2 is a secreted factor promoting leukocyte apoptosis (32). Serum amyloid A3 is a secreted lipoprotein whose induction is indicative of the acute phase response, or activation of the innate immune system (33). Interestingly, other features of this response include upregulation of some antiproteolytic enzymes and cell matrix proteins (33), which also occurred in ß-cells exposed to palmitate.
Many genes encoding signal transduction enzymes were also lipid-regulated, including categories involved in protein phosphorylation and G-protein function. The observed upregulation of phospholipase D1 (PLD1) and PAP is of potential interest because these enzymes catalyze the two-step formation of diacylglycerol from precursor phospholipids. This implies increased activity of the diacylglycerol/protein kinase C (PKC) pathway in lipid-treated cells. There was also a (generally) increased expression of genes for Ca2+ and phospholipid-binding proteins. One of these, calcyclin, whose expression was most sensitive to palmitate exposure, has been directly implicated in the control of insulin secretion (34,35). SNAP25, an essential component of the exocytotic machinery (36), was also increased in the lipid-treated cells. Increased expression of calcyclin and SNAP25 suggests that the distal exocytotic pathway might be upregulated in cells pretreated with palmitate. Such a possibility would also be consistent with enhancement of genes relating to protein processing and export, such as chaperone proteins, disulphide isomerases, and an endoplasmic reticulum translocon protein. Indeed, there was also a general upregulation of genes for secreted products and hormones. The increased expression of islet hormones, such as pancreatic polypeptide and glucagon, which are not normally found in ß-cells, might point to a relative loss of cellular differentiation induced by FA exposure.
To address the prediction that lipid pretreatment would lead to a sensitization of distal secretory pathways, functional responses to a variety of nonnutrient secretagogues were next examined. As shown in Fig. 3, pretreatment with palmitate for 48 h markedly enhanced secretion due to agents acting at the level of activation of PKC (the phorbol ester 12-O-tetradecanoylphorbol-13-acetate [TPA]), the cAMP-dependent protein kinase (forskolin), and Ca2+-dependent secretion elicited with a depolarizing concentration of KCl. In cells correspondingly pretreated with oleate, only TPA-induced secretion was augmented to an extent equivalent to that seen with chronic palmitate treatment, although the forskolin response was very slightly sensitized relative to control. Ca2+-dependent secretion was not sensitized at all by oleate pretreatment. These observations would be consistent with the finding that whereas oleate and palmitate both elevated PLD1, expression of SNAP25 and calcyclin were more markedly upregulated by palmitate.
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DISCUSSION |
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Should this modest augmentation of glucose-induced insulin secretion in palmitate-treated cells be viewed as a sensitization or, alternatively, as a desensitization if the maximal response, is calculated as the diminished fold increase over an elevated baseline? In our view, this question is no longer especially relevant, since it is based on a rather artificial protocol in which ß-cells are pretreated with FAs and then challenged with glucose in the absence of FAs. In contrast, our studies suggest that under physiological conditions of constant exposure to FAs, the degree of resultant sensitization to the combined stimuli would far outweigh any small effect seen on the response to glucose alone. Such a conclusion would be consistent with the findings of one earlier study, in which the effect of a combined challenge of glucose plus FA was enhanced in lipid-pretreated islets (17). However, the experimental design of that study did not allow for a full appreciation of the multiple influences at work. On the other hand, oleate-pretreated INS-1 cells did not show an enhanced acute response to a subsequent challenge with oleate (20), but this might represent a difference in the potency of palmitate versus oleate in the sensitization process. Moreover, our findings are supported by in vivo evidence that secretory responses to nonglucose stimuli were upregulated in fat-fed mice and in several animal models of diabetes (3739). However, it is uncertain whether these enhanced responses, and hence the palmitate-induced sensitization described here, are ultimately beneficial in terms of compensatory insulin secretion, or whether they are implicated in a pathophysiological overstimulation of the ß-cell, and hence progression to diabetes. In the latter context, it needs to be stressed that there was a depletion of intracellular insulin content in MIN6 cells pretreated with either oleate or palmitate. This is consistent with previous studies using islets, and the knowledge that lipids are unable to stimulate transcriptional or translational pathways for insulin synthesis to compensate for chronically stimulated secretion (7,10,17).
The further utility of the MIN6 cell model for transcript profiling is witnessed by the fact that four genes, GLUT2, carnitine palmitoyl transferase 1 (CPT1), c-fos, and PC, previously known to be lipid-regulated in ß-cells, were also detected in our screen (10,11,13,40). Similarly, expression of the ß-cellspecific transcription factor pancreatic duodenal homeobox-1 (PDX-1), known to be downregulated in islets by palmitate treatment (10), was also inhibited in the MIN6 cells by 1.4 ± 0.1-fold. Although this decrease was too small to be included in Table 1, it is likely to be functionally significant. Many other genes known to be altered during ß-cell dysfunction were also shown here to be regulated by palmitate (see below). However, we failed to detect changes in some other ß-cell genes previously reported to be lipid-regulated, including: acetyl CoA carboxylase (12) and nur-77 (13) (not present on the microarrays), glucokinase (10), inducible NO synthase (9), and uncoupling protein 2 (14), as well as insulin (10). The latter was expressed so highly in MIN6 cells that it saturated even the "mismatch" probe sets on the array and was therefore paradoxically called as absent. Glucokinase is downregulated by only 25% by palmitate in islets (15), which was probably too a small a difference to be detected here. Uncoupling protein 2 was well expressed in MIN6 cells, but its levels were unaltered by lipid pretreatment (not shown), in contrast to a previous study using INS-1 cells (14). The discrepancy is possibly due to differences in treatment times (18 vs. 48 h here) and the fact that we used a much lower effective FA concentration (molar FA-to-BSA ratio of 3:1, in contrast to 6:1 in the previous study). We were also unable to demonstrate upregulation of NO synthase by lipid, but this has not been previously shown using homogenous ß-cells. Macrophages present in the islet might be important for induction of this gene in situ.
Our results provide novel insights into the effects of chronic FA exposure on ß-cell function in terms of the distal sensitization (described here) as well as the enhancement of basal secretion and disruption of glucose-dependent stimulus-secretion coupling that have been previously reported. The increased expression of SNAP25, calcyclin, and PLD in palmitate-treated cells is consistent with the observed sensitization to distally acting stimuli, especially those activating Ca2+-dependent secretion. SNAP25 is essential for targeting insulin secretory granules to the sites of exocytosis, and inhibition of its function blocks insulin secretion (36). Similarly, calcyclin is also required for Ca2+-dependent insulin secretion, and, crucially, its overexpression is sufficient for upregulation of the secretory response (34,35). Enhanced PLD signaling should also be stimulatory, either by activation of PKC or at the level of vesicular trafficking. Most importantly, the relative capacities of palmitate versus oleate to alter these genes is consistent with their observed effects on distal secretory pathways. Thus, oleate pretreatment only sensitized the MIN6 cells to PKC-dependent secretion, in keeping with the accompanying increases in PLD1 expression, relatively modest effects on calcyclin, and no effect at all on SNAP25. However, causative relationships between these alterations in gene expression and secretory function will need to be substantiated in future experiments.
Upregulation of SNAP25, calcyclin, and PLD1 might also contribute to the enhancement of basal secretion that accompanies lipid pretreatment of ß-cells. However, the observed decrease in the expression of short chain L-3-hydroxyacyl CoA dehydrogenase (SCHAD) is also of particular interest because an inactivating mutation in that enzyme has very recently been causally associated with basal insulin hypersecretion (41). On the other hand, downregulation of GLUT2, PC, and mGPDH might be expected to contribute specifically to the decoupling of glucose metabolism from proximal signaling pathways (2123). Moreover, the unexpected induction of two forms of FBPase, by both palmitate and oleate, might be particularly relevant to the loss of glucose sensitivity because these enzymes could divert glycolytic flux into alternative metabolic pathways. Indeed, upregulation of one of these branch pathways, the HBP, is sufficient to inhibit glucose-stimulated insulin release (42). Other evidence implicates the HBP in the ß-cell apoptosis that accompanies prolonged exposure to high glucose concentrations (43). It is noteworthy that another enzyme in the HBP pathway, GPAT, was also upregulated in palmitate-pretreated MIN6 cells. Interpreting these earlier observations in the light of our new findings, it is conceivable that augmented flux through the HBP could contribute both to the secretory defects and the apoptotic response that accompany chronic ß-cell exposure to lipids.
Importantly, our results also demonstrate that palmitate pretreatment is sufficient to alter ß-cell gene expression in a similar fashion to that occurring in models of type 2 diabetes. The coregulated genes include GLUT2 (3,44), mGPDH (44,45), LDH (44), PC (45), SNAP25 (46,47), p21 (48), ATF3 (25), ICER (49), and C/EBPß (50). The fact that highly abundant ß-cell genes like GLUT2, PC, and mGPDH were downregulated after lipid treatment, whereas poorly expressed ones like LDH and FBPase (as well as those for hormones other than insulin) were induced, is suggestive of a loss of ß-cell differentiation. Upregulation of C/EBPß might be important in this context (50,51) because this also occurs with supraphysiological glucose levels in vitro and is causally associated with decreased expression of PDX-1 (30). Indeed, a primary role for C/EBPß in de-differentiating ß-cells has been postulated as an underlying cause of type 2 diabetes (51). Our results would be consistent with this hypothesis, but they suggest that other transcriptional regulators are also involved. ATF3 is induced in ß-cells as a result of oxidative stress, and its ectopic overexpression leads to abnormal islet development (25). Likewise, induction of ICER correlates with transcriptional repression of the insulin gene in vitro (31), and expression of ICER is increased in the islets of diabetic GK rats relative to nondiabetic controls (49).
Another major finding was the upregulation by both lipids of proinflammatory genes encoding chemokines and mediators of the acute phase response. Although the latter is known to be triggered by cellular stress, its induction in ß-cells by the physiologically relevant FA concentrations used here is surprising. On the other hand, type 2 diabetes is associated with increases in circulating markers of the acute phase response, and a causative role of a hypersensitive innate immune system in the progression of the disease has been hypothesized (52). According to this hypothesis, however, ß-cells are passive victims of this response and not, as our results might now suggest, themselves playing a more active role. Whether this, along with chemokine release, could form part of self-destructive loop will obviously require further investigation. However, the fact that binding elements for C/EBPß are found in the promoters of many acute-phase genes (29) suggests, at the very least, that ß-cell dysfunction and the acute phase response share a common etiology.
In conclusion, we have demonstrated that palmitate and oleate induce profound, but differing, alterations in ß-cell gene expression. Our results suggest novel explanations, such as induction of FBPase and upregulation of the HBP, for the secretory defects that also occur in lipid-treated islets. Moreover, palmitate pretreatment was sufficient to reproduce many of the alterations in gene expression previously documented in models of ß-cell dysfunction. This is in marked contrast to those genes regulated by glucose, as assessed in a comparable study (25). However, palmitate pretreatment also increased expression of genes controlling distal secretory processes and was shown functionally to sensitize ß-cells to secretory stimuli. Our study highlights the contribution of regulated gene expression to the phenotypic alterations induced in ß-cells after FA exposure, but our results additionally demonstrate that these phenotypic alterations are multiple and complex. The balance between them is likely to be influenced by genetic factors. Allelic variation in some of the lipid-regulated genes that we have documented here might therefore predispose to type 2 diabetes.
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ACKNOWLEDGMENTS |
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We thank Andrew Sutherland for help with the array hybridization and Dr. Naras Lapsys and Megan Lim-Fraser for technical advice. We also thank Drs. Christopher Ormandy, Lee Carpenter, and Christopher Mitchell for critically reading the manuscript. The MIN6 cells were a generous gift from Dr. Jun-ichi Miyazaki.
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FOOTNOTES |
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Received for publication 21 September 2001 and accepted in revised form 8 January 2002.
C/EBP, CCAAT/enhancer binding protein; DMEM, Dulbeccos modified Eagles medium; FA, fatty acid; FBPase, fructose 1,6-bisphosphatase; GPAT, glucosamine-phosphate N-acetyl transferase; HBP, hexosamine biosynthesis pathway; ICER, inducible cAMP early repressor; KRB, Krebs-Ringer bicarbonate; LDH, lactate dehydrogenase; mGPDH, mitochondrial glycerol 3-phosphate dehydrogenase; PAP2, phosphatidic acid phosphohydrolase 2; PC, pyruvate carboxylase; PDX-1, pancreatic duodenal homeobox-1; PKC, protein kinase C; PLD, phospholipase D; SNAP25, 25-kDa synaptosomal-associated protein; TPA, 12-O-tetradecanoylphorbol-13-acetate.
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REFERENCES |
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