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Expression Profiling of Palmitate Expression Profiling of Palmitate- and Oleate-Regulated Genes Provides Novel Insights Into the Effects of Chronic Lipid Exposure on Pancreatic ␤-Cell Function Anna K. Busch,1 Damien Cordery,1 Gareth S. Denyer,2 and Trevor J. Biden1 Chronic lipid exposure is implicated in ␤-cell dysfunc- tion in type 2 diabetes. We therefore used oligonucleo- tide arrays to define global alterations in gene irculating nutrients regulate the function of pan- expression in MIN6 cells after 48-h pretreatment with creatic ␤-cells at multiple levels: they acutely oleate or palmitate. Altogether, 126 genes were altered stimulate insulin secretion, modulate insulin >1.9-fold by palmitate, 62 by oleate, and 46 by both biosynthesis, and, over the longer term, bring lipids. Importantly, nine of the palmitate-regulated C about adaptive changes in gene expression (1). Dysregu- genes are known to be correspondingly changed in ␤ lation of any of these aspects of the nutrient response models of type 2 diabetes. A tendency toward -cell ␤ de-differentiation was also apparent with palmitate: and/or alterations in -cell differentiation and survival are pyruvate carboxylase and mitochondrial glycerol potentially implicated in the onset of type 2 diabetes. This 3-phosphate dehydrogenase were downregulated, disease is associated with peripheral insulin resistance but whereas lactate dehydrogenase and fructose 1,6- only develops in conjunction with a failure of ␤-cell bisphosphatases were induced. Increases in the latter compensation, which otherwise counteracts the insulin (also seen with oleate), along with glucosamine-phos- resistance (2–5). Type 2 diabetic patients display reduc- phate N-acetyl transferase, imply upregulation of the tions in ␤-cell mass as compared with insulin-resistant hexosamine biosynthesis pathway in palmitate-treated cells. However, palmitate also increased expression of nondiabetic individuals, as well as a number of clearly calcyclin and 25-kDa synaptosomal-associated protein defined secretory abnormalities. The latter include both an (SNAP25), which control distal secretory processes. elevated fasting secretion and a selective loss of sensitivity Consistent with these findings, secretory responses to to glucose (2). noncarbohydrate stimuli, especially palmitate itself, Although ongoing hyperglycemia is undoubtedly a major were upregulated in palmitate-treated cells (much less contributor to ␤-cell decompensation, it is apparent that so with oleate). Indeed, glucose-stimulated secretion chronic elevations in circulating fatty acids (FAs) also was slightly sensitized by chronic palmitate exposure accompany the progression to type 2 diabetes (3–5). Since but inhibited by oleate treatment, whereas both lipids enhanced basal secretion. Oleate and palmitate also FAs themselves stimulate insulin secretion, a key role in induced expression of chemokines (MCP-1 and GRO1 secretory compensation has been ascribed to their eleva- oncogene) and genes of the acute phase response (se- tion (4,6). Carried to extremes, however, or in association rum amyloid A3). Increases in transcriptional modula- with an underlying genetic predisposition, prolonged ex- tors such as ATF3, CCAAT/enhancer binding protein-␤ posure to FAs could lead to “overstimulation” or “␤-cell (C/EBP␤), C/EBP␦, and c-fos were also seen. The results exhaustion” (5). Moreover, chronic exposure of ␤-cells to highlight links between regulated gene expression and elevated FAs, both in vivo and in vitro, leads to an elevated phenotypic alterations in palmitate versus oleate-pre- basal secretion and a blunted response to glucose, both of treated ␤-cells. Diabetes 51:977–987, 2002 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 ␤-cell lipid metabolism that also characterize those models (3,8). Finally, elevated FAs might also contribute to the reduc- From the 1Garvan Institute of Medical Research, St. Vincent’s Hospital, Sydney, Australia; and the 2Department of Biochemistry, University of Sydney, tion in ␤-cell mass that accompanies diabetes (9). Al- Sydney, Australia. though initial studies of ␤-cell dysfunction due to chronic Address correspondence and reprint requests to Dr. Trevor Biden, Cell Signalling Group, Garvan Institute of Medical Research, 384 Victoria St., lipid exposure focused on metabolic alterations (5,7), Darlinghurst, Sydney 2010, Australia. Email: [email protected]. more recent emphasis has been on changes in ␤-cell gene Received for publication 21 September 2001 and accepted in revised form 8 expression. Around a dozen genes have now been identi- January 2002. C/EBP, CCAAT/enhancer binding protein; DMEM, Dulbecco’s modified fied as being lipid-regulated in ␤-cells (10–14). Eagle’s medium; FA, fatty acid; FBPase, fructose 1,6-bisphosphatase; GPAT, At present, however, there is no comprehensive docu- glucosamine-phosphate N-acetyl transferase; HBP, hexosamine biosynthesis pathway; ICER, inducible cAMP early repressor; KRB, Krebs-Ringer bicarbon- mentation of the global alterations in gene expression that ate; LDH, lactate dehydrogenase; mGPDH, mitochondrial glycerol 3-phos- would occur in lipid-treated ␤-cells. In contrast, a host of phate dehydrogenase; PAP2, phosphatidic acid phosphohydrolase 2; PC, glucose-regulated genes have recently been identified by pyruvate carboxylase; PDX-1, pancreatic duodenal homeobox-1; PKC, protein ␤ kinase C; PLD, phospholipase D; SNAP25, 25-kDa synaptosomal-associated transcript profiling of -cells using high-density oligonu- protein; TPA, 12-O-tetradecanoylphorbol-13-acetate. cleotide arrays (15). That study made use of the highly DIABETES, VOL. 51, APRIL 2002 977 LIPID-REGULATED ␤-CELL GENES differentiated and glucose-responsive cell line MIN6 as a the original version U74A arrays, now known to contain some faulty probe source of homogenous ␤-cells (16). Our present aim was sets. The arrays were then washed on an Affymetrix fluidics station and stained with streptavidin-phycoerythrin according to the manufacturer’s in- to undertake a comparable analysis of lipid-regulated structions. Probe sets were visualized on a Hewlett-Packard GeneArray genes. Transcript profiling of expressed genes revealed scanner and then quantified for intensity and comparison between experimen- novel and multiple effects of the major circulating FAs tal groups using Affymetrix Genechip software. This raw data were further oleate and palmitate, including induction of pro-inflamma- analyzed using Phenzomix, a data management tool developed in-house for ␤ use with these microarrays. Based on FileMaker Pro 5.3, this tool facilitated tory genes and, at least with palmitate, a partial -cell cluster formation based on both inputs from the arrays (mRNA intensity, fold de-differentiation. In addition, a palmitate-induced upregu- change, and present/absent calls) and known functionality. For the current lation of genes encoding a number of exocytotic and study, we assembled clusters of palmitate- or oleate-regulated genes satisfying signaling enzymes suggested a previously unappreciated the following criteria: 1) genes were called as “changed” by the Genechip sensitization to noncarbohydrate stimuli, which was con- software and showed alterations of at least 1.9-fold by lipid treatment, 2) those called “increased” or “marginally increased” were also described as firmed functionally. Most importantly, palmitate exposure “present” in the experimental group, and 3) those called “decreased” or was sufficient to reproduce many changes in ␤-cell gene “marginally decreased” were also “present” in the control group. Gene clusters expression that have been previously shown to accom- satisfying these conditions in two independent experiments for each lipid pany the progression of diabetes. were saved and combined in multiple-comparison statements, using Phenzo- mix 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 RESEARCH DESIGN AND METHODS ␮g per reaction was converted to cDNA using a Superscript cDNA kit Materials. Culture media, TRIzol, and the Superscript Choice system were according to the manufacturer’s instructions. Real-time PCR was undertaken from Gibco BRL (Gaithersburg, MD). Falcon plasticware was obtained from on a LightCycler (Roche) with a commercial kit containing 10 mmol/l MgCl2 Becton Dickinson (Franklin Lakes, NJ). The molecular biological reagents and 1 ␮l cDNA in a PCR of 45–55 cycles (annealing temperature of 55°C for all used (and their sources) were: RNeasy mini kits (Qiagen, Melbourne, Austra- primers). Standards for each transcript were prepared using appropriate lia), Wizard PCR preps (Promega, Madison, WI), High Yield RNA transcript primers in a conventional PCR and purified using Wizard PCR preps. For labeling kits (Enzo Biochem, New York), Faststart DNA Master kit and experimental analyses, PCR products were quantified fluorometrically using SYBR-Green II (Roche Diagnostics, Castle Hill, Australia). U74A murine SYBR-Green II. Concentrations were then calculated from standard curves as genome microarrays and T2 test arrays were purchased from Affymetrix copies per microliter. These PCR products of known concentration for each (Santa Clara, CA). Kits for radioimmunoassay of rat insulin were obtained primer pair were then used in the corresponding LightCycler PCR to provide from Linco
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