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Research Resource: Nuclear Hormone Receptor Expression in the Endocrine Pancreas

Departments of Physiology (J.-C.C., J.-Y.C., J.J.R.) and Internal Medicine (J.J.R.), University of Texas Southwestern Medical Center, Dallas, Texas 75390-9077; Department of Internal Medicine, Division of Endocrinology and Metabolism (J.C.G), University of Virginia Health System, Charlottesville, Virginia 22903; and Department of Pediatrics and the Wells Center for Pediatric Research (R.G.M.), Indiana University School of Medicine, Indianapolis, Indiana 46202

Address all correspondence and requests for reprints to: Joyce J. Repa, Ph.D., Departments of Physiology and Internal Medicine, Touchstone Center for Diabetes Research, Division for Hypothalamic Research-Y6.322C, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390-9077. E-mail: joyce.repa{at}utsouthwestern.edu.

ABSTRACT

The endocrine pancreas comprises the islets of Langerhans, tiny clusters of cells that contribute only about 2% to the total pancreas mass. However, this little endocrine organ plays a critical role in maintaining glucose homeostasis by the regulated secretion of insulin (by β-cells) and glucagon (by -cells). The rapid increase in the incidence of diabetes worldwide has spurred renewed interest in islet cell biology. Some of the most widely prescribed oral drugs for treating type 2 diabetes include agents that bind and activate the nuclear hormone receptor, peroxisome proliferator-activated receptor-. As a first step in addressing potential roles of peroxisome proliferator-activated receptor- and other nuclear hormone receptors (NHRs) in the biology of the endocrine pancreas, we have used quantitative real-time PCR to profile the expression of all 49 members of the mouse NHR superfamily in primary islets, and cell lines that represent -cells (TC1) and β-cells (βTC6 and MIN6). In summary, 19 NHR members were highly expressed in both - and β-cell lines, 13 receptors showed predominant expression (at least an 8-fold difference) in - vs. β-cell lines, and 10 NHRs were not expressed in the endocrine pancreas. In addition we evaluated the relative expression of these transcription factors during hyperglycemia and found that 16 NHRs showed significantly altered mRNA levels in mouse islets. A similar survey was conducted in primary human islets to reveal several significant differences in NHR expression between mouse and man. These data identify potential therapeutic targets in the endocrine pancreas for the treatment of diabetes mellitus.

THE INCIDENCE OF diabetes mellitus is increasing at an alarming rate. The World Health Organization estimates that more than 180 million people worldwide have diabetes. In the United States alone, the American Diabetes Association reports that approximately 7% of the population has diabetes. Some of the most widely prescribed oral drugs for treating type 2 diabetes include agents that bind and activate the peroxisome proliferator-activated receptor- (PPAR) (1). PPAR is one of the 48 members of the human nuclear hormone receptor (NHR) superfamily of transcription factors (mice have 49 members). In animal studies, additional NHR ligands have been shown to have potent serum glucose-lowering effects. Synthetic agonists for the retinoid X receptor [RXR (2)], liver X receptor [LXR (3, 4)], PPAR (5), and farnesoid X receptor [FXR (6)] all have been reported to lower serum glucose levels in rodent models of diabetes.

Although many of the positive effects of these NHR agonists have been attributed to improved insulin sensitivity and glucose clearance by peripheral tissues, there has been increased interest in their potential role in the endocrine pancreas: can they improve glucose responsiveness, insulin secretion or survival of β-cells, or alter glucagon production and secretion by -cells? As a first step in answering these questions, we have performed a comprehensive survey to identify the complement of NHR expressed in mouse primary islets and cell lines and in human islets of Langerhans.

RESULTS

The expression of NHR in mouse islets and cell lines recognized as models for cells of the endocrine pancreas was determined by quantitative real-time PCR (qPCR). This technique is extremely sensitive, can utilize small amounts of RNA (thus allowing measurement with RNA from islets), provides a quantitative assessment of RNA species across a large linear range of values, and can analyze sufficient sample numbers to depict RNA levels as an average with biological variance (7). In all qPCR analyses that were performed for our NHR survey, four tissues from young adult, male A129/SvJ mice were included as positive controls. NHR play a critical role in metabolism (adipose, liver), reproduction (testis), and nervous system (whole brain), and among these four tissues all 49 mouse NHRs are expressed at appreciable levels (8). Cyclophilin was selected as the housekeeping gene to use as the invariant control for each tissue, because previous Northern analyses demonstrated that cyclophilin is expressed at equivalent levels in these four organs (9), and we determined that it was similarly expressed in mouse islets and cell lines relative to total RNA content (data not shown).

This survey of NHR expression is first presented by subfamily classification showing mRNA levels in islets and cell lines relative to various mouse tissues. This format is consistent with previous NHR surveys and provides a basis of comparison to tissues commonly associated with each receptor type. This receptor classification system is based on sequence similarity and phylogenetic tree construction and most often correlates with DNA-binding and dimerization characteristics [Figs. 1, 2, and 3 (10, 11)]. Additional general information on any of the NHRs can be found in reviews (11, 12) or at the web site for the Nuclear Receptor Signaling Atlas (http://www.nursa.org/index.cfm). In addition, because this survey focused on expression in a select few cell types (islets, cell lines), we could analyze RNA levels for all receptors in a single assay for direct comparison of NHR levels within a given tissue. This rank order of expression is provided in Fig. 4.

View larger version (36K): [in this window] [in a new window]   Fig. 1. Nuclear Receptors Expressed in the Mouse Endocrine Pancreas: Subfamilies 1, 5, 6, and 0 The relative mRNA levels are depicted for mouse liver (L), brain (B), testis (T) epididymal white adipose tissue (W, WAT), islets (I), -cell line (TC1, ), and β-cell lines (β-TC6, βT; and MIN6, βM). All values are expressed relative to cyclophilin and arithmetically adjusted to depict the lowest-expressing sample as a unit of 1. Values represent the means and SEM of three independent samples for each tissue or cell line, thus portraying biological variance, and the results shown are representative of two independent studies. Note that as these data are portrayed, comparisons can only be made between different tissues for a single NHR, not between various receptors (see Fig. 4 for this comparison). CAR, Constitutive androstane receptor; SHP, short heterodimer partner.

View larger version (25K): [in this window] [in a new window]   Fig. 2. Nuclear Receptors Expressed in the Mouse Endocrine Pancreas: Subfamily 2 Refer to the legend of Fig. 1 for details.

View larger version (26K): [in this window] [in a new window]   Fig. 3. Nuclear Receptors Expressed in the Mouse Endocrine Pancreas: Subfamilies 3 and 4 Refer to the legend of Fig. 1 for details.

View larger version (23K): [in this window] [in a new window]   Fig. 4. Comparative Expression Levels of the 49 NHRs for Mouse Islets and Cell Lines, and 48 NHRs in Human Islets The relative mRNA levels are depicted for mouse islets, β-cells (MIN6 cell line) and -cells (TC1 cell line), and human islets. All values are expressed relative to cyclophilin and arithmetically adjusted to depict the highest-expressed NHR for each tissue/cell line as a unit of 100. Values represent the means and SEM of three independent samples for each tissue or cell line for mouse. Setting arbitrary cutoffs at CT < 26 (abundant); 26 < CT < 30 (present); CT > 30 (absent) as shown by broken lines in the mouse islet panel, reveals that 19 NHRs are highly expressed in both - and β-cell lines; five NHRs are predominant (>8-fold difference in RNA level) in β-cell lines; eight only in the -cell line; and 10 not expressed in the mouse endocrine pancreas or cell lines. For the analysis of human islets (a representative analysis depicting NHR pattern in islets (90% purity, maintained in 5.6 mM glucose) of a 55-yr-old Caucasian female is provided), these cutoffs correspond to bars of height greater or equal to RAR (CT 26, abundant) and less than PPAR (CT = 30, absent).

Mouse-NHR Subfamily 1 (Fig. 1)
The majority of these receptors function as RXR heterodimer partners [TRs, retinoic acid receptors (RARs), PPARs, LXRs, FXRs, vitamin D receptor (VDR), pregnane X receptor, and constitutive androstane receptor] that respond to small lipophilic ligands of dietary origin to regulate gene expression (13). Thyroid receptors are expressed in the endocrine pancreas at much lower levels than in other tissues; however, among the receptors of the islet, thyroid hormone receptor (TR) is found at appreciable levels and TRβ is expressed at moderate amounts (Fig. 4). RAR is the most abundant of the RAR subtypes in the islet and β-cell (Figs. 1 and 4). PPAR is highly expressed in mouse islets, -cells, and β-cells, and is the most prominent of the PPARs. LXRβ is more highly expressed than LXR in β-cells and islets, in agreement with previous reports (14). FXR mRNA is present in intact mouse islets but there is no evidence that this receptor is found in the - or β-cell lines examined in this study. This suggests that FXR is present in one of the minor cell types of the islet (-cells, -cells, or PP-cells) or that these immortalized cell lines have lost expression of this receptor. Further confirmation that islets express FXR was provided by the finding that islets exposed to synthetic FXR agonists alter the expression of several recognized FXR target genes (Kjalarsdottir, L., and J. J. Repa, unpublished data). FXRβ, a NHR unique to mice [a pseudogene in humans (15, 16)] is not expressed in the endocrine pancreas. The vitamin D receptor is highly expressed in islets and represents the fourth most abundant NHR, based on its level of RNA expression (Fig. 4).

The remaining members of subfamily 1 are thought to function as constitutive repressors (RevERBs) or activators [retinoid-related orphan receptors (RORs)] that play an important role in the regulation of circadian rhythm (17). RevERBβ and RevERB are highly expressed in -cells, and RevERBβ is also expressed in β-cells and islets. All ROR members are expressed, again showing the highest expression in -cells. In fact, the RORβ RNA level in -cells is equivalent to that of brain, the mouse tissue exhibiting highest expression of this receptor (8).

In summary, a large number of the lipid-activated receptors are present in cells of the endocrine pancreas, and several reports have appeared to suggest that ligands for some of these receptors affect insulin secretion, including the LXRs (14, 18), PPAR (19, 20, 21), PPAR (22), and VDR (23). In addition, receptors associated with development and circadian rhythm in other organs are likewise expressed in the adult mouse islet. This suggests that these, and perhaps additional, NHRs of this family play critical roles in islet function.

Mouse-NHR Subfamily 2 (Fig. 2)
Hepatocyte nuclear factor (HNF)4 plays a fundamental role in the function and development of the mouse islet (24, 25, 26). Inactivating mutations in the human HNF4 are responsible for maturity onset diabetes of youth type 1 (27). HNF4 is expressed in β-cells of the mouse islet at levels far below that seen in liver (Fig. 2) and represent the HNF47 and 8 isoforms, rather than the HNF41 and 2 isoforms of mature hepatocytes (Ref. 28 and Chuang, J.-C., and J. J. Repa, data not shown) The closely related receptor, HNF4, is expressed in islets, exclusively in β-cells (Ref. 29 , Fig. 2, and Chuang, J.-C., and J. J. Repa, unpublished results). The common heterodimer partner, RXR, is expressed in islets, and the isoforms are found in the rank order RXR >>RXRβ>>>RXR (Fig. 4). The testicular orphan receptors, TR2 and TR4, which function as homodimers and heterodimers, are highly expressed in islets; in fact, TR4 exhibits the highest relative RNA level of all the 49 mouse NHRs (Fig. 4). Tailless homolog (TLX) is not expressed, and photoreceptor-specific nuclear receptor (PNR) is found at very low levels in the endocrine pancreas. The orphan receptors, chicken ovalbumin upstream promoter transcription factors (COUP-TFs), are expressed with the following relative abundance: COUP-TFIII > COUP-TFII > COUP-TFI.

Mouse-NHR Subfamilies 3 and 4 (Fig. 3)
Subfamily 3 contains the steroid receptors. The estrogen receptors (ERs) are expressed in islets and cell lines at very low levels relative to other mouse tissues. GR is highly expressed in -cells. The rank order of the steroid receptors in mouse islets is mineralocorticoid receptor (MR)>>glucocorticoid receptor (GR)>>androgen receptor (AR) = progesterone receptor (PR) = ER>ERβ. Among the ER-related receptors (ERRs), ERR and ERR are highly expressed in islets, particularly in -cells.

The NR4 members of the NHR superfamily are expressed in the following rank order: nerve growth factor (NGF)IB >>> NURR1 = neuron-derived orphan receptor 1 (NOR1), and are found in intact islets as well as both β- and -cell lines.

Mouse-NHR Subfamilies 5, 6, and 0 (Fig. 1)
There is no detectable RNA for steroidogenic factor 1 in the mouse endocrine pancreas. The highly related gene, liver receptor homolog, LRH-1, was originally cloned from the pancreas (30), and its distribution has been evaluated by in situ hybridization to reveal that it is highly expressed in the exocrine pancreas (31). Our results confirm the lack of LRH-1 expression in β-cells, but indicate that LRH-1 mRNA is present in an -cell line at a level equivalent to that seen in liver tissue.

The germ cell nuclear factor is the sole member of subfamily 6 and plays critical roles in development and reproduction (32). There are detectable levels of germ cell nuclear factor mRNA in mouse islets and in - and β-cells.

The receptors of subfamily 0 are unique in that they lack the conventional DNA-binding domain that defines the NHR family of transcription factors. DAX-1 is not expressed in the mouse endocrine pancreas, and short heterodimer partner is found at very low levels in β-cells.

NHR Rank Order of Expression (Fig. 4) and Comparison with mRNA Levels in Human Islets
The design of these studies allowed for a rank order determination of RNA levels for NHR within a given cell type. The PCR primers were designed to provide equivalent PCR amplification efficiency (7) and showed no evidence of product formation in the absence of template cDNA. Therefore, because all of these analyses were performed on the same triplicate samples for each tissue and cell type, we could compare the relative expression of all 49 NHRs to one another within a given tissue (Fig. 4). In mouse islets the six most abundant receptors are TR4, RevERBβ, ROR, VDR, RevERB, and RXR.

When the NHRs of the β-cell line (MIN6) and the -cell line are placed in a similar arrangement, one can easily see the differences in relative expression of the receptors in these cells. MIN6 cells express relatively lower levels of ROR, VDR, RevERB, and RXR than intact islets. The -cell line expresses no VDR, and higher levels of ERRβ, NURR1, and TR than islets or the β-cell lines.

A similar survey of NHR transcripts in human islets was performed and revealed significant differences to the receptor expression pattern observed in mouse (Fig. 4). The most abundant receptor is human islets is LRH-1, which is present at mRNA levels equivalent to the housekeeping gene cyclophilin (C_T 21.5). This increase may be partly attributable to the larger contribution of -cells in the human islet (33), but also suggests that LRH-1 may play a more important role in the development and/or function of the endocrine pancreas in humans than rodents. The second most abundant NHR is COUP-TFIII, which falls into subtype rank order (COUP-TFIII > TFII > TFI) similar to that observed in mouse islets. ER, HNF4, and GR mRNA species are present at levels similar to the housekeeping gene HPRT1 (hypoxanthine-guanine phosphoribosyl transferase 1)(C_T = 24), and receptors indicated by bars with heights greater than that shown for RAR (C_T = 26) would be declared abundant by the criteria used to rank receptor expression in the mouse islet.

Glucose Regulation of NHR Expression in Mouse Islets and β-Cells (Fig. 5)
One of the most important functions of the endocrine pancreas is to respond to elevated circulating glucose by secreting insulin. Therefore, we also evaluated the expression of the 49 mouse NHRs in islets to determine whether changes in RNA levels occur during hyperglycemia, thus suggesting they may play a role in glucose responses by this organ. Islets were exposed to low-glucose (5 mM) or high-glucose (17.5 mM) conditions for 16 h before RNA isolation. Although our aim was to evaluate the impact of elevated glucose on NHR gene expression, the 16-h high-glucose exposure resulted in very high insulin levels in the culture media (insulin concentrations of 37 ng/ml for the low-glucose-treated islets and 408 ng/ml, or approximately 70 nM, for the high-glucose-treated islets). Therefore, genes up-regulated under high-glucose conditions in islets could be either glucose regulated or insulin regulated. In the course of these studies, we also evaluated the mRNA levels of several common housekeeping genes in islets at various times after high-glucose administration and found that most (β-actin, HPRT, 36B4, RPL19) remained constant; however, cyclophilin and glyceraldehydes-3-phosphate dehydrogenase showed a gradual increase with time, making them inappropriate for use as invariant calibrator genes in the studies shown in Fig. 5.

View larger version (30K): [in this window] [in a new window]   Fig. 5. The Effect of Elevated Glucose on NHR Expression in the Mouse Endocrine Pancreas Mouse islets and cell-lines were incubated in 3 mM glucose for 8 h, and then shifted to 5 mM (low glucose) or 17.5 mM glucose (high glucose) for 16 h before RNA isolation. The efficacy of this treatment protocol is illustrated by the robust changes in RNA levels for insulin and representative glucose-responsive genes. A, The gene structures for mouse insulin I and insulin II genes are provided, and the respective fragments amplified by qPCR are shown for total mature insulin (insulin I/II primer set, red), native insulin II (NAT, green), an insulin II splice variant (SPV, blue (53 ), and heterogenous nuclear insulin II, hnINSII, yellow). mRNA levels relative to the housekeeping gene HPRT are shown for mouse islets (B) and MIN6 cells (C). The three upper panels in panels B and C display the results of both low-glucose (open bars) and high-glucose (hatched or black bars) treatments for insulin, glucose-responsive genes, and LXR-target genes. Values depict the means ± SEM of three independent samples. Below the results for each RNA species we have provided the qPCR cycle number (Ct) for the low-glucose group to allow for an appreciation of relative expression among various genes. In the lower panel the 16 NHRs, of the 49 tested, that showed significantly different mRNA levels by high-glucose treatment in mouse islets are shown. Note, whereas all 16 were significantly different in mouse islets, only VDR, ER, and NGFIB were significantly altered in the MIN6 cell line. Statistical significance was determined by Student’s t test, n = 3; *, P < 0.05. chr., Chromosome.

Of the 49 mouse NHRs, 16 exhibited significantly altered RNA levels in islets exposed to high glucose. Reduced RNA levels were observed for TR, PPAR [as previously reported (35)], PPAR, RevERBs, ROR, TR2, ERβ, and MR. Significantly increased RNA levels were observed for RORβ, FXR, VDR, the early response genes NGFIB, NURR1, and NOR1, and ER. A similar increase of ER mRNA was observed by microarray analysis of human islets exposed to high glucose (data not shown).

A similar evaluation of glucose-mediated changes in gene expression in MIN6 cells demonstrates the limitations of insulinoma cell lines. Typically, insulinoma cell lines have dampened capacity for glucose responsiveness and insulin secretion, and efforts are underway to identify new cell lines or subclones with improved glucose-stimulated insulin secretion capacity (36, 37, 38). High glucose exposure did not increase mature insulin RNA levels, although the primary INSII (hnINSII) transcript levels were modestly elevated and the Chop10 mRNA levels were altered. There were no changes in RNA levels for LXR target genes. Of the 16 NHRs that showed altered expression in mouse islets, only three exhibited significant changes in the MIN6 cells (VDR, ER, and NGFIB).

DISCUSSION

Global gene expression profiling by microarray analysis has now been widely used to identify RNA species in a variety of mouse and human tissues (39, 40, 41, 42). In these analyses, pancreas was included among the many tissues examined. However, the islets of Langerhans make up only about 2% by mass of the pancreas, so extrapolating the results from whole pancreas to endocrine pancreas is difficult. The comprehensive evaluation of transcription factor expression in fetal and adult human pancreas has identified potential regulators of pancreatic development (43), but again those factors expressed in the endocrine vs. exocrine compartment are difficult to distinguish.

NHRs are one of the largest transcription factor families in the mammalian genome. These receptors are clinically important, because many are bound and activated by orally available lipophilic compounds, and represent about 3% of the druggable genome in humans (44). The earliest surveys of NHR expression relied on Northern analysis (9), thus requiring fairly large quantities of RNA. Subsequently, RT-PCR was used to identify NHRs in selected tissues, e.g. the mouse small intestine (45). Most recently the distribution of NHR RNA has been performed using qPCR. By this method the quantitative assessment of NHR RNA levels has been established for various mouse tissues including the pancreas (8), mouse macrophages (46), mouse adipocytes (47), and during the circadian cycle in mouse liver, adipose, and muscle (17).

In this report we provide the first survey of NHR expression in the adult mouse endocrine pancreas and commonly used mouse cell lines resembling β-cells and -cells. The mouse islet typically contains about 1500 cells, consisting of 75% β-cells and 20% -cells (33). Previous work by Gu et al. (48) to elucidate gene expression changes in the mouse endocrine pancreas during development reported three findings relevant to NHR: 1) LXR and COUP-TFII are enriched in endoderm (vs. mesoderm and ectoderm) in the E7.5 mouse; 2) LXR is enriched in mature islets relative to early-stage pancreas; and 3) RXR is down-regulated as endocrine pancreas develops. Our current findings also suggest that the expression of various NHRs in the mouse islet can be affected by extracellular glucose levels. In addition, we provide a survey of NHR expression in human islets obtained from a cadaveric pancreas of a nondiabetic adult. Of the 18 receptors that are abundantly expressed in human islet, nine also fall into this category in mouse islet and eight others are clearly present in mouse islet. The obvious exception is LRH-1, which is highly expressed in human islet, unlike the mouse islet where it is predicted to reside in the -cell compartment, based on the expression in the TC1 cell line.

In summary, our results identify NHR present in cells of the mouse and human endocrine pancreas and suggest potential therapeutic targets to affect islet physiology. Future work that takes advantage of NHR ligands and genetically altered mouse strains should further reveal roles for these proteins in islet physiology, and ultimately these findings will need to be confirmed in human islets.

MATERIALS AND METHODS

Animals
All tissues and islets were obtained from 3-month-old, male A129/SvJ mice. Mice were maintained in a temperature-controlled room (23 ± 1 C) with 12-h light (0700 h–1900 h), 12-h dark cycle and ad libitum access to water, a standard rodent diet (Harlan Teklad Premier Laboratory Diet no. 7001), and housed with sanitized wood-chip bedding (Sani-Chips, P.J. Murphy Forest Products, Montville, NJ). Tissues were harvested in early morning with mice in the fed state. All experiments were performed with the approval of the Institutional Animal Care and Use Committee of the University of Texas Southwestern Medical Center, which assures that all animal use adheres to federal regulations as published in the Animal Welfare Act, the Guide for the Care and Use of Laboratory Animals (Guide), the Public Health Service Policy, and the US Government Principles Regarding the Care and Use of Animals (http://www.utsouthwestern.edu/utsw/cda/ dept41600/files/54634.html).

Mouse Islet Isolation
The mouse pancreas was perfused and digested with liberase R1 (Roche, Indianapolis, IN). Islets were then isolated using Ficoll gradient centrifugation and hand selection under a stereomicroscope for transfer to RPMI 1640 medium (11.1 mM glucose) supplemented with 10% (vol/vol) heat-inactivated fetal bovine serum (FBS), 100 IU/ml penicillin, and 100 µg/ml streptomycin (Invitrogen, Carlsbad, CA). These culture conditions are routinely used for murine islet studies to avoid apoptotic cell death and preserve optimal glucose-stimulated insulin capacity. Islets were allowed to recover overnight (37 C, 5% CO₂) before further use. Typically 10–12 mice were used to isolate islets that would be pooled for each group and then distributed evenly among multiple wells (200 islets per well) for each assay condition. To determine the effect of glucose, mouse islets and cell lines were incubated in 3 mM glucose for 8 h, and then shifted to 5 mM (low glucose) or 17.5 mM glucose (high glucose) for 16 h before RNA isolation. All experiments were performed a minimum of two times.

Human Islet Experiments
Human islets were isolated from three cadaveric donors at the University of Virginia. Two donors were male and one was female; their ages ranged from 34–55 yr. Purity of the islet preparation ranged from 40–90%. Upon receipt, islets were immediately placed in RPMI medium containing 11 mM glucose, 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin (Invitrogen) and incubated overnight at 37 C and 5% CO₂. Islets were transferred to 5.6 mM glucose media for 16 h before RNA isolation.

Cell Culture
The insulinoma cell line β-TC-6 [CRL-11506 (49)] and the adenoma-derived glucagonoma cell line TC1-clone 9 [CRL-2350 (50)] were obtained from American Type Culture Collection (Manassas, VA). The MIN6 cell line, passage 24 (51), was kindly provided by Melanie Cobb (University of Texas Southwestern). Cells were maintained in their optimal culture conditions unless indicated otherwise. β-TC6 cells were routinely cultured in DMEM (4.5 g/liter glucose, 4 mM L-glutamine) with 15% heat-inactivated FBS. MIN6 cells were maintained in DMEM (4.5 g/liter glucose); 2 mM L-glutamine, 1 mM sodium pyruvate, and 10% heat-inactivated FBS. -TC1 cells were cultured in DMEM with 4 mM L-glutamine adjusted to contain 1.5 g/liter sodium bicarbonate and 3 g/liter glucose with 10% heat-inactivated dialyzed FBS, further supplemented with 15 mM HEPES, 0.1 mM nonessential amino acids, and 0.02% BSA.

RNA Measurement
RNA was isolated from tissue samples or cultured cells using RNA STAT-60 (Tel-Test Inc., Friendswood, TX), and 2 µg of total RNA was treated with ribonuclease-free deoxyribonuclease (Roche), and then reverse transcribed with random hexamers using SuperScript II (Invitrogen), as previously described in detail (52).

qPCR was performed using an Applied Biosystem Prism 7900HT sequence detection system (Applied Biosystems, Foster City, CA) and SYBR-green chemistry (7, 52). Gene-specific primers were designed using Primer Express Software (PerkinElmer Life Sciences, Boston, MA) and validated by analysis of template titration and dissociation curves. From the titration curve, a plot of log input amount vs. C_T was generated, and primers were deemed valid if the resulting line had a slope of –3.3 ± 0.1 to establish equal efficiency of amplification for all target and reference genes. Primer sequences are provided in supplemental Table 1 (published as supplemental data on The Endocrine Society’s Journals Online web site address at http://mend.endojournals.org) for mouse genes. Primer sequences used for the measurements of human NHR mRNA are available at www.nursa.org/10/1621/datasets.04011. qPCRs (10 µl) contained 25 ng of reverse-transcribed RNA, each primer (150 nM), and 5 µl of 2X SYBR Green PCR master mix (Applied Biosystems). Multiple housekeeping genes were evaluated in each assay to ensure that their RNA levels were invariant under the experimental conditions of each study. Results of qPCR were evaluated by the comparative Ct method [user bulletin no. 2, PerkinElmer Life Sciences (52)] using hypoxanthine-guanine phosphoribosyl transferase (HPRT) or cyclophilin as the invariant control gene.

Statistics
Values shown reflect the mean ± SEM, n = 3 samples per tissue or cell line. Two-tailed Student’s t test was performed to compare low- and high-glucose-treated samples (Fig. 5), and significance was established at P < 0.05.

ACKNOWLEDGMENTS

We thank Angie Bookout and Drs. Raymond MacDonald and Philipp Scherer for critically reading the manuscript. We thank Mark A. Valasek, Stephen L. Clarke, and Chunmei Yang for their assistance with animal studies and for thoughtful discussions.

FOOTNOTES

This work was supported by Research Award 7-04-RA-94 from the American Diabetes Association (to J.J.R.) and National Institutes of Health Grant R01 DK60581 (to R.G.M.).

Disclosure Statement: The authors have nothing to disclose.

First Published Online August 7, 2008

Abbreviations: AR, Androgen receptor; COUP-TF, Chicken ovalbumin upstream promoter transcription factor; ER, estrogen receptor; ERR, ER-related receptor; FBS, fetal bovine serum; FXR, farnesoid X receptor; GR, glucocorticoid receptor; HNF, hepatocyte nuclear factor; hnINSII, heterogenous, nuclear insulin II transcript; HPRT1, hypoxanthine-guanine phosphoribosyl transferase 1; LRH-1, liver receptor homolog 1; LXR, liver X receptor; MR, mineralocorticoid receptor; NGF, nerve growth factor; NHR, nuclear hormone receptor; NOR1, neuron-derived orphan receptor 1; PPAR, peroxisome proliferator-activated receptor; PR, progesterone receptor; qPCR, quantitative real-time PCR; RAR, retinoic acid receptor; ROR, retinoid-related orphan receptor; RXR, retinoid X receptor; TR, thyroid hormone receptor; VDR, vitamin D receptor.

Received for publication December 19, 2007. Accepted for publication July 25, 2008.

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