- Open Access
Feeding and fasting controls liver expression of a regulator of G protein signaling (Rgs16) in periportal hepatocytes
© Huang et al; licensee BioMed Central Ltd. 2006
- Received: 07 May 2006
- Accepted: 23 November 2006
- Published: 23 November 2006
Heterotrimeric G protein signaling in liver helps maintain carbohydrate and lipid homeostasis. G protein signaling is activated by binding of extracellular ligands to G protein coupled receptors and inhibited inside cells by regulators of G protein signaling (RGS) proteins. RGS proteins are GTPase activating proteins, and thereby regulate Gi and/or Gq class G proteins. RGS gene expression can be induced by the ligands they feedback regulate, and RGS gene expression can be used to mark tissues and cell-types when and where Gi/q signaling occurs. We characterized the expression of mouse RGS genes in liver during fasting and refeeding to identify novel signaling pathways controlling changes in liver metabolism.
Rgs16 is the only RGS gene that is diurnally regulated in liver of ad libitum fed mice. Rgs16 transcription, mRNA and protein are up regulated during fasting and rapidly down regulated after refeeding. Rgs16 is expressed in periportal hepatocytes, the oxygen-rich zone of the liver where lipolysis and gluconeogenesis predominates. Restricting feeding to 4 hr of the light phase entrained Rgs16 expression in liver but did not affect circadian regulation of Rgs16 expression in the suprachiasmatic nuclei (SCN).
Rgs16 is one of a subset of genes that is circadian regulated both in SCN and liver. Rgs16 mRNA expression in liver responds rapidly to changes in feeding schedule, coincident with key transcription factors controlling the circadian clock. Rgs16 expression can be used as a marker to identify and investigate novel G-protein mediated metabolic and circadian pathways, in specific zones within the liver.
- Circadian Clock
- Light Phase
- Restricted Feeding
- Mating Pheromone
- Fast Mouse
Body weight homeostasis is maintained through complex communications between the brain and peripheral organs [1–8]. Declining body weight during fasting promotes increased food intake and decreased energy expenditure. By contrast, weight gain following several large meals is compensated by decreased food intake and increased energy expenditure. Many of the orexigenic and anorexigenic signals providing dynamic control of energy and body weight homeostasis are conveyed by G protein coupled receptors (GPCRs) in the brain and periphery [5, 9, 10]. Here, we investigate a novel approach to understand how G protein signaling in liver regulates metabolic activity to maintain body weight and energy balance.
The activity cycle of heterotrimeric G proteins revolves around receptor-catalyzed guanine nucleotide exchange and GTP hydrolysis on the Gα subunit. In the inactive state, GαGDP forms a heterotrimeric complex with Gβγ. Hormone binding to GPCRs activates intracellular signaling by catalyzing guanine nucleotide exchange on the Gα subunit . Active GαGTP and Gβγ subunits dissociate to regulate effector proteins and the subsequent production of second messengers that provoke cellular responses to physiologic stimuli. GTP hydrolysis on Gα restores the inactive heterotrimeric complex of GαGDPβγ. Regulators of G protein signaling (RGS) proteins are GTPase activating proteins (GAPs) for Gi and Gq class α subunits [12–15], and are distantly related to rgRGS proteins that accelerate GTP hydrolysis on G12 class α subunits [16, 17]. RGS proteins regulate the specificity, intensity and duration of Gi and Gq signaling . RGS proteins of the R4 family, such as Rgs16, are feedback inhibitors that can terminate signaling by uncoupling hormone binding from effector protein activation [19–21].
A useful characteristic of RGS gene expression is that it can be induced by GPCR agonists and second messengers [22, 23]. A paradigm for feedback regulation of G protein signaling by RGS proteins was established by analysis of the yeast mating response . Mating pheromones are GPCR ligands that stimulate cell cycle arrest in haploid cells. The G protein alpha subunit (GPA-1) releases Gβγ to stimulate a MAP kinase cascade resulting in the transcriptional activation of the mating response pathway [25–27]. Interestingly, transcription of the yeast RGS gene Sst-2 is also induced by mating pheromone . SST-2 is the GAP for GPA-1 and it is the most important gene product for pheromone desensitization and re-entry into the cell cycle [28, 29]. We applied this paradigm of GPCR-ligand induced RGS gene expression to identify and characterize G protein signaling in liver during fasting and refeeding because we had made several observations indicating that Gq and RGS proteins influenced and responded to changes in liver metabolism.
We found that knockout mice deficient in either Gαq or its close paralog, Gα11 , exhibited abnormalities in liver regeneration following partial hepatectomy (Yu and Wilkie, unpublished observation). Given that RGS proteins are essential regulators of Ca+2 signaling evoked by Gq/11-coupled agonists [19, 21, 31], we reasoned that a RGS gene might be induced in response to activation of Gq/11 signaling during liver regeneration. We found Rgs16 was rapidly up regulated following partial hepatectomy, suggesting a role in the metabolic response to the sudden loss of 70% of the liver. Therefore, we screened the livers of fasted and refed mice for differential regulation of RGS genes. Interestingly, of the 20 cloned RGS genes, only Rgs16 mRNA was induced in liver during fasting. Our studies described herein demonstrate that Rgs16 is a diurnally regulated gene in periportal hepatocytes of the liver, its expression is regulated by feeding and dietary constituents, and Rgs16 can be used as a biomarker to investigate G-protein pathways in liver regulating energy homeostasis.
Diurnal regulation of Rgs16 mRNA in liver
Rgs16 is one of a small subset of genes that is diurnally regulated in both liver and the hypothalamic suprachiasmatic nuclei (SCN), site of the central circadian pacemaker [33, 34]. We showed by in situ hybridization that Rgs16 mRNA is expressed in the SCN of C57BL/6 female mice in our colony in the same temporal pattern as previously reported (Fig. 1B; ). Furthermore, this temporal pattern of Rgs16 expression in the SCN is maintained in ad libitum fed mice housed in constant darkness (CD) for two days prior to sacrifice on the third day; expression is elevated at 6 hr into the presumptive light phase (CD6) and declines to basal levels by 2 hr into the presumptive dark phase (CD14; Fig. 1C). Thus, Rgs16 expression in the SCN appears to be entrained similarly to other circadian regulated genes [35, 36].
Regulation of Rgs16 by fasting and refeeding
Rgs16 mRNA in periportal hepatocytes
Restricted feeding shifts circadian expression of Rgs16 in liver but not SCN
On the first day of RF, Rgs16 mRNA expression in liver is no higher at ZT5 than in ad libitum fed mice (see Fig. 1, lane 1, 2), even though the RF mice were fasted through the dark phase and had lost 2.5 gm from their starting weight (Fig. 4, middle panel). This may reflect an underlying circadian regulation of liver gene expression as clock genes show a similar response on the first day of RF [35, 36, 38]. During the first 4 hr feeding period (day 1), female mice ate about 1.5 gm of chow and regained about 1 gm body weight (Fig. 4, bottom panel). However, they ate about half as much on day 1 as on subsequent days, presumably because they had not yet learned to anticipate fasting during the dark phase and feeding during the light phase. By day 2 RF, prior to feeding at ZT5, mice lost about 3.5 gm of their original weight, but they were alert and active, and ate voraciously upon refeeding, consuming about 0.7 gm chow within the first 30 min, 2.5 gm during 4 hr. This behavior was repeated days 3 and 4 of RF, and each day mice regained body weight to within about 1 gm of their starting weight.
Oscillations in Rgs16 mRNA expression are slightly delayed in the liver relative to the SCN in ad libitum fed mice maintained in a 12 hr light-dark cycle [35, 36, 38]. By contrast, restricted feeding during the light phase advances and intensifies Rgs16 expression in liver but does not alter expression in SCN (Fig. 4, top panel) presumably reflecting separate inputs of nutrients and light regulating Rgs16 expression in liver and SCN.
Transcriptional control of Rgs16 gene expression
Rgs16 mRNA and protein are rapidly down regulated after feeding
Rgs16 is one of the few genes whose expression oscillates in a circadian pattern in both liver and SCN (Figs. 1, 2, 3, 4) . Rgs16 and other oscillatory genes in liver are synchronized by feeding time, whereas SCN neurons presumably respond to light stimulated neurotransmitter release at synaptic junctions with neurons from the retinal-hypothalamic tract. Many of the genes that oscillate both in liver and SCN are transcription factors integral to the circadian clock [35, 36, 38, 39], whereas Rgs16 is a GAP for G alpha proteins of the Gi and Gq class [31, 40, 41]. Rgs16 and closely related RGS proteins in the R4 family negatively regulate G protein signaling , and can feedback inhibit Gi/Gq signaling by uncoupling hormone binding from effector protein activation in a variety of primary cell types [19, 21, 31]. The expression pattern and enzymatic activity of Rgs16 identifies it as a candidate regulator of Gi/Gq signaling during the transitions from fasting to feeding in liver and between light and dark phases in SCN.
G protein-coupled ligands stimulate expression of RGS genes in eukaryotes from mammals to yeast [18, 22–24]. For example, the yeast mating pheromones are GPCR-ligands that induce both cell cycle arrest and the transcription/translation of the yeast RGS protein SST-2, which is required for rapid desensitization to mating pheromone and re-entry to the cell cycle . Because the early phase of liver regeneration is impaired in mutant mice deficient in Gq signaling (data not shown), we applied the yeast paradigm as our rationale for screening the expression of all RGS genes in liver of fed and fasted mice to identify Gi and/or Gq signaling pathways regulating liver metabolism.
We found that Rgs16 is the only RGS gene (of 20 total ) that is diurnally expressed in liver of ad libitum fed mice (Fig. 1). Mice are nocturnal, and when provided ad libitum access to food and water, they eat nearly 80% of their daily food during the dark phase in a 12 hr light:dark (12hL:D) cycle. Rgs16 mRNA accumulates in liver in anticipation of a meal, either at the end of the light phase in mice maintained on a 12 hr L:D cycle (Figs. 1, 2), or prior to feeding at ZT5 on day 2 and subsequent days of restricted feeding (RF; Fig. 4). Furthermore, the amplitude of daily oscillations in Rgs16 mRNA and protein expression are modulated by energy deficiency, either increased in fasted mice whose body weight is significantly below set point (Fig. 4) or decreased in over weight mice maintained on a high fat diet (Fig. 6A). Importantly, the rate of Rgs16 gene transcription is induced by fasting and declines to basal levels shortly after feeding (Fig. 5). Coincident with these changes in transcription, Rgs16 mRNA and protein levels decline within 20 minutes after feeding begins, and drop to basal levels within 120 minutes (Fig. 6). Interestingly, if mice are not allowed to continue eating, but are restricted to the amount they can consume within 30 minutes of refeeding, Rgs16 mRNA expression returns within 120 min (data not shown). The tight localization of expression to Zone 1 periportal hepatocytes (Fig. 3) places Rgs16 at an ideal location to be regulated by orexigenic and satiety signals from the gut, the peripheral and/or central nervous system. The dynamic and localized expression of Rgs16 could be regulated by hormones and/or metabolites controlling liver metabolism.
The liver helps maintain whole body energy homeostasis in part by metabolism of glucose and fatty acids. Catabolic and anabolic metabolism in liver is regulated in response to daily repetitions of fasting and feeding, and the availability of energy stores in liver, adipose, and muscle. Glucagon and insulin are classical hormone regulators of the fasted and fed states 42, 43]. During fasting, glucagon simultaneously stimulates gluconeogenesis while inhibiting fatty acid synthesis in liver . Glucagon signaling in hepatocytes is transduced by Gαs, which stimulates adenylyl cyclase to produce the second messenger cAMP, thereby activating the cAMP responsive transcription factor CREB [45, 46]. However, Rgs16 is not a Gs-GAP and does not directly regulate Gs activity ; rather, Rgs16 may help integrate Gi- and/or Gq-signaling with glucagon or insulin signaling in liver.
Guided by the paradigm of the yeast mating pathway, we hypothesize that Rgs16 expression is induced in liver during fasting by activation of the Gi/Gq pathway(s) that Rgs16 protein negatively regulates. We propose that a preprandial agonist activates a hypothetical GPCR pathway that induces Rgs16 gene transcription and, possibly, promotes Rgs16 mRNA stability. Curiously, Rgs16 protein is not abundantly expressed in liver of fasted mice. We note that Rgs16 and other RGS proteins of the R4 subfamily typically are weakly expressed in many cell types and tissues despite abundant mRNA expression . One possible explanation for this pattern is Rgs16 mRNA accumulates during fasting and is poised for translation, dependent on other dietary factors.
Rgs16 is specifically expressed in periportal hepatocytes, the oxygen-rich zone of the liver where lipolysis and gluconeogenesis predominates, suggests Rgs16 may regulate Gi/Gq pathway(s) that stimulate fatty acid oxidation and/or glucose production in liver. Because refeeding rapidly terminates Rgs16 gene transcription and allows Rgs16 mRNA and protein degradation, Rgs16 might inhibit Gi/Gq signaling during the transition from the fasted to fed states. In this model, Rgs16 functions as a switch, turning off preprandial signaling in liver once feeding has commenced. Given that the hepatic expression of Rgs16 and circadian clock genes rapidly and coordinately adjust to changes in feeding schedules, Rgs16 might regulate the feeding cues that reset the circadian oscillator in liver.
Rgs16 is the only RGS gene (of 20 total) that is diurnally expressed in liver of ad libitum fed mice. Restricting feeding entrained Rgs16 transcription and mRNA expression in liver but did not affect circadian regulation of Rgs16 expression in the SCN. Thus, separate inputs regulate Rgs16 expression in liver and SCN. Rgs16 gene transcription, mRNA and protein levels are rapidly down regulated by feeding. The expression pattern and enzymatic activity of Rgs16 identifies it as a candidate regulator of Gi/Gq signaling during the transitions from fasting to feeding in liver and between light and dark phases in SCN.
[32P]-dCTP was purchased from Amersham Bioscience (Piscataway, NJ). TRIZOL was purchased from Invitrogen (Carlsbad, California) and GeneScreen nylon membranes were obtained from New England Nuclear (Shelton, CT). All other lab supplies were purchased from Sigma (St Louis, MO) or Fisher (Hampton, NH).
Animal and colony conditions
Mice were maintained at 20°C under a standard 12-hour light:dark cycle (12h L:D, lights on at 5 am). C57BL/6 mice were purchased from Jackson Laboratories (Bar Harbor, Maine). Female C57BL/6 mice in feeding studies were pair-caged unless otherwise indicated; male mice were caged individually during restricted feeding studies. Mice were ad libitum fed standard mouse chow (Normal Chow) containing 6% total energy as fat (Teklad 7002, Harlan Teklad Laboratories, Indianapolis, IN), except where noted. The high-fat diet contains 41% calories as fat-40% carbohydrate-19% protein (Teklad #96001). Mice were six to eight weeks of age before being switched from a normal chow to high fat diet. Experimental research on animals followed internationally recognized guidelines and had UT Southwestern IACUC approval (APN#0602-05-01-2).
Real-time quantitative PCR
mRNA expression levels were determined using real time quantitative polymerase chain reaction. Cyclophilin was used as the normalizing gene. QPCR primers were designed using the Primer Express Software v 2.0 (Applied Biosystems, Foster City, CA) from published mRNA sequences. Rgs16 (NM_011267) QPCR primers: Forward – 5'cctggtacttgctactcgctttt3'; Reverse – 5'agcacgtcgtggagaggat3'; Cyclophilin (M60456) QPCR primers: Forward – 5'tggagagcaccaagacagaca3'; Reverse – 5'tgccggagtcgacaatgat3'. Total RNA was extracted from 50 mg liver as described below in Northern Blot Hybridization. Following DNase treatment, cDNA was synthesized from total RNA (2 μg) in 100 μL reactions using Superscript II reverse transcriptase kit (Invitrogen, Carlsbad, CA) and stored at -20°C until use. Polymerase chain reaction amplifications were performed in 96-well optical reaction plates (ABI) on the ABI Prism 7000 Sequence Detection system using the SYBR-Green (ABI) reaction conditions (1 cycle at 50°C for 2 min, 1 cycle at 95°C for 10 min, 40 cycles at 95°C for 15 sec and 60°C for 1 min), the baseline and threshold were set to experimentally determined values and the data were analyzed using the Comparative CT method () as described . Relative Rgs16 mRNA fold induction was calculated compared to pooled RNA from fed mice (as in Fig. 2A, condition 2).
Northern blot hybridization
At time of collection, mice were sacrificed by cervical dislocation and individual livers were dissected, frozen in liquid nitrogen immediately, and stored at -80°C for future analysis. RNA extraction was performed using TRIZOL according to manufacture's protocol and liver RNA (20 μg per lane) was used for Northern analysis. Briefly, RNA samples were size separated by electrophoresis on a 1.0% agarose denaturing gel, transferred to a nylon membrane overnight, and probed with Rgs16 cDNA in 50% formamide. Radionucleotide hybridization probes were either random primed (Rgs16 cDNA; complete open reading frame) or end-labeled (18s rRNA oligonucleotide [GCCGTGCGTACTTAGACATGCATG]) as described . Northern blot hybridization filters were prehybridized at 42°C for six hours, hybridized overnight at 42°C, and then the membrane was washed twice in 2X SSC at 25°C, once in 2X SSC/2% SDS at 55°C, and once in 0.1X SSC at 25°C; filters were dried and exposed to Fuji RA film for at least 16 hours. Fold change (Δ) in Rgs16 mRNA levels are relative to basal expression at D12 (assayed by densitometry).
In situ hybridization
Mice were sacrificed by cervical dislocation and the livers were rapidly frozen on dry ice and stored at -80°C. Fresh frozen sections were cut in a cryostat and thaw-mounted onto SuperFrost Plus glass slides (Fisher Scientific, Pittsburgh, PA). Sections were pre-treated as described, including fixation, acetic anhydride and defatting steps . Slides with cover slip were incubated for 18 hrs at 60°C in a humidified chamber with buffer containing denatured salmon sperm DNA (0.033 mg/mL), yeast tRNA (0.15 mg/mL), dithiothreitol (40 μM), and a cRNA at 1 × 107 cpm/mL, other conditions as described . Riboprobes were generated using an in vitro transcription kit (Ambion; Austin, TX) by using T3 or T7 RNA polymerase in the presence of 32P-UTP, and purified using RNA quickspin columns. Sections were then treated with RNase A (20 mg/mL, 30 min at 45°C) and washed in descending concentrations of sodium citrate buffer to a stringency of 0.1X SSC at 60°C, and air-dried. Tissue sections were exposed to Biomax MR film (Kodak, Rochester, NY) followed by dipping in autoradiographic emulsion (NTB2, Kodak), exposed for an appropriate duration, developed, fixed, counterstained with cresyl violet acetate, a cover slip applied with DPX mounting media and visualized under bright and dark field microscopy (Olympus, Melville, NY). Adjacent sections from fresh-frozen livers were used as controls for the effects of perfusion on mRNA integrity and hybridization to sense probes.
Livers were rapidly excised and immediately frozen in liquid nitrogen. Liver (50 mg) was resuspended in 1% SDS that contained a cocktail of protease inhibitors, including leupeptin (10 μg/mL), soybean trypsin inhibitor (10 μg/mL), MG132 (16 μg/mL), phenylmethylsulfonyl fluoride (16 μg/mL), tosyl lysine choromethyl ketone (16 μg/mL), and tosyl phenylalanine choromethyl ketone (16 μg/mL). The samples were sonicated (Virtis, Gardiner, NY) and immediately boiled for three minutes. A Lowry protein assay was employed to quantitate the protein and ensure equal loading. The samples were separated by SDS-PAGE and transferred to nitrocellulose paper. Proteins were detected using Rgs16 antiserum (a gift from Carol Beadling, Cornell University). Rgs16 antiserum does not cross react with recombinant mouse Rgs4 or Rgs8 protein. Enhanced Chemiluminescence (Amersham-Pharmacia) was utilized to detect the rabbit HA-tagged secondary antibody. The membrane was exposed to ML film (Kodak Biomax) for 2–15 minutes. Autoradiographs were scanned with a BioRad Fluor-S MultiImager to determine signal intensities.
Nuclei from mouse livers were isolated as described , resuspend in glycerol storage buffer (50 mM Tris-HCl pH 8.3, 5 mM MgCl2, 0.1 mM EDTA pH 8.0 and 40% glycerol) and flash-frozen in liquid nitrogen. Nuclei were stored at -80°C and used within 4 weeks. Mouse Rgs16, mouse Rgs8, rat GAPDH cDNA or empty plasmid vectors (5 μg) were slot-blotted onto nylon GeneScreen membrane (NEF 983, NEN life Science Products, Inc) according the manufacturer's protocol. The nuclear RNA elongation reaction, isolation of newly synthesized 32P-RNA, and hybridization to cDNA plasmids were performed as described 51]. The amount of hybridizing 32P-RNA was quantitated by densitometric scanning (using a Fujifilm FLA-5100 image reader) of phosphorimager screens exposed for 24 h. Data from different fasting and refeeding conditions were normalized to transcription of the GAPDH gene.
Experiments were conducted in groups of two or three mice per condition and repeated at least twice. Quantitative data for each condition or time point are represented as mean values ± SEM. GraphPad Prism software (GraphPad, San Diego, CA) was used to perform all statistical analyses. The two-tailed, unpaired Student's t-test was used to determine statistically significant differences (P < 0.05) between mean values.
We thank John Shelton, Jim Richardson, Liping Zhao, and Brian Potts for in situ hybridization, Bob Hammer for providing the PEPCK in situ probe, Carol Beadling for Rgs16 antiserum, Susanne Mumby for advice on protein preparation from tissues for Westerns, Joyce Repa for generously sharing expertise and machine time for Real-Time quantitative PCR, David Sierra for technical assistance, and the many colleagues mentioned above or in Discovery Biology for discussions and comments on the manuscript. This work was supported by NIH (GM61395; PAR-98-057) and Welch Foundation (I-1382) grants to TMW and a NIH postdoctoral fellowship (HL072551) to DMK.
- Spiegelman BM, Flier JS: Obesity and the regulation of energy balance. Cell. 2001, 104: 531-543. 10.1016/S0092-8674(01)00240-9.View ArticlePubMedGoogle Scholar
- Schwartz MW, Porte D: Diabetes, obesity, and the brain. Science. 2005, 307: 375-379. 10.1126/science.1104344.View ArticlePubMedGoogle Scholar
- Coppari R, Ichinose M, Lee CE, Pullen AE, Kenny CD, McGovern RA, Tang V, Liu SM, Ludwig T, Chua SC, Lowell BB, Elmquist JK: The hypothalamic arcuate nucleus: a key site for mediating leptin's effects on glucose homeostasis and locomotor activity. Cell Metab. 2005, 1: 63-72. 10.1016/j.cmet.2004.12.004.View ArticlePubMedGoogle Scholar
- Henry M, Ghibaudi L, Gao J, Hwa JJ: Energy metabolic profile of mice after chronic activation of central NPY Y1, Y2, or Y5 receptors. Obes Res. 2005, 13: 36-47.View ArticlePubMedGoogle Scholar
- Ramos EJ, Meguid MM, Campos AC, Coelho JC: Neuropeptide Y, alpha-melanocyte-stimulating hormone, and monoamines in food intake regulation. Nutrition. 2005, 21: 269-279. 10.1016/j.nut.2004.06.021.View ArticlePubMedGoogle Scholar
- Kishi T, Elmquist JK: Body weight is regulated by the brain: a link between feeding and emotion. Mol Psychiatry. 2005, 10: 132-146. 10.1038/sj.mp.4001638.View ArticlePubMedGoogle Scholar
- Kim S, Sohn I, Ahn JI, Lee KH, Lee YS: Hepatic gene expression profiles in a long-term high-fat diet-induced obesity mouse model. Gene. 2004, 340: 99-109. 10.1016/j.gene.2004.06.015.View ArticlePubMedGoogle Scholar
- Gavrila A, Chan JL, Miller LC, Heist K, Yiannakouris N, Mantzoros CS: Circulating melanin-concentrating hormone, agouti-related protein, and alpha-melanocyte-stimulating hormone levels in relation to body composition: alterations in response to food deprivation and recombinant human leptin administration. J Clin Endocrinol Metab. 2005, 90: 1047-1054. 10.1210/jc.2004-1124.View ArticlePubMedGoogle Scholar
- Farooqi IS, O'Rahilly S: Monogenic obesity in humans. Annu Rev Med. 2005, 56: 443-458. 10.1146/annurev.med.56.062904.144924.View ArticlePubMedGoogle Scholar
- Cone RD: Anatomy and regulation of the central melanocortin system. Nat Neurosci. 2005, 8: 571-578. 10.1038/nn1455.View ArticlePubMedGoogle Scholar
- Gilman AG: G proteins: transducers of receptor-generated signals. Annu Rev Biochem. 1987, 56: 615-649. 10.1146/annurev.bi.56.070187.003151.View ArticlePubMedGoogle Scholar
- Berman DM, Wilkie TM, Gilman AG: GAIP and RGS4 are GTPase-activating proteins for the Gi subfamily of G protein alpha subunits. Cell. 1996, 86: 445-452. 10.1016/S0092-8674(00)80117-8.View ArticlePubMedGoogle Scholar
- Watson N, Linder ME, Druey KM, Kehrl JH, Blumer KJ: RGS family members: GTPase-activating proteins for heterotrimeric G- protein alpha-subunits. Nature. 1996, 383: 172-175. 10.1038/383172a0.View ArticlePubMedGoogle Scholar
- Hepler JR, Berman DM, Gilman AG, Kozasa T: RGS4 and GAIP are GTPase-activating proteins for Gq alpha and block activation of phospholipase C beta by gamma-thio-GTP-Gq alpha. Proc Natl Acad Sci U S A. 1997, 94: 428-432. 10.1073/pnas.94.2.428.PubMed CentralView ArticlePubMedGoogle Scholar
- Hunt TW, Fields TA, Casey PJ, Peralta EG: RGS10 is a selective activator of G alpha i GTPase activity. Nature. 1996, 383: 175-177. 10.1038/383175a0.View ArticlePubMedGoogle Scholar
- Kozasa T, Jiang X, Hart MJ, Sternweis PM, Singer WD, Gilman AG, Bollag G, Sternweis PC: p115 RhoGEF, a GTPase activating protein for Galpha12 and Galpha13. Science. 1998, 280: 2109-2111. 10.1126/science.280.5372.2109.View ArticlePubMedGoogle Scholar
- Wilkie TMK: New Roles for G alpha and RGS Proteins:Communication Continues despitePulling Sisters Apart. Current Biology. 2005, ,Google Scholar
- Ross EM, Wilkie TM: GTPase-activating proteins for heterotrimeric G proteins: regulators of G protein signaling (RGS) and RGS-like proteins. Annu Rev Biochem. 2000, 69: 795-827. 10.1146/annurev.biochem.69.1.795.View ArticlePubMedGoogle Scholar
- Zeng W, Xu X, Popov S, Mukhopadhyay S, Chidiac P, Swistok J, Danho W, Yagaloff KA, Fisher SL, Ross EM, Muallem S, Wilkie TM: The N-terminal domain of RGS4 confers receptor-selective inhibition of G protein signaling. J Biol Chem. 1998, 273: 34687-34690. 10.1074/jbc.273.52.34687.View ArticlePubMedGoogle Scholar
- Popov SG, Krishna UM, Falck JR, Wilkie TM: Ca2+/Calmodulin reverses phosphatidylinositol 3,4, 5-trisphosphate-dependent inhibition of regulators of G protein-signaling GTPase-activating protein activity. J Biol Chem. 2000, 275: 18962-18968. 10.1074/jbc.M001128200.View ArticlePubMedGoogle Scholar
- Luo X, Popov S, Bera AK, Wilkie TM, Muallem S: RGS proteins provide biochemical control of agonist-evoked [Ca2+]i oscillations. Mol Cell. 2001, 7: 651-660. 10.1016/S1097-2765(01)00211-8.View ArticlePubMedGoogle Scholar
- Hollinger S, Hepler JR: Cellular regulation of RGS proteins: modulators and integrators of G protein signaling. Pharmacol Rev. 2002, 54: 527-559. 10.1124/pr.54.3.527.View ArticlePubMedGoogle Scholar
- De Vries L, Zheng B, Fischer T, Elenko E, Farquhar MG: The regulator of G protein signaling family. Annu Rev Pharmacol Toxicol. 2000, 40: 235-271. 10.1146/annurev.pharmtox.40.1.235.View ArticlePubMedGoogle Scholar
- Dohlman HG, Thorner J: RGS proteins and signaling by heterotrimeric G proteins. J Biol Chem. 1997, 272: 3871-3874. 10.1074/jbc.272.7.3871.View ArticlePubMedGoogle Scholar
- Whiteway M, Hougan L, Dignard D, Thomas DY, Bell L, Saari GC, Grant FJ, O'Hara P, MacKay VL: The STE4 and STE18 genes of yeast encode potential beta and gamma subunits of the mating factor receptor-coupled G protein. Cell. 1989, 56: 467-477. 10.1016/0092-8674(89)90249-3.View ArticlePubMedGoogle Scholar
- Herskowitz I: MAP kinase pathways in yeast: for mating and more. Cell. 1995, 80: 187-197. 10.1016/0092-8674(95)90402-6.View ArticlePubMedGoogle Scholar
- Dohlman HG, Thorner JW: Regulation of G protein-initiated signal transduction in yeast: paradigms and principles. Annu Rev Biochem. 2001, 70: 703-754. 10.1146/annurev.biochem.70.1.703.View ArticlePubMedGoogle Scholar
- Dohlman HG, Song J, Ma D, Courchesne WE, Thorner J: Sst2, a negative regulator of pheromone signaling in the yeast Saccharomyces cerevisiae: expression, localization, and genetic interaction and physical association with Gpa1 (the G-protein alpha subunit). Mol Cell Biol. 1996, 16: 5194-5209.PubMed CentralView ArticlePubMedGoogle Scholar
- Apanovitch DM, Slep KC, Sigler PB, Dohlman HG: Sst2 is a GTPase-activating protein for Gpa1: purification and characterization of a cognate RGS-Galpha protein pair in yeast. Biochemistry. 1998, 37: 4815-4822. 10.1021/bi9729965.View ArticlePubMedGoogle Scholar
- Offermanns S, Zhao LP, Gohla A, Sarosi I, Simon MI, Wilkie TM: Embryonic cardiomyocyte hypoplasia and craniofacial defects in G alpha q/G alpha 11-mutant mice. Embo J. 1998, 17: 4304-4312. 10.1093/emboj/17.15.4304.PubMed CentralView ArticlePubMedGoogle Scholar
- Xu X, Zeng W, Popov S, Berman DM, Davignon I, Yu K, Yowe D, Offermanns S, Muallem S, Wilkie TM: RGS proteins determine signaling specificity of Gq-coupled receptors. J Biol Chem. 1999, 274: 3549-3556. 10.1074/jbc.274.6.3549.View ArticlePubMedGoogle Scholar
- Kurrasch DM, Huang J, Wilkie TM, Repa JJ: Quantitative real-time polymerase chain reaction measurement of regulators of G-protein signaling mRNA levels in mouse tissues. Methods Enzymol. 2004, 389: 3-15.View ArticlePubMedGoogle Scholar
- Ralph MR, Foster RG, Davis FC, Menaker M: Transplanted suprachiasmatic nucleus determines circadian period. Science. 1990, 247: 975-978. 10.1126/science.2305266.View ArticlePubMedGoogle Scholar
- Rusak B, Zucker I: Neural regulation of circadian rhythms. Physiol Rev. 1979, 59: 449-526.PubMedGoogle Scholar
- Panda S, Antoch MP, Miller BH, Su AI, Schook AB, Straume M, Schultz PG, Kay SA, Takahashi JS, Hogenesch JB: Coordinated transcription of key pathways in the mouse by the circadian clock. Cell. 2002, 109: 307-320. 10.1016/S0092-8674(02)00722-5.View ArticlePubMedGoogle Scholar
- Damiola F, Le Minh N, Preitner N, Kornmann B, Fleury-Olela F, Schibler U: Restricted feeding uncouples circadian oscillators in peripheral tissues from the central pacemaker in the suprachiasmatic nucleus. Genes Dev. 2000, 14: 2950-2961. 10.1101/gad.183500.PubMed CentralView ArticlePubMedGoogle Scholar
- Jungermann K, Katz N: Functional specialization of different hepatocyte populations. Physiol Rev. 1989, 69: 708-764.PubMedGoogle Scholar
- Stokkan KA, Yamazaki S, Tei H, Sakaki Y, Menaker M: Entrainment of the circadian clock in the liver by feeding. Science. 2001, 291: 490-493. 10.1126/science.291.5503.490.View ArticlePubMedGoogle Scholar
- Ueda HR, Chen W, Adachi A, Wakamatsu H, Hayashi S, Takasugi T, Nagano M, Nakahama K, Suzuki Y, Sugano S, Iino M, Shigeyoshi Y, Hashimoto S: A transcription factor response element for gene expression during circadian night. Nature. 2002, 418: 534-539. 10.1038/nature00906.View ArticlePubMedGoogle Scholar
- Chen C, Lin SC: The core domain of RGS16 retains G-protein binding and GAP activity in vitro, but is not functional in vivo. FEBS Lett. 1998, 422: 359-362. 10.1016/S0014-5793(98)00042-8.View ArticlePubMedGoogle Scholar
- Druey KM, Ugur O, Caron JM, Chen CK, Backlund PS, Jones TL: Amino-terminal cysteine residues of RGS16 are required for palmitoylation and modulation of Gi- and Gq-mediated signaling. J Biol Chem. 1999, 274: 18836-18842. 10.1074/jbc.274.26.18836.View ArticlePubMedGoogle Scholar
- Cherrington AD: Control of glucose production in vivo by insulin and glucagon. Handbook of Physiology The Endocrine System; The Endocrine Pancreas and Regulation of Metabolism. 2001, Bethesda, MD, Am. Physiol. Soc., vol. II, chapt. 26: 759–785-Google Scholar
- Raju B, Cryer PE: Maintenance of the postabsorptive plasma glucose concentration: insulin or insulin plus glucagon?. Am J Physiol Endocrinol Metab. 2005, 289: E181-6. 10.1152/ajpendo.00460.2004.View ArticlePubMedGoogle Scholar
- Jungermann K: Role of intralobular compartmentation in hepatic metabolism. Diabete Metab. 1992, 18: 81-86.PubMedGoogle Scholar
- Iyengar R, Rich KA, Herberg JT, Premont RT, Codina J: Glucagon receptor-mediated activation of Gs is accompanied by subunit dissociation. J Biol Chem. 1988, 263: 15348-15353.PubMedGoogle Scholar
- Herzig S, Long F, Jhala US, Hedrick S, Quinn R, Bauer A, Rudolph D, Schutz G, Yoon C, Puigserver P, Spiegelman B, Montminy M: CREB regulates hepatic gluconeogenesis through the coactivator PGC-1. Nature. 2001, 413: 179-183. 10.1038/35093131.View ArticlePubMedGoogle Scholar
- Krumins AM, Barker SA, Huang C, Sunahara RK, Yu K, Wilkie TM, Gold SJ, Mumby SM: Differentially regulated expression of endogenous RGS4 and RGS7. J Biol Chem. 2004, 279: 2593-2599. 10.1074/jbc.M311600200.View ArticlePubMedGoogle Scholar
- Russell DW: Molecular Cloning: A laboratory manual. Edited by: Sambrook J RD. 2001, Cold Spring Harbor, NY, Cold Spring Harbor Press, 3: 3rdGoogle Scholar
- Seroogy KB HJP: In situ hybridization approaches to the study of the nervous system. Neurochemistry: A practical approach. Edited by: Turner AJ BHS. 1997, Oxford, Oxford Univ Press, 121-150.Google Scholar
- Hattori M, Tugores A, Veloz L, Karin M, Brenner DA: A simplified method for the preparation of transcriptionally active liver nuclear extracts. DNA Cell Biol. 1990, 9: 777-781.View ArticlePubMedGoogle Scholar
- Srivastava RAKS: Measurements of rate of transcription in isolated nuclei bynuclear "run-off" assay. The Nucleic Acid Protocol Handbook. Edited by: Rapley R. 2000, Totowa, New Jersey, Humana Press, 207 -2212.Google Scholar
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