Clustering of GLUT4, TUG, and RUVBL2 protein levels correlate with myosin heavy chain isoform pattern in skeletal muscles, but AS160 and TBC1D1 levels do not

Skeletal muscle is a heterogeneous tissue. To further elucidate this heterogeneity, we probed relationships between myosin heavy chain (MHC) isoform composition and abundance of GLUT4 and four other proteins that are established or putative GLUT4 regulators [Akt substrate of 160 kDa (AS160), Tre-2/Bub2/Cdc 16-domain member 1 (TBC1D1), Tethering protein containing an UBX-domain for GLUT4 (TUG), and RuvB-like protein two (RUVBL2)] in 12 skeletal muscles or muscle regions from Wistar rats [adductor longus, extensor digitorum longus, epitrochlearis, gastrocnemius (mixed, red, and white), plantaris, soleus, tibialis anterior (red and white), tensor fasciae latae, and white vastus lateralis]. Key results were 1) significant differences found among the muscles (range of muscle expression values) for GLUT4 (2.5-fold), TUG (1.7-fold), RUVBL2 (2.0-fold), and TBC1D1 (2.7-fold), but not AS160; 2) significant positive correlations for pairs of proteins: GLUT4 vs. TUG (R = 0.699), GLUT4 vs. RUVBL2 (R = 0.613), TUG vs. RUVBL2 (R = 0.564), AS160 vs. TBC1D1 (R = 0.293), and AS160 vs. TUG (R = 0.246); 3) significant positive correlations for %MHC-I: GLUT4 (R = 0.460), TUG (R = 0.538), and RUVBL2 (R = 0.511); 4) significant positive correlations for %MHC-IIa: GLUT4 (R = 0.293) and RUVBL2 (R = 0.204); 5) significant negative correlations for %MHC-IIb vs. GLUT4 (R = -0.642), TUG (R = -0.626), and RUVBL2 (R = -0.692); and 6) neither AS160 nor TBC1D1 significantly correlated with MHC isoforms. In 12 rat muscles, GLUT4 abundance tracked with TUG and RUVBL2 and correlated with MHC isoform expression, but was unrelated to AS160 or TBC1D1. Our working hypothesis is that some of the mechanisms that regulate GLUT4 abundance in rat skeletal muscle also influence TUG and RUVBL2 abundance.     

Insulin-stimulated Phosphorylation of the Rab GTPase-activating Protein TBC1D1 Regulates GLUT4 Translocation

Insulin stimulates the translocation of the glucose transporter GLUT4 from intracellular locations to the plasma membrane in adipose and muscle cells. Prior studies have shown that Akt phosphorylation of the Rab GTPase-activating protein, AS160 (160-kDa Akt substrate; also known as TBC1D4), triggers GLUT4 translocation, most likely by suppressing its Rab GTPase-activating protein activity. However, the regulation of a very similar protein, TBC1D1 (TBC domain family, member 1), which is mainly found in muscle, in insulin-stimulated GLUT4 translocation has been unclear. In the present study, we have identified likely Akt sites of insulin-stimulated phosphorylation of TBC1D1 in C2C12 myotubes. We show that a mutant of TBC1D1, in which several Akt sites have been converted to alanine, is considerably more inhibitory to insulin-stimulated GLUT4 translocation than wild-type TBC1D1. This result thus indicates that similar to AS160, Akt phosphorylation of TBC1D1 enables GLUT4 translocation. We also show that in addition to Akt activation, activation of the AMP-dependent protein kinase partially relieves the inhibition of GLUT4 translocation by TBC1D1. Finally, we show that the R125W variant of TBC1D1, which has been genetically associated with obesity, is equally inhibitory to insulin-stimulated GLUT4 translocation, as is wild-type TBC1D1, and that healthy and type 2 diabetic individuals express approximately the same level of TBC1D1 in biopsies of vastus lateralis muscle. In conclusion, phosphorylation of TBC1D1 is required for GLUT4 translocation. Thus, the regulation of TBC1D1 resembles that of its paralog, AS160.Insulin stimulates glucose transport into adipose and muscle cells by increasing the amount of the GLUT4 glucose transporter at the cell surface by a process termed GLUT4 translocation (1, 2). Unstimulated adipocytes and myotubes sequester GLUT4 in intracellular compartments. Insulin activates signaling cascades that lead to the trafficking of specialized GLUT4 vesicles to the cell membrane and fusion of the vesicles therewith. A key signaling pathway for GLUT4 translocation proceeds from the insulin receptor through the activation of the protein kinase Akt. One Akt substrate that connects signaling to GLUT4 trafficking is the Rab GTPase-activating protein (GAP)³ known as AS160. There is now considerable evidence for the following scheme (2, 3): under basal conditions, AS160 acts as a brake on GLUT4 translocation by maintaining one or more Rab proteins required for translocation in their inactive GDP state; in response to insulin, Akt phosphorylates AS160 and thereby suppresses its GAP activity; as a consequence, the elevation of the GTP form of the Rab proteins occurs, leading to the increased docking and subsequent fusion of the GLUT4 vesicles at the plasma membrane.More recently, we and others have characterized a paralog of AS160 known as TBC1D1 (4–7). Overall, TBC1D1 is 47% identical to AS160, with the GAP domain being 79% identical (4). Its GAP domain has the same Rab specificity as the GAP domain of AS160 (4). TBC1D1 is predominantly expressed in skeletal muscle; its expression in adipocytes is very low (5, 6). Nevertheless, 3T3-L1 adipocytes are a convenient cell type in which to examine the role of proteins in GLUT4 translocation, because insulin causes an ∼10-fold increase in GLUT4 at the cell surface. Previously, we examined the role of TBC1D1 in GLUT4 translocation by overexpressing it in 3T3-L1 adipocytes. Surprisingly, even though insulin led to phosphorylation of TBC1D1 on Akt site(s), ectopic TBC1D1 potently inhibited GLUT4 translocation (4, 5). By contrast, overexpression of AS160 did not inhibit GLUT4 translocation (8). This difference suggested that the regulation of TBC1D1 might be fundamentally different from that of AS160. In the present study, we show that this is not the case. By reducing the level of ectopic TBC1D1, we have obtained evidence that phosphorylation of TBC1D1 on several likely Akt sites relieves the inhibitory effect on GLUT4 translocation. In addition, we have examined the effect of a variant of TBC1D1 genetically associated with obesity on GLUT4 translocation and determined the relative levels of TBC1D1 in muscle biopsies from healthy and type 2 diabetic individuals.     

PKC and Rab13 mediate Ca2+ signal-regulated GLUT4 traffic

Exercise/muscle contraction increases cell surface glucose transporter 4 (GLUT4), leading to glucose uptake to regulate blood glucose level. Elevating cytosolic Ca²⁺ mediates this effect, but the detailed mechanism is not clear yet. We used calcium ionophore ionomycin to raise intracellular cytosolic Ca²⁺ level to explore the underlying mechanism. We showed that in L6 myoblast muscle cells stably expressing GLUT4myc, ionomycin increased cell surface GLUT4myc levels and the phosphorylation of AS160, TBC1D1. siPKCα and siPKCθ but not siPKCδ and siPKCε inhibited the ionomycin-increased cell surface GLUT4myc level. siPKCα, siPKCθ inhibited the phosphorylation of AS160 and TBC1D1 induced by ionomycin. siPKCα and siPKCθ prevented ionomycin-inhibited endocytosis of GLUT4myc. siPKCθ, but not siPKCα inhibited ionomycin-stimulated exocytosis of GLUT4myc. siRab13 but not siRab8a, siRab10 and siRab14 inhibited the exocytosis of GLUT4myc promoted by ionomycin. In summary, ionomycin-promoted exocytosis of GLUT4 is partly reversed by siPKCθ, whereas ionomycin-inhibited endocytosis of GLUT4 requires both siPKCα and siPKCθ. PKCα and PKCθ contribute to ionomycin-induced phosphorylation of AS160 and TBC1D1. Rab13 is required for ionomycin-regulated GLUT4 exocytosis.     

Muscle cells engage Rab8A and myosin Vb in insulin-dependent GLUT4 translocation

Insulin causes translocation of glucose transporter 4 (GLUT4) to the membrane of muscle and fat cells, a process requiring Akt activation. Two Rab-GTPase-activating proteins (Rab-GAP), AS160 and TBC1D1, were identified as Akt substrates. AS160 phosphorylation is required for insulin-stimulated GLUT4 translocation, but the participation of TBC1D1 on muscle cell GLUT4 is unknown. Moreover, there is controversy as to the AS160/TBC1D1 target Rabs in fat and muscle cells, and Rab effectors are unknown. Here we examined the effect of knockdown of AS160, TBC1D1, and Rabs 8A, 8B, 10, and 14 (in vitro substrates of AS160 and TBC1D1 Rab-GAP activities) on insulin-induced GLUT4 translocation in L6 muscle cells. Silencing AS160 or TBC1D1 increased surface GLUT4 in unstimulated cells but did not prevent insulin-induced GLUT4 translocation. Knockdown of Rab8A and Rab14, but not of Rab8B or Rab10, inhibited insulin-induced GLUT4 translocation. Furthermore, silencing Rab8A or Rab14 but not Rab8B or Rab10 restored the basal-state intracellular retention of GLUT4 impaired by AS160 or TBC1D1 knockdown. Lastly, overexpression of a fragment of myosin Vb, a recently identified Rab8A-interacting protein, inhibited insulin-induced GLUT4 translocation and altered the subcellular distribution of GTP-loaded Rab8A. These results support a model whereby AS160, Rab8A, and myosin Vb are required for insulin-induced GLUT4 translocation in muscle cells, potentially as part of a linear signaling cascade. glucose transporter 4; insulin signaling; Rab guanosine 5'-triphosphatases; Rab-guanosine 5'-triphosphatase-activating protein; myosin Vb     

The Rab GTPase-Activating Protein TBC1D4/AS160 Contains an Atypical Phosphotyrosine-Binding Domain That Interacts with Plasma Membrane Phospholipids To Facilitate GLUT4 Trafficking in Adipocytes

The Rab GTPase-activating protein TBC1D4/AS160 regulates GLUT4 trafficking in adipocytes. Nonphosphorylated AS160 binds to GLUT4 vesicles and inhibits GLUT4 translocation, and AS160 phosphorylation overcomes this inhibitory effect. In the present study we detected several new functional features of AS160. The second phosphotyrosine-binding domain in AS160 encodes a phospholipid-binding domain that facilitates plasma membrane (PM) targeting of AS160, and this function is conserved in other related RabGAP/Tre-2/Bub2/Cdc16 (TBC) proteins and an AS160 ortholog in Drosophila. This region also contains a nonoverlapping intracellular GLUT4-containing storage vesicle (GSV) cargo-binding site. The interaction of AS160 with GSVs and not with the PM confers the inhibitory effect of AS160 on insulin-dependent GLUT4 translocation. Constitutive targeting of AS160 to the PM increased the surface GLUT4 levels, and this was attributed to both enhanced AS160 phosphorylation and 14-3-3 binding and inhibition of AS160 GAP activity. We propose a model wherein AS160 acts as a regulatory switch in the docking and/or fusion of GSVs with the PM.     

Insulin sensitivity and inhibition by forskolin,dipyridamole and pentobarbital of glucose transport in three L6 muscle cell lines

L6 skeletal muscle myoblasts stably overexpressing glucose transporter GLUT1 or GLUT4 with exofacial myc-epitope tags were characterized for their response to insulin. In clonally selected cultures, 2-deoxyglucose uptake into L6-GLUT1myc myoblasts and myotubes was linear within the time of study. In L6-GLUT1myc and L6-GLUT4myc myoblasts, 100 nmol/L insulin treatment increased the GLUT1 content of the plasma membrane by 1.58±0.01 fold and the GLUT4 content 1.96±0.11 fold, as well as the 2-deoxyglucose uptake 1.53±0.09 and 1.86±0.17 fold respectively, all by a wortmannin-inhibitable manner. The phosphorylation of Akt in these two cell lines was increased by insulin. L6-GLUT1myc myoblasts showed a dose-dependent stimulation of glucose uptake by insulin, with unaltered sensitivity and maximal responsiveness compared with wild type cells. By contrast, the improved insulin responsiveness and sensitivity of glucose uptake were observed in L6-GLUT4myc myoblasts. Earlier studies indicated that forskolin might affect insulin-stimulated GLUT4 translocation. A 65% decrease of insulin-stimulated 2-deoxyglucose uptake in GLUT4myc cells was not due to an effect on GLUT4 mobilization to the plasma membrane, but instead on direct inhibition of GLUT4. Forskolin and dipyridamole are more potent inhibitors of GLUT4 than GLUT1. Alternatively, pentobarbital inhibits GLUT1 more than GLUT4. The use of these inhibitors confirmed that the overexpressed GLUT1 or GLUT4 are the major functional glucose transporters in unstimulated and insulin-stimulated L6 myoblasts. Therefore, L6-GLUT1myc and L6-GLUT4myc cells provide a platform to screen compounds that may have differential effects on GLUT isoform activity or may influence GLUT isoform mobilization to the cell surface of muscle cells.     

Insulin sensitivity and inhibition by forskolin, dipyridamole and pentobarbital of glucose transport in three L6 muscle cell lines

L6 skeletal muscle myoblasts stably overexpressing glucose transporter GLUT1 or GLUT4 with exofa- cial myc-epitope tags were characterized for their response to insulin. In clonally selected cultures, 2-deoxyglucose uptake into L6-GLUT1myc myoblasts and myotubes was linear within the time of study. In L6-GLUT1myc and L6-GLUT4myc myoblasts, 100 nmol/L insulin treatment increased the GLUT1 content of the plasma membrane by 1.58±0.01 fold and the GLUT4 content 1.96±0.11 fold, as well as the 2-deoxyglucose uptake 1.53±0.09 and 1.86±0.17 fold respectively, all by a wortmannin-inhibitable manner. The phosphorylation of Akt in these two cell lines was increased by insulin. L6-GLUT1myc myoblasts showed a dose-dependent stimulation of glucose uptake by insulin, with unaltered sensitiv- ity and maximal responsiveness compared with wild type cells. By contrast, the improved insulin re- sponsiveness and sensitivity of glucose uptake were observed in L6-GLUT4myc myoblasts. Earlier studies indicated that forskolin might affect insulin-stimulated GLUT4 translocation. A 65% decrease of insulin-stimulated 2-deoxyglucose uptake in GLUT4myc cells was not due to an effect on GLUT4 mobi- lization to the plasma membrane, but instead on direct inhibition of GLUT4. Forskolin and dipyridamole are more potent inhibitors of GLUT4 than GLUT1. Alternatively, pentobarbital inhibits GLUT1 more than GLUT4. The use of these inhibitors confirmed that the overexpressed GLUT1 or GLUT4 are the major functional glucose transporters in unstimulated and insulin-stimulated L6 myoblasts. Therefore, L6-GLUT1myc and L6-GLUT4myc cells provide a platform to screen compounds that may have differ- ential effects on GLUT isoform activity or may influence GLUT isoform mobilization to the cell surface of muscle cells.     

Akt substrate TBC1D1 regulates GLUT1 expression through the mTOR pathway in 3T3-L1 adipocytes

Multiple studies have suggested that the protein kinase Akt/PKB (protein kinase B) is required for insulin-stimulated glucose transport in skeletal muscle and adipose cells. In an attempt to understand links between Akt activation and glucose transport regulation, we applied mass spectrometry-based proteomics and bioinformatics approaches to identify potential Akt substrates containing the phospho-Akt substrate motif RXRXXpS/T. The present study describes the identification of the Rab GAP (GTPase-activating protein)-domain containing protein TBC1D1 [TBC (Tre-2/Bub2/Cdc16) domain family, member 1], which is closely related to TBC1D4 [TBC domain family, member 4, also denoted AS160 (Akt substrate of 160 kDa)], as an Akt substrate that is phosphorylated at Thr(590). RNAi (RNA interference)-mediated silencing of TBC1D1 elevated basal deoxyglucose uptake by approx. 61% in 3T3-L1 mouse embryo adipocytes, while the suppression of TBC1D4 and RapGAP220 under the same conditions had little effect on basal and insulin-stimulated deoxyglucose uptake. Silencing of TBC1D1 strongly increased expression of the GLUT1 glucose transporter but not GLUT4 in cultured adipocytes, whereas the decrease in TBC1D4 had no effect. Remarkably, loss of TBC1D1 in 3T3-L1 adipocytes activated the mTOR (mammalian target of rapamycin)-p70 S6 protein kinase pathway, and the increase in GLUT1 expression in the cells treated with TBC1D1 siRNA (small interfering RNA) was blocked by the mTOR inhibitor rapamycin. Furthermore, overexpression of the mutant TBC1D1-T590A, lacking the putative Akt/PKB phosphorylation site, inhibited insulin stimulation of p70 S6 kinase phosphorylation at Thr(389), a phosphorylation induced by mTOR. Taken together, our data suggest that TBC1D1 may be involved in controlling GLUT1 glucose transporter expression through the mTOR-p70 S6 kinase pathway.     

Protein kinase WNK1 promotes cell surface expression of glucose transporter GLUT1 by regulating a Tre-2/USP6-BUB2-Cdc16 domain family member 4 (TBC1D4)-Rab8A complex

One mechanism by which mammalian cells regulate the uptake of glucose is the number of glucose transporter proteins (GLUT) present at the plasma membrane. In insulin-responsive cells types, GLUT4 is released from intracellular stores through inactivation of the Rab GTPase activating protein Tre-2/USP6-BUB2-Cdc16 domain family member 4 (TBC1D4) (also known as AS160). Here we describe that TBC1D4 forms a protein complex with protein kinase WNK1 in human embryonic kidney (HEK293) cells. We show that WNK1 phosphorylates TBC1D4 in vitro and that the expression levels of WNK1 in these cells regulate surface expression of the constitutive glucose transporter GLUT1. WNK1 was found to increase the binding of TBC1D4 to regulatory 14-3-3 proteins while reducing its interaction with the exocytic small GTPase Rab8A. These effects were dependent on the catalytic activity because expression of a kinase-dead WNK1 mutant had no effect on binding of 14-3-3 and Rab8A, or on surface GLUT1 levels. Together, the data describe a pathway regulating constitutive glucose uptake via GLUT1, the expression level of which is related to several human diseases.     

Unraveling the complex genetic model for cystic fibrosis: pleiotropic effects of modifier genes on early cystic fibrosis-related morbidities

The existence of pleiotropy in disorders with multi-organ involvement can suggest therapeutic targets that could ameliorate overall disease severity. Here we assessed pleiotropy of modifier genes in cystic fibrosis (CF). CF, caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, affects the lungs, liver, pancreas and intestines. However, modifier genes contribute to variable disease severity across affected organs, even in individuals with the same CFTR genotype. We sought to determine whether SLC26A9, SLC9A3 and SLC6A14, that contribute to meconium ileus in CF, are pleiotropic for other early-affecting CF co-morbidities. In the Canadian CF population, we assessed evidence for pleiotropic effects on (1) pediatric lung disease severity (n = 815), (2) age at first acquisition of Pseudomonas aeruginosa (P. aeruginosa) (n = 730), and (3) prenatal pancreatic damage measured by immunoreactive trypsinogen (n = 126). A multiple-phenotype analytic strategy assessed evidence for pleiotropy in the presence of phenotypic correlation. We required the same alleles to be associated with detrimental effects. SLC26A9 was pleiotropic for meconium ileus and pancreatic damage (p = 0.002 at rs7512462), SLC9A3 for meconium ileus and lung disease (p = 1.5 × 10^?6 at rs17563161), and SLC6A14 for meconium ileus and both lung disease and age at first P. aeruginosa infection (p = 0.0002 and p = 0.006 at rs3788766, respectively). The meconium ileus risk alleles in SLC26A9, SLC9A3 and SLC6A14 are pleiotropic, increasing risk for other early CF co-morbidities. Furthermore, co-morbidities affecting the same organ tended to associate with the same genes. The existence of pleiotropy within this single disorder suggests that complementary therapeutic strategies to augment solute transport will benefit multiple CF-associated tissues.     

Regulation of glucose transporter 4 translocation by the Rab guanosine triphosphatase-activating protein AS160/TBC1D4: role of phosphorylation and membrane association

Insulin-stimulated translocation of the glucose transporter GLUT4 to the plasma membrane in muscle and fat cells depends on the phosphatidylinositide 3-kinase/Akt pathway. The downstream target AS160/TBC1D4 is phosphorylated upon insulin stimulation and contains a TBC domain (Tre-2/Bub2/Cdc16) that is present in most Rab guanosine triphosphatase-activating proteins. TBC1D4 associates with GLUT4-containing membranes under basal conditions and dissociates from membranes with insulin. Here we show that the association of TBC1D4 with membranes is required for its inhibitory action on GLUT4 translocation under basal conditions. Whereas the insulin-dependent dissociation of TBC1D4 from membranes was not required for GLUT4 translocation, its phosphorylation was essential. Many agonists that stimulate GLUT4 translocation failed to trigger TBC1D4 translocation to the cytosol, but in most cases these agonists stimulated TBC1D4 phosphorylation at T642, and their effects on GLUT4 translocation were inhibited by overexpression of the TBC1D4 phosphorylation mutant (TBC1D4-4P). We postulate that TBC1D4 acts to impede GLUT4 translocation by disarming a Rab protein found on GLUT4-containing-membranes and that phosphorylation of TBC1D4 per se is sufficient to overcome this effect, allowing GLUT4 translocation to the cell surface to proceed.     

Peripheral clock system circadian abnormalities in Cushing’s disease

ABSTRACT

In Cushing’s syndrome, the cortisol rhythm is impaired and can be associated with the disruption in the rhythmic expression of clock genes. In this study, we evaluated the expression of CLOCK, BMAL1, CRY1, CRY2, PER1, PER2, PER3 genes in peripheral blood leukocytes of healthy individuals (n = 13) and Cushing’s disease (CD) patients (n = 12). Participants underwent salivary cortisol measurement at 0900 h and 2300 h. Peripheral blood samples were obtained at 0900 h, 1300 h, 1700 h, and 2300 h for assessing clock gene expression by qPCR. Gene expression circadian variations were evaluated by the Cosinor method. In healthy controls, a circadian variation in the expression of CLOCK, BMAL1, CRY1, PER2, and PER3 was observed, whereas the expression of PER1 and CRY2 followed no specific pattern. The expression of PER2 and PER3 in healthy leukocytes presented a late afternoon acrophase, similarly to CLOCK, whereas CRY1 showed night acrophase, similarly to BMAL1. In CD patients, the circadian variation in the expression of clock genes was lost, along with the abolition of cortisol circadian rhythm. However, CRY2 exhibited a circadian variation with acrophase during the dark phase in patients. In conclusion, our data suggest that Cushing’s disease, which is characterized by hypercortisolism, is associated with abnormalities in the circadian pattern of clock genes. Higher expression of CRY2 at night outlines its putative role in the cortisol circadian rhythm disruption.     

The porcine <Emphasis Type="Italic">TBC1D1</Emphasis> gene: mapping,SNP identification,and association study with meat,carcass and production traits in Italian heavy pigs

TBC1D1 [TBC1 (tre-2/USP6, BUB2, cdc16) domain family, member 1] is a Rab-GTPase-activating related protein implicated in regulating the trafficking of glucose transporter 4 (GLUT4 or SLC2A4) storage vesicles to the cell surface in response to insulin and AMPK-activating stimuli in skeletal muscle. Mutations in the human and mouse TBC1D1 genes confer risk of obesity or leanness. We identified five single nucleotide polymorphisms (SNPs) in the porcine TBC1D1 gene. One of them (FN677935:g.219G>A) was genotyped either by high resolution melting and PCR-RFLP analyses to study allele frequencies in a few pig breeds and evaluate association with meat production and carcass traits in five groups of sib-tested pigs of Italian Large White and Italian Duroc breeds. The g.219G>A SNP was associated (P < 0.05) with ham weight, back fat thickness and lean cuts content in Italian Large White and with visible intermuscular fat in Italian Duroc pigs.     

Evaluation of housekeeping genes as references for quantitative real-time PCR analysis of European eel,Anguilla anguilla

Eels are important aquaculture species for which an increasing number of reference genes are being identified and applied. In this study, five housekeeping genes [RPL7 (ribosomal protein L7), 18 S (18 S ribosomal RNA), EF1A (elongation factor 1α), ACTB (β-actin) and GAPDH (glyceraldehyde-3-phosphate dehydrogenase)] were chosen to evaluate their reliability as reference genes for quantitative real-time PCR (qPCR) for the study of Anguilla anguilla. The expression of the selected genes in different eel tissues was determined using qPCR at different growth stages or upon challenge by Anguillid herpesvirus (AngHV), and the expression levels of these genes were then compared and evaluated using the geNorm and NormFinder algorithms. Then, RefFinder was used to comprehensively rank the examined housekeeping genes. Interestingly, the expression of the evaluated housekeeping genes exhibited tissue-dependent and treatment-dependent variations. In different growth periods A. anguilla tissues, the most stable genes were the following: ACTB in mucus; 18 S in skin and kidney; RPL7 in muscle, gill, intestine and brain; EF1A in heart and liver; and GAPDH in spleen. In contrast, in AngHV-challenged A. anguilla tissues, the most stable genes were the following: 18 S in mucus; RPL7 in skin, gill, heart, spleen, kidney and intestine; EF1A in muscle and liver; and ACTB in brain. Further comparison analysis indicated that the expression of RPL7 and EF1A was stable in multiple A. anguilla tissues in different growth periods and in eels challenged by AngHV. Nonetheless, the expression level of GAPDH in eel tissues was lower, and it was unstable in several tissues. These results indicated that the selection of reference genes for qPCR analysis in A. anguilla should be made in accordance with experimental parameters, and both RPL7 and EF1A could be used as reference genes for qPCR study of A. anguilla at different growth stages or upon challenge by AngHV. The reference genes identified in this study could improve the accuracy of qPCR data and facilitate further studies aimed at understanding the biology of eels.