Growth hormone resistance induced by amino acid deprivation in fao cells is independent of FGF21.

Adequate dietary intake of amino acids is imperative for normal animal growth. Our previous work using rat hepatocarcinoma Fao cells demonstrated that growth hormone (GH) resistance, coupled with a concurrent reduction in insulin-like growth factor 1 (Igf1) mRNA levels, may underlie the growth retardation associated with a low-protein diet (LPD). In this study, we investigated whether FGF21 contributes to liver GH resistance in Fao rat hepatoma cells under amino acid deprivation conditions. Mice subjected to an LPD exhibited growth retardation, compromised GH signaling in the liver, and decreased blood IGF-1 levels compared with those on a control diet. To assess the potential involvement of fibroblast growth factor (FGF) 21, produced in response to amino acid deficiency, in the development of GH resistance, we examined GH signaling and Igf1 mRNA levels in Fao cells cultured in amino acid-deprived medium. Despite the inhibition of Fgf21 expression by the integrated stress response inhibitor, an inhibitor of the eukaryotic initiation factor 2-activating transcription factor 4 pathway, GH resistance persisted in response to amino acid deprivation. Additionally, the introduction of FGF21 into the control medium did not impair either GH signaling or GH-induced Igf1 transcription. These data suggest that, in Fao cells, amino acid deprivation induces GH resistance independently of FGF21 activity. By shedding light on the mechanisms behind growth retardation-associated GH resistance linked to amino acid deficiencies, our findings provide valuable insights for clinicians in formulating effective treatment strategies for individuals facing these challenges.

Somatic GJA4 gain-of-function mutation in orbital cavernous venous malformations.

Orbital cavernous venous malformation (OCVM) is a sporadic vascular anomaly of uncertain etiology characterized by abnormally dilated vascular channels. Here, we identify a somatic missense mutation, c.121G > T (p.Gly41Cys) in GJA4, which encodes a transmembrane protein that is a component of gap junctions and hemichannels in the vascular system, in OCVM tissues from 25/26 (96.2%) individuals with OCVM. GJA4 expression was detected in OCVM tissue including endothelial cells and the stroma, through immunohistochemistry. Within OCVM tissue, the mutation allele frequency was higher in endothelial cell-enriched fractions obtained using magnetic-activated cell sorting. Whole-cell voltage clamp analysis in Xenopus oocytes revealed that GJA4 c.121G > T (p.Gly41Cys) is a gain-of-function mutation that leads to the formation of a hyperactive hemichannel. Overexpression of the mutant protein in human umbilical vein endothelial cells led to a loss of cellular integrity, which was rescued by carbenoxolone, a non-specific gap junction/hemichannel inhibitor. Our data suggest that GJA4 c.121G > T (p.Gly41Cys) is a potential driver gene mutation for OCVM. We propose that hyperactive hemichannel plays a role in the development of this vascular phenotype.

The IGF-Independent Role of IRS-2 in the Secretion of MMP-9 Enhances the Growth of Prostate Carcinoma Cell Line PC3.

Insulin receptor substrate-2 (IRS-2), a substrate of the insulin-like growth factor (IGF)-I receptor, is highly expressed in the prostate cancer cell line, PC3. We recently demonstrated that extracellular signal-regulated kinase (Erk1/2), a kinase downstream of IGF signaling, is activated in PC3 cells under serum starvation, and this activation can be inhibited by IRS-2 knockdown. Here, we observed that adding an IGF-I-neutralizing antibody to the culture medium inhibited the activation of Erk1/2. Suppression of Erk1/2 in IRS-2 knockdown cells was restored by the addition of a PC3 serum-free conditioned medium. In contrast, the IRS-2-silenced PC3 conditioned medium could not restore Erk1/2 activation, suggesting that IRS-2 promotes the secretion of proteins that activate the IGF signaling pathway. Furthermore, gelatin zymography analysis of the conditioned medium showed that matrix metalloproteinase-9 (MMP-9) was secreted extracellularly in an IRS-2 dependent manner when PC3 was cultured under serum starvation conditions. Moreover, MMP-9 knockdown suppressed Erk1/2 activation, DNA synthesis, and migratory activity. The IRS-2 levels were positively correlated with Gleason grade in human prostate cancer tissues. These data suggest that highly expressed IRS-2 activates IGF signaling by enabling the secretion of MMP-9, which is associated with hyperproliferation and malignancy of prostate cancer cell line, PC3.

Dietary lysine restriction induces lipid accumulation in skeletal muscle through an increase in serum threonine levels in rats.

We previously reported that dietary amino acid restriction induces the accumulation of triglycerides (TAG) in the liver of growing rats. However, differences in TAG accumulation in individual cell types or other tissues were not examined. In this study, we show that TAG also accumulates in the muscle and adipose tissues of rats fed a low amino acid (low-AA) diet. In addition, dietary lysine restriction (low-Lys) induces lipid accumulation in muscle and adipose tissues. In adjusting the nitrogen content to that of the control diet, we found that glutamic acid supplementation to the low-AA diet blocked lipid accumulation, but supplementation with the low-Lys diet did not, suggesting that a shortage of nitrogen caused lipids to accumulate in the skeletal muscle in the rats fed a low-AA diet. Serum amino acid measurement revealed that, in rats fed a low-Lys diet, serum lysine levels were decreased, while serum threonine levels were significantly increased compared with the control rats. When the threonine content was restricted in the low-Lys diet, TAG accumulation induced by the low-Lys diet was completely abolished in skeletal muscle. Moreover, in L6 myotubes cultured in medium containing high threonine and low lysine, fatty acid uptake was enhanced compared with that in cells cultured in control medium. These findings suggest that the increased serum threonine in rats fed a low-Lys diet resulted in lipid incorporation into skeletal muscle, leading to the formation of fatty muscle tissue. Collectively, we propose conceptual hypothesis that "amino-acid signal" based on lysine and threonine regulates lipid metabolism.

A translation repressor, 4E-BP1, regulates the triglyceride level in rat liver during protein deprivation.

Protein deprivation has been shown to induce fatty liver in humans and animals, but the molecular mechanisms underlying such induction are largely unknown. Our previous studies have shown that a low-protein diet increases eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) protein and triglyceride (TG) levels in rat liver. 4E-BP1 is known to repress translation by binding to eIF4E. There is also evidence indicating that 4E-BP1 regulates lipid metabolism. Here, we examined the role of 4E-BP1 on TG accumulation in the livers of rats under protein deprivation. The low-protein diet rapidly increased the hepatic 4E-BP1 mRNA level within 1 day, followed by the induction of hepatic TG accumulation. The knockdown of hepatic 4E-BP1 attenuated the TG accumulation in rat liver induced by the low-protein diet. 4E-BP1 knockdown also increased the protein level of carnitine palmitoyltransferase 1A (CPT1A), a regulator of fatty acid oxidation, in the liver of rats fed a low-protein diet. These results indicate that a low-protein diet increases the amount of 4E-BP1, leading to TG accumulation in rat liver. We thus conclude that 4E-BP1 plays an important role in inducing hepatic steatosis under protein deprivation.

Lysosomal targeting of SIDT2 via multiple YxxΦ motifs is required for SIDT2 function in the process of RNautophagy.

RNA degradation is an essential process for maintaining cellular homeostasis. Previously, we discovered a novel RNA degradation system, RNautophagy, during which direct import of RNA into lysosomes in an ATP-dependent manner followed by degradation takes place. The putative nucleic acid transporter SID-1 transmembrane family member 2 (SIDT2) predominantly localizes to lysosomes and mediates the translocation of RNA into lysosomes during RNautophagy. However, little is known about the mechanisms of sorting SIDT2 to lysosomes. Here, we show that three cytosolic YxxΦ motifs (in which x is any amino acid and Φ is an amino acid with a bulky hydrophobic side chain) are required for the lysosomal localization of SIDT2, and that SIDT2 interacts with adaptor protein complexes AP-1 and AP-2. We also find that localization to lysosomes by these three motifs is necessary for SIDT2 function in the process of RNautophagy, and that SIDT2 strikingly increases endogenous RNA degradation at the cellular level. To our knowledge, this is the first study to report an endogenous intracellular protein for which overexpression substantially increased intracellular RNA degradation. This study provides new insight into lysosomal targeting of proteins and intracellular RNA degradation, and further confirms the critical function of SIDT2 in RNautophagy.This article has an associated First Person interview with the first author of the paper.

SM22α (Smooth Muscle Protein 22-α) Promoter-Driven IGF1R (Insulin-Like Growth Factor 1 Receptor) Deficiency Promotes Atherosclerosis.

Objective- IGF-1 (insulin-like growth factor 1) is a major autocrine/paracrine growth factor, which promotes cell proliferation, migration, and survival. We have shown previously that IGF-1 reduced atherosclerosis and promoted features of stable atherosclerotic plaque in Apoe-/- mice-an animal model of atherosclerosis. The aim of this study was to assess effects of smooth muscle cell (SMC) IGF-1 signaling on the atherosclerotic plaque. Approach and Results- We generated Apoe-/- mice with IGF1R (IGF-1 receptor) deficiency in SMC and fibroblasts (SM22α [smooth muscle protein 22 α]-CreKI/IGF1R-flox mice). IGF1R was decreased in the aorta and adventitia of SM22α-CreKI/IGF1R-flox mice and also in aortic SMC, embryonic, skin, and lung fibroblasts isolated from SM22α-CreKI/IGF1R-flox mice. IGF1R deficiency downregulated collagen mRNA-binding protein LARP6 (La ribonucleoprotein domain family, member 6) and vascular collagen, and mice exhibited growth retardation. The high-fat diet-fed SM22α-CreKI/IGF1R-flox mice had increased atherosclerotic burden and inflammatory responses. α-SMA (α-smooth muscle actin)-positive plaque cells had reduced proliferation and elevated apoptosis. SMC/fibroblast-targeted decline in IGF-1 signaling decreased atherosclerotic plaque SMC, markedly depleted collagen, reduced plaque fibrous cap, and increased plaque necrotic cores. Aortic SMC isolated from SM22α-CreKI/IGF1R-flox mice had decreased cell proliferation, migration, increased sensitivity to apoptosis, and these effects were associated with disruption of IGF-1-induced Akt signaling. Conclusions- IGF-1 signaling in SMC and in fibroblast is a critical determinant of normal vascular wall development and atheroprotection.

Low-protein diet enhances adiponectin secretion in rats.

Previous studies including ours have shown that a low-protein diet up-regulates insulin signaling in the liver and muscle and induces fatty liver in rats. Adiponectin is known as an insulin-sensitizing adipocytokine. We, therefore, examined the effect of a low-protein diet on the adiponectin levels in rats. The low-protein diet significantly increased serum adiponectin level. However, mRNA and protein levels of adiponectin in white adipose tissue (WAT) were not changed by the low-protein diet. Since it is known that oligomerization is important to control serum adiponectin level, we examined the population of adiponectin oligomeric forms in WAT and found that low-protein diet did not change it. Despite these events, the amount of its secretion was significantly increased in the adipocytes isolated from WAT of low-protein diet-fed rats. These results indicate that a low-protein diet enhances adiponectin secretion, which is not due to the increased intracellular amount and oligomerization of adiponectin.

USP40 gene knockdown disrupts glomerular permeability in zebrafish.

Unbiased transcriptome profiling and functional genomics approaches have identified ubiquitin-specific protease 40 (USP40) as a highly specific glomerular transcript. This gene product remains uncharacterized, and its biological function is completely unknown. Here, we showed that mouse and rat glomeruli exhibit specific expression of the USP40 protein, which migrated at 150 kDa and was exclusively localized in the podocyte cytoplasm of the adult kidney. Double-labeling immunofluorescence staining and confocal microscopy analysis of fetal and neonate kidney samples revealed that USP40 was also expressed in the vasculature, including in glomerular endothelial cells at the premature stage. USP40 in cultured glomerular endothelial cells and podocytes was specifically localized to the intermediate filament protein nestin. In glomerular endothelial cells, immunoprecipitation confirmed actual protein-protein binding of USP40 with nestin, and USP40-small-interfering RNA transfection revealed significant reduction of nestin. In a rat model of minimal-change nephrotic syndrome, USP40 expression was apparently reduced, which was also associated with the reduction of nestin. Zebrafish morphants lacking Usp40 exhibited disorganized glomeruli with the reduction of the cell junction in the endothelium and foot process effacement in the podocytes. Permeability studies in these zebrafish morphants demonstrated a disruption of the selective glomerular permeability filter. These data indicate that USP40/Usp40 is a novel protein that might play a crucial role in glomerulogenesis and the glomerular integrity after birth through the modulation of intermediate filament protein homeostasis.

Triglyceride synthesis in hepatocytes isolated from rats fed a low-protein diet is enhanced independently of upregulation of insulin signaling.

It is known that protein malnutrition develops fatty liver in rats. However, the mechanisms by which protein malnutrition enhances lipid accumulation in the liver are not fully understood. Our previous studies have demonstrated that protein malnutrition upregulates insulin signaling with an increase in TG levels in rat livers. Here, we examined whether the upregulated insulin signaling contributes to an enhancement of TG accumulation under protein malnutrition. As it is difficult to analyze insulin-induced hepatic TG synthesis in vivo, the isolated hepatocytes derived from rats fed a low-protein diet were used. The hepatocytes were isolated from rats fed a 15% casein diet (15C) as a control diet or a 5% casein diet (5C) as a low-protein diet and then treated with insulin. As shown in vivo, insulin signaling was upregulated in isolated hepatocytes from 5C-fed rats (5C hepatocytes). However, the insulin-induced increase in the mRNA levels of lipogenic enzymes, including acetyl-CoA carboxylase 1 (ACC1) and fatty acid synthase (FAS), was similar in both groups. The amounts of TG synthesized from both glucose and palmitate, as well as ACC1 and FAS protein levels, were increased at the basal state in 5C hepatocytes, but were not further increased by insulin. These results indicate that TG synthesis via both de novo fatty acid synthesis and esterification is enhanced in 5C hepatocytes, which is independent of the upregulation of insulin signaling.

USP15 attenuates IGF-I signaling by antagonizing Nedd4-induced IRS-2 ubiquitination.

Insulin receptor substrates (IRSs) are phosphorylated by IGF-I receptor tyrosine kinase in a ligand-dependent manner. In turn, they bind to and activate effector proteins such as PI3K, leading to various cell responses including cell proliferation. We had reported that ubiquitin ligase Nedd4 induces mono-ubiquitination of IRS-2, thereby enhancing IRS-2 tyrosine phosphorylation, leading to increased IGF signaling and mitogenic activity. Here we show that ubiquitin-specific protease 15 (USP15) antagonizes the effect of Nedd4 on IRS-2. We identified USP15 as a protein that preferentially bound to IRS-2 when IRS-2 was conjugated with ubiquitin. In HEK293 cells, Nedd4 overexpression induced IRS-2 ubiquitination, which was decreased by USP15 co-expression while increased by USP15 knockdown. Nedd4 overexpression enhanced IGF-I-dependent IRS-2 tyrosine phosphorylation, and USP15 co-expression suppressed it. Conversely, USP15 knockdown increased IRS-2 tyrosine phosphorylation and downstream signaling in prostate cancer PC-3 cells. We concluded that USP15 attenuates IGF-I signaling by antagonizing Nedd4-induced IRS-2 ubiquitination.

Roles of chondroitin sulfate proteoglycan 4 in fibrogenic/adipogenic differentiation in skeletal muscle tissues.

Intramuscular adipose tissue and fibrous tissue are observed in some skeletal muscle pathologies such as Duchenne muscular dystrophy and sarcopenia, and affect muscle strength and myogenesis. They originate from common fibrogenic/adipogenic cells in the skeletal muscle. Thus, elucidating the regulatory mechanisms underlying fibrogenic/adipogenic cell differentiation is an important step toward the mediation of these disorders. Previously, we established a highly adipogenic progenitor clone, 2G11, from rat skeletal muscle and showed that basic fibroblast growth factor (bFGF) is pro-adipogenic in these cells. Here, we demonstrated that 2G11 cells give rise to fibroblasts upon transforming growth factor (TGF)-ß1 stimulation, indicating that they possess mesenchymal progenitor cells (MPC)-like characteristics. The previously reported MPC marker PDGFRα is expressed in other cell populations. Accordingly, we produced monoclonal antibodies that specifically bind to 2G11 cell surface antigens and identified chondroitin sulfate proteoglycan 4 (CSPG4) as a potential MPC marker. Based on an RNA interference analysis, we found that CSPG4 is involved in both the pro-adipogenic effect of bFGF and in TGF-ß-induced alpha smooth muscle actin expression and stress fiber formation. By establishing an additional marker for MPC detection and characterizing its role in fibrogenic/adipogenic differentiation, these results will facilitate the development of effective treatments for skeletal muscle pathologies.

Glucose deprivation attenuates sortilin levels in skeletal muscle cells.

In skeletal muscle, sortilin plays a predominant role in the sorting of glucose transporter 4 (Glut4), thereby controlling glucose uptake. Moreover, our previous study suggested that the sortilin expression levels are also implicated in myogenesis. Despite the importance of sortilin in skeletal muscle, however, the regulation of sortilin expression has not been completely understood. In the present study, we analyzed if the sortilin expression is regulated by glucose in C2C12 myocytes and rat skeletal muscles in vivo. Sortilin protein expression was elevated upon C2C12 cell differentiation and was further enhanced in the presence of a high concentration of glucose. The gene expression and protein degradation of sortilin were not affected by glucose. On the other hand, rapamycin partially reduced sortilin induction by a high concentration of glucose, which suggested that sortilin translation could be regulated by glucose, at least in part. We also examined if the sortilin regulation by glucose was also observed in skeletal muscles that were obtained from fed or fasted rats. Sortilin expression in both gastrocnemius and extensor digitorum longus (EDL) muscle was significantly decreased by 17-18h of starvation. On the other hand, pathological levels of high blood glucose did not alter the sortilin expression in rat skeletal muscle. Overall, the present study suggests that sortilin protein levels are reduced under hypoglycemic conditions by post-transcriptional control in skeletal muscles.

RNautophagy/DNautophagy possesses selectivity for RNA/DNA substrates.

Lysosomes can degrade various biological macromolecules, including nucleic acids, proteins and lipids. Recently, we identified novel nucleic acid-degradation systems termed RNautophagy/DNautophagy (abbreviated as RDA), in which RNA and DNA are directly taken up by lysosomes in an ATP-dependent manner and degraded. We also found that a lysosomal membrane protein, LAMP2C, the cytoplasmic region of which binds to RNA and DNA, functions, at least in part, as an RNA/DNA receptor in the process of RDA. However, it has been unclear whether RDA possesses selectivity for RNA/DNA substrates and the RNA/DNA sequences that are recognized by LAMP2C have not been determined. In the present study, we found that the cytosolic region of LAMP2C binds to poly-G/dG, but not to poly-A/dA, poly-C/dC, poly-dT or poly-U. Consistent with this binding activity, poly-G/dG was transported into isolated lysosomes via RDA, while poly-A/dA, poly-C/dC, poly-dT and poly-U were not. GGGGGG or d(GGGG) sequences are essential for the interaction between poly-G/dG and LAMP2C. In addition to poly-G/dG, G/dG-rich sequences, such as a repeated GGGGCC sequence, interacted with the cytosolic region of LAMP2C. Our findings indicate that RDA does possess selectivity for RNA/DNA substrates and that at least some consecutive G/dG sequence(s) can mediate RDA.

Tumor necrosis factor (TNF)-α-induced repression of GKAP42 protein levels through cGMP-dependent kinase (cGK)-Iα causes insulin resistance in 3T3-L1 adipocytes.

Insulin receptor substrates (IRSs) have been shown to be major mediators of insulin signaling. Recently, we found that IRSs form high-molecular weight complexes, and here, we identify by yeast two-hybrid screening a novel IRS-1-associated protein: a 42-kDa cGMP-dependent protein kinase-anchoring protein (GKAP42). GKAP42 knockdown in 3T3-L1 adipocytes suppressed insulin-dependent IRS-1 tyrosine phosphorylation and downstream signaling, resulting in suppression of GLUT4 translocation to plasma membrane induced by insulin. In addition, GLUT4 translocation was also suppressed in cells overexpressing GKAP42-N (the IRS-1 binding region of GKAP42), which competed with GKAP42 for IRS-1, indicating that GKAP42 binding to IRS-1 is required for insulin-induced GLUT4 translocation. Long term treatment of 3T3-L1 adipocytes with TNF-α, which induced insulin resistance, significantly decreased the GKAP42 protein level. We then investigated the roles of cGMP-dependent kinase (cGK)-Iα, which bound to GKAP42, in these changes. cGK-Iα knockdown partially rescued TNF-α-induced decrease in GKAP42 and impairment of insulin signals. These data indicated that TNF-α-induced repression of GKAP42 via cGK-Iα caused reduction of insulin-induced IRS-1 tyrosine phosphorylation at least in part. The present study describes analysis of the novel TNF-α-induced pathway, cGK-Iα-GKAP42, which regulates insulin-dependent signals and GLUT4 translocation.

Prolyl isomerase Pin1 negatively regulates AMP-activated protein kinase (AMPK) by associating with the CBS domain in the γ subunit.

AMP-activated protein kinase (AMPK) plays a critical role in metabolic regulation. In this study, first, it was revealed that Pin1 associates with any isoform of γ, but not with either the α or the ß subunit, of AMPK. The association between Pin1 and the AMPK γ1 subunit is mediated by the WW domain of Pin1 and the Thr(211)-Pro-containing motif located in the CBS domain of the γ1 subunit. Importantly, overexpression of Pin1 suppressed AMPK phosphorylation in response to either 2-deoxyglucose or biguanide stimulation, whereas Pin1 knockdown by siRNAs or treatment with Pin1 inhibitors enhanced it. The experiments using recombinant Pin1, AMPK, LKB1, and PP2C proteins revealed that the protective effect of AMP against PP2C-induced AMPKα subunit dephosphorylation was markedly suppressed by the addition of Pin1. In good agreement with the in vitro data, the level of AMPK phosphorylation as well as the expressions of mitochondria-related genes, such as PGC-1α, which are known to be positively regulated by AMPK, were markedly higher with reduced triglyceride accumulation in the muscles of Pin1 KO mice as compared with controls. These findings suggest that Pin1 plays an important role in the pathogenic mechanisms underlying impaired glucose and lipid metabolism, functioning as a negative regulator of AMPK.

Novel repressor regulates insulin sensitivity through interaction with Foxo1.

Forkhead box-containing protein o (Foxo) 1 is a key transcription factor in insulin and glucose metabolism. We identified a Foxo1-CoRepressor (FCoR) protein in mouse adipose tissue that inhibits Foxo1's activity by enhancing acetylation via impairment of the interaction between Foxo1 and the deacetylase Sirt1 and via direct acetylation. FCoR is phosphorylated at Threonine 93 by catalytic subunit of protein kinase A and is translocated into nucleus, making it possible to bind to Foxo1 in both cytosol and nucleus. Knockdown of FCoR in 3T3-F442A cells enhanced expression of Foxo target and inhibited adipocyte differentiation. Overexpression of FCoR in white adipose tissue decreased expression of Foxo-target genes and adipocyte size and increased insulin sensitivity in Lepr(db/db) mice and in mice fed a high-fat diet. In contrast, Fcor knockout mice were lean, glucose intolerant, and had decreased insulin sensitivity that was accompanied by increased expression levels of Foxo-target genes and enlarged adipocytes. Taken together, these data suggest that FCoR is a novel repressor that regulates insulin sensitivity and energy metabolism in adipose tissue by acting to fine-tune Foxo1 activity.

In vivo loss of function study reveals the short stature homeobox-containing (shox) gene plays indispensable roles in early embryonic growth and bone formation in zebrafish.

BACKGROUND: Congenital loss of the SHOX gene is considered to be a genetic cause of short stature phenotype in Turner syndrome and Leri-Weill dyschondrosteosis patients. Though SHOX expression initiates during early fetal development, little is known about the embryonic roles of SHOX. The evolutionary conservation of the zebrafish shox gene and the convenience of the early developmental stages for analyses make zebrafish a preferred model. Here, we characterized structure, expression, and developmental roles of zebrafish shox through a loss-of-function approach. RESULTS: We found a previously undiscovered Shox protein that has both a homeodomain and an OAR-domain in zebrafish. The shox transcript emerged during the segmentation period and it increased in later stages. The predominant domains of shox expression were mandibular arch, pectoral fin, anterior notochord, rhombencephalon, and mesencephalon, suggesting that Shox is involved in bone and neural development. Translational blockade of Shox mRNA by an antisense morpholino oligo delayed embryonic growth, which was restored by the co-overexpression of morpholino-resistant Shox mRNA. At later stages, impaired Shox expression markedly delayed the calcification process in the anterior vertebral column and craniofacial bones. CONCLUSIONS: Our data demonstrate evolutionarily conserved Shox plays roles in early embryonic growth and in later bone formation.

Rapid increase in fibroblast growth factor 21 in protein malnutrition and its impact on growth and lipid metabolism.

Protein malnutrition promotes hepatic steatosis, decreases insulin-like growth factor (IGF)-I production and retards growth. To identify new molecules involved in such changes, we conducted DNA microarray analysis on liver samples from rats fed an isoenergetic low-protein diet for 8 h. We identified the fibroblast growth factor 21 gene (Fgf21) as one of the most strongly up-regulated genes under conditions of acute protein malnutrition (P<0·05, false-discovery rate<0·001). In addition, amino acid deprivation increased Fgf21 mRNA levels in rat liver-derived RL-34 cells (P<0·01). These results suggested that amino acid limitation directly increases Fgf21 expression. FGF21 is a polypeptide hormone that regulates glucose and lipid metabolism. FGF21 also promotes a growth hormone-resistance state and suppresses IGF-I in transgenic mice. Therefore, to determine further whether Fgf21 up-regulation causes hepatic steatosis and growth retardation after IGF-I decrease in protein malnutrition, we fed an isoenergetic low-protein diet to Fgf21-knockout (KO) mice. Fgf21-KO did not rescue growth retardation and reduced plasma IGF-I concentration in these mice. Fgf21-KO mice showed greater epididymal white adipose tissue weight and increased hepatic TAG and cholesterol levels under protein malnutrition conditions (P<0·05). Overall, the results showed that protein deprivation directly increased Fgf21 expression. However, growth retardation and decreased IGF-I were not mediated by increased FGF21 expression in protein malnutrition. Furthermore, FGF21 up-regulation rather appears to have a protective effect against obesity and hepatic steatosis in protein-malnourished animals.

Something old, something new and something borrowed: emerging paradigm of insulin-like growth factor type 1 receptor (IGF-1R) signaling regulation.

The insulin-like growth factor type 1 receptor (IGF-1R) plays a key role in the development and progression of cancer; however, therapeutics targeting it have had disappointing results in the clinic. As a receptor tyrosine kinase (RTK), IGF-1R is traditionally described as an ON/OFF system, with ligand stabilizing the ON state and exclusive kinase-dependent signaling activation. Newly added to the traditional model, ubiquitin-mediated receptor downregulation and degradation was originally described as a response to ligand/receptor interaction and thus inseparable from kinase signaling activation. Yet, the classical model has proven over-simplified and insufficient to explain experimental evidence accumulated over the last decade, including kinase-independent signaling, unbalanced signaling, or dissociation between signaling and receptor downregulation. Based on the recent findings that IGF-1R "borrows" components of G-protein coupled receptor (GPCR) signaling, including ß-arrestins and G-protein-related kinases, we discuss the emerging paradigm for the IGF-1R as a functional RTK/GPCR hybrid, which integrates the kinase signaling with the IGF-1R canonical GPCR characteristics. The contradictions to the classical IGF-1R signaling concept as well as the design of anti-IGF-1R therapeutics treatment are considered in the light of this paradigm shift and we advocate recognition of IGF-1R as a valid target for cancer treatment.