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.

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.

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.

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.

Steroid hormones are novel nucleoside transport inhibitors by competition with nucleosides for their transporters.

Nucleoside transport is important for nucleic acid synthesis in cells that cannot synthesize nucleosides de novo, and for entry of many cytotoxic nucleoside analog drugs used in chemotherapy. This study demonstrates that various steroid hormones induce inhibition of nucleoside transport in mammalian cells. We analyzed the inhibitory effects of estradiol (E2) on nucleoside transport using SH-SY5Y human neuroblastoma cells. We observed inhibitory effects after acute treatment with E2, which lasted in the presence of E2. However, when E2 was removed, the effect immediately disappeared, suggesting that E2 effects are not mediated through the canonical regulatory pathway of steroid hormones, such as transcriptional regulation. We also discovered that E2 could competitively inhibit thymidine uptake and binding of the labeled nucleoside transporter inhibitor, S-[4-nitrobenzyl]-6-thioinosine (NBTI), indicating that E2 binds to endogenous nucleoside transporters, leading to inhibition of nucleoside transport. We then tested the effects of various steroids on nucleoside uptake in NBTI-sensitive cells, SH-SY5Y and NBTI-insensitive cells H9c2 rat cardiomyoblasts. We found E2 and progesterone clearly inhibited both NBTI-sensitive and insensitive uptake at micromolar concentrations. Taken together, we concluded that steroid hormones function as novel nucleoside transport inhibitors by competition with nucleosides for their transporters.

Aspp2 negatively regulates body growth but not developmental timing by modulating IRS signaling in zebrafish embryos.

The growth and developmental rate of developing embryos and fetus are tightly controlled and coordinated to maintain proper body shape and size. The insulin receptor substrate (IRS) proteins, key intracellular transducers of insulin and insulin-like growth factor signaling, play essential roles in the regulation of growth and development. A short isoform of apoptosis-stimulating protein of p53 2 (ASPP2) was recently identified as a binding partner of IRS-1 and IRS-2 in mammalian cells in vitro. However, it is unclear whether ASPP2 plays any role in vertebrate embryonic growth and development. Here, we show that zebrafish Aspp2a and Aspp2b negatively regulate embryonic growth without affecting developmental rate. Human ASPP2 had similar effects on body growth in zebrafish embryos. Aspp2a and 2b inhibit Akt signaling. This inhibition was reversed by coinjection of myr-Akt1, a constitutively active form of Akt1. Zebrafish Aspp2a and Aspp2b physically bound with Irs-1, and the growth inhibitory effects of ASPP2/Aspp2 depend on the presence of their ankyrin repeats and SH3 domains. These findings uncover a novel role of Aspp2 in regulating vertebrate embryonic growth.

Insulin injection restored increased insulin receptor substrate (IRS)-2 protein during short-term protein restriction but did not affect reduced insulin-like growth factor (IGF)-I mRNA or increased triglyceride accumulation in the liver of rats.

Dietary protein restriction reduces insulin-like growth factor (IGF)-I synthesis and impairs growth. Moreover, insulin secretion is impaired and hepatic insulin signaling is activated presumably through upregulation of insulin receptor substrate (IRS)-2, which can stimulate lipogenesis thereby resulting in steatosis. In order to determine whether impaired insulin secretion is the primary cause of these changes, we injected insulin into protein-restricted rats and compensated for the reduction in insulin secretion for 1 and 7 d. Insulin infusion did not overcome the reduction in liver IGF-I mRNA nor the hepatic triglyceride accumulation. In contrast, it clearly suppressed the upregulation of hepatic IRS-2 on day 1, but not on day 7. Furthermore, insulin elimination increased IRS-2 in H4IIE-C3 cells. In summary, we found that reduced insulin secretion during protein restriction directly increased hepatic IRS-2 as a rapid response on day 1, while additional mechanisms contributed to the upregulation of IRS-2 on day 7.

Tissue-specific effects of protein malnutrition on insulin signaling pathway and lipid accumulation in growing rats.

Our previous studies have revealed that protein malnutrition enhances insulin signaling in rat liver and muscle in response to a bolus insulin injection. However, it has not been established whether protein malnutrition up-regulates insulin signaling under physiological conditions, such as feeding. Here, we studied the effects of protein malnutrition on insulin signaling after feeding in rat liver, muscle and white adipose tissue (WAT). Six-week-old rats were fed a 15% casein diet (15C) or a calorie-matched 5% casein diet (5C) for 8 h/day during 14 days. On the 15th day, blood and tissues were collected at various time points after feeding. Feeding-induced insulin secretion was reduced in 5C-fed rats compared to 15C-fed rats. The 5C-feeding suppressed immediate activation of insulin receptor after feeding in the liver, muscle, and WAT. However, 5C-feeding constantly increased tyrosine phosphorylation of insulin receptor substrate (IRS)-2 and threonine phosphorylation of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) in the liver during the examined periods, corresponding to the changes of their amounts. In skeletal muscle, 5C-feeding did not appreciably alter insulin signaling. In WAT, 5C-feeding decreased tyrosine phosphorylation of IRS-1 compared to 15C-feeding. Furthermore, hepatic triglyceride content was increased and feeding-induced acetyl-CoA carboxylase 1 gene expression was enhanced in 5C-fed rats. The 5C-feeding decreased insulin-dependent glucose uptake in adipocytes. These results suggest that enhanced insulin signaling through increased IRS-2 and 4E-BP1 levels in the liver and repressed insulin signaling through decreased IRS-1 levels in WAT contribute to the preferential hepatic lipid accumulation under protein malnutrition.

Metabolic Effects of Short-Term High-Fat Intake Vary Depending on Dietary Amino Acid Composition.

Background: It is generally accepted that excessive fat intake has undesirable effects on the energy metabolism of our body. Dietary amino acid composition is also critical to the regulation of lipid metabolism. Objectives: This study aimed to investigate whether high-fat diets (HFDs) with different amino acid deficiencies lead to different metabolic outcomes. Methods: Six-wk-old male Wistar rats were fed either a control diet (CN; 3.7 kcal/g, 12% calories from fat) or HFDs (5.1 kcal/g, 60% calories from fat) with 7 different amino acid compositions [control or methionine, arginine, histidine, lysine, threonine, or branched-chain amino acids (BCAAs) deficient], for 7 d. Tissue weights and lipid accumulation in the liver, skeletal muscle, and adipose tissue were measured, and serum biochemical parameters were analyzed. Results: Although the food intake of the HFD groups was a little less than that of the CN group, the total calorie intakes were comparable among the groups, except for histidine-deficient and BCAA-deficient groups. In rats fed am HFD with a control amino acid composition (HFCN), dramatic increase in triglyceride (TG) accumulation in the liver and serum LDL cholesterol concentration were observed compared with the CN group. However, when the arginine content in the diet was reduced, liver TG accumulation was completely inhibited, with no apparent effects on serum lipoprotein-cholesterol concentrations. Meanwhile, deficiency of the other amino acids, such as threonine, reversed HFD-induced upregulation of serum LDL cholesterol. Conclusions: It is observed that although the rats ingested an excessive amount of fat, neither ectopic fat accumulation nor dyslipidemia were always induced at least in the short term; hence, the consequent metabolic change was dependent on the dietary amino acid composition. These findings introduce an important perspective regarding HFD regimens in both scientific and clinical contexts.

Advanced Interferometry with 3-D Structured Illumination Reveals the Surface Fine Structure of Complex Biospecimens.

Interference reflection microscopy (IRM) is a powerful, label-free technique to visualize the surface structure of biospecimens. However, stray light outside a focal plane obscures the surface fine structures beyond the diffraction limit (dxy ≈ 200 nm). Here, we developed an advanced interferometry approach to visualize the surface fine structure of complex biospecimens, ranging from protein assemblies to single cells. Compared to 2-D, our unique 3-D structure illumination introduced to IRM enabled successful visualization of fine structures and the dynamics of protein crystal growth under lateral (dx-y ≈ 110 nm) and axial (dx-z ≤ 5 nm) resolutions and dynamical adhesion of microtubule fiber networks with lateral resolution (dx-y ≈ 120 nm), 10 times greater than unstructured IRM (dx-y ≈ 1000 nm). Simultaneous reflection/fluorescence imaging provides new physical fingerprints for studying complex biospecimens and biological processes such as myogenic differentiation and highlights the potential use of advanced interferometry to study key nanostructures of complex biospecimens.

Phosphatidylinositol 3-kinase (PI3K) activity bound to insulin-like growth factor-I (IGF-I) receptor, which is continuously sustained by IGF-I stimulation, is required for IGF-I-induced cell proliferation.

Continuous stimulation of cells with insulin-like growth factors (IGFs) in G(1) phase is a well established requirement for IGF-induced cell proliferation; however, the molecular components of this prolonged signaling pathway that is essential for cell cycle progression from G(1) to S phase are unclear. IGF-I activates IGF-I receptor (IGF-IR) tyrosine kinase, followed by phosphorylation of substrates such as insulin receptor substrates (IRS) leading to binding of signaling molecules containing SH2 domains, including phosphatidylinositol 3-kinase (PI3K) to IRS and activation of the downstream signaling pathways. In this study, we found prolonged (>9 h) association of PI3K with IGF-IR induced by IGF-I stimulation. PI3K activity was present in this complex in thyrocytes and fibroblasts, although tyrosine phosphorylation of IRS was not yet evident after 9 h of IGF-I stimulation. IGF-I withdrawal in mid-G(1) phase impaired the association of PI3K with IGF-IR and suppressed DNA synthesis the same as when PI3K inhibitor was added. Furthermore, we demonstrated that Tyr(1316)-X-X-Met of IGF-IR functioned as a PI3K binding sequence when this tyrosine is phosphorylated. We then analyzed IGF signaling and proliferation of IGF-IR(-/-) fibroblasts expressing exogenous mutant IGF-IR in which Tyr(1316) was substituted with Phe (Y1316F). In these cells, IGF-I stimulation induced tyrosine phosphorylation of IGF-IR and IRS-1/2, but mutated IGF-IR failed to bind PI3K and to induce maximal phosphorylation of GSK3ß and cell proliferation in response to IGF-I. Based on these results, we concluded that PI3K activity bound to IGF-IR, which is continuously sustained by IGF-I stimulation, is required for IGF-I-induced cell proliferation.