The ability to predict pregnancy loss in cattle with ELISAs that detect pregnancy associated glycoproteins is antibody dependent.

The concentration of circulating pregnancy associated glycoproteins (PAGs) early in pregnancy may serve as markers to predict late embryonic mortality or fetal mortality in cattle. In this study, pregnancies were established in dairy cows, by either fixed-time AI (FTAI) or fixed-time embryo transfer (FTET) with in vitro produced embryos. Circulating PAGs were measured with different combinations of antibodies in either a laboratory-based ELISA or a commercial ELISA. For the in-house ELISA, three monoclonal 'trapping' antibodies (A6, J2, and L4) and two polyclonal 'detection' antisera (antibodies F2 or 45) were used to quantify PAGs in serum from the same cows. The different assays were identified as follows: 'Mix-45' (A6, J2, and L4 with 45), 'Mix-F2' (A6, J2, and L4 with F2), and 'L4-F2': (L4 with F2); the commercial assay was from IDEXX. Ovulation was synchronized and FTAI or FTET was performed on day 0 or 7, respectively. Ultrasound-based diagnosis of pregnancy and serum collections occurred on day 30. The proportion of cows that subsequently experienced pregnancy loss between days 30 and 60 was 23% (43 of 183) and 16% (21 of 131) for the FTAI or FTET groups, respectively. In the FTAI group, mean serum concentration of PAGs detected with Mix-45 was higher in cows that maintained pregnancy (9.2 ±â€¯0.4 ng/ml; mean ±â€¯SEM) compared with cows that experienced pregnancy failure (3.9 ±â€¯0.6 ng/ml) between day 30-60 (P < .001). However, there was no difference (P > .69) in circulating concentrations of PAGs between cows that experienced loss or survival between days 30 and 60 when Mix-F2 or L4-F2 were used in an in-house ELISA. Likewise, a commercial assay also did not result in measurable differences in PAG concentrations between those animals that experienced loss or survival. Following FTET, circulating concentrations of PAGs on day 30 were lower (P < .001) in cows that experienced pregnancy failure compared to cows that maintained pregnancy when the Mix-45 and the commercial assay were used, but not with the other antibody combinations. A receiver operating characteristic curve showed that only the Mix-45 antibody combination was predictive (95% accuracy) of pregnancy loss but not the other antibody combinations following FTAI. However, both Mix-45 and the commercial assay were predictive of losses following FTET. In summary, although multiple PAG assay formats have been shown to accurately detect pregnancy, the ability to predict embryo survival during early gestation appears to be antibody dependent.

Effects of Serial Skin Testing with Purified Protein Derivative on the Level and Quality of Antibodies to Complex and Defined Antigens in Mycobacterium bovis-Infected Cattle.

Several serological tests designed to detect antibodies to immunodominant Mycobacterium bovis antigens have recently emerged as ancillary tests for the detection of bovine tuberculosis in cattle, particularly when used after the injection of purified protein derivative (PPD) for skin testing, which significantly boosts M. bovis-specific antibody responses. The present findings demonstrate the onset and duration of boosted antibody responses after the injection of M. bovis PPD for the caudal fold test (CFT) and Mycobacterium avium and M. bovis PPDs for the comparative cervical test (CCT), administered in series in cattle experimentally infected with M. bovis. While skin tests boosted the responses to certain antigens (i.e., MPB83 and MPB70), they did not affect the responses to other antigens (e.g., ESAT-6, CFP10, MPB59, and MPB64). Administration of the CCT 105 days after the CFT resulted in an even greater secondary boost in antibody responses to MPB83 and MPB70 and to a proteinase K-digested whole-cell sonicate (WCS-PK) of M. bovis. Both IgM and IgG contributed to the initial boost in the MPB83/MPB70-specific antibody response after the CFT. The secondary boost after the CCT was primarily due to increased IgG levels. Also, the avidity of antibodies to MPB83 and MPB70 increased after the CCT in M. bovis-infected cattle. The avidity of antibodies to the WCS-PK antigens increased in the interval between the CFT and the CCT but did not increase further after the CCT. Together, these findings demonstrate that the administration of PPDs for skin tests results in additive enhancement (i.e., when the CFT and CCT are performed in series), both qualitative and quantitative, of MPB83/MPB70-specific antibody responses.

A pregnancy detection assay using milk samples: evaluation and considerations.

Two experiments were conducted to evaluate a pregnancy-detection assay based on the measurement of pregnancy-associated glycoproteins (PAG) in milk samples. In experiment 1, milk samples were collected on the day of first pregnancy check (33-52 d postinsemination; n=119) or second check (60-74 d postinsemination; n=60). The accuracy in identification of pregnant and nonpregnant cows was 99% at first check. Only 6% of samples were found to be within an intermediate range of PAG concentrations and classified as requiring recheck by the assay. At second check, the accuracy of the assay was 98%. Fifteen percent of these samples were classified as requiring recheck. In experiments 2a (n=17 cows) and 2b (n=16 cows), milk and plasma samples were collected from cows at weekly intervals beginning 2 (experiment 2a) or 4 d (experiment 2b) after insemination. The earliest time point at which pregnant cows were accurately classified as pregnant by the assay was on d 30 postinsemination. A transient decline in PAG levels into the intermediate range was observed on d 46 to 72 postinsemination. This coincides with the time of recheck in experiment 1. Results obtained with the plasma samples were essentially the same. The accuracy of pregnancy identification based on milk samples from nonpregnant and pregnant cows was 99%. Levels of PAG in milk were useful in identifying 6 incidences of embryonic mortality. No consistent relationship was noted between the timing of the decline in PAG levels and the timing of luteal regression in this small number of cows.

Strategic use of serology for the diagnosis of bovine tuberculosis after intradermal skin testing.

Diagnostic tests based on cell-mediated immunity are used in programmes for eradication of bovine tuberculosis (Mycobacterium bovis). Serological assays could be applied as ancillary methods to detect infected animals. Our objective was to evaluate two serological techniques: M. bovis Ab Test (IDEXX, USA) and Enferplex™ TB assay (Enfer, Ireland) in animals tested simultaneously with the single and comparative intradermal tests and the interferon-gamma assay. This work was performed at two stages. First, a preliminary panel of samples collected prior to intradermal tests from tuberculosis-free (n=60) and M. bovis-infected herds (n=78) was assayed, obtaining high specificity: 100% (M. bovis Ab Test) and 98.3% (Enferplex TB assay) but low sensitivity (detection of M. bovis infected animals): 23.9% (M. bovis Ab Test) and 32.6% (Enferplex TB assay). Subsequently, the use of serological techniques was further studied in two herds with M. bovis infection (n=77) using samples collected prior to, and 72 h and 15 days after PPD inoculation. The highest level of detection of infected animals for serology was achieved at 15 days post-intradermal tests taking advantage of the anamnestic effect: 70.4% and 85.2% in herd A, and 66.7% and 83.3% in herd B, using M. bovis Ab Test and Enferplex TB assay, respectively. Quantitative results (average values obtained with M. bovis Ab Test ELISA and degree of positivity obtained with Enferplex TB assay) were higher in animals showing lesions compatible with tuberculosis. No significant differences were observed in the number of confirmed infected animals detected with either serological technique.

Evaluation of a commercial enzyme-linked immunosorbent assay for the diagnosis of bovine tuberculosis from milk samples from dairy cows.

Milk samples from dairy cows provide a ready source of material for measuring antibody responses to Mycobacterium bovis antigens. In this study, we evaluated the IDEXX enzyme-linked immunosorbent assay (ELISA) for the measurement of antibody responses to M. bovis antigens MPB70 and MPB83 in milk samples from New Zealand cattle. Test sensitivities for individual milk and serum samples were assessed in samples collected from 44 M. bovis-infected cows, and test specificities were assessed in milk samples collected from 356 cows from tuberculosis (TB)-free herds. Milk vat samples were collected from 505 herds from regions with relatively high or low prevalences of infection. The ELISA had a sensitivity of 50% and a specificity of 97.5% for milk samples, and the test sensitivities for milk and serum samples were the same. Dilution of the positive test milk samples in milk from noninfected cows at 1/10, 1/20, and 1/50 dilutions reduced the proportions of positive responses to 13/21, 9/21, and 4/21, respectively. Small differences were observed in the ELISA responses of milk samples from individual TB-free cows collected at different times during lactation. No significant differences were detected in the ELISA responses of milk vat samples collected from infected and noninfected herds. This study shows that milk samples can be substituted for serum samples for screening individual cows for M. bovis infection, and pooling of milk samples from 10 to 20 animals can result in a reduction in the sensitivity by approximately 50%. However, screening of milk vat samples is unlikely to be useful in countries with low prevalences of M. bovis in cattle and large herd sizes.

Lipin 1 represses NFATc4 transcriptional activity in adipocytes to inhibit secretion of inflammatory factors.

Lipin 1 is a bifunctional protein that regulates gene transcription and, as a Mg(2+)-dependent phosphatidic acid phosphatase (PAP), is a key enzyme in the biosynthesis of phospholipids and triacylglycerol. We describe here the functional interaction between lipin 1 and the nuclear factor of activated T cells c4 (NFATc4). Lipin 1 represses NFATc4 transcriptional activity through protein-protein interaction, and lipin 1 is present at the promoters of NFATc4 transcriptional targets in vivo. Catalytically active and inactive lipin 1 can suppress NFATc4 transcriptional activity, and this suppression may involve recruitment of histone deacetylases to target promoters. In fat pads from mice deficient for lipin 1 (fld mice) and in 3T3-L1 adipocytes depleted of lipin 1 there is increased expression of several NFAT target genes including tumor necrosis factor alpha, resistin, FABP4, and PPARgamma. Finally, both lipin 1 protein and total PAP activity are decreased with increasing adiposity in the visceral, but not subcutaneous, fat pads of ob/ob mice. These observations place lipin 1 as a potentially important link between triacylglycerol synthesis and adipose tissue inflammation.

Fat cell-specific ablation of rictor in mice impairs insulin-regulated fat cell and whole-body glucose and lipid metabolism.

OBJECTIVE: Rictor is an essential component of mammalian target of rapamycin (mTOR) complex (mTORC) 2, a kinase that phosphorylates and activates Akt, an insulin signaling intermediary that regulates glucose and lipid metabolism in adipose tissue, skeletal muscle, and liver. To determine the physiological role of rictor/mTORC2 in insulin signaling and action in fat cells, we developed fat cell-specific rictor knockout (FRic(-/-)) mice. RESEARCH DESIGN AND METHODS: Insulin signaling and glucose and lipid metabolism were studied in FRic(-/-) fat cells. In vivo glucose metabolism was evaluated by hyperinsulinemic-euglycemic clamp. RESULTS: Loss of rictor in fat cells prevents insulin-stimulated phosphorylation of Akt at S473, which, in turn, impairs the phosphorylation of downstream targets such as FoxO3a at T32 and AS160 at T642. However, glycogen synthase kinase-3beta phosphorylation at S9 is not affected. The signaling defects in FRic(-/-) fat cells lead to impaired insulin-stimulated GLUT4 translocation to the plasma membrane and decreased glucose transport. Furthermore, rictor-null fat cells are unable to suppress lipolysis in response to insulin, leading to elevated circulating free fatty acids and glycerol. These metabolic perturbations are likely to account for defects observed at the whole-body level of FRic(-/-) mice, including glucose intolerance, marked hyperinsulinemia, insulin resistance in skeletal muscle and liver, and hepatic steatosis. CONCLUSIONS: Rictor/mTORC2 in fat cells plays an important role in whole-body energy homeostasis by mediating signaling necessary for the regulation of glucose and lipid metabolism in fat cells.

Mammalian target of rapamycin complex 1 (mTORC1) activity is associated with phosphorylation of raptor by mTOR.

mTORC1 contains multiple proteins and plays a central role in cell growth and metabolism. Raptor (regulatory-associated protein of mammalian target of rapamycin (mTOR)), a constitutively binding protein of mTORC1, is essential for mTORC1 activity and critical for the regulation of mTORC1 activity in response to insulin signaling and nutrient and energy sufficiency. Herein we demonstrate that mTOR phosphorylates raptor in vitro and in vivo. The phosphorylated residues were identified by using phosphopeptide mapping and mutagenesis. The phosphorylation of raptor is stimulated by insulin and inhibited by rapamycin. Importantly, the site-directed mutation of raptor at one phosphorylation site, Ser(863), reduced mTORC1 activity both in vitro and in vivo. Moreover, the Ser(863) mutant prevented small GTP-binding protein Rheb from enhancing the phosphorylation of S6 kinase (S6K) in cells. Therefore, our findings indicate that mTOR-mediated raptor phosphorylation plays an important role on activation of mTORC1.

Alterations in hepatic metabolism in fld mice reveal a role for lipin 1 in regulating VLDL-triacylglyceride secretion.

OBJECTIVE: Lipin 1 controls fatty acid metabolism in the nucleus as a transcriptional regulator and in the cytosol as an enzyme catalyzing the penultimate step in phosphoglycerol triacylglyceride (TAG) synthesis. We sought to evaluate the effects of lipin 1 on hepatic TAG synthesis and secretion by gain-of-function and loss-of-function approaches. METHODS AND RESULTS: Rates of TAG synthesis were not impaired in hepatocytes isolated from adult lipin 1-deficient (fld) mice and were actually increased in 14-day-old fld mice. Additionally, compared to littermate controls, VLDL-TAG secretion rates were markedly increased in fld mice of both ages. Lipin 1 overexpression did not alter TAG synthesis rates but significantly suppressed VLDL-TAG secretion. The lipin 1-mediated suppression of VLDL-TAG secretion was linked to the peptide motif mediating its transcriptional-regulatory effects. However, the expression of candidate genes required for VLDL assembly and secretion was unaltered by lipin 1 activation or deficiency. Finally, the hepatic expression of lipin 1 was diminished in obese insulin-resistant mice, whereas adenoviral-mediated overexpression of lipin 1 in liver of these mice inhibits VLDL-TAG secretion and improves hepatic insulin signaling. CONCLUSIONS: Collectively, these studies reveal new and unexpected effects of lipin 1 on hepatic TAG metabolism and obesity-related hepatic insulin resistance.

Regulation of proline-rich Akt substrate of 40 kDa (PRAS40) function by mammalian target of rapamycin complex 1 (mTORC1)-mediated phosphorylation.

The rapamycin-sensitive mammalian target of rapamycin (mTOR) complex 1 (mTORC1) contains mTOR, raptor, mLST8, and PRAS40 (proline-rich Akt substrate of 40 kDa). PRAS40 functions as a negative regulator when bound to mTORC1, and it dissociates from mTORC1 in response to insulin. PRAS40 has been demonstrated to be a substrate of mTORC1, and one phosphorylation site, Ser-183, has been identified. In this study, we used two-dimensional phosphopeptide mapping in conjunction with mutational analysis to show that in addition to Ser-183, mTORC1 also phosphorylates Ser-212 and Ser-221 in PRAS40 when assayed in vitro. Mutation of all three residues to Ala markedly reduces mTORC1-mediated phosphorylation of PRAS40 in vitro. All three sites were confirmed to be phosphorylated in vivo by [(32)P]orthophosphate labeling and peptide mapping. Phosphorylation of Ser-221 and Ser-183 but not Ser-212 is sensitive to rapamycin treatment. Furthermore, we demonstrate that mutation of Ser-221 to Ala reduces the interaction with 14-3-3 to the same extent as mutation of Thr-246, the Akt/protein kinase B-phosphorylated site. We also find that mutation of Ser-221 to Ala increases the inhibitory activity of PRAS40 toward mTORC1. We propose that after mTORC1 kinase activation by upstream regulators, PRAS40 is phosphorylated directly by mTOR, thus contributing to the relief of PRAS40-mediated substrate competition.

Muscle-specific deletion of rictor impairs insulin-stimulated glucose transport and enhances Basal glycogen synthase activity.

Rictor is an essential component of mTOR (mammalian target of rapamycin) complex 2 (mTORC2), a kinase complex that phosphorylates Akt at Ser473 upon activation of phosphatidylinositol 3-kinase (PI-3 kinase). Since little is known about the role of either rictor or mTORC2 in PI-3 kinase-mediated physiological processes in adult animals, we generated muscle-specific rictor knockout mice. Muscle from male rictor knockout mice exhibited decreased insulin-stimulated glucose uptake, and the mice showed glucose intolerance. In muscle lacking rictor, the phosphorylation of Akt at Ser473 was reduced dramatically in response to insulin. Furthermore, insulin-stimulated phosphorylation of the Akt substrate AS160 at Thr642 was reduced in rictor knockout muscle, indicating a defect in insulin signaling to stimulate glucose transport. However, the phosphorylation of Akt at Thr308 was normal and sufficient to mediate the phosphorylation of glycogen synthase kinase 3 (GSK-3). Basal glycogen synthase activity in muscle lacking rictor was increased to that of insulin-stimulated controls. Consistent with this, we observed a decrease in basal levels of phosphorylated glycogen synthase at a GSK-3/protein phosphatase 1 (PP1)-regulated site in rictor knockout muscle. This change in glycogen synthase phosphorylation was associated with an increase in the catalytic activity of glycogen-associated PP1 but not increased GSK-3 inactivation. Thus, rictor in muscle tissue contributes to glucose homeostasis by positively regulating insulin-stimulated glucose uptake and negatively regulating basal glycogen synthase activity.

PRAS40 regulates mTORC1 kinase activity by functioning as a direct inhibitor of substrate binding.

Mammalian target of rapamycin (mTOR) functions in two distinct signaling complexes, mTORC1 and mTORC2. In response to insulin and nutrients, mTORC1, consisting of mTOR, raptor (regulatory-associated protein of mTOR), and mLST8, is activated and phosphorylates eukaryotic initiation factor 4E-binding protein (4EBP) and p70 S6 kinase to promote protein synthesis and cell size. Previously we found that activation of mTOR kinase in response to insulin was associated with increased 4EBP1 binding to raptor. Here we identify prolinerich Akt substrate 40 (PRAS40) as a binding partner for mTORC1. A putative TOR signaling motif, FVMDE, is identified in PRAS40 and shown to be required for interaction with raptor. Insulin stimulation markedly decreases the level of PRAS40 bound by mTORC1. Recombinant PRAS40 inhibits mTORC1 kinase activity in vivo and in vitro, and this inhibition depends on PRAS40 association with raptor. Furthermore, decreasing PRAS40 expression by short hairpin RNA enhances 4E-BP1 binding to raptor, and recombinant PRAS40 competes with 4E-BP1 binding to raptor. We, therefore, propose that PRAS40 regulates mTORC1 kinase activity by functioning as a direct inhibitor of substrate binding.

A conserved phosphatase cascade that regulates nuclear membrane biogenesis.

A newly emerging family of phosphatases that are members of the haloacid dehalogenase superfamily contains the catalytic motif DXDX(T/V). A member of this DXDX(T/V) phosphatase family known as Dullard was recently shown to be a potential regulator of neural tube development in Xenopus [Satow R, Chan TC, Asashima M (2002) Biochem Biophys Res Commun 295:85-91]. Herein, we demonstrate that human Dullard and the yeast protein Nem1p perform similar functions in mammalian cells and yeast cells, respectively. In addition to similarity in primary sequence, Dullard and Nem1p possess similar domains and show similar substrate preferences, and both localize to the nuclear envelope. Additionally, we show that human Dullard can rescue the aberrant nuclear envelope morphology of nem1Delta yeast cells, functionally replacing Nem1p. Finally, Nem1p, has been shown to deposphorylate the yeast phosphatidic acid phosphatase Smp2p [Santos-Rosa H, Leung J, Grimsey N, Peak-Chew S, Siniossoglou S (2005) EMBO J 24:1931-1941], and we show that Dullard dephosphorylates the mammalian phospatidic acid phosphatase, lipin. Therefore, we propose that Dullard participates in a unique phosphatase cascade regulating nuclear membrane biogenesis, and that this cascade is conserved from yeast to mammals.

Insulin controls subcellular localization and multisite phosphorylation of the phosphatidic acid phosphatase, lipin 1.

Brain, liver, kidney, heart, and skeletal muscle from fatty liver dystrophy (fld/fld) mice, which do not express lipin 1 (lipin), contained much less Mg(2+)-dependent phosphatidic acid phosphatase (PAP) activity than tissues from wild type mice. Lipin harboring the fld(2j) (Gly(84) --> Arg) mutation exhibited relatively little PAP activity. These results indicate that lipin is a major PAP in vivo and that the loss of PAP activity contributes to the fld phenotype. PAP activity was readily detected in immune complexes of lipin from 3T3-L1 adipocytes, where the protein was found both as a microsomal form and a soluble, more highly phosphorylated, form. Fifteen phosphorylation sites were identified by mass spectrometric analyses. Insulin increased the phosphorylation of multiple sites and promoted a gel shift that was due in part to phosphorylation of Ser(106). In contrast, epinephrine and oleic acid promoted dephosphorylation of lipin. The PAP-specific activity of lipin was not affected by the hormones or by dephosphorylation of lipin with protein phosphatase 1. However, the ratio of soluble to microsomal lipin was markedly increased in response to insulin and decreased in response to epinephrine and oleic acid. The results suggest that insulin and epinephrine control lipin primarily by changing localization rather than intrinsic PAP activity.

Lipin 1 is an inducible amplifier of the hepatic PGC-1alpha/PPARalpha regulatory pathway.

Perturbations in hepatic lipid homeostasis are linked to the development of obesity-related steatohepatitis. Mutations in the gene encoding lipin 1 cause hepatic steatosis in fld mice, a genetic model of lipodystrophy. However, the molecular function of lipin 1 is unclear. Herein, we demonstrate that the expression of lipin 1 is induced by peroxisome proliferator-activated receptor gamma (PPARgamma) coactivator 1alpha (PGC-1alpha), a transcriptional coactivator controlling several key hepatic metabolic pathways. Gain-of-function and loss-of-function strategies demonstrated that lipin selectively activates a subset of PGC-1alpha target pathways, including fatty acid oxidation and mitochondrial oxidative phosphorylation, while suppressing the lipogenic program and lowering circulating lipid levels. Lipin activates mitochondrial fatty acid oxidative metabolism by inducing expression of the nuclear receptor PPARalpha, a known PGC-1alpha target, and via direct physical interactions with PPARalpha and PGC-1alpha. These results identify lipin 1 as a selective physiological amplifier of the PGC-1alpha/PPARalpha-mediated control of hepatic lipid metabolism.

Activation of mammalian target of rapamycin (mTOR) by insulin is associated with stimulation of 4EBP1 binding to dimeric mTOR complex 1.

Insulin stimulates protein synthesis by promoting phosphorylation of the eIF4E-binding protein, 4EBP1. This effect is rapamycin-sensitive and mediated by mammalian target of rapamycin (mTOR) complex 1 (mTORC1), a signaling complex containing mTOR, raptor, and mLST8. Here we demonstrate that insulin produces a stable increase in the kinase activity of mTORC1 in 3T3-L1 adipocytes. The response was associated with a marked increase in 4EBP1 binding to raptor in mTORC1, and it was abolished by disrupting the TOR signaling motif in 4EBP1. The stimulatory effects of insulin on both 4EBP1 kinase activity and binding occurred rapidly and at physiological concentrations of insulin, and both effects required an intact mTORC1. Results of experiments involving size exclusion chromatography and coimmunoprecipitation of epitope-tagged subunits provide evidence that the major insulin-responsive form is dimeric mTORC1, a structure containing two heterotrimers of mTOR, raptor, and mLST8.

mTOR-dependent stimulation of the association of eIF4G and eIF3 by insulin.

Insulin stimulates protein synthesis by increasing translation initiation. This response is mediated by mTOR and is believed to result from 4EBP1 phosphorylation, which allows eIF4E to bind eIF4G. Here, we present evidence that mTOR interacts directly with eIF3 and that mTOR controls the association of eIF3 and eIF4G. Activating mTOR signaling with insulin increased by as much as five-fold the amount of eIF4G bound to eIF3. This novel effect was blocked by rapamycin and other inhibitors of mTOR, and it required neither eIF4E binding to eIF4G nor eIF3 binding to the 40S ribosomal subunit. The increase in eIF4G associated with eIF3 occurred rapidly and at physiological concentrations of insulin. Moreover, the magnitude of the response was similar to the increase in eIF4E binding to eIF4G produced by insulin. Thus, increasing eIF4G association with eIF3 represents a potentially important mechanism by which insulin, as well as amino acids and growth factors that activate mTOR, stimulate translation.

Farnesylthiosalicylic acid inhibits mammalian target of rapamycin (mTOR) activity both in cells and in vitro by promoting dissociation of the mTOR-raptor complex.

The mammalian target of rapamycin (mTOR) functions with raptor and mLST8 in a signaling complex that controls rates of cell growth and proliferation. Recent results indicate that an inhibitor of the Ras signaling pathway, farnesylthiosalicylic acid (FTS), decreased phosphorylation of the mTOR effectors, PHAS-I and S6K1, in breast cancer cells. Here we show that incubating 293T cells with FTS produced a stable change in mTOR activity that could be measured in immune complex kinase assays using purified PHAS-I as substrate. Similarly, FTS decreased the PHAS-I kinase activity of mTOR when added to cell extracts or to immune complexes containing mTOR. Incubating either cells or extracts with FTS also decreased the amount of raptor that coimmunoprecipitated with mTOR, although having relatively little effect on the amount of mLST8 that coimmunoprecipitated. The concentration effect curves of FTS for inhibition of mTOR activity and for dissociation of the raptor-mTOR complex were almost identical. Caffeine, wortmannin, LY294002, and rapamycin-FKBP12 also markedly inhibited mTOR activity in vitro, but unlike FTS, none of the other mTOR inhibitors appreciably changed the amount of raptor associated with mTOR. Thus, our findings indicate that FTS represents a new type of mTOR inhibitor, which acts by dissociating the functional mTOR-raptor signaling complex.

Effects of insulin and transgenic overexpression of UDP-glucose pyrophosphorylase on UDP-glucose and glycogen accumulation in skeletal muscle fibers.

UDP-glucose (UDP-Glc) and glycogen levels in skeletal muscle fibers of defined fiber type were measured using microanalytical methods. Infusing rats with insulin increased glycogen in both Type I and Type II fibers. Insulin was without effect on UDP-Glc in Type I fibers but decreased UDP-Glc by 35-40% in Type IIA/D and Type IIB fibers. The reduction in UDP-Glc suggested that UDP-Glc pyrophosphorylase (PPL) activity might limit glycogen synthesis in response to insulin. To explore this possibility, we generated mice overexpressing a UDP-Glc PPL transgene in skeletal muscle. The transgene increased both UDP-Glc PPL activity and levels of UDP-Glc in skeletal muscles by approximately 3-fold. However, overexpression of UDP-Glc PPL was without effect on either the levels of skeletal muscle glycogen or glucose tolerance in vivo. The transgene was also without effect on either control or insulin-stimulated rates of (14)C-glucose incorporation into glycogen in muscles incubated in vitro. The results indicate that UDP-Glc PPL activity is not limiting for glycogen synthesis.

Insulin receptor substrate-2 proteasomal degradation mediated by a mammalian target of rapamycin (mTOR)-induced negative feedback down-regulates protein kinase B-mediated signaling pathway in beta-cells.

Regulation of insulin receptor substrate (IRS)-2 expression is critical to beta-cell survival, but the mechanisms that control this are complex and undefined. Here in pancreatic beta-cells (INS-1), chronic exposure (>8 h) to 15 mm glucose and/or 5 nm IGF-1, increased Ser/Thr phosphorylation of IRS-2, which correlated with decreased IRS-2 levels. This glucose/IGF-1-induced decrease in IRS-2 levels was prevented by the proteasomal inhibitor, lactacystin. In addition, the glucose/IGF-1-induced increase in Ser/Thr phosphorylation of IRS-2 and the subsequent decrease in INS-1 cell IRS-2 protein levels was thwarted by the mammalian target of rapamycin(mTOR) inhibitor, rapamycin. Moreover, adenoviral-mediated expression of constitutively active mTOR (mTORDelta) further increased glucose/IGF-1-induced Ser/Thr phosphorylation of IRS-2 and decreased IRS-2 protein levels, whereas adenoviral-mediated expression of "kinase-dead" mTOR (mTOR-KD) conversely reduced Ser/Thr phosphorylation of IRS-2 and maintained IRS-2 protein levels. In adenoviral-infected beta-cells expressing mTORDelta, the decrease in IRS-2 protein levels was also prevented by rapamycin or lactacystin, further indicating a proteasomal mediated degradation of IRS-2 mediated via mTOR-induced Ser/Thr phosphorylation of IRS-2. Finally, we found that chronic activation of mTOR leading to decreased levels of IRS-2 in INS-1 cells led to a significant decrease in PKB activation and consequently increased beta-cell apoptosis. Thus, chronic activation of mTOR by glucose (and/or IGF-1) in beta-cells leads to increased Ser/Thr phosphorylation of IRS-2 that targets it for proteasomal degradation, resulting in decreased IRS-2 expression and increased beta-cell apoptosis. This may be a contributing mechanism as to how beta-cell mass is decreased by chronic hyperglycemia in the pathogenesis of type-2 diabetes.