Chronic mTOR inhibition by rapamycin induces muscle insulin resistance despite weight loss in rats.

BACKGROUND AND PURPOSE: mTOR inhibitors are currently used as immunosuppressants in transplanted patients and as promising anti-cancer agents. However, new-onset diabetes is a frequent complication occurring in patients treated with mTOR inhibitors such as rapamycin (Sirolimus). Here, we investigated the mechanisms associated with the diabetogenic effects of chronic Sirolimus administration in rats and in in vitro cell cultures. EXPERIMENTAL APPROACH: Sirolimus was administered to rats fed either a standard or high-fat diet for 21 days. Metabolic parameters were measured in vivo and in ex vivo tissues. Insulin sensitivity was assessed by glucose tolerance tests and euglycaemic hyperinsulinaemic clamps. Rapamycin effects on glucose metabolism and insulin signalling were further evaluated in cultured myotubes. KEY RESULTS: Sirolimus induced a decrease in food intake and concomitant weight loss. It also induced specific fat mass loss that was independent of changes in food intake. Despite these beneficial effects, Sirolimus-treated rats were glucose intolerant, hyperinsulinaemic and hyperglycaemic, but not hyperlipidaemic. The euglycaemic hyperinsulinaemic clamp measurements showed skeletal muscle is a major site of Sirolimus-induced insulin resistance. At the molecular level, long-term Sirolimus administration attenuated glucose uptake and metabolism in skeletal muscle by preventing full insulin-induced Akt activation and altering the expression and translocation of glucose transporters to the plasma membrane. In rats fed a high-fat diet, these metabolic defects were exacerbated, although Sirolimus-treated animals were protected from diet-induced obesity. CONCLUSIONS AND IMPLICATIONS: Taken together, our data demonstrate that the diabetogenic effect of chronic rapamycin administration is due to an impaired insulin action on glucose metabolism in skeletal muscles.

[Validation of a screening test of language and learning disorders for 6-year old children (ERTLA6)--a prospective study]. / Validité des epreuves du repérage des troubles du langage et des apprentissages de l'enfant de 6 ans (ERTLA6)--etude prospective.

Medical practitioners are, like the other health, education and childhood professionals, important actors of the language and learning disorders' screening. Six years old--the age at which children start the elementary school--is a key age for this screening. At the request of practitioners, a multidisciplinary staff had developed a screening tool: ERTLA6 (Epreuves de repérage des troubles du langage et des apprentissages de l'enfant de 6 ans). The objective was to validate the capacity of ERTLA6 to predict the school performance. A sample of 187 children was randomly constituted among the whole population of last year nursery school children in an area of France (the Académie de Nancy-Metz). Those children, aged from 5 to 6, were screened with ERTLA6 by the school practitioner during a medical visit (score from 0 [the best] to 18 [the worse]). The School outcomes (considered as judgment criteria) were assessed 2 or 3 years later, after two years of elementary school. 148 children had completed their follow-up (the others: 27 moving house, 6 absents the day of evaluation, 2 missing data). Mean age was 5; 10 years. With a threshold > or = 7, ERTLA6 sensibility and specificity were respectively 79% [63-94] and 87% [81-93]; the positive predictive value was 58% [42-74], the negative predictive value was 95% [90-99]. The percentage of well classified children was 84% [69-99]. To our knowledge, ERTLA6 is the first validated tool in France for screening language and learning disorders which can be used by practitioners for the prediction of school outcomes.

An arginine to cysteine(252) mutation in insulin receptors from a patient with severe insulin resistance inhibits receptor internalisation but preserves signalling events.

AIMS/HYPOTHESIS: We examined the properties of a mutant insulin receptor (IR) with an Arg(252) to Cys (IR(R252C)) substitution in the alpha-subunit originally identified in a patient with extreme insulin resistance and acanthosis nigricans. METHODS: We studied IR cell biology and signalling pathways in Chinese Hamster Ovary cells overexpressing this IR(R252C). RESULTS: Our investigation showed an impairment in insulin binding to IR(R252C) related mostly to a reduced affinity of the receptor for insulin and to a reduced rate of IR(R252C) maturation; an inhibition of IR(R252C)-mediated endocytosis resulting in a decreased insulin degradation and insulin-induced receptor down-regulation; a maintenance of IR(R252C) on microvilli even in the presence of insulin; a similar autophosphorylation of mutant IR(R252C) followed by IRS 1/IRS 2 phosphorylation, p85 association with IRS 1 and IRS 2 and Akt phosphorylation similar to those observed in cells expressing wild type IR (IRwt); and finally, a reduced insulin-induced Shc phosphorylation accompanied by decreased ERK1/2 phosphorylation and activity and of thymidine incorporation into DNA in cells expressing IR(R252C) as compared to cells expressing IRwt. CONCLUSION/INTERPRETATION: These observations suggest that: parameters other than tyrosine kinase activation participate in or control the first steps of IR internalisation or both; IR-mediated IRS 1/2 phosphorylation can be achieved from the cell surface and microvilli in particular; Shc phosphorylation and its subsequent signalling pathway might require IR internalisation; defective IR endocytosis correlates with an enhancement of some biological responses to insulin and attenuation of others.

Detecting language disorders in 4-year-old French children. An application of the ERTL-4.

This paper discusses the development of the ERTL-4 (Epreuve de repérage des troubles du langage lors du bilan medical de l'enfant de quatre ans), a measure developed in Nancy, France specifically for the purpose of identifying children with language difficulties in the 3.9-4.6 years age range. The test has been designed to identify 10-15% of the population and allows the assessing primary care doctor to ascertain whether difficulties occur in language, voice, fluency, hearing or perception on the basis of a 5-min assessment.

Measurement of bilirubin partition coefficients in bile salt micelle/aqueous buffer solutions by micellar electrokinetic chromatography.

The partition coefficients for the distribution of bilirubin between aqueous phosphateborate buffer and cholic, taurocholic, taurodeoxycholic, and taurochenodeoxycholic micelles have been measured by micellar electrokinetic chromatography at pH 8.5. Determination of the partition coefficients required that the critical micelle concentration and partial specific volumes be determined for each bile salt. Critical micelle concentrations were slightly higher for the trihydroxy bile salts. Partial specific volumes of the bile salt micelles differed very little from each other, and for each bile salt they were constant over the concentration range studied, which was typically from slightly above the critical micelle concentration to 35 mM. Capacity factors were corrected for the effects of applied voltage by extrapolation of the capacity factor to zero applied volts. The free solution mobility of bilirubin, determined in the absence of bile salt, was also corrected for the effects of applied voltage. Plots of extrapolated capacity factor versus phase ratio yield the partition coefficient as the slope of a linear fit to the data. Partition coefficients for bilirubin were significantly higher for dihydroxy bile salts than for trihydroxy bile salts.

[Diagnostic value of ERTL4: a screening test of language disorders in 4-year-old children]. / Valeur diagnostique de ERTL4: un test de repérage des troubles du langage chez l'enfant de 4 ans.

BACKGROUND: Language disorders in children are frequent and may sometimes severely affect their development. ERTL4 (screening test for language disorders of children aged 4) is the first test for screening language disorders to be used by doctors. It has not yet been evaluated. AIM: The objective of this study is to assess and optimize the diagnostic value of the ERTL4 test and to evaluate its potential interest as a screening test. MATERIAL AND METHODS: A sample of 370 children was randomly constituted among children aged between 3 years 9 months and 4 years 6 months from schools in Nancy, France and surrounding areas. These children undertook both the ERTL4 test and a standardized examination of language and speech as a reference. RESULTS: Among the 325 children included in the analysis, 34.1% presented with disorders requiring speech and language therapy. ERTL4 sensitivity and specificity were 88.6% and 66.8%, respectively. Varying thresholds for disorder diagnoses and proposals for a modified test led to a sensitivity of 72.9% and a specificity of 91.0%. The proportion of well-classified subjects improved from 73.8% to 85.3% (P < 10(-3)). The positive predictive value was 78.7% and the negative predictive value was 88.1% (with the observed frequency in the sample). CONCLUSION: ERTL4 is a good test for doctors to screening of children's language disorders and their management.

Soluble form of complement C3b/C4b receptor (CR1) results from a proteolytic cleavage in the C-terminal region of CR1 transmembrane domain.

The complement C3b/C4b receptor (CR1) is an integral protein, anchored in the plasma membrane through a hydrophobic domain of 25 amino acids, but is also found in the plasma in soluble form (sCR1). A recombinant, soluble form of CR1 has been demonstrated to reduce complement-dependent tissue injury in animal models of ischaemia/reperfusion. In view of the important pathophysiological relevance of sCR1, we have investigated the mechanisms governing CR1 release by using various mutated and chimaeric receptors transiently expressed in COS cells. Pulse-chase experiments revealed that (1) sCR1 is produced by a proteolytic process, (2) the cleavage site lies within the C-terminus of CR1 transmembrane domain, (3) the proteolytic process involves a fully glycosylated CR1 form and (4) this process takes place in late secretory vesicles or at the plasma membrane.

Dual role of a dileucine motif in insulin receptor endocytosis.

Two leucines (Leu986 and Leu987) have recently been shown to take part in the control of human insulin receptor (HIR) internalization (Renfrew-Haft, C., Klausner, R. D., and Taylor, S. I. (1994) J. Biol. Chem. 269, 26286-26294). The aim of the present study was to further investigate the exact mechanism of this control process. Constitutive and insulin-induced HIR internalizations were studied biochemically and morphologically in NIH 3T3 cells overexpressing either a double alanine (amino acid residues 986-987) mutant HIR (HIR AA1) or HIR truncated at either amino acid residue 981 (HIR Delta981) or 1000 (HIR Delta1000). Data collected indicate that: (a) the three mutant HIR show a reduced association with microvilli as compared with HIR wild-type; (b) the two receptors containing the dileucine motif (HIR WT and HIR Delta1000) show the highest propensity to associate with clathrin-coated pits, independently of kinase activation; (c) the two receptors lacking the dileucine motif but containing two tyrosine-based motifs, previously described as participating in clathrin-coated pit segregation, associate with these surface domains with a lower affinity than the two others, (d) in the presence of the kinase domain, an unmasking of the tyrosine-based motifs mediated by kinase activation is required. These results indicate that the dileucine motif is not sufficient by itself, but participates in anchoring HIR on microvilli and that another sequence, located downstream from position 1000 is crucial for this event. This dileucine motif also plays a role in HIR segregation in clathrin-coated pits. This latter function is additive with that of the tyrosine-based motifs but the role of the dileucine motif predominates. Eventually, the clathrin-coated pit anchoring function of the dileucine motif is independent of receptor kinase activation in contrast to the tyrosine-based motifs.

Cells with osteoblastic phenotypes can be explanted from human gingiva and periodontal ligament.

Considerable phenotypic heterogeneity has been reported in gingival fibroblasts. Similarly, cells from the periodontal ligament (PDL) can be isolated with different phenotypes. Although it has been suggested that cells from the gingiva do not contribute to the formation of hard tissue, it is theoretically possible that under appropriate stimuli, immature mesenchymal cells in gingiva could differentiate along an osteoblastic pathway. Differentiation of immature mesenchymal cells into osteoblasts following stimulation with osteoinductive factors has been demonstrated in muscle. We undertook experiments to establish whether cells with osteoblastic characteristics could be identified from human gingiva as well as from human periodontal ligament. Some cell populations from each of these tissues were found to have high basal alkaline phosphatase activity, to release osteocalcin in response to 1,25(OH)2 VitD3, and to form a mineralized matrix. Thus, cells can be isolated from the gingiva and PDL that exhibit phenotypic markers, which taken together are characteristic of osteoblastic cells. Other cell populations derived from the PDL and gingival connective tissue were isolated that had fibroblastic characteristics. These studies support the concept that gingival tissue can give rise to cells which may differentiate along either a fibroblastic or an osteoblastic pathway.