A Prospective Study of Arterial Spin Labelling in Paediatric Posterior Fossa Tumour Survivors: A Correlation with Neurocognitive Impairment.

AIMS: Posterior fossa tumours (PFTs), which account for two-thirds of paediatric brain tumours, are successfully treated in about 70% of patients, but most survivors experience long-term cognitive impairment. We evaluated arterial spin labelling (ASL), a common, non-invasive magnetic resonance imaging (MRI) technique, as a biomarker of cognitive impairment in a paediatric PFT survivor population. MATERIALS AND METHODS: Sixty participants were prospectively analysed. PFT survivors were at least 5 years post-treatment and had been treated as appropriate for their age and type of tumour. Group 1 had received radiotherapy and Group 2 had not. Group 3 were healthy controls matched to Group 1 for age, sex and handedness. All participants underwent cognitive assessment and multimodal MRI, including an ASL perfusion sequence. We used semi-quantitative ASL methods to assess differences in mean perfusion in the thalamus, caudate, putamen and hippocampus. RESULTS: Statistically, no significant associations between cognitive data and radiation doses were identified. Compared with healthy controls, Group 1 patients had significantly lower overall mean perfusion values (20-30% lower, depending on the cerebral structure) and Group 2 had slightly lower mean perfusion values (5-10% lower). Perfusion values did not correlate with total prescribed irradiation doses nor with doses received by different cerebral structures. Episodic and semantic memory test scores were significantly lower in Group 1 and correlated with lower mean absolute perfusion values in the hippocampus (P < 0.04). CONCLUSIONS: These preliminary results indicate that radiotherapy affects the perfusion of specific cerebral structures and identify perfusion as a potential biomarker of hippocampus-dependent memory deficit.

Chimeras in Merlot grapevine revealed by phased assembly.

Chimerism is the phenomenon when several genotypes coexist in a single individual. Used to understand plant ontogenesis they also have been valorised through new cultivar breeding. Viticulture has been taking economic advantage out of chimeras when the variant induced an important modification of wine type such as berry skin colour. Crucial agronomic characters may also be impacted by chimeras that aren't identified yet. Periclinal chimera where the variant has entirely colonised a cell layer is the most stable and can be propagated through cuttings. In grapevine, leaves are derived from both meristem layers, L1 and L2. However, lateral roots are formed from the L2 cell layer only. Thus, comparing DNA sequences of roots and leaves allows chimera detection. In this study we used new generation Hifi long reads sequencing, recent bioinformatics tools and trio-binning with parental sequences to detect periclinal chimeras on 'Merlot' grapevine cultivar. Sequencing of cv. 'Magdeleine Noire des Charentes' and 'Cabernet Franc', the parents of cv. 'Merlot', allowed haplotype resolved assembly. Pseudomolecules were built with a total of 33 to 47 contigs and in few occasions a unique contig for one chromosome. This high resolution allowed haplotype comparison. Annotation was transferred from PN40024 VCost.v3 to all pseudomolecules. After strong selection of variants, 51 and 53 'Merlot' specific periclinal chimeras were found on the Merlot-haplotype-CF and Merlot-haplotype-MG respectively, 9 and 7 been located in a coding region. A subset of positions was analysed using Molecular Inversion Probes (MIPseq) and 69% were unambiguously validated, 25% are doubtful because of technological noise or weak depth and 6% invalidated. These results open new perspectives on chimera detection as an important resource to improve cultivars through clonal selection or breeding.

Standard Diffusion-Weighted Imaging in the Brain Can Detect Cervical Internal Carotid Artery Dissections.

BACKGROUND AND PURPOSE: The ICA is the most common site of cervical artery dissection. Prompt and reliable identification of the mural hematoma is warranted when a dissection is clinically suspected. The purpose of this study was to assess to capacity of a standard DWI sequence acquired routinely on the brain to detect dissecting hematoma related to cervical ICA dissections. MATERIALS AND METHODS: This was a retrospective study of a cohort of 110 patients younger than 55 years of age (40 women; mean age, 46.79 years) admitted at the acute phase of a neurologic deficit, headache, or neck pain and investigated by at least a standard 3T diffusion-weighted sequence of the brain. Among them were 50 patients (14 women; mean age, 46.72 years) with subsequently confirmed ICA dissection. In the whole anonymized cohort, both a senior and junior radiologist separately assessed, on the DWI sequences only, the presence of a crescent-shaped or circular hypersignal projecting on the subpetrosal segment of the ICA arteries, assuming that it would correspond to a mural hematoma related to an ICA dissection. RESULTS: The senior radiologist found 46 subpetrosal hyperintensities in 43/50 patients with ICA dissection and none in patients without dissection (sensitivity, 86%; specificity, 100%). The junior radiologist found 48 subpetrosal hyperintensities in 45/50 patients with dissection and none in patients without dissection (sensitivity, 90%; specificity, 100%). CONCLUSIONS: In our cohort, a standard DWI sequence performed on the brain at the acute phase of a stroke or for a clinical suspicion of dissection detected nearly 90% of cervical ICA dissections.

Hemorrhagic transformation after stroke: inter- and intrarater agreement.

BACKGROUND AND PURPOSE: Hemorrhagic transformation (HT) is a complication of stroke that can occur spontaneously or after treatment. We aimed to assess the inter- and intrarater reliability of HT diagnosis. METHODS: Studies assessing the reliability of the European Cooperative Acute Stroke Study (ECASS) classification of HT or of the presence (yes/no) of HT were systematically reviewed. A total of 18 raters independently examined 30 post-thrombectomy computed tomography scans selected from the Aspiration versus STEnt-Retriever (ASTER) trial. They were asked whether there was HT (yes/no), what the ECASS classification of the particular scan (0/HI1/HI2/PH1/PH2) (HI indicates hemorrhagic infarctions and PH indicates parenchymal hematomas) was and whether they would prescribe an antiplatelet agent if it was otherwise indicated. Agreement was measured with Fleiss' and Cohen's κ statistics. RESULTS: The systematic review yielded four studies involving few (≤3) raters with heterogeneous results. In our 18-rater study, agreement for the presence of HT was moderate [κ = 0.55; 95% confidence interval (CI), 0.41-0.68]. Agreement for ECASS classification was only fair for all five categories, but agreement improved to substantial (κ = 0.72; 95% CI, 0.69-0.75) after dichotomizing the ECASS classification into 0/HI1/HI2/PH1 versus PH2. The inter-rater agreement for the decision to reintroduce antiplatelet therapy was moderate for all raters, but substantial among vascular neurologists (κ = 0.70; 95% CI, 0.57-0.84). CONCLUSION: The ECASS classification may involve too many categories and the diagnosis of HT may not be easily replicable, except in the presence of a large parenchymal hematoma.

Prognostic value of baseline total metabolic tumor volume (TMTV0) measured on FDG-PET/CT in patients with peripheral T-cell lymphoma (PTCL).

BACKGROUND: Most peripheral T-cell lymphoma (PTCL) patients have a poor outcome and the identification of prognostic factors at diagnosis is needed. PATIENTS AND METHODS: The prognostic impact of total metabolic tumor volume (TMTV0), measured on baseline [(18)F]2-fluoro-2-deoxy-d-glucose positron emission tomography/computed tomography, was evaluated in a retrospective study including 108 PTCL patients (27 PTCL not otherwise specified, 43 angioimmunoblastic T-cell lymphomas and 38 anaplastic large-cell lymphomas). All received anthracycline-based chemotherapy. TMTV0 was computed with the 41% maximum standardized uptake value threshold method and an optimal cut-off point for binary outcomes was determined and compared with others prognostic factors. RESULTS: With a median follow-up of 23 months, 2-year progression-free survival (PFS) was 49% and 2-year overall survival (OS) was 67%. High TMTV0 was significantly associated with a worse prognosis. At 2 years, PFS was 26% in patients with a high TMTV0 (>230 cm(3), n = 53) versus 71% for those with a low TMTV0, [P < 0.0001, hazard ratio (HR) = 4], whereas OS was 50% versus 80%, respectively, (P = 0.0005, HR = 3.1). In multivariate analysis, TMTV0 was the only significant independent parameter for both PFS and OS. TMTV0, combined with PIT, discriminated even better than TMTV0 alone, patients with an adverse outcome (TMTV0 >230 cm(3) and PIT >1, n = 33,) from those with good prognosis (TMTV0 ≤230 cm(3) and PIT ≤1, n = 40): 19% versus 73% 2-year PFS (P < 0.0001) and 43% versus 81% 2-year OS, respectively (P = 0.0002). Thirty-one patients (other TMTV0-PIT combinations) had an intermediate outcome, 50% 2-year PFS and 68% 2-year OS. CONCLUSION: TMTV0 appears as an independent predictor of PTCL outcome. Combined with PIT, it could identify different risk categories at diagnosis and warrants further validation as a prognostic marker.

A retrospective study of the prescribing and outcomes of tyrosine kinase inhibitors in chronic myeloid leukaemia over a period of more than 10 years.

WHAT IS KNOWN AND OBJECTIVE: Since their introduction, tyrosine kinase inhibitors (TKIs) have been increasingly used in clinical practice. We describe the prescribing and the clinical and biological consequences of two such inhibitors, imatinib and erlotinib, in patients with chronic myeloid leukaemia (CML) in a practice setting over a period of more than 10 years. METHODS: All patients who received at least one TKI for chronic phase CML between 2001 and 2012 in our university hospital were included in the study. RESULTS AND DISCUSSION: Of the 139 patients, with a median age of 57 years, who were surveyed, imatinib and nilotinib were prescribed as the first TKI in 131 (94%) and 8 (6%) patients, respectively. With a median follow-up of 6 years, 342 treatment modifications were observed: 113 (33%) increased doses, 109 (32%) decreased doses, 89 (26%) TKI changes, 14 (4%) definitive discontinuations, 13 (4%) temporary discontinuations and 4 (1%) additions of IFN-α. The main reasons for the 342 treatment modifications were adverse events (n = 112, 33%), long-term optimal response (n = 58, 17%) and failure (n = 57, 17%). Eighty-five (61%), 31 (22%), 18 (13%) and 5 (4%) patients had no, 1, 2 and 3 TKI changes, respectively. Imatinib was the most prescribed TKI (75%). Adverse events resulting in treatment modifications occurred in 18% of patients for imatinib, 49% for nilotinib and 41% for dasatinib (P < 0·001). Median time to TKI change whatever the reason was >50 months (not achieved) for imatinib, 22 months for nilotinib and 27 months for dasatinib (log-rank test, P < 0·001). WHAT IS NEW AND CONCLUSION: Imatinib was the most prescribed TKI both in the first and in subsequent therapeutic lines for chronic phase CML. Our study showed a very good efficacy-safety profile for imatinib at a median follow-up of 6 years in an unselected French population.

The reversed halo sign: pathognomonic pattern of pulmonary mucormycosis in leukemic patients with neutropenia?

BACKGROUND: Pulmonary mucormycosis (PM) is a life-threatening fungal infection with an increasing incidence among patients with acute leukemia. In some immunocompromised hosts, the reversed halo sign (RHS) has been described on the pulmonary computed tomographic (CT) scan of patients with mucormycosis. METHODS: This study reports a single-center experience with PM exclusively in patients with acute leukemia. Clinical records, laboratory results, and CT scans were retrospectively analyzed to evaluate the clinical usefulness of the RHS for the early identification and treatment of PM, with regard to outcomes in these patients. RESULTS: Between 2003 and 2012, 16 cases of proven PM were diagnosed among 752 consecutive patients receiving chemotherapy for acute myeloblastic or lymphoblastic leukemia. At the time PM was diagnosed, all patients but one were neutropenic. The study of sequential thoracic CT scans showed that during the first week of the disease, the RHS was observed in 15 of 16 patients (94%). Initially, other radiologic findings (multiple nodules and pleural effusion) were less frequent, but appeared later in the course of the disease (6% and 12% before vs 64% and 55% after the first week). After the diagnosis of PM, median overall survival was 25 weeks (range, 3-193 weeks), and 6 patients (38%) died before day 90. CONCLUSIONS: In the particular setting of neutropenic leukemia patients with pulmonary infection, the presence of the RHS on CT was a strong indicator of PM. It could allow the early initiation of appropriate therapy and thus improve the outcome.

High prevalence of diabetes mellitus and hospital-related hyperglycaemia in French general wards.

AIM: The study evaluated the in-hospital prevalence of diabetes and hospital-related hyperglycaemia in a variety of French general wards. METHODS: The multicentre cross-sectional study involving nine French hospitals measured venous fasting plasma glucose (FPG) on a single day in patients hospitalized in adult medical and surgical short-term wards. Diabetes status and length of stay were recorded. RESULTS: Of the 2141 inpatients included in the study, 355 (16.5%) had known diabetes, 156 (7.3%) had screened diabetes (FPG ≥7 mmol/L with no diabetes history), 515 (24.1%) had impaired fasting glucose (IFG; FPG 5.5-6.9 mmol/L) and 1115 (52.1%) had normal glucose values (FPG < 5.5 mmol/L). Diabetes prevalence varied from 11% in hospitals in the west of France to 21% in hospitals in northern and eastern regions. The highest known diabetes prevalence was observed in units for cardiovascular surgery (33%), infectious diseases (27%) and kidney disorders (26%). In cancer units, one-fifth of patients had screened diabetes and one-sixth had known diabetes. Among the known diabetes patients, 127 (36%) were already being treated with insulin, while an additional 41 (12%) started insulin therapy during their hospital stay. Patients with known and screened diabetes were older (70.8 ± 12.2 and 71.1 ± 15.6 years, respectively) than the normal-glucose patients (65.6 ± 18.9 years; P<0.001). Average length of stay was no different between known diabetes and normal-glucose patients after adjusting for age (11.3 ± 7.7 vs 10.0 ± 7.4 days; NS). CONCLUSION: Overall, metabolic glucose disorders (known or screened diabetes and IFG) were found in 48% of inpatients in various French hospital general wards.

The effectiveness of a pyriprole (125 mg/ml) and a metaflumizone (150 mg/ml) combined with amitraz (150 mg/ml) spot-on treatment in preventing Phlebotomus perniciosus from feeding on dogs.

A controlled clinical trial was performed to assess the effectiveness of a pyriprole (125 mg/ml) and a metaflumizone (150 mg/ml) combined with amitraz (150 mg/ml) spot-on treatment (recommended dosage) in preventing adult female sandflies (Phlebotomus perniciosus) from feeding on dogs. Sandfly mortality was also assessed. Twelve beagle dogs were used in the study. Prior to treatment they were checked for their attractiveness to sandflies, ranked accordingly to generate partner triplets of equivalent sensitivity to sandflies: four control dogs, four treated with the pyriprole and four with the metaflumizone spot-on. The dogs were challenged with 50 unfed adult female sandflies (8-10 days old), in cages for one hour on Day 1 and Day 7. The sandflies were checked for blood feeding and mortality at one hour, 24 hours and 48 hours after exposure to the dogs. A very poor anti-feeding effect (near 7%) was seen on sandflies with the metaflumizone combined with amitraz and no antifeeding effect was seen with pyriprole. The sandfly mortality effect as a result of exposure to treated dogs was under 20% for the two spot-on. The two formulations could not be proposed in a leishmaniosis prevention program.

WY-14643 and 9- cis-retinoic acid induce IRS-2/PI 3-kinase signalling pathway and increase glucose transport in human skeletal muscle cells: differential effect in myotubes from healthy subjects and Type 2 diabetic patients.

AIMS/HYPOTHESIS: To determine the effects of peroxisome proliferator-activated receptor alpha (PPARalpha) and retinoid X receptor (RXR) agonists on insulin action, we investigated the effects of Wy-14643 and 9- cis-retinoic acid (9- cis-RA) on insulin signalling and glucose uptake in human myotubes. METHODS: Primary cultures of differentiated human skeletal muscle cells, established from healthy subjects and Type 2 diabetic patients, were used to study the effects of Wy-14643 and 9- cis-RA on the expression and activity of proteins involved in the insulin signalling cascade. Glucose transport was assessed by measuring the rate of [(3)H]2-deoxyglucose uptake. RESULTS: Wy-14643 and 9- cis-RA increased IRS-2 and p85alpha phosphatidylinositol 3-kinase (PI 3-kinase) mRNA and protein expression in myotubes from non-diabetic and Type 2 diabetic subjects. This resulted in increased insulin stimulation of protein kinase B phosphorylation and increased glucose uptake in cells from control subjects. Myotubes from diabetic patients displayed marked alterations in the stimulation by insulin of the IRS-1/PI 3-kinase pathway. These alterations were associated with blunted stimulation of glucose transport. Treatment with Wy-14643 and 9- cis-RA did not restore these defects but increased the basal rate of glucose uptake. CONCLUSIONS/INTERPRETATION: These results demonstrate that PPARalpha and RXR agonists can directly affect insulin signalling in human muscle cells. They also indicate that an increase in the IRS-2/PI 3-kinase pathway does not overcome the impaired stimulation of the IRS-1-dependent pathway and does not restore insulin-stimulated glucose uptake in myotubes from Type 2 diabetic patients.

WY-14643 and 9-cis-retinoic acid induce IRS-2/PI 3-kinase signalling pathway and increase glucose transport in human skeletal muscle cells: differential effect in myotubes from healthy subjects and Type 2 diabetic patients.

AIMS/HYPOTHESIS: To determine the effects of peroxisome proliferator-activated receptor α (PPARα) and retinoid X receptor (RXR) agonists on insulin action, we investigated the effects of Wy-14643 and 9-cis-retinoic acid (9-cis-RA) on insulin signalling and glucose uptake in human myotubes. METHODS: Primary cultures of differentiated human skeletal muscle cells, established from healthy subjects and Type 2 diabetic patients, were used to study the effects of Wy-14643 and 9-cis-RA on the expression and activity of proteins involved in the insulin signalling cascade. Glucose transport was assessed by measuring the rate of [3H]2-deoxyglucose uptake. RESULTS: Wy-14643 and 9-cis-RA increased IRS-2 and p85α phosphatidylinositol 3-kinase (PI 3-kinase) mRNA and protein expression in myotubes from non-diabetic and Type 2 diabetic subjects. This resulted in increased insulin stimulation of protein kinase B phosphorylation and increased glucose uptake in cells from control subjects. Myotubes from diabetic patients displayed marked alterations in the stimulation by insulin of the IRS-1/PI 3-kinase pathway. These alterations were associated with blunted stimulation of glucose transport. Treatment with Wy-14643 and 9-cis-RA did not restore these defects but increased the basal rate of glucose uptake. CONCLUSIONS/INTERPRETATION: These results demonstrate that PPARα and RXR agonists can directly affect insulin signalling in human muscle cells. They also indicate that an increase in the IRS-2/PI 3-kinase pathway does not overcome the impaired stimulation of the IRS-1-dependent pathway and does not restore insulin-stimulated glucose uptake in myotubes from Type 2 diabetic patients.

Morphological hysteresis of the small airways.

The resistance to airflow that develops in most obstructive processes takes place in the small airways. The aim of the present paper is to describe bronchial hysteresis morphometrically in a respiratory cycle model. As a working hypothesis, it is proposed that the changes that take place in the respiratory tract during the respiratory cycle are related to the bronchial size. Specimen rat lungs were organized into five groups: In the first group, the lungs were filled with a liquid fixative to 25 cm of H2O transpulmonary pressure. The following four groups were inflated with air and fixed through the pulmonary artery. Groups 2 and 3 were fixed at 10 and 20 cm transpulmonary pressure in inflation. The last two groups were fixed in deflation and, for this purpose, the transpulmonary pressure was increased to 27 cm and decreased to 20 and 10 cm, respectively. The lungs were processed for morphometrical study and the following variables were quantified: pulmonary volume, internal area, internal perimeter, wall area, internal area radius and bronchial wall radius. The diameter of the airways studied varied between 84.06 microm and 526.4 microm. The results were classified into three subgroups consisting of small, medium-sized and large bronchi. With a single exception--the internal area in the medium-sized bronchi inflated to 20 cm--all the results obtained in deflation were higher than those obtained in inflation. The internal area increased or decreased significantly upon raising or lowering the transpulmonary pressure respectively, in the small and medium-sized bronchi. The wall area in the large bronchi showed significant differences between inflation and deflation at 10 and 20 cm transpulmonary pressure. The wall area was modified significantly in the lungs fixed at 20 cm in the small bronchi and at 10 cm in medium-sized bronchi. The bronchial wall radius was significantly greater in the large bronchi and smaller in the small bronchi. The lumen of the medium-sized and small bronchi increases in inspiration and decreases in expiration. The wall thickness displayed differences between inflation and deflation. The most marked hysteresis was presented by the bronchial wall in the large bronchi. Our results suggest that the behavior of the bronchi varies according to their size.

Pressure volume curve and alveolar recruitment/de-recruitment. A morphometric model of the respiratory cycle.

HYPOTHESIS: The changes in pulmonary volume taking place during respiration are accompanied by the opening and closing of the alveoli, with the number of alveoli open, at the same transpulmonary pressure (TPP) differing, depending on whether the lung is insufflated or deflated. MATERIAL AND METHODS: Seventy 344 Fischer rats divided into five groups. Group 1 lungs were fixed by instilling 10% formalin through the trachea to a pressure of 25 cm H2O. The lungs of the next four groups were air-filled and fixed via the pulmonary artery: group 2 lungs were fixed in inflation at 10 cm H2O TPP; group 3 lungs were fixed in inflation at 20 cm. H2O TPP; the lungs of groups 4 and 5 were fixed in deflation and, therefore, were inflated with air up to 27 cm. H2O to drop to 20 cm in group 4 and to 10 cm in group 5. The lungs were processed for light microscopy, carrying out a morphometric study. The results were statistically processed. RESULTS: The lungs insufflated with liquid fixative at 25 cm of TPP reached higher values in the variables Pulmonary Volume, Internal Alveolar Surface (IAS) and Number of Alveoli, being statistically significant (p < 0.05) in comparison with the other four groups. In the lungs fixed in deflation, the pulmonary volume, IAS and number of alveoli were greater than in those fixed in inflation. The lungs fixed to 20 cm in deflation displayed significant statistical differences compared with those fixed to 20 cm in inflation. The IAS and number of alveoli gave good rates in relation with the pulmonary volume (r > or = 0.65). Three variables were used to measure the size of the alveoli, alveolar cord, alveolar surface and Lm, but none showed significant modifications. CONCLUSION: This study supports the hypothesis that changes in lung volume are related to the increase/decrease in the number of alveoli that are open/closed and not to the modification in the size of the alveoli. Alveolar recruitment is the microscopic expression of pulmonary hysteresis, since the number of alveoli open in deflation is greater than the number open during inflation.

Expression of the splice variants of the p85alpha regulatory subunit of phosphoinositide 3-kinase in muscle and adipose tissue of healthy subjects and type 2 diabetic patients.

The regulation by insulin of the expression of the p85alpha regulatory subunit of phosphoinositide 3-kinase (PI 3-kinase) is impaired in skeletal muscle and adipose tissue of type 2 diabetic patients. The gene encoding p85alpha (named grb-1) can generate several variants by alternative splicing, all being able to activate the p110 catalytic subunits of PI 3-kinase. Our aims were (i) to determine the mRNA expression profiles of these variants in human skeletal muscle and adipose tissue; (ii) to investigate the effect of insulin on their expression in vivo and in vitro in muscle and (iii) to verify whether this regulation is defective in type 2 diabetes. We determined the human genomic organization of grb-1 and set up reverse transcriptase competitive PCR assays for the quantification of each mRNA variant. In muscle, p85alpha and p50alpha mRNAs were the most abundant, and p55alpha represented less than 20% of all grb-1-derived mRNAs. In adipose tissue, p85alpha was expressed predominantly and p55alpha mRNA was not detectable. These expression profiles were not different in type 2 diabetics. During a 3 h hyperinsulinaemic clamp, insulin increased the mRNA expression of the three variants in muscle of control subjects. In diabetic patients, the effect of insulin on p85alpha and p50alpha mRNAs was blunted, and largely reduced on p55alpha transcripts. In cultured human myotubes, up-regulation of p85alpha, p55alpha and p50alpha mRNAs by insulin was abolished by LY294002 (10 microM) and by rapamycin (50 nM), suggesting that the PI 3-kinase/protein kinase B/p70 S6 kinase pathway could be involved in the stimulation of grb-1 gene expression by insulin in human muscle cells.

Regulation of p85alpha phosphatidylinositol-3-kinase expression by peroxisome proliferator-activated receptors (PPARs) in human muscle cells.

Regulation of p85a phosphatidylinositol-3-kinase (p85alphaPI-3K) expression by peroxisome proliferator-activated receptor (PPAR) activators was studied in human skeletal muscle cells. Activation of PPARgamma or PPARbeta did not modify the expression of p85alphaPI-3K. In contrast, activation of PPARalpha increased p85alphaPI-3K mRNA. This effect was potentiated by 9-cis-retinoic acid, an activator of RXR. Up-regulation of p85alphaPI-3K gene expression resulted in a rise in p85alphaPI-3K protein level and in an increase in insulin-induced PI3-kinase activity. According to the role of p85alphaPI-3K in insulin action, these results suggest that drugs with dual action on both PPARgamma and PPARalpha can be of interest for the treatment of insulin resistance.

The regulation of uncoupling protein-2 gene expression by omega-6 polyunsaturated fatty acids in human skeletal muscle cells involves multiple pathways, including the nuclear receptor peroxisome proliferator-activated receptor beta.

Fatty acids have been postulated to regulate uncoupling protein (UCP) gene expression in skeletal muscle in vivo. We have identified, at least in part, the mechanism by which polyunsaturated fatty acids increase UCP-2 expression in primary culture of human muscle cells. omega-6 fatty acids and arachidonic acid induced a 3-fold rise in UCP-2 mRNA levels possibly through transcriptional activation. This effect was prevented by indomethacin and mimicked by prostaglandin (PG) E(2) and carbaprostacyclin PGI(2), consistent with a cyclooxygenase-mediated process. Incubation of myotubes for 6 h with 100 micrometer arachidonic acid resulted in a 150-fold increase in PGE(2) and a 15-fold increase in PGI(2) in the culture medium. Consistent with a role of cAMP and protein kinase A, both prostaglandins induced a marked accumulation of cAMP in human myotubes, and forskolin reproduced the effect of arachidonic acid on UCP-2 mRNA expression. Inhibition of protein kinase A with H-89 suppressed the effect of PGE(2), whereas cPGI(2) and arachidonic acid were still able to increase ucp-2 gene expression, suggesting additional mechanisms. We found, however, that the MAP kinase pathway was not involved. Prostaglandins, particularly PGI(2), are potent activators of the peroxisome proliferator-activated receptors. A specific agonist of peroxisome proliferator-activated receptor (PPAR) beta (L165041) increased UCP-2 mRNA levels in myotubes, whereas activation of PPARalpha or PPARgamma was ineffective. These results suggest thus that ucp-2 gene expression is regulated by omega-6 fatty acids in human muscle cells through mechanisms involving at least protein kinase A and the nuclear receptor PPARbeta.

Triiodothyronine-mediated up-regulation of UCP2 and UCP3 mRNA expression in human skeletal muscle without coordinated induction of mitochondrial respiratory chain genes.

Triiodothyronine (T3) increases mitochondrial respiration and promotes the uncoupling between oxygen consumption and ATP synthesis. T3 effect is mediated partly through transcriptional control of genes encoding mitochondrial proteins. We determined the effect of T3 on mRNA levels of uncoupling proteins (UCP) and proteins involved in the biogenesis of the respiratory chain in human skeletal muscle and on UCP2 mRNA expression in adipose tissue. Ten young, healthy males received 75 to 100 5g of T3 per day for 14 days. The increase in plasma-free T3 levels was associated with an increase of resting metabolic rate and a decrease of respiratory quotient. In skeletal muscle, treatment with T3 induced a twofold increase of both UCP2 and UCP3 mRNA levels (p c oxidase subunits 2 and 4, nuclear respiratory factor 1, mitochondrial transcription factor A, and the co-activator PGC1 did not change during the treatment. In adipose tissue, UCP2 mRNA levels increased threefold. The direct effect of T3 on skeletal muscle an d adipose tissue UCP2 and UCP3 mRNA expression was demonstrated in vitro in human primary cultures. Our data show that T3 induces UCP2 and UCP3 mRNA expression in humans. In skeletal muscle, UCP regulation by T3 is not associated with the transcriptional regulation of respiratory chain proteins.

A phosphatidylinositol 3-Kinase/p70 ribosomal S6 protein kinase pathway is required for the regulation by insulin of the p85alpha regulatory subunit of phosphatidylinositol 3-kinase gene expression in human muscle cells.

Insulin acutely up-regulates p85alpha phosphatidylinositol 3-kinase (p85alphaPI 3-K) mRNA levels in human skeletal muscle (Laville, M., Auboeuf, D., Khalfallah, Y., Vega, N., Riou, J. P., and Vidal, H. (1996) J. Clin. Invest. 98, 43-49). In the present work, we attempted to elucidate the mechanism of action of insulin in primary cultures of human muscle cells. Insulin (10(-7) M, 6 h of incubation) induced a 2-fold increase in p85alphaPI 3-K mRNA abundances (118 +/- 12 versus 233 +/- 35 amol/microgram total RNA, n = 5, p < 0.01) without changing the expression levels of insulin receptor, IRS-1, glycogen synthase, and Glut 4 mRNAs in differentiated myotubes from healthy subjects. The effect is most probably due to a transcriptional activation of the p85alphaPI 3-K gene because the half-life of the mRNA was not affected by insulin treatment (4.0 +/- 0.8 versus 3.1 +/- 0.4 h). PD98059 (50 microM) did not modify the insulin response but increased p85alphaPI 3-K mRNA levels in the absence of insulin, suggesting that the mitogen-activated protein kinase pathway exerts a negative effect on p85alphaPI 3-K mRNA expression in the absence of the hormone. On the other hand, the insulin effect was totally abolished by LY294002 (10 microM) and rapamycin (50 nM). In addition, overexpression of a constitutively active protein kinase B increased p85alphaPI 3-K mRNA levels. These results indicate that the phosphatidylinositol 3-kinase/PKB/p70S6 kinase pathway is required for the stimulation by insulin of p85alphaPI 3-K gene expression in human muscle cells.

Insulin acutely regulates the expression of the peroxisome proliferator-activated receptor-gamma in human adipocytes.

Peroxisome proliferator-activated receptor (PPAR)-gamma is one of the key actors of adipocyte differentiation. This study demonstrates 1) that PPAR-gamma mRNA expression is not altered in subcutaneous adipose tissue (n = 44) or in skeletal muscle (n = 19) of subjects spanning a wide range of BMIs (20-53 kg/m2) and 2) that insulin acutely increases PPAR-gamma mRNA expression in human adipocytes both in vivo and in vitro. The effect of insulin was investigated in abdominal subcutaneous biopsies obtained before and at the end of a 3-h euglycemic-hyperinsulinemic clamp. Insulin significantly increased PPAR-gamma mRNA levels in lean subjects (88 +/- 17%, n = 6), in type 2 diabetic patients (100 +/- 19%, n = 6), and in nondiabetic obese patients (91 +/- 20%, n = 6). Both PPAR-gamma1 and PPAR-gamma2 mRNA variants were increased (P < 0.05) after insulin infusion. In isolated human adipocytes, insulin induced the two PPAR-gamma mRNAs in a dose-dependent manner, with half-maximal stimulation at a concentration of approximately 1-5 nmol/l. However, PPAR-gamma2 mRNA was rapidly (2 h) and transiently increased, whereas a slow and more progressive induction of PPAR-gamma1 was observed during the 6 h of incubation. In explants of human adipose tissue, PPAR-gamma protein levels were significantly increased (42 +/- 3%, P < 0.05) after 12 h of incubation with insulin. These data demonstrate that PPAR-gamma belongs to the list of the insulin-regulated genes and that obesity and type 2 diabetes are not associated with alteration in the expression of this nuclear receptor in adipose tissue.