PDK1 selectively phosphorylates Thr(308) on Akt and contributes to human platelet functional responses.

3-phosphoinositide-dependent protein kinase 1 (PDK1), a member of the protein A,G and C (AGC) family of proteins, is a Ser/Thr protein kinase that can phosphorylate and activate other protein kinases from the AGC family, including Akt at Thr308, all of which play important roles in mediating cellular responses. The functional role of PDK1 or the importance of phosphorylation of Akt on Thr308 for its activity has not been investigated in human platelets. In this study, we tested two pharmacological inhibitors of PDK1, BX795 and BX912, to assess the role of Thr308 phosphorylation on Akt. PAR4-induced phosphorylation of Akt on Thr308 was inhibited by BX795 without affecting phosphorylation of Akt on Ser473. The lack of Thr308 phosphorylation on Akt also led to the inhibition of PAR4-induced phosphorylation of two downstream substrates of Akt, viz. GSK3ß and PRAS40. In vitro kinase activity of Akt was completely abolished if Thr308 on Akt was not phosphorylated. BX795 caused inhibition of 2-MeSADP-induced or collagen-induced aggregation, ATP secretion and thromboxane generation. Primary aggregation induced by 2-MeSADP was also inhibited in the presence of BX795. PDK1 inhibition also resulted in reduced clot retraction indicating its role in outside-in signalling. These results demonstrate that PDK1 selectively phosphorylates Thr308 on Akt thereby regulating its activity and plays a positive regulatory role in platelet physiological responses.

Heparin but not citrate anticoagulation of blood preserves platelet function for prolonged periods.

BACKGROUND: Current guidelines state that platelet aggregation studies should be conducted within 4 h of venepuncture because of the decline in sensitivity to platelet agonists. This constrains studies of platelet activity in clinical situations where samples need to be transported or there are unavoidable delays prior to assessment. OBJECTIVES: The aim of the present study was to compare systematically the responses of platelets stored in the presence of either citrate or heparin, the two most widely used anti-coagulants, using a range of standard techniques. METHODS: Blood was taken from healthy volunteers and either assessed immediately or stored at ambient temperature (18-25 degrees C) for 24 h. Platelet reactivity to a range of agonists was determined by a combination of 96-well plate techniques; light transmission aggregometry, thrombi adhesion, ATP and ADP release, and TxA(2) release; by whole blood aggregometry; and by PFA-100. RESULTS AND CONCLUSIONS: Testing using 96-well plate techniques allowed for the simultaneous measurement of responses to multiple concentrations of multiple agonists. The responses of platelets from blood anti-coagulated with heparin were predominantly preserved in all assays after 24 h storage, whereas, responses of platelets stored in blood anti-coagulated with citrate were greatly diminished. Consequently, anti-coagulation with heparin, but not citrate, preserves platelet responses for up to 24 h as determined by a range of techniques.

The in vivo effects of the Pro12Ala PPARgamma2 polymorphism on adipose tissue NEFA metabolism: the first use of the Oxford Biobank.

AIMS/HYPOTHESIS: To investigate the phenotypic effects of common polymorphisms on adipose tissue metabolism and cardiovascular risk factors, we set out to establish a biobank with the unique feature of allowing a prospective recruit-by-genotype approach. The first use of this biobank investigates the effects of the peroxisome proliferator-activated receptor (PPAR) Pro12Ala polymorphism on integrative tissue-specific physiology. We hypothesised that Ala12 allele carriers demonstrate greater adipose tissue metabolic flexibility and insulin sensitivity. MATERIALS AND METHODS: From a comprehensive population register, subjects were recruited into a biobank, which was genotyped for the Pro12Ala polymorphism. Twelve healthy male Ala12 carriers and 12 matched Pro12 homozygotes underwent detailed physiological phenotyping using stable isotope techniques, and measurements of blood flow and arteriovenous differences in adipose tissue and muscle in response to a mixed meal containing [1,1,1-(13)C]tripalmitin. RESULTS: Of 6,148 invited subjects, 1,072 were suitable for inclusion in the biobank. Among Pro12 homozygotes, insulin sensitivity correlated with HDL-cholesterol concentrations, and inversely correlated with blood pressure, apolipoprotein B, triglyceride and total cholesterol concentrations. Ala12 carriers showed no such correlations. In the meal study, Ala12 carriers had lower plasma NEFA concentrations, higher adipose tissue and muscle blood flow, and greater insulin-mediated postprandial hormone-sensitive lipase suppression along with greater insulin sensitivity than Pro12 homozygotes. CONCLUSIONS/INTERPRETATION: This study shows that a recruit-by-genotype approach is feasible and describes the biobank's first application, providing tissue-specific physiological findings consistent with the epidemiological observation that the PPAR Ala12 allele protects against the development of type 2 diabetes.

Normal tension glaucoma is not associated with the common apolipoprotein E gene polymorphisms.

BACKGROUND: /aims: In normal tension glaucoma (NTG) factors other than raised intraocular pressure have a role in the pathogenesis of the optic neuropathy. Because particular apolipoprotein E (ApoE) gene polymorphisms have been associated with cell death and survival in neurological degenerative diseases, the purpose of this study was to determine the ApoE allele frequencies in patients with normal tension glaucoma. METHODS: The apolipoprotein E genotype of 155 patients with normal tension glaucoma was compared to that of 349 non-affected, control subjects from the same geographical area. A similar comparison was made between 53 patients with normal tension glaucoma who demonstrated progressive visual field loss, and control subjects. The frequencies of genotypes was compared with the chi(2) test and Mantel-Haenszel coefficent. RESULTS: There was no significant difference in the frequency of ApoE alleles or genotypes in the normal tension glaucoma population compared to the control group. The ApoE alleles and genotypes in NTG patients with progressive disease were not different from the control group. CONCLUSION: ApoE gene polymorphisms are not linked to normal tension glaucoma, suggesting that this gene does not have a role in the pathogenesis of optic neuropathy in this disease.

Blunted glucose metabolism in anorexia nervosa.

Only few studies have specifically investigated diet-induced thermogenesis in anorexia nervosa. Twenty women, 10 anorectics (body mass index [BMI] = 14.98 +/- 1.02 kg/m(2)) and 10 controls (BMI = 22.53 +/- 0.75 kg/m(2)) were studied. Body composition was evaluated by isotopic dilution. Respiratory gas exchange was measured by indirect calorimetry. An oral glucose load (75 g) was administered to the anorectics (A) and the controls (CA). The controls underwent a second load (CB) with a higher glucose amount (1.85 +/- 0.11 g/kg body weight [BW]) to compare with the load taken by anorectics. Glucose-induced thermogenesis (GIT) was computed for 300 minutes following the load as the percent increase of energy expenditure (EE) above resting-EE (REE). Serum glucose levels were lower in anorectic patients both in fasting (3.46 +/- 0.66 v 5.23 +/- 0.23 in CA, P <.01 v 5.32 +/- 0.34 mmol in CB, P <.01) and in the postprandial state (glucose area under the curve [AUC] 175.51 +/- 6.40 v 289.80 +/- 7.30 in CA, P <.01 v 324.65 mmol in CB, P <.001); insulin AUC was lower, 1,926 +/- 452 versus 41,148 +/- 2,071 in CA, P <.0001 versus 60,765.5 pmol in CB, P <.0001. REE, normalized by fat-free mass (FFM), was similar between groups. GIT was lower in anorectics (3.58 +/- 1.20 v 5.45 +/- 1.83 in CA, P <.05 v 9.09% +/- 1.05% in CB, P <.01). Glucose oxidation was higher in anorectics than in CA (689.44 +/- 72.22 v 333.32 +/- 32.98 micromol/L/min, P <.001), but similar to CB. Lipid oxidation become negative after 30 minutes in anorectics (postprandial lipid oxidation = -93.58 +/- 39.86 v 370.61 +/- 21.73 in CA, P <.0001 v 119.01 +/- 12.32 micromol/L/300 min in CB, P <.0001). Anorectic patients displayed a low REE and GIT. Carbohydrate oxidation was similar between groups; lipid oxidation was extremely reduced. An increased protein catabolism was observed.

Glucose disposal in morbidly obese patients in the early post-operative period.

BACKGROUND: In surgical patients, operative stress causes protein catabolism, muscle mass loss, and impaired glucose tolerance. We investigated fuel metabolism and glucose and protein turnover in 15 obese subjects who underwent biliopancreatic diversion (BPD) by using stable labelled isotopes. METHODS: 6 males and 9 females (age 45.11 +/- 8.9 years, BMI of 48.85 +/- 4.43 kg/m2) who underwent BPD were studied, and the APACHE II score was calculated. Patients were studied 3 times (before BPD, 1st and 3rd postoperative day). Glycemia was stable--maintained by continuously infusing Rapid insulin. Each day of the study, the patients received a primed, constant infusion of [15N2] urea, and, 180 min after, a [6.6-2H] glucose infusion started and continued for 3 hours. Indirect calorimetry was performed during the study, under TPN (30 kcal/kg). RESULTS: The APACHE score was lower on the 3rd postoperative day than on the 1st postoperative day (7.4 +/- 2.7 vs 6.3 +/- 2.6, p < 0.05). The npRQ was different throughout the post-operative period (0.82 +/- 0.03 vs 0.9 +/- 0.06, p < 0.05), while urinary nitrogen excretion, energy expenditure and glycemia did not change. The insulin amount infused was lower during the 3rd post-operative day (44.25 +/- 12.3 vs 64.12 +/- 11 UI on the 1st one, p < 0.05). Insulinemia was lower during the 3rd than during the 1st postoperative day (66.4 +/- 9.49 vs 117.44 +/- 8.49 microU/ml, p < 0.05). Non-essential fatty acid levels were higher on the 3rd post-operative day than on the 1st one (0.98 +/- 0.6 vs 0.45 +/- 0.34 mmol/L, p < 0.01). No differences were observed in glucose and urea turn-over. CONCLUSION: The metabolic pattern of morbidly obese patients operated by BPD was similar to that of other critically ill patients previously studied in the literature. Furthermore, the increased glucose oxidation rate observed on the 3rd post-operative day was coupled with an improved clinical condition.

Intestinal permeability increases with the severity of abdominal trauma: a comparison between gas liquid chromatographic and enzymatic method.

BACKGROUND/AIMS: Increased intestinal permeability was found in humans after multiple trauma, burn injury and major vascular surgery. However, no data are reported regarding possible correlation between trauma and intestinal permeability degree. This study was undertaken to compare gas-liquid chromatographic and enzymatic method for the evaluation of intestinal permeability impairment in patients after severe abdominal trauma. METHODOLOGY: Five traumatized patients with an injury severity score of more than 24 and 5 cross-matched healthy volunteers were studied. The intestinal permeability was performed using a test solution, containing 10 g lactulose and 5 g mannitol. Gas-liquid chromatographic method was applied to measure sugar standards and 5-hour urine samples and the results were compared with those obtained employing a specific enzymatic method. RESULTS: Linearity of myoinositol, lactulose and mannitol measured by gas-liquid chromatographic method was from 0.2-1 microgram injected. Using the enzymatic method, the response was linear between mannitol concentrations of 0.34 and 5.49 mM. Linearity of lactulose standard was from 0.18-2.92 mM. The gas-liquid chromatographic and enzymatic methods showed a good agreement using the Bland-Altman procedure. The mean lactulose/mannitol ratio was 0.085 +/- 0.025 in patients and 0.009 +/- 0.001 in controls (P < 0.001). The higher the injury severity score (30.8 +/- 5) the larger the ratio of lactulose to mannitol (R2 = 0.74). CONCLUSIONS: The enzymatic method--inexpensive, easy-to-perform and timesaving--is suitable for intestinal permeability studies. An abdominal trauma, without injury requiring surgical operation, modifies the intestinal mucosa permeability possibly favoring passage of bacteria and subsequent sepsis.

Acetyl-L-carnitine infusion increases glucose disposal in type 2 diabetic patients.

Little information is available in the literature on the effect of L-carnitine to improve glucose disposal in healthy control subjects and type 2 diabetic patients. No data are reported on the pharmacological properties of acetyl-L-carnitine (ALC) in type 2 diabetes mellitus. The present study evaluates glucose uptake and oxidation rates with either ALC or placebo administration in 18 type 2 diabetic patients. On different days, each patient received both a primed-constant infusion of ALC (5 mg/kg body weight [BW] priming bolus and either 0.025, 0.1, or 1.0 mg/kg BW/min constant infusion) and a comparable placebo formulation. During the infusion period, continuous indirect calorimetric monitoring and a euglycemic-hyperinsulinemic clamp (EHC) study were performed. The total end-clamp glucose tissue uptake (M value) was significantly increased by the administration of ALC (from 3.8 to 5.2 mg/kg/min, P = .006), and the dose dependence of this effect reached borderline statistical significance (P = .037). The increase in the M/I ratio was also highly significant after ALC administration (from 3.9 to 5.8 x 10(-2) mg/kg/min/(microUI/mL, P < .001), while no statistically significant effect was attributable to the different dosages. The increase in the M value was related to increased glucose storage (highly significant effect of ALC) rather than increased glucose oxidation (no statistical significance). In conclusion, the effect of ALC on glucose disposal has no relationship to the amount administered. This could be due to an effect of ALC on the enzymes involved in both the glycolytic and gluconeogenetic pathways, and a possible reversibility of glycogen synthase inhibition in diabetic subjects.

[Malformations of the midline. A case-control study]. / Malformazioni della linea mediana. Uno studio caso-controllo.

Defects of the middle line are an heterogeneous group of congenital malformations due to commune pathogenetic mechanisms. We have made a case-control study about 150 newborns, who have at least 1 defect of the middle line. Results prove an excess of males between the cases, due mostly to hypospadias. We haven't found families with defects of the middle line with x-linked manner of hereditary transmission. We haven't found any particular risk present in cases and not in controls. We haven't found any case with 2 or more middle line defects.