Pathways of Acetyl-CoA Metabolism Involved in the Reversal of Palmitate-Induced Glucose Production by Metformin and Salicylate.

The pathways through which fatty acids induce insulin resistance have been the subject of much research. We hypothesise that by focussing on the reversal of insulin resistance, novel insights can be made regarding the mechanisms by which insulin resistance can be overcome. Using global gene and lipid expression profiling, we aimed to identify biological pathways altered during the prevention of palmitate-induced glucose production in hepatocytes using metformin and sodium salicylate. FAO hepatoma cells were treated with palmitate (0.075 mM, 48 h) with or without metformin (0.25 mM) and sodium salicylate (2 mM) in the final 24 h of palmitate treatment, and effects on glucose production were determined. RNA microarray measurements followed by gene set enrichment analysis were performed to investigate pathway regulation. Lipidomic analysis and measurement of secreted bile acids and cholesterol were also performed. Reversal of palmitate-induced glucose production by metformin and sodium salicylate was characterised by co-ordinated down-regulated expression of pathways regulating acetyl-CoA to cholesterol and bile acid biosynthesis. All 20 enzymes that regulate the conversion of acetyl-CoA to cholesterol were reduced following metformin and sodium salicylate. Selected findings were confirmed using primary mouse hepatocytes. Although total intracellular levels of diacylglycerol, triacylglycerol and cholesterol esters increased with palmitate, these were not, however, further altered by metformin and sodium salicylate. 6 individual diacylglycerol, triacylglycerol and cholesterol ester species containing 18:0 and 18:1 side-chains were reduced by metformin and sodium salicylate. These results implicate acetyl-CoA metabolism and C18 lipid species as modulators of hepatic glucose production that could be targeted to improve glucose homeostasis.

The effect of insulin and exercise on c-Cbl protein abundance and phosphorylation in insulin-resistant skeletal muscle in lean and obese Zucker rats.

AIMS/HYPOTHESIS: Recruitment of the protein c-Cbl to the insulin receptor (IR) and its tyrosine phosphorylation via a pathway that is independent from phosphatidylinositol 3'-kinase is necessary for insulin-stimulated GLUT4 translocation in 3T3-L1 adipocytes. The activation of this pathway by insulin or exercise has yet to be reported in skeletal muscle. METHODS: Lean and obese Zucker rats were randomly assigned to one of three treatment groups: (i). control, (ii). insulin-stimulated or (iii). acute, exhaustive exercise. Hind limb skeletal muscle was removed and the phosphorylation state of IR, Akt and c-Cbl measured. RESULTS: Insulin receptor phosphorylation was increased 12-fold after insulin stimulation ( p<0.0001) in lean rats and threefold in obese rats. Acute exercise had no effect on IR tyrosine phosphorylation. Similar results were found for serine phosphorylation of Akt. Exercise did not alter c-Cbl tyrosine phosphorylation in skeletal muscle of lean or obese rats. However, in contrast to previous studies in adipocytes, c-Cbl tyrosine phosphorylation was reduced after insulin treatment ( p<0.001). CONCLUSIONS/INTERPRETATION: We also found that c-Cbl associating protein expression is relatively low in skeletal muscle of Zucker rats compared to 3T3-L1 adipocytes and this could account for the reduced c-Cbl tyrosine phosphorylation after insulin treatment. Interestingly, basal levels of c-Cbl tyrosine phosphorylation were higher in skeletal muscle from insulin-resistant Zucker rats ( p<0.05), but the physiological relevance is not clear. We conclude that the regulation of c-Cbl phosphorylation in skeletal muscle differs from that previously reported in adipocytes.

Evaluation of a novel monoclonal enzyme immunoassay for detection of Helicobacter pylori antigen in stool from children.

BACKGROUND: Reliable non-invasive methods for detection of Helicobacter pylori infection are required to investigate the incidence, transmission, and clearance of infection in childhood. AIM: To evaluate a new monoclonal enzyme immunoassay (EIA) (FemtoLab H pylori Cnx) for detection of H pylori antigen in stool in a large cohort of children compared with invasive diagnostic methods and the (13)C urea breath test. PATIENTS AND METHODS: A total of 302 symptomatic previously untreated children (aged 0.5-18.7 years; 148 girls) were recruited at three centres. H pylori status was defined by results of culture, histology, the rapid urease test, and the (13)C urea breath test. Stool samples were investigated locally by the EIA using two different production lots. According to the manufacturer's recommendations, an optical density (OD) of 0.150 was used as a cut off value. RESULTS: OD values clearly differentiated between the 92 H pylori infected and the 210 non-infected children (median (5th-95th percentiles) 2.729 (0.232->4.000) v 0.021 (0.009-0.075)). Only two false positive and two false negative results occurred, giving a sensitivity, specificity, positive predictive value, and negative predictive value of 98%, 99%, 98%, and 99%, respectively. No significant relation was found between age and OD values in infected or non-infected children. CONCLUSIONS: The monoclonal stool antigen EIA was excellent in diagnosing H pylori infection in symptomatic children. Accuracy was independent of the laboratory, production lot used, or the child's age. Because only 18/116 children <6 years of age were infected with H pylori, further validation of the test is needed in young infected children.

Chemokine receptor 5 expression in gastric mucosa of Helicobacter pylori-infected and noninfected children.

Experimental data from human adults or animal models indicate that the Helicobacter pylori-specific immune response is dominated by inflammatory T cells of the Th1 type. To investigate whether a Th1 immune response is established in early H. pylori infection, gastric biopsy samples from 70 children were subjected to immunohistochemical analysis. To this end, T cells, B cells, monocytes, neutrophils, and chemokine receptor 5 (CCR5)-expressing (CCR5(+)) cells, which are associated with Th1 immune responses, were quantified. Children were classified according to H. pylori status and clinical, laboratory, and macroscopic (during endoscopy) findings, without knowledge of histological findings. Group 1 included 31 H. pylori-infected children, group 2 contained 24 children with other conditions possibly affecting the stomach, and group 3 contained 15 children without verifiable pathological findings in the stomach. Lymphoid follicles were present in 90% of biopsy samples from group 1 and 48% of those from group 2 but absent in group 3 biopsy samples. Intraepithelial T cells and CCR5(+) cells were regularly detected in all groups without significant differences. B cells, monocytes, and neutrophils were not found. In contrast, the numbers of lamina propria T cells (P < 0.003) and CCR5(+) cells (P < 0.001) were increased significantly in H. pylori-infected children. B cells (in 13 of 66 children) were detected in children with active (n = 11) or previously cleared (n = 2) H. pylori infections but were absent in healthy children. The numbers of monocytes (in 10 of 67 children) did not differ among the groups. Calculations indicated that the majority of gastric T cells express CCR5; this finding is in contrast to the low percentage of CCR5(+) T cells in the peripheral circulation. Thus, an increase in the numbers of CCR5(+) cells in H. pylori-infected stomach mucosa suggests that this molecule may play an important role in gastric immune responses.

Evaluation of two commercial enzyme immunoassays, testing immunoglobulin G (IgG) and IgA responses, for diagnosis of Helicobacter pylori infection in children.

Serological testing to diagnose Helicobacter pylori infection in children is still controversial, although commonly used in clinical practice. We compared the immunoglobulin G (IgG) and IgA results of two commercially available enzyme immunoassays (EIAs) (Pyloriset IgG and IgA and Enzygnost II IgG and IgA) for 175 children with abdominal symptoms divided into three age groups (0 to < or =6 years, n = 47; >6 to < or =12 years, n = 77; >12 years, n = 51). A child was considered H. pylori infected if at least two of three tests (histology, rapid urease test, 13C-urea breath test) or culture were positive and noninfected if all results were concordantly negative. Of 175 children, 93 (53%) were H. pylori negative and 82 were H. pylori positive. With the recommended cutoff values, the overall specificity was excellent for all four EIAs (95.7 to 97.8%) regardless of age. Sensitivity varied markedly between tests and was 92.7, 70.7, 47.5, and 24.4% for Enzygnost II IgG, Pyloriset IgG, Enzygnost II IgA, and Pyloriset IgA, respectively. Sensitivity was low in the youngest age group (25 to 33.3%), except for Enzygnost II IgG (91.6%). Receiver-operating curve analyses revealed that lower cutoff values would improve the accuracy of all of the tests except Enzygnost II IgG. Measurement of specific IgA, in addition to IgG, antibodies hardly improved the sensitivity. The specificity of commercial serological tests is high in children when the cutoff values obtained from adults are used. In contrast, sensitivity is variable, with a strong age dependence in some, but not all, tests. We speculate that young children may have a different immune response to H. pylori, with preferable responses to certain antigens, as well as lower titers than adults. The Pyloriset test may fail to recognize these specific antibodies.

Reversal of obesity- and diet-induced insulin resistance with salicylates or targeted disruption of Ikkbeta.

We show that high doses of salicylates reverse hyperglycemia, hyperinsulinemia, and dyslipidemia in obese rodents by sensitizing insulin signaling. Activation or overexpression of the IkappaB kinase beta (IKKbeta) attenuated insulin signaling in cultured cells, whereas IKKbeta inhibition reversed insulin resistance. Thus, IKKbeta, rather than the cyclooxygenases, appears to be the relevant molecular target. Heterozygous deletion (Ikkbeta+/-) protected against the development of insulin resistance during high-fat feeding and in obese Lep(ob/ob) mice. These findings implicate an inflammatory process in the pathogenesis of insulin resistance in obesity and type 2 diabetes mellitus and identify the IKKbeta pathway as a target for insulin sensitization.

Evaluation of the Helicobacter pylori stool antigen test (HpSA) for detection of Helicobacter pylori infection in children.

OBJECTIVE: Helicobacter pylori (H. pylori) infection is usually acquired in early childhood. Noninvasive methods for detection of H. pylori infection are required to study its incidence, transmission, and clearance. They should be easy to perform, inexpensive, and have a high diagnostic accuracy, especially in infants and toddlers. Both serology and the 13C-urea breath test (13C-UBT) do not fulfill all these requirements. The aim of this study was to evaluate a new enzyme immunoassay for detection of H. pylori antigen in stool (Premier Platinum HpSA, Meridian Diagnostics, Cincinnati, OH) in a large cohort of children and to compare it to invasive techniques and the 13C-UBT. METHODS: HpSA was performed in 310 stool samples of 274 children divided into three groups. Group A consisted of 145 children and adolescents (0.5-19.8 yr, 66/145 <6 yr) who underwent upper endoscopy for various gastrointestinal symptoms. H. pylori status was defined by histology, culture, and rapid urease test from biopsies of the antrum and corpus. A 13C-UBT was performed in 133 of 145 children. Group B consisted of 22 patients (5.7-16.1 yr) who were retested with both noninvasive tests 8 wk after anti-H. pylori triple therapy. Group C consisted of 129 healthy infants and toddlers (0.9-3.1 yr) who were tested with the 13C-UBT. Children with discrepant or positive test results were retested after 2 and 12 months. Results of the HpSA were read at 450/620 nm by spectrophotometry. An optical density <0.100 was defined as negative, >0.120 as positive, and values between 0.100 and 0.120 were considered as equivocal. RESULTS: In Group A, the HpSA gave false-negative results in five of 45 infected children and false-positive results in four of 100 noninfected children, whereas four patients (2.8%) showed equivocal results. In both infected and noninfected children, no relation between the optical density values and age was found. The 13C-UBT was correct in 132 of 133 children tested. In Group B, there was complete concordance between the HpSA and 13C-UBT: 19 children tested negative and three positive. In Group C, concordant results between the two noninvasive methods were found in 124 of 129 (96%) toddlers (122 negative and two positive). Retesting of five children with discrepant results revealed that, on initial testing, the HpSA was incorrect in two (one false-positive, one false-negative), and the 13C-UBT was incorrect in three (always false-positive). CONCLUSIONS: In symptomatic children, the HpSA revealed a sensitivity of 88.9% (95% CI 77.3-96.3) and a specificity of 94.0% (88.1-97.7) compared to the 13C-UBT, 100% (94.0-100) and 98.9% (94.7-100), respectively. However, in healthy toddlers, the HpSA performed as well as the 13C-UBT with excellent concordance between the two noninvasive tests. There was no age dependency of the stool test results, and changing the cutoff would not have improved accuracy. Thus, the HpSA test seems suitable to monitor the success of anti-H. pylori therapy.

Stoichiometry, kinetic and binding analysis of the interaction between epidermal growth factor (EGF) and the extracellular domain of the EGF receptor.

The kinetics, binding equilibria and stoichiometry of the interaction between epidermal growth factor and the soluble extracellular domain of the epidermal growth factor receptor (sEGFR), produced in CHO cells using a bioreactor, have been studied by three methods: analytical ultracentrifugation, biosensor analysis using surface plasmon resonance detection (BIAcore 2000) and fluorescence anisotropy. These studies were performed with an sEGFR preparation purified in the absence of detergent using a mild two step chromatographic procedure employing anion exchange and size exclusion HPLC. The fluorescence anisotropy and analytical ultracentrifugation data indicated a 1:1 molar binding ratio between EGF and the sEGFR. Analytical ultracentrifugation further indicated that the complex comprised 2EGF:2sEGFR, consistent with the model proposed recently by Lemmon et al. (1997). Global analysis of the BIAcore binding data showed that a simple Langmuirian interaction does not adequately describe the EGF:sEGFR interaction and that more complex interaction mechanisms are operative. Furthermore, analysis of solution binding data using either fluorescence anisotropy or the biosensor, to determine directly the concentration of free sEGFR in solution competition experiments, yielded Scatchard plots which were biphasic and Hill coefficients of less than unity. Taken together our data indicate that in solution there are two sEGFR populations; one which binds EGF with a KD of 2-20 nM and the other with a KD of 400-550 nM.

Reduced cell attachment and phosphorylation of focal adhesion kinase associated with expression of a mutant insulin receptor.

Insulin signaling results in rapid changes to the cell cytoskeleton, and it has recently been shown that insulin stimulates the dephosphorylation of the cytoskeletal-associated tyrosine kinase, focal adhesion kinase (pp125(FAK)). We report here that mutation of two tryptic cleavage sites (Lys164 and Lys582 --> Asn; 2N) in the insulin receptor alpha-subunit results in a cell-line (CHO.2N-10) with altered morphology associated with an increase in cell size, a decrease in cell adhesiveness, and a decrease in pp125(FAK) tyrosine phosphorylation in the absence of insulin (45.2 +/- 9.7% compared to nontransfected Chinese hamster ovary (CHO) cells). In contrast to pp125(FAK), paxillin phosphorylation was similar in all cell lines despite lower levels (61.0 +/- 10.4% compared to CHO cells) of paxillin protein in CHO.2N-10 cells. We observed comparable protein levels of pp125(FAK) and the structural focal adhesion protein, vinculin, in all cell lines. Despite underphosphorylation of pp125(FAK) in the basal state, insulin stimulation of CHO.2N-10 cells still resulted in dephosphorylation of pp125(FAK). CHO.2N-10 and CHO.T (overexpress wild-type insulin receptor) cells have similar insulin binding characteristics, insulin-stimulated autokinase and peptide phosphorylation, and insulin-stimulated pp185/IRS-1 phosphorylation. Our results suggest that the insulin receptor may play an important role in cell-matrix interactions, such as modulating cell adhesion and inducing cell architecture changes.

Insulin and insulin-like growth factor-1 stimulate dephosphorylation of paxillin in parallel with focal adhesion kinase.

Paxillin and focal adhesion kinase (pp125FAK) co-localize in focal adhesions; recently insulin has been shown to stimulate the dephosphorylation of pp125FAK; however, its effect on paxillin is unknown. We show that insulin and IGF-1 can stimulate the dephosphorylation of paxillin in CHOT (overexpress human insulin receptors) and CHO delta CT69 (overexpress insulin receptors lacking C-terminal 69 amino acids) cells. Furthermore, the insulin-receptor C-terminus is not needed for either insulin or IGF-1 to stimulate paxillin or pp125FAK dephosphorylation in the CHO (Chinese-hamster ovary) cell lines used.

Insulin receptor autophosphorylation and exogenous substrate phosphorylation: role of receptor C-terminus and effects of mild reduction.

A mutant insulin receptor lacking the final 69 amino acids of the beta-subunit (delta 69) was used to examine the role of the receptor C-terminal domain in kinase activation. With increasing deletion of the C-terminus from 43 to 69 amino acids we show that exogenous peptide kinase activity is lost before autokinase activity. Despite this, phosphorylation of an in vivo insulin receptor substrate, IRS-1, and insulin bioeffects are similar to wild-type. In addition, with the exception of insulin-stimulated peptide phosphorylation, the reductant glutathione modified kinase activity in a similar manner for both wild-type and mutant delta 69 receptors. These results suggest that conformational changes proposed to occur within the receptor C-terminus upon insulin binding may not be necessary for kinase activation under a variety of conditions.

Sulphydryl agents modulate insulin- and epidermal growth factor (EGF)-receptor kinase via reaction with intracellular receptor domains: differential effects on basal versus activated receptors.

Sulphydryl reagents have been shown to produce a variety of effects on insulin-receptor structure and function. However, localization of these effects to specific receptor domains has not been attempted. We have investigated this question with insulin- and epidermal growth factor (EGF)-receptors (both are receptor tyrosine kinases but have different sulphydryl/disulphide structures within the external domain), and the insulin receptor kinase (IRK) protein consisting solely of the insulin-receptor cytoplasmic domain and exhibiting constitutive kinase activity. Results showed a differential response between basal and activated receptors. The physiological reductant GSH stimulated basal receptor autophosphorylation, but was either without effect (EGF) or inhibited (insulin) activated receptors, and occurred without visible reduction of receptor structure. These results contrast with those obtained with dithiothreitol which appears to activate phosphorylation in association with reduction of the extracellular insulin-receptor disulphides, but is without effect on the EGF receptor or the IRK protein. Alkylating agents N-ethylmaleimide (NEM) and iodoacetamide (IAM) had opposing effects on receptor autophosphorylation. However, only in the basal state was IAM able to protect receptors from the inhibitory effect of NEM. Our results suggest that complex sulphydryl interactions can occur within the cytoplasmic domain of insulin- and EGF-receptors to alter receptor kinase activity. The basal and activated state of receptors is not the same with respect to sulphydryl reagent action, possibly due to conformational change in the receptor induced by ligand (insulin, EGF) or constitutive (IRK) activation.