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PubMed1995

High-level expression of endogenous acid phosphatase inhibits growth and vectorial secretion in Saccharomyces cerevisiae.

The secretion pathway of Saccharomyces cerevisiae was challenged by constitutively overexpressing plasmid-encoded acid phosphatase, a secreted endogenous glycoprotein. A 2-microns-based multicopy plasmid carrying the coding sequence of acid phosphatase under the control of a truncated variant of the strong constitutive glyceraldehyde-3-phosphate dehydrogenase promoter was used for expression. Selection for the promoterless dLEU2 marker leads to a growth arrest. This is not per se due to leucine starvation, but due to intracellular accumulation of highly glycosylated enzymatically active acid phosphatase. Immunofluorescence and cytological analysis indicate that intracellular accumulation of acid phosphatase occurs in a subpopulation of cells. By Ludox-AM density centrifugation, these cells can be enriched on the basis of their higher density. The dense accumulating cells have a higher average plasmid copy number and produce more acid phosphatase than non-accumulating cells of low density. These cells are defective in directed secretion and bud formation, therefore can no longer grow and show dramatic changes in cell morphology. We suggest that the secretion pathway in these cells is overloaded with the high level of acid phosphatase leading to a shutdown in vectorial secretion, subsequently to a standstill in growth and to the intracellular accumulation of further expressed acid phosphatase. We have indications that accumulation of acid phosphatase occurs in the late Golgi, suggesting a limitation of the overall secretion at this stage.

Acid Phosphatase↗
PubMed1985

Bile lipid secretion in obese and non-obese individuals with and without gallstones.

Biliary lipid secretion rates were measured in non-obese and obese individuals with and without cholesterol gallstones, using a steady-state, amino acid duodenal perfusion method. In addition, biliary lipid secretion rates were measured in five obese gallstone patients receiving high-dose chenodeoxycholic acid therapy (16-22 mg day-1 kg-1). Bile acid secretion rates in the non-obese patients with cholesterol gallstones (563 +/- SEM 70 mumol/h, n = 6) were significantly lower than in the non-obese controls (1078 +/- 210 mumol/h, n = 10, P less than 0.05), whereas cholesterol secretion rates were similar in the non-obese individuals with and without gallstones (51 +/- 7 and 42 +/- 4 mumol/h respectively). In the obese, both with and without gallstones, the major abnormality was hypersecretion of cholesterol (107 +/- 7 mumol/h, n = 7, and 81 +/- 15 mumol/h, n = 7, respectively). Both these values were significantly greater than those in the non-obese controls (P less than 0.01-0.02). Biliary cholesterol secretion rates correlated significantly with bile acid secretion rates but, for every mole of bile acid secreted, the obese secreted more cholesterol than the non-obese. Chenodeoxycholic acid treatment lowered biliary cholesterol saturation in obese gallstone patients by reducing biliary cholesterol secretion. These results suggest that there are two major types of defect in biliary lipid secretion in gallstone patients: reduced biliary bile acid secretion in non-obese gallstone patients and excessive biliary cholesterol secretion in the obese.

Adult↗
PubMed1999

Rapid-acting insulin secretagogues: a clinical need?

The history of achieving adequate insulin secretion in Type 2 diabetes has interesting parallels with Type 1 diabetes. Initial insulin secretagogue therapy involved short-acting sulphonylureas--tolbutamide being introduced 40 years ago--and early insulin therapy used unmodified (soluble) insulin. Subsequently, long-acting sulphonylureas and insulin were introduced, but more recently short-acting agents have become popular again. This approach was endorsed by the European non-insulin-dependent diabetes mellitus guidelines of 1989/1993. The trend at present is to match modern rapid-acting agents (such as repaglinide or the rapid-acting insulin analogues) to physiological meal-time insulin requirements in both types of diabetes. The fundamental reasons for tailoring therapy to meal-times fall into two categories: a pathophysiological rationale and a behavioural rationale. The pathophysiological rationale for tailoring treatment to mealtimes is based on the importance of restoring the mealtime insulin secretion profiles of patients with Type 2 diabetes to physiological levels to re-establish the tight control of blood glucose levels seen in healthy individuals. The behavioural rationale is derived from the observation that most people with Type 2 diabetes are overweight and would like to reduce calorie consumption, but that the risk of hypoglycaemia does not allow them to be flexible over their day-to-day calorie intake. These observations suggest that the matching of a meal-time pharmacodynamic profile to a patient's eating habits would provide a knowledge-based rationale for achieving good blood glucose control once dietary means alone are inadequate. Evidently, this can be usefully combined with other pharmacological approaches (insulin sensitizers or basal insulin). The need for combination therapy is increasingly likely as the defect in insulin secretion progresses.

Blood Glucose↗
PubMed2001

Prediction of the long-term metabolic success of the pancreatic graft function.

BACKGROUND: Strategies to prevent the return to the diabetic state for graft loss or failure or any other cause after pancreas transplantation require the identification of the subjects at risk. This study evaluated whether daily glucose, insulin, and c-peptide profiles and studies of insulin sensitivity and secretion after transplantation predict pancreatic graft failure. METHODS: Fifty-three subjects with type 1 diabetes with end-stage renal failure who received a combined pancreas and kidney transplant underwent the following procedures 1 year after transplantation: 1-day metabolic profiles, sampling every 2 hours for plasma glucose, serum insulin, and c-peptide (n=51); an intravenous glucose tolerance test (IVGTT) to evaluate insulin secretion (n=48); and an euglycemic insulin clamp to evaluate insulin sensitivity (M value, n=14). The recipients were then followed up to 8 years (mean follow-up 4.8+/-0.3 years) to evaluate the return to the diabetic state. RESULTS: Survival analysis showed that plasma glucose in the profiles and insulin secretion in IVGTT were strongly related to the risk of returning to the diabetic state. A cutoff value of mean daily plasma glucose >127 mg/dL, corresponding to the top quartile of the mean plasma glucose distribution in the profiles, predicted the return to the diabetic state within 4 years from transplantation with a 93% specificity and a 100% sensitivity. A cutoff value of insulin delta peak <32 microU/ml in the IVGTT predicted the return to the diabetic state within 4 years from transplantation with a 75% specificity and a 75% sensitivity. In contrast, the M value in the clamp was devoid of predictive value. CONCLUSIONS: This study indicates that the mean 24-h plasma glucose 1 year after transplantation is the strongest predictor of the return to the diabetic state. The risk is related to defects in insulin secretion and not to insulin resistance. Metabolic profiles can be used to screen the subjects at risk to strictly monitor the graft function and to investigate early determinants of graft failure.

Adult↗
PubMed2004

Impaired insulin sensitivity, insulin secretion, and glucose effectiveness predict future development of impaired glucose tolerance and type 2 diabetes in pre-diabetic African Americans: implications for primary diabetes prevention.

OBJECTIVE: We examined the determinants of impaired glucose tolerance (IGT) and type 2 diabetes in first-degree relatives of African-American type 2 diabetic patients over 5-8 years (median 6). RESEARCH DESIGN AND METHODS: A total of 81 healthy subjects (age 41.5 +/- 4.8 years; BMI 31.3 +/- 3.6 kg/m(2)) participated in the study. Each subject underwent an oral glucose tolerance test (OGTT) and a frequently sampled intravenous glucose tolerance test at baseline. Insulin sensitivity index (S(i)) and glucose effectiveness index (S(g)) were determined by the minimal model method. Homeostasis model assessment (HOMA) was used to estimate insulin resistance (HOMA-IR) and beta-cell function (HOMA-%B). A total of 18 subjects progressed to either IGT or type 2 diabetes (progressors), whereas 19 subjects maintained normal glucose tolerance (nonprogressors). RESULTS: Comparing the progressors and nonprogressors, mean fasting serum glucose levels (95 +/- 8 vs. 80 +/- 14 mg/dl, P < 0.01) and 2-h serum glucose levels (149 +/- 27 vs. 100 +/- 60 mg/dl, P < 0.01) as well as 2-h serum insulin levels (117 +/- 81 vs. 72 +/- 87 microU/ml, P < 0.01) during OGTT were higher at baseline. Mean acute first-phase insulin secretion (205 +/- 217 vs. 305 +/- 230 microU/ml), HOMA-%B (148 +/- 60 vs. 346 +/- 372, P < 01), S(i) (1.61 +/- 1.13 vs. 2.48 +/- 1.25 x 10(-4). min(-1) [microU/ml](-1)), and S(g) (1.48 +/- 0.61 vs. 2.30 +/- 0.97 x 10(-2). min(-1)) were lower in the progressors than in the nonprogressors at baseline. Mean HOMA-IR (3.31 +/- 1.64 vs. 2.36 +/- 1.64) was significantly greater in the progressors than the nonprogressors. At the time of diagnosis of glucose intolerance (IGT + diabetes), HOMA-%B (101 +/- 48 vs. 148 +/- 60, P < 0.001) and HOMA-IR (5.44 +/- 2.55 vs. 3.31 +/- 1.64, P < 0.003) deteriorated in the progressors versus baseline. CONCLUSIONS: We conclude that nondiabetic, first-degree relatives of African-American type 2 diabetic patients who progressed to IGT and type 2 diabetes manifest triple defects (decreased insulin secretion, insulin action, and glucose effectiveness) that antecede the disease.

Adult↗
PubMed1994

Defective protein phosphorylation and Ca2+ mobilization in a low secreting variant of the rat basophilic leukemia cell line.

High and low secreting variants of the rat basophilic leukemia cell line represent powerful tools to study the molecular basis of stimulus/secretion coupling via the high affinity receptor (Fc epsilon R1) complex for immunoglobulin E since an identification of the differences between these subclones may produce important information concerning the signaling pathways involved. A comparison between a variant supporting high mediator secretion (> 50%) and one with a 10-fold reduced response to antigen shows that the latter is associated with a defect in threonine and tyrosine phosphorylation of the subunits of the Fc epsilon R1 complex. The delayed onset and reduced mediator release in the low secretor facilitated a slow motion study of the early events following receptor activation. It showed that tyrosine phosphorylation of a 72-kDa protein is an early event preceding threonine and subsequent tyrosine phosphorylation of the gamma-chain. This points to the activation of both protein-tyrosine kinases and protein kinase(s) C as early events in signal transduction. The retarded onset and low intensity of phosphorylation in the low secreting variant is associated with reduced levels of inositol phosphate production, and this and the lack of the Ca2+ mobilization from intracellular stores indicate a defect upstream of teh activation of phospholipase C.

Animals↗
PubMedDate not supplied

Investigation of a novel, heritable bleeding diathesis of Thoroughbred horses and development of a screening assay.

BACKGROUND: Bleeding in racing horses associated with exercise appears to be multifactorial, and clinical investigation into severe cases rarely occurs. Previously, we reported a severe bleeding diathesis in a Thoroughbred mare. Herein, we describe the cellular physiology of this defect, provide a diagnostic tool for identifying it, and demonstrate that the dysfunction is heritable. HYPOTHESIS: The subject has a heritable defect in platelet secretion that reduces thrombin generation in the absence of additional plasma factors and delays the onset of thrombin production even in the presence of these factors. ANIMALS: The study included 3 clinically normal Thoroughbred horses: the subject and her offspring. METHODS: Washed platelets were examined for their ability to (1) translocate phosphatidylserine to the outer leaflet of the platelet membrane as determined by annexin-V binding, (2) generate thrombin as assessed by the activity of the prothrombinase enzyme complex, and (3) bind fibrinogen and form aggregates as determined by flow cytometry. RESULTS: Subject and offspring platelets created procoagulant surfaces by translocating phosphatidylserine. The subject's platelets demonstrated reduced prothrombinase activity, resulting in decreased production of thrombin relative to control platelets. Subject and offspring platelets bound less fibrinogen than control platelets when stimulated with thrombin. CONCLUSIONS AND CLINICAL IMPORTANCE: The subject mare has a transmissible defect that involves reduced generation of thrombin by activated platelets, resulting in decreased aggregation and ineffective clotting. A flow cytometric assay of fibrinogen binding to washed platelets discriminates individuals with this platelet dysfunction and may be useful for discerning subclinical congenital or acquired platelet dysfunctions.

Animals↗
PubMed1987

Growth hormone releasing hormone in the assessment and long-term treatment of growth hormone deficiency.

The secretion of hGH after the administration of the analogue of growth hormone releasing hormone, GHRH (1-29)NH2, to 8 normal adults and 41 short children has been studied. The children were classified on the basis of their hGH response to insulin-induced hypoglycaemia; 28 had severe hGH deficiency (peak serum hGH less than 7 mIU/litre) and 13 had simple short stature (peak serum hGH greater than 15 mIU/litre). The hGH response to GHRH was similar in normal adults and short stature children, but significantly lower in the hGH deficient children. In 23 (82%) of the hGH deficient children the peak serum hGH in response to GHRH was greater than 7 mIU/litre (the maximum value seen during hypoglycaemia), and in 14 (50%) the peak serum hGH in response to GHRH was greater than 15 mIU/litre. This suggests that in the majority of hGH deficient children the defect in hGH secretion results from hypothalamic GHRH deficiency. The hGH responses of the short stature children to insulin-induced hypoglycaemia were mainly in the low range of normal, and the majority showed normal hGH responses to GHRH. Eighteen prepubertal children with definite hGH deficiency have been treated for 3-18 months with twice daily, subcutaneous injections of GHRH. This has promoted linear growth in 12 children, of whom 8 showed an increment in height velocity of 2-11 cm/year. GHRH provides a valuable method for the assessment of hGH secretion, but by itself it cannot be used to establish deficient hGH secretion; this requires a stimulation test that promotes hypothalamic GHRH secretion, such as insulin-induced hypoglycaemia. GHRH is a practical alternative therapy to hGH for some hGH-deficient children.

Adult↗
PubMed1976

Kinetics of insulin secretion in chronic pancreatitis and mild maturity onset diabetes. (Evidence for "gut hormone" action beyond glucoreceptor and cyclic adenosine monophosphate mediated insulin release).

We studied insulin responses to glucose with and without cholecystokinin-pancreozymin and aminophyllin infusions in normal, chronic pancreatitic and genetic (maturity-onset) diabetic subjects. Glucose was given alone as separate 5 and 10 g boluses followed by infusion at 250 mg/min. and 500 mg/min., respectively. Chronic pancreatitis patients and genetic diabetic patients had decreased Imax values, indicating a decreased insulin reserve. Sensitivity to glucose was normal in pancreatitic subjects, but the diabetic subjects had a raised G50 value, compatible with glucoreceptor dysfunction. Infusions of aminophyllin enhanced insulin responses (Imax) to glucose injection in normal subjects and to a lesser degree in pancreatitic subjects, but decreased sensitivity to glucose (increase in G50) in both groups. Although the Imax value in pancreatitic subjects was significantly lower than in the control subjects during the glucose plus aminophyllin infusion, the blood glucose concentration in the pancreatitic subjects was nonetheless decreased. This suggests that pancreatitic subjects have increased endogenous insulin sensitivity. Aminophyllin had no effect in diabetic subjects. Crude cholecystokinin-pancreozymin changed the shape of the glucose/insulin dose response curve in normal, pancreatitic and diabetic subjects. These findings further suggest that the defect in insulin secretion in pancreatitic subjects is partly situated at the cyclic adenosine monophosphate stage of insulin release. Crude cholecystokin-pancreozymin seems to affect insulin release at a point beyond the cyclic adenosine monophosphate stage.

Adult↗
PubMed2001

[Endocrinology 1999-2000].

Long-lasting problem on the differentiation of adenohypophyseal cell, which prepares them for their specific tasks (somatotropic, lactotropic ect.), becomes elucidated after recognition of the differentiational effect of transcription factor Pit-1. Expression of that factor in somatotrops results in STH secretion, contrary to lactotrops producing prolactin. Subclinical hypothyreosis (increased TSH with normal T3 and T4) endangers vessel not because of hypercholesterolemia, but because of changes in the dynamics of the blood flow. The idea of cardiotropic effect of thyroidal hormones is supported by the finding that administration of trijodthyronine to children after the surgical correction of heart malformations (cardiopulmonary bypass) improves myocardial function--it elevates cardiac output and decreases requirements on the intensive care. Receptors for hormones in tissues are flexible, they can be "heterooligomers" for dopamine and somatostatin. Mutations of mineralocorticoid receptor may cause hypertension in pregnancy and progesterone receptors have several isoforms. Receptors can be also activated by short exposition to a hormone. Glucocorticoids have probably also membrane receptors. Diabetes mellitus "type I" needn't to be immunogenic and DM type II not only results from down-regulation of receptors and subsequent insulin resistance, but it can be also caused by defects in insulin secretion. Insulin has receptors in the brain and participates in the appetite regulation. The attempt to use "desensibilisation" by peroraly administered insulin in patients with immunogenic DM had no effect. Stress affects memory mechanisms, heavy emotional stress during gravidity can bring congenital malformations. The decrease of mental functions in aged women depends on the level of free estradiol (the fraction, which is not bound to plasma proteins). Activation of dopaminergic neurons can be achieved by neurotropic growth factors. Nesiritide is a recombinant brain natriuretic hormone successfully tested in heart failure. The role of leptin in the appetite regulation in man is still not clear, other signalling molecules may have also an effect, e.g., ghrelin, which primarily stimulates STH secretion and brings about weight gain. Sildenafil influences nitrergic neurons elsewhere than in penis, for example it has positive effects in patients with oesophageal achalasia.

Endocrine System Diseases↗
PubMed1998

Central nervous system nitric oxide synthase activity regulates insulin secretion and insulin action.

Systemic inhibition of nitric oxide synthase (NOS) with NG-monomethyl-L-arginine (L-NMMA) causes acute insulin resistance (IR), but the mechanism is unknown. We tested whether L-NMMA-induced IR occurs via NOS blockade in the central nervous system (CNS). Six groups of Sprague-Dawley rats were studied after chronic implantation of an intracerebroventricular (ICV) catheter into the lateral ventricle and catheters into the carotid artery and jugular vein. Animals were studied after overnight food deprivation, awake, unrestrained, and unstressed; all ICV infusion of L-NMMA or D-NMMA (control) were performed with artificial cerebrospinal fluid. ICV administration of L-NMMA resulted in a 30% rise in the basal glucose level after 2 h, while ICV D-NMMA had no effect on glucose levels. Insulin, epinephrine, and norepinephrine levels were unchanged from baseline in both groups. Tracer (3H-3-glucose)-determined glucose disposal rates during 2 h euglycemic hyperinsulinemic (300 microU/ml) clamps performed after ICV administration of L-NMMA were reduced by 22% compared with D-NMMA. Insulin secretory responses to a hyperglycemic clamp and to a superimposed arginine bolus were reduced by 28% in L-NMMA-infused rats compared with D-NMMA. In conclusion, ICV administration of L-NMMA causes hyperglycemia via the induction of defects in insulin secretion and insulin action, thus recapitulating abnormalities observed in type 2 diabetes. The data suggest the novel concept that central NOS-dependent pathways may control peripheral insulin action and secretion. This control is not likely to be mediated via adrenergic mechanisms and could occur via nonadrenergic, noncholinergic nitrergic neural and/or endocrine pathways. These data support previously published data suggesting that CNS mechanisms may be involved in the pathogenesis of some forms of insulin resistance and type 2 diabetes independent of adiposity.

Animals↗
PubMed2006

Relationships between glycaemic abnormalities, obesity and insulin resistance in nondiabetic Polynesians of New Caledonia.

OBJECTIVE: Polynesians in New Caledonia have an increased risk for developing diabetes, compared to Melanesians or Europeans. They are also more prone to obesity. The aim of this study was to analyse differences in the pre-diabetic state that may explain the varying susceptibility to diabetes between these three ethnic groups, focusing on the balance between insulin resistance and capacity of pancreatic cells to secrete insulin. DESIGN AND SUBJECTS: The CALDIA Study is a population-based cross-sectional survey of diabetes prevalence conducted in New Caledonia. All participants who did not have diabetes, according to the results of a 0-2 h oral glucose tolerance test (n=392), were selected for analysis. RESULTS: Compared to Europeans, Polynesians and Melanesians had significantly higher body mass indices (BMI) and waist-to-hip ratios (WHRs). Polynesians had higher fasting plasma glucose values than Europeans or Melanesians (6.03 mmol/l, vs 5.78 and 5.46, respectively; P<0.0001). Fasting plasma insulin level and the estimate of insulin resistance by homeostasis model assessment were not significantly different between the three ethnic groups. Homeostasis model assessment estimate of beta-cell secretory capacity was lower in Polynesians compared to the two other ethnic groups (83.1 mU/mmol, vs 119.3 and 125.2, respectively; P<0.02). CONCLUSION: Despite a high prevalence of central obesity, as judged by high BMI and WHR, in Polynesians of New Caledonia, their high risk of diabetes may be more strongly related to a defect in insulin secretion capacity than to insulin resistance.

Blood Glucose↗
PubMed1987

Intact microtubules are necessary for complete processing, storage and regulated secretion of von Willebrand factor by endothelial cells.

The importance of intact microtubules in the processing, storage and regulated secretion of von Willebrand factor (vWf) from Weibel-Palade bodies in endothelial cells was investigated. Human umbilical vein endothelial cells treated for 1 h with colchicine (10(-6) M) or nocodozole (10(-6) M) lost their organized microtubular network. Stimulation of these cells with secretagogues (A23187, thrombin) produced only 30% release of vWf in comparison to control cells containing intact microtubules. The nocodazole treatment was reversible. One-hour incubation in the absence of the drug was sufficient for microtubules to reform and restore the full capacity of the cells to release vWf. Long-term incubation (24 h) of endothelial cells with microtubule-destabilizing agents had a profound effect on vWf distribution. In control cells, vWf was localized to organelles in the perinuclear region (i.e., endoplasmic reticulum and Golgi apparatus) and to Weibel-Palade bodies. In drug-treated cells vWf staining was dispersed throughout the cytoplasm, and Weibel-Palade bodies were absent. The vWf synthesized in the absence of microtubules contained significantly less large multimers than that produced by control cells. Since Weibel-Palade bodies specifically contain the large multimers, we hypothesize that the structural defect in vWf secreted by cells in the absence of microtubules is due to the lack of Weibel-Palade bodies in these cultures.

Benzimidazoles↗
PubMed2005

Phospholipid transfer protein deficiency impairs apolipoprotein-B secretion from hepatocytes by stimulating a proteolytic pathway through a relative deficiency of vitamin E and an increase in intracellular oxidants.

Genetic deficiency of the plasma phospholipid transfer protein (PLTP) in mice unexpectedly causes a substantial impairment in liver secretion of apolipoprotein-B (apoB), the major protein of atherogenic lipoproteins. To explore the mechanism, we examined the three known pathways for hepatic apoB secretory control, namely endoplasmic reticulum (ER)/proteasome-associated degradation (ERAD), post-ER pre-secretory proteolysis (PERPP), and receptor-mediated degradation, also known as re-uptake. First, we found that ERAD and cell surface re-uptake were not active in PLTP-null hepatocytes. Moreover, ER-to-Golgi blockade by brefeldin A, which enhances ERAD, equalized total apoB recovery from PLTP-null and wild-type cells, indicating that the relevant process occurs post-ER. Second, because PERPP can be stimulated by intracellular reactive oxygen species (ROS), we examined hepatic redox status. Although we found previously that PLTP-null mice exhibit elevated plasma concentrations of vitamin E, a lipid anti-oxidant, we now discovered that their livers contain significantly less vitamin E and significantly more lipid peroxides than do livers of wild-type mice. Third, to establish a causal connection, the addition of vitamin E or treatment with an inhibitor of intracellular iron-dependent peroxidation, desferrioxamine, abolished the elevation in cellular ROS as well as the defect in apoB secretion from PLTP-null hepatocytes. Overall, we conclude that PLTP deficiency decreases liver vitamin E content, increases hepatic oxidant tone, and substantially enhances ROS-dependent destruction of newly synthesized apoB via a post-ER process. These findings are likely to be broadly relevant to hepatic apoB secretory control in vivo.

Animals↗
PubMed2004

Phosphatase under-producer mutants have altered phosphorus relations.

Phosphorus (P) acquisition and partitioning are essential for plant homeostasis. P is available for plant uptake when in its inorganic form (H2PO4-, or Pi), but Pi is often limiting in soils. Plants secrete acid phosphatases (APases) into the apoplastic space, which may be important for obtaining Pi from organic P sources; however, the relative importance of these enzymes for plant P nutrition has yet to be determined. We demonstrate that the root-associated APase pool is increased in Arabidopsis when Pi is limiting and document five APase isoforms secreted from Arabidopsis roots. Previously, we presented the identification of the phosphatase under-producer (pup) mutants, which have decreased in vivo root APase staining when grown under low P conditions. Here, we present the characterization of one of these, pup3, and further studies with pup1. pup3 has 49%, 38%, and 37% less specific APase activity in exudates, roots, and shoots, respectively. Root-associated APase activity is decreased by 16% in pup1 and 25% in pup3, regardless of P treatment. Two APase activity isoforms are reduced in pup3 exudates, and root and shoot isoforms are also affected. One of the two exudate isoforms is recognized by a polyclonal antibody raised to an Arabidopsis purple APase recombinant protein (AtPAP12); however, AtPAP12 transcript levels are unaffected in the mutant. The pup3 mutation was mapped to 68.4 +/- 6.0 centimorgans on chromosome 5. Although P concentrations were not altered in pup1 and pup3 tissues when grown in nutrient solution in which Pi was the sole source of P, the mutants had 10% (pup1) and 17% (pup3) lower shoot P concentrations when grown in a peat-vermiculite mix in which the majority of the total P was present as organic P. Therefore, the pup defects, which include secreted APases, are functionally important for plant P nutrition.

Acid Phosphatase↗
PubMed1996

Absence of the canalicular isoform of the MRP gene-encoded conjugate export pump from the hepatocytes in Dubin-Johnson syndrome.

The Dubin-Johnson syndrome is characterized by an inherited defect in the secretion of amphiphilic anionic conjugates from hepatocytes into the bile. We have recently identified the membrane protein mediating the adenosine triphosphate (ATP)-dependent transport of glutathione and glucuronate conjugates as a multidrug-resistance protein (MRP) and localized it to the canalicular as well as to the lateral hepatocyte plasma membrane. In the present study we show the selective absence of the canalicular isoform of MRP (cMRP) from the hepatocytes in a patient with Dubin-Johnson syndrome by double-label immunofluorescence and confocal laser scanning microscopy using antibodies directed against MRP and dipeptidyl-peptidase IV (DPPIV). Another isoform of MRP was detected, however, in the lateral hepatocyte membrane of the patient. Moreover, MRP was present on immunoblots of erythrocyte membranes from Dubin-Johnson syndrome and normal humans. These findings are analogous to our recent observations on the localization of the rat homolog of MRP and its canalicular isoform, cMrp, in normal and transport-deficient GY/TR- Wistar rat liver. The elucidation of the selective absence of an isoform of MRP and from the canalicular membrane domain in conjunction with the defined substrate specificity of the MRP and cMRP gene-encoded conjugate export pumps contributes to the molecular definition of the transport defect in Dubin-Johnson syndrome.

ATP-Binding Cassette Transporters↗
PubMed2006

Insulin sensitivity, glucose effectiveness, and free fatty acid dynamics after human islet transplantation for type 1 diabetes.

CONTEXT: Islet transplantation results in impaired insulin secretion, but whether defects in insulin sensitivity contribute to impaired glucose disposal after islet transplantation under modern immunosuppression is not known. OBJECTIVE: Our objective was to evaluate insulin sensitivity after islet transplantation performed under tacrolimus-based immunosuppression that used minimal steroids. SETTING: This study was conducted at the University of Pennsylvania General Clinical Research Center. PARTICIPANTS: Eight islet transplant recipients, six type 1 diabetic (T1D), and 10 nondiabetic control subjects participated. INTERVENTION: We performed an insulin-modified frequently sampled iv glucose tolerance test to measure insulin sensitivity (S(I)), glucose effectiveness, and free fatty acid (FFA) dynamics. RESULTS: S(I) was significantly greater in the islet transplant and control groups, compared with the T1D group (P < 0.05 for both comparisons). Glucose effectiveness was not significantly different across all three groups but was lower by trend in the T1D and islet transplant groups, compared with the control group (P = 0.07 overall ANOVA). FFA levels suppressed normally in the transplant recipients, but the timing and magnitude of FFA suppression were significantly impaired in the T1D group, compared with the islet transplant and control groups (P < 0.05 for all comparisons). The acute insulin response to glucose and the disposition index (D(I) = acute insulin response to glucose x S(I)) were significantly lower in the islet transplant group, compared with the control group (P < 0.05 for all comparisons). CONCLUSIONS: These data suggest that even modest restoration of insulin secretion in islet transplant recipients may result in improved insulin sensitivity and FFA dynamics.

Adult↗
PubMed1998

Actions of adenosine A1 and A2 receptor antagonists on CFTR antibody-inhibited beta-adrenergic mucin secretion response.

1. The cystic fibrosis gene protein, the cystic fibrosis transmembrane conductance regulator (CFTR) acts as a chloride channel and is a key regulator of mucin secretion. The mechanism by which 3-isobutyl-1-methylxanthine (IBMX) corrects the defect in CFTR mediated beta-adrenergic stimulation of mucin secretion has not been determined. The present study has investigated the actions of adenosine A1 and A2 receptor antagonists to determine whether ability to stimulate mucin secretion correlates with correction of CFTR antibody inhibited beta-adrenergic response and whether excessive cyclic AMP rise is required. 2. CFTR antibodies were introduced into living rat submandibular acini by hypotonic swelling. Following recovery, mucin secretion in response to isoproterenol was measured. 3. The adenosine A1 receptor antagonist, 8 cyclopentyltheophylline (CPT) was a less potent stimulator of mucin secretion than was the A2 receptor antagonist dimethylpropargylxanthine (DMPX). A concentration of CPT close to the Ki for A1 receptor antagonism (10 nM) did not stimulate mucin secretion. 4. DMPX, although a potent stimulator of mucin secretion, did not correct CFTR antibody inhibited mucin secretion. 5. CPT corrected defective CFTR antibody inhibited mucin secretion at a high (1 mM) concentration, suggesting a mechanism other than adenosine receptor antagonism. 6. DMPX potentiated the isoproterenol induced cyclic AMP rise, whereas CPT did not. 7. Correction of the defective CFTR mucin secretion response did not correlate with ability to stimulate mucin secretion and did not require potentiation of beta-adrenergic induced increases in cyclic AMP. This affords real promise for the development of a selective drug treatment for cystic fibrosis.

1-Methyl-3-isobutylxanthine↗