Contribution of epithelial innate immunity to systemic protection afforded by prolyl hydroxylase inhibition in murine colitis.

Pharmacological stabilization of hypoxia-inducible factor (HIF) through prolyl hydroxylase (PHD) inhibition limits mucosal damage associated with models of murine colitis. However, little is known about how PHD inhibitors (PHDi) influence systemic immune function during mucosal inflammation or the relative importance of immunological changes to mucosal protection. We hypothesized that PHDi enhances systemic innate immune responses to colitis-associated bacteremia. Mice with colitis induced by trinitrobenzene sulfonic acid were treated with AKB-4924, a new HIF-1 isoform-predominant PHDi, and clinical, immunological, and biochemical endpoints were assessed. Administration of AKB-4924 led to significantly reduced weight loss and disease activity compared with vehicle controls. Treated groups were pyrexic but did not become subsequently hypothermic. PHDi treatment augmented epithelial barrier function and led to an approximately 50-fold reduction in serum endotoxin during colitis. AKB-4924 also decreased cytokines involved in pyrogenesis and hypothermia, significantly reducing serum levels of interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α while increasing IL-10. Treatment offered no protection against colitis in epithelial-specific HIF-1α-deficient mice, strongly implicating epithelial HIF-1α as the tissue target for AKB-4924-mediated protection. Taken together, these results indicate that inhibition of prolyl hydroxylase with AKB-4924 enhances innate immunity and identifies that the epithelium is a central site of inflammatory protection afforded by PHDi in murine colitis.

Study of the pharmacokinetic interaction between simvastatin and prescription omega-3-acid ethyl esters.

The coadministration of prescription omega-3-acid ethyl esters (P-OM3) with a statin may present a treatment option for patients with mixed hyperlipidemia. This open-label, randomized, 2-way crossover, drug-drug interaction study evaluated the impact of P-OM3 capsules on plasma simvastatin pharmacokinetics in 24 healthy volunteers. Under fasted conditions, 80 mg simvastatin was administered with or without 4 g P-OM3 for two 14-day periods. After 14 days of dosing to achieve steady state, no significant differences were found in either the extent (AUC(tau)) or rate (Cmax) of exposure to simvastatin or its major beta-hydroxy metabolite after coadministration of P-OM3 with simvastatin compared with administration of simvastatin alone. At steady state, the coadministration of P-OM3 capsules did not appear to affect the pharmacokinetics of simvastatin tablets. The combination of P-OM3 capsules and simvastatin appeared to be well tolerated.

Lactoferrin protects neonatal rats from gut-related systemic infection.

Lactoferrin is a milk protein that reportedly protects infants from gut-related, systemic infection. Proof for this concept is limited and was addressed during in vivo and in vitro studies. Neonatal rats pretreated orally with recombinant human lactoferrin (rh-LF) had less bacteremia and lower disease severity scores (P < 0.001) after intestinal infection with Escherichia coli. Control animals had 1,000-fold more colony-forming units of E. coli per milliliter of blood than treated animals (P < 0.001). Liver cultures from control animals had a twofold increase in bacterial counts compared with cultures from rh-LF-treated pups (P < 0.02). Oral therapy with rh-LF + FeSO(4) did not alter the protective effect. In vitro studies confirmed that rh-LF interacted with the infecting bacterium and rat macrophages. An in vitro assay showed that rh-LF did not kill E. coli, but a combination of rh-LF + lysozyme was microbicidal. In vitro studies showed that rat macrophages released escalating amounts of nitric oxide and tumor necrosis factor-alpha when stimulated with increasing concentrations of rh-LF. The in vitro studies suggest that rh-LF may act with other "natural peptide antibiotics" or may prime macrophages to kill E. coli in vivo.

Multicenter study of surfactant (beractant) use in the treatment of term infants with severe respiratory failure. Survanta in Term Infants Study Group.

OBJECTIVE: The purpose of this study was to determine whether surfactant (beractant) administration to term newborns in respiratory failure and at risk for requiring extracorporeal membrane oxygenation (ECMO) treatment would significantly reduce the incidence of severe complications through 28 days of age and the need for ECMO. STUDY DESIGN: A multicenter (n = 44), randomized, double-blind, placebo-controlled trial was conducted. Infants weighing 2000 gm or more with gestational ages of 36 weeks or greater were stratified by diagnosis (meconium aspiration syndrome, sepsis, or idiopathic persistent pulmonary hypertension of the newborn) and oxygenation index (15 to 22, 23 to 30, 31 to 39) and then randomly assigned to receive four doses of beractant, 100 mg/kg (n = 167), or air placebo (n = 161) before ECMO treatment and four additional doses during ECMO, if ECMO was required. The incidence of untoward effects (including hemorrhagic, neurologic, pulmonary, renal, cardiovascular, infectious, metabolic, and technical complications) occurring before and after randomization and through 28 days of age or discharge were recorded. RESULTS: The two treatment groups were comparable in baseline parameters, including birth weight, sex, gestational age, oxygenation index, and primary diagnosis. There was no difference in the incidence of severe complications. The need for ECMO therapy was significantly less in the surfactant group than in the placebo group (p = 0.038); this effect was greatest within the lowest oxygenation index stratum (15 to 22; p = 0.013). CONCLUSIONS: Use of surfactant, particularly in the early phase of respiratory failure, significantly decreases the need for ECMO in the treatment of term newborns with respiratory failure, without increasing the risk of complications.

Time course for effects of hypoglycemia on insulin gene transcription in vivo.

The purpose of these studies was to determine the time course for onset of effects of hypoglycemia on insulin gene transcription in vivo. Using insulin infusions, we found that insulin-induced hypoglycemia decreased levels of precursors for insulin mRNA, reflecting changes in new mRNA synthesis, to new steady-state values within 100 min. These changes were followed by declines in processed insulin mRNA. An alternate infusion technique was developed to lower plasma glucose levels from a constant level of 120-130 to 50-60 mg/dl in < 10 min without changing insulin levels from those maintained during a preceding 1-h control period. Using this protocol, we found that levels of precursors for insulin mRNA remained constant for the first 20 min of hypoglycemia, then decreased rapidly at 40 and 60 min. The initial delay followed by rapid decline suggests that the decrease of insulin gene transcription in response to hypoglycemia is an active process requiring one or more inductive events before implementation.

Use of 2H2O to study labeling in plasma glucose and hepatic glycogen during a hyperglycemic clamp.

In this study, we demonstrate the use of 2H2O, in a manner analogous to 3H2O, to study gluconeogenic flux (deuterium labeling at the carbon-6 position of glucose) relative to overall flux through glucose 6-phosphate (deuterium labeling at the carbon-2 position of glucose) into glucose output and glycogen synthesis during hyperglycemia. Before the study (4 days), jugular and carotid catheters were placed. Rats were fasted for 17 h before the study. 2H2O was infused for 2 h at 3 ml/h, with a subsequent 1-h equilibration period. A hyperglycemic clamp at 180 mg/dl (10 mM) was then performed for 90 min (plasma samples obtained at 10-min intervals). At the end of the experiment, anesthesia was induced and the liver removed. Gas chromatography-mass spectroscopy isotopomer analysis of four different mass clusters from glucose was used to determine deuterium enrichment on the carbon-2 (E2D) and carbon-6 (E6D) positions of plasma glucose and glycogen-glucose. The results show that the labeling pattern in glycogen and plasma glucose was virtually identical. In addition, the E6D-to-E2D ratio in plasma glucose did not change during hyperglycemia. Additional studies were performed to show that the E6D-to-E2D ratio was decreased in the fed state and that the fed animal, compared with the fasted rat, had a marked increase in the ratio when given an epinephrine infusion. Thus it was concluded that this was a robust new technique for analyzing glucose and glycogen metabolism in rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Hypoglycemia but not hyperglycemia induces rapid changes in pancreatic beta-cell gene transcription.

The purpose of these studies was to quantify several mRNAs expressed specifically in pancreatic islet cells and known or postulated to be important for insulin release after acute well defined alterations in levels of plasma glucose. Glucose levels were maintained at 50, 120, or 180 mg/dl (2.8, 6.7, or 10 mM) for 3 h in conscious unrestrained rats. Hypoglycemia (for 3 h) caused significant decreases in pancreatic content of mRNAs for insulin 2 and GLUT-2 to 55 and 34% of control values, respectively. There were no significant changes in insulin 1, amylin, glucokinase, or glucagon mRNAs. Unprocessed insulin 1 and 2 mRNA precursors were decreased to 17 and 10% of levels in controls, consistent with effects of short-term hypoglycemia on new mRNA synthesis. Hyperglycemia (for 3 h) caused no increase in pancreatic content of any mRNA measured. To discriminate between effects of hypoglycemia and hyperinsulinemia in the hypoglycemic animals, rats were made hypoglycemic by infusion with etomoxir, a carnitine palmitoyltransferase I inhibitor that lowers glucose in the fasted (glycogen-depleted) state by inhibiting hepatic gluconeogenesis. A single dose of this agent caused a decrease in glucose from 120 mg/dl (6.7 mM) to 80 mg/dl (4.4 mM) and significantly decreased insulin mRNA and pre-mRNA. These results are consistent with the hypothesis that glucose modulates islet cell gene transcription directly. They indicate that the range of glucose concentrations that modulate gene transcription differs from the levels of glucose that alter both insulin biosynthetic and secretion rates.

In vivo NMR imaging and spectroscopic investigation of renal pathology in lean and obese rat kidneys.

Diabetic nephropathy is a major cause of end-stage renal failure. While our understanding of the pathogenesis of nephropathy is incomplete, progressive glomerular injury appears to play a significant role in the decline of renal function. Proton NMR spectroscopy and imaging techniques were used to address changes in renal pathology associated with glomerular mesangial expansion in vivo in kidneys from spontaneously obese and lean (control) littermate Zucker rats. Fully functioning rat kidneys were surgically exposed and externalized for direct NMR signal detection via a coil placed around the organ. High-resolution (78 microns in plane) proton images were obtained at 4.7 T magnetic field strength revealing fine structure within the well-defined cortical and medullary regions. The obese rat kidney images were distinct in appearance from the lean kidney images and exhibited marked cortical expansion as well as increased overall kidney size. Enlargement of mean glomerular diameter was verified histologically in the obese kidneys as compared with the lean kidneys. Proton T1 and T2 relaxation times were determined from the entire kidney using standard spectroscopic techniques, and from specific regions within the kidney from multiple T1- and T-2 weighted images. Additionally, image contrast enhancement resulting from saturation transfer between protons in restricted-mobility environments and mobile water protons within the kidney was investigated in the lean and obese rat kidneys using magnetization-transfer imaging techniques. At the early stage of renal injury examined in this study, diseased and healthy kidneys could not be differentiated on the basis of relaxation times alone. The magnitude of saturation transfer obtained in cortical tissue in the lean and obese kidneys was also not statistically significantly different. However, the magnitude of saturation transfer achieved in the medullary tissue of obese kidneys was statistically significantly less than that achieved in lean kidneys.

The characterization of radioimmunoassay for rat pancreatic polypeptide in serum.

A radioimmunoassay for the measurement of rat pancreatic polypeptide (RPP) in serum or plasma has been developed and characterized using a new guinea-pig anti-rat-PP antibody. The assay provides a high degree of sensitivity and lacks cross-reactivity (CR less than 0.01%) to neuropeptide Y and peptide YY. It also does not interact with PPs of other species or peptide hormones namely, amylin, glucagon, human insulin, human-PP, human-proinsulin, rat C-peptide and rat insulin. The assay employs synthetic rat PP as standards from concentrations of 21-2100 pg/ml (i.e., 5-500 pM) and produces a sensitivity limit of 19 pg/ml (4.5 pM) PP at +/- 3 S.D. The intra- and interassay % coefficient of variations are 6.4% and 5.9%, respectively. The % recovery of RPP added to rat serum samples ranges from 98% to 103%. Assay of serum volumes ranging from 25 microliters to 100 microliters does not significantly alter the expected RPP level. The migration patterns of rat serum PP and that of a synthetic RPP are identical by Sephadex G-50 chromatographic analysis. The mean values of fasting and a 2 h post-feeding plasma RPP levels in normal rats are 40 +/- 2 and 80 +/- 10 pg/ml (9.5 pM and 19.0 pM), respectively. Rat-PP release during insulin induced hypoglycemia in conscious rats rises from 38 +/- 5 pg/ml to 261 +/- 34 pg/ml (9.0 to 62.1 pM, P less than 0.005) by 30 min. Additionally, the antibody used in this study cross-reacts well with mouse-PP as determined by linear serum dilution curves, thus making it useful in the measurement of murine-PP. In conclusion, we have developed and validated a sensitive and specific rat-PP assay. This assay provides a new tool for the reliable measurement of PP in physiologic studies using rat and mouse animal models.

Prevalence and consequences of nocturnal hypoglycemia among conventionally treated children with diabetes mellitus.

To determine the prevalence and predictors of, and the glucose responses after, nocturnal hypoglycemia, we studied 135 pediatric patients with insulin-dependent diabetes mellitus on 388 nights. The frequencies of blood glucose values less than 60, 50, and 40 mg/dl (3.3, 2.8, and 2.2 mmol/L) at 2 AM were 14.4%, 7.0%, and 2.1%, and at 6 AM were 6.7%, 2.6%, and 0.5%, respectively. Longer duration of diabetes, higher daily insulin doses, and lower glycosylated hemoglobin values were all significant but weak predictors of 2 AM hypoglycemia (glucose less than or equal to 60 mg/dl (less than or equal to 3.3 mmol/L). A 10 PM glucose concentration less than or equal to 100 mg/dl (less than or equal to 5.6 mmol/L) was present on 48% of nights with 2 AM glucose values less than or equal to 60 mg/dl (less than or equal to 3.3 mmol/L), but only 24% of nights with 10 PM blood glucose values less than or equal to 100 mg/dl (less than or equal to 5.6 mmol/L) were followed by 2 AM hypoglycemia. After treatment of 70 episodes of 2 AM glucose concentrations less than or equal to 60 mg/dl (less than or equal to 3.3 mmol/L), mean 6 AM glucose concentration was 95 +/- 6 mg/dl (5.7 +/- 0.3 mmol/L) and less than or equal to 100 mg/dl in 68.6%. In only 4.3% of these cases was the 6 AM glucose concentration greater than 200 mg/dl (greater than 11.1 mmol/L). Among patients who experienced 2 AM hypoglycemia, after-breakfast glucose values were not greater on days with 2 AM hypoglycemia than on days without it. These data indicate that 2 AM hypoglycemia is relatively common in patients with insulin-dependent diabetes mellitus, is frequently preceded by a 10 PM glucose value less than or equal to 5.6 mmol/L, and is less well predicted by other factors. Appropriate treatment of 2 AM hypoglycemia seldom results in either before-breakfast or after-breakfast blood glucose values greater than 200 mg/dl (greater than 11.1 mmol/L). Early-morning hypoglycemia is an uncommon cause of otherwise unexplained, prebreakfast hyperglycemia in children with insulin-dependent diabetes mellitus.

Hepatic glycogen synthesis from duodenal glucose and alanine. An in situ 13C NMR study.

An in situ and in vivo surface coil 13C NMR study was performed to study hepatic glycogen synthesis from [3-13C]alanine and [1-13C]glucose administered by intraduodenal infusion in 18-h fasted male Sprague-Dawley rats. Combined, equimolar amounts of alanine and glucose were given. Hepatic appearance and disappearance of substrate and concurrent glycogen synthesis was followed over 150 min, with 5-min time resolution. Active glycogen synthesis from glucose via the direct (glucose----glycogen) and indirect (glucose----lactate----glycogen) pathways and from alanine via gluconeogenesis was observed. The indirect pathway of glycogen synthesis from [1-13C]glucose accounted for 30% (+/- 6 S.E.) of total glycogen formed from labeled glucose. This estimate does not take into account dilution of label in the hepatic oxaloacetate pool and is, therefore, somewhat uncertain. Hepatic levels of [3-13C]alanine achieved were significantly lower than levels of [1-13C]glucose in the liver, and the period of active glycogen synthesis from [3-13C]alanine was longer than from glucose. However, the overall pseudo-first-order rate constant during the period of active glycogen synthesis from [3-13C]alanine (0.075 min-1 +/- 0.026 S.E.) was almost 3 times that from [1-13C]glucose via the direct pathway (0.025 min-1 +/- 0.005 S.E.). The most likely reason for the small rate constant governing direct glycogen formation from duodenally administered glucose compared to that from duodenally administered alanine is a low level of glucose phosphorylating capacity in the liver.

Visibility of mammalian hepatic glycogen to the NMR experiment, in vivo.

An in vivo 13C NMR study was performed to investigate whether the relaxation properties of the C-1 carbon of the glucopyranose residues of glycogen changed as the molecule increased in size. There was no change in resonance intensity or linewidth. Therefore, no significant change in T1 or T2 was observed.

Deuterium nuclear magnetic resonance measurements of blood flow and tissue perfusion employing 2H2O as a freely diffusible tracer.

The use of deuterium oxide (2H2O) is proposed as a freely diffusible nuclear magnetic resonance (NMR) blood flow and tissue perfusion tracer of potential clinical utility. Deuterium is a stable, nonradiative isotope commercially available as 2H2O at enrichment levels of essentially 100%--i.e., 110 molar equivalent deuterium. This high concentration, together with the short relaxation time of the spin 1 (quadrupole) deuterium nuclide, provides substantial sensitivity for NMR spectroscopy. As a result, when 2H2O is administered in a bolus fashion to a specific tissue or organ in vivo, the deuterium NMR intensity time course can be analyzed, using mathematical models developed by others for radiolabeled tracers, to measure the rate of blood flow and tissue perfusion. Such an application is demonstrated herein at a static magnetic field of 8.5 tesla. Using single-compartment flow modeling, hepatic blood flow and tissue perfusion in fasted (18 hr) male Sprague-Dawley rats was determined to be 61 +/- 17 (mean +/- SD) ml/100 g per min (n = 5).

The effect of puberty on the development of early diabetic microvascular disease in insulin-dependent diabetes.

We studied the prevalence of early diabetic retinopathy and nephropathy in 21 prepubertal and 55 late-pubertal subjects with insulin-dependent diabetes (IDD). All subjects had IDD of 5-7 years duration at the time of evaluation. The prevalence of early diabetic retinopathy was significantly greater in the late-pubertal subjects than prepubertal subjects (33% vs. 9.5%, P = 0.05), despite similar glycosylated hemoglobin values between the two groups (11.7 +/- 2.7% vs. 10.1 +/- 1.6%) at the time of evaluation. Nephropathy was infrequent in late-pubertal subjects (9%), and absent in the prepubertal subjects. We hypothesize that puberty plays an important role in the development of microvascular complications of IDD, and that increases in growth factors, sex hormones and deterioration in glycemic control at the time of puberty may each enhance the development of diabetic microvascular disease.

Effects of hormone and glucose administration on hepatic glucose and glycogen metabolism in vivo. A 13C NMR study.

Effects of peripheral venous injection of glucagon and insulin on [1-13C]glucose incorporation into hepatic glycogen of rats were studied by 13C NMR in vivo. Each animal was given a continuous somatostatin infusion and a 100-mg intravenous injection of [1-13C] glucose in NMR experiments or unlabeled glucose in parallel experiments for determination of serum glucose. Insulin administration caused serum glucose to fall below basal levels and accelerated the loss of hepatic [1-13C]glucose; these effects were counteracted by the addition of glucagon. Glucagon administration alone did not affect serum glucose or hepatic [1-13C] glucose but caused the loss of [1-13C]glucose from glycogen and inhibited [1-13C]glucose incorporation into glycogen. Insulin did not alter [1-13C]glucose incorporation into glycogen when given alone or in combination with glucagon. The data are consistent with a model in which liver glycogen synthesis increases linearly with hepatic glucose concentration above a threshold glucose concentration. Insulin did not alter the rate constant or the threshold for synthesis.