Habitat selection and refuge-use by a color polymorphic salamander reveal behavioral niche differences.

Color polymorphic species provide an excellent opportunity to investigate the ecology and evolution of intraspecific niche differences. The red-backed salamander, Plethodon cinereus, is a fully terrestrial lungless salamander with two common color forms, striped and unstriped. Previous research suggests the morphs may be differentially adapted to surface and subsurface microhabitats, with the unstriped morph being more fossorial. This hypothesis predicts that the unstriped morph should be more sensitive to the risks of surface activity (e.g., thermal stress, dehydration, predation), and therefore be more selective than striped morphs when choosing soil surface microhabitats. To test this hypothesis, we experimentally manipulated leaf litter mass in small forest patches (~0.45 m2). Leaf litter addition reduced soil temperatures, buffered against changes in air temperature, and likely provided physical protection from predators. Over 3 years, we found that unstriped adults responded positively to leaf litter addition, but striped adults did not. In addition, unstriped morphs spent significantly more time in protective refuges (opaque, moistened tubes) than striped morphs in laboratory assays. Taken together, the field and laboratory results support the hypothesis that the unstriped morph is more sensitive to the risks of surface activity, and therefore is more likely to be fossorial. This difference in microhabitat use, combined with spatiotemporal variation in leaf litter accumulation on the forest floor, may play an important role in the maintenance of the polymorphism.

Intra-patient variability in adalimumab drug levels within and between cycles in Crohn's disease.

BACKGROUND: Whether therapeutic drug monitoring for adalimumab needs to be performed at trough has not been defined. AIM: To determine intra-patient adalimumab drug-level variation and to identify modulating patient and disease factors. METHODS: In this prospective observational study, adult patients with Crohn's disease established on maintenance adalimumab had drug levels measured repeatedly according to pre-defined schedules (visit 1: day 4-6, visit 2: day 7-9, trough: day 13-14) across two consecutive fortnightly cycles. Disease activity was assessed using Harvey-Bradshaw Index, C-reactive protein and faecal calprotectin. For this analysis, trough levels ≥4.9 µg/mL were considered therapeutic. RESULTS: Nineteen patients underwent 111 evaluations. Mean intra-patient drug levels from paired visits between cycles did not differ (visit1 cycle1: 4.81, cycle2: 5.21 µg/mL, P = 0.24, visit2 cycle1: 4.86, cycle2: 4.82, P = 0.91 and trough cycle1: 3.95, cycle2: 3.95, P = 0.99), irrespective of disease activity. Drug levels were stable over the first 9 days (visit 1-2), but declined to trough by a mean 1.06 and 0.89 µg/mL between visit 1 or 2, respectively (P < 0.001). Models using nontemporal factors (smoking, syringe delivery device) and levels at earlier visits accounted for 66-80% of the variance in trough levels. On receiver-operating curve analysis, thresholds identified in the first 9 days that predicted a therapeutic trough level were similar to the trough threshold itself, with high sensitivity but modest specificity. CONCLUSION: While therapeutic drug monitoring should be performed at trough, a drug level ≥4.9 µg/mL obtained during the first 9 days predicts a therapeutic trough drug level with reasonable confidence.

Features of syndrome X develop in transgenic rats expressing a non-insulin responsive phosphoenolpyruvate carboxykinase gene.

AIMS/HYPOTHESIS: Obesity, glucose intolerance, dyslipidaemia and hypertension are a cluster of disorders (syndrome X) affecting many people. It has been hypothesised that these abnormalities are caused by insulin resistance, but definitive proof is lacking. We have developed transgenic rats in which the rate-limiting gluconeogenic enzyme, phosphoenolpyruvate carboxykinase, is non-insulin responsive. The aim of our study was to investigate whether syndrome X develops in these animals and if a high-fat diet interacts with this genetic defect. METHODS: Chow-fed transgenic and control rats aged 1, 3, 6 and 17 months and a subgroup of transgenic and control rats fed chow plus cafeteria foods for 6 months were examined for features of syndrome X. RESULTS: At 3 months, transgenic rats had fasting and postprandial hyperinsulinaemia, mild obesity (in abdominal and, to a lesser extent, peripheral regions) and fasting hypercholesterolaemia. Hypertriglyceridaemia was evident after 6 months while hyperglycaemia was apparent at 17 months. Hypertension had not developed by 17 months. The effect of a high-fat diet on insulin, glucose, body weight and body fat was more dramatic than the effect of the transgene alone while the effect of a high-fat diet on cholesterol and triglyceride was similar to the transgene. This illustrates that a high-fat diet is a potent catalyst for many abnormalities associated with syndrome X. There was no evidence of an additive effect of the high-fat diet plus transgene. CONCLUSION/INTERPRETATION: Therefore rats genetically-engineered with a non-insulin responsive gluconeogenic enzyme develop several aspects of syndrome X, supporting the hypothesis that insulin resistance initiates this cluster of disorders.

Impaired regulation of hepatic fructose-1,6-biphosphatase in the New Zealand Obese mouse: an acquired defect.

Increased hepatic glucose production, a feature of (non-insulin-dependent diabetes mellitus [NIDDM]), is present at an early age in the New Zealand Obese (NZO) mouse and is associated with impaired suppression of the gluconeogenic enzyme, fructose-1,6-bisphosphatase (FBPase). The aim of this study was to further characterize the abnormality in the regulation of hepatic FBPase in NZO mice versus New Zealand Chocolate (NZC) control mice. At 20 weeks of age, NZO mice have elevated FBPase activity (65.3 +/- 7.9 v 46.7 +/- 5.0 micromol/min/mg protein, P =.07) and protein levels (31.7 +/- 3.1 v 22.5 +/- 2.8 arbitrary units, P < .05), but not mRNA levels (0.18 +/- 0.03 v 0.16 +/- 0.03 arbitrary units). Elevated FBPase activity and protein levels in NZO mice were also shown at 4 to 6 weeks of age, but not in 1-day-old mice, suggesting that the increase occurs between birth and weaning. The Km of the enzyme was the same in NZO and NZC mice (3.7 +/- 0.5 v 5.0 +/- 0.9 micromol/L, NZO v NZC). The regulation of FBPase by the competitive inhibitor, fructose-2,6-bisphosphate ([Fru(2,6)Pz] 5 micromol/L) measured over a range of substrate concentrations (2.5 to 80 micromol/L) was similar between NZO and control mice (Km in the presence of Fru(2,6)Pz, 10.8 +/- v 1.9 v 13.2 +/- 3.3 micromol/L, NZO v NZC). It is concluded that increased FBPase activity in the NZO mouse is due to elevated protein levels, and that this appears to be due to a failure of the normal decrease that occurs following birth in control animals.

Understanding the pathogenesis of type 2 diabetes: can we get off the metabolic merry-go-rounds?

The aetiology of non-insulin-dependent diabetes mellitus (NIDDM) is not known. The concordance of NIDDM in identical twins and differences in the prevalence rate of NIDDM between different racial groups suggest a genetic cause. Hyperglycaemia in established diabetes is caused by a combination of hepatic insulin resistance, impaired peripheral (muscle and fat) glucose uptake and a defect in glucose-mediated insulin secretion. However, it is not known if these defects are all inherited or if one can cause the others. This uncertainty is due to the fact that hyperglycaemia per se can cause defects in insulin action and insulin secretion that resemble those found in NIDDM. Furthermore the elevated free fatty acid (FFA) levels found when NIDDM is associated with obesity are known to cause both peripheral and hepatic insulin resistance. Recently we have demonstrated the mechanism by which elevated FFA levels can cause hepatic insulin resistance. However, we also have evidence that the converse holds in that genetically engineered hepatic insulin resistance in a transgenic rat model leads to obesity. Thus an understanding of the pathogenesis of NIDDM is complicated by the fact that hyperglycaemia and obesity can be both causes and consequences of insulin resistance. To overcome these difficulties, studies in young euglycaemic diabetes-prone subjects have been conducted. Results suggest that there may be different causes for NIDDM in different racial groups.

Impaired glucose tolerance and increased weight gain in transgenic rats overexpressing a non-insulin-responsive phosphoenolpyruvate carboxykinase gene.

The effects of an overexpressed, non-insulin-responsive gluconeogenic enzyme, phosphoenolpyruvate carboxykinase (GTP) (PEPCK; EC 4.1.1.32), on glucose homeostasis were investigated. Transgenic rats harboring a metallothionein-driven PEPCK gene (lacking the entire PEPCK upstream-regulatory region) expressed transgene PEPCK mRNA in the key gluconeogenic tissues, liver and kidney. Female transgenic rats, studied at 10 weeks of age, showed mild fasting hyperglycemia (6.9 +/- 0.2 vs. 5.9 +/- 0.1 mM P = 0.002 n = 6), hyperinsulinemia (92.2 +/- 4.0 vs. 54.0 +/- 6.6 pM, P = 0.001, n = 6), impaired glucose tolerance and increased weight gain (178.3 +/- 3.2 vs. 153.4 +/- 2.5 g, P = 0.001, n = 16 and n = 13 transgenic and control rats, respectively). Despite hyperinsulinemia at this age, kidneys of transgenic rats maintained a significant 20% elevation of total PEPCK enzyme activity, while total liver PEPCK activity was not reduced. This study suggests that an insulin-resistant step in the gluconeogenic pathway can lead to glucose intolerance and an increase in weight. These rats offer the unique opportunity to study the metabolic consequences of chronic, mild excess glucose supply, as seen in non-insulin-dependent diabetes.

Butyrate is a potent inhibitor of urokinase secretion by normal colonic epithelium in vitro.

BACKGROUND/AIMS: Because the neutral protease urokinase is important in control of cell adhesion and migration, the effects of the physiologically relevant fermentation product butyrate on urokinase secretion by colonic epithelium were examined. METHODS: Secreted and cell-associated levels of urokinase and plasminogen activator inhibitor 1 were measured in colonic crypt cells within 24 hours of isolation from macroscopically normal mucosa of normal or cancer-bearing colons. RESULTS: Butyrate caused a concentration-dependent inhibition of both secreted (56% +/- 4% inhibition after 24-hour exposure to 1 mmol/L butyrate; n = 20; mean +/- SEM; P < 0.001) and cell-associated urokinase content (35% +/- 6%; P = 0.003). Acetate and propionate had minimal effects. Butyrate also stimulated plasminogen activator inhibitor 1 secretion by 25% +/- 7% (P = 0.013). Net urokinase activities were suppressed in supernates and cell homogenates by butyrate. Levels of transcripts for urokinase and the inhibitor changed with butyrate exposure in parallel to the levels of secretion of the respective proteins. Cells from the cancer group showed significantly reduced inhibitor secretion and abnormal responses to butyrate (greater inhibition of urokinase secretion and no stimulation of inhibitor secretion), probably reflecting the diffuse disturbance of colonic epithelial biology associated with colorectal cancer. CONCLUSIONS: Butyrate has dual effects in markedly reducing colonic epithelial urokinase activity, and these may have important implications to understanding colonic epithelial physiology and the pathogenesis and treatment of colonic diseases.

Secretion of urokinase and plasminogen activator inhibitor-1 by normal colonic epithelium in vitro.

Urokinase is a neutral protease whose major site of action is the external surface of the plasma membrane of cells and whose major function seems to be modulation of cell adhesion, such as that which occurs during cell migration. This study aimed to determine whether colonic epithelium is involved with the urokinase system. The contents of urokinase and one of its specific inhibitors, plasminogen activator inhibitor-1, were measured in culture supernatant and cell homogenates of isolated human colonic crypt cells. The amounts of both factors increased in supernatants over 24 hours, and approximately twice the amount was found in supernatants than in autologous cell homogenates. The secretion of both factors was similar in serum free and serum containing media. Northern blot analysis showed that messenger ribonucleic acid specific for urokinase and plasminogen activator inhibitor-1 was present in colonic crypt cells and that expression over 18 hours of culture was increased 12 fold for urokinase type plasminogen activator and two to fourfold for the inhibitor compared with values found in autologous freshly isolated cells. Urokinase activity was detected in crypt cell homogenates and supernatants indicating that it was present in excess of its inhibitors. Control experiments indicated that the epithelial cells themselves were responsible for the observations and excluded artefactual effects of the isolation procedure. In conclusion, isolated human colonic epithelial cells secrete urokinase and at least one of its specific inhibitors. Further investigation of the role of urokinase in the physiology and pathophysiology of colonic epithelium is indicated.

Impaired regulation of hepatic fructose-1,6-bisphosphatase in the New Zealand obese mouse model of NIDDM.

The New Zealand obese mouse, a model of NIDDM, is characterized by hyperglycemia, hyperinsulinemia, and hepatic and peripheral insulin resistance. The aim of this study was to investigate the biochemical basis of hepatic insulin resistance in NZO mice. Glycolytic and gluconeogenic enzyme activities were measured in fed and overnight fasted 19- to 20-wk-old NZO and control New Zealand chocolate mice. The NZO mice were twice as heavy as the NZC mice. The activity of the glycolytic enzymes glucokinase and pyruvate kinase was higher, whereas that of the gluconeogenic enzymes PEPCK and glucose-6-phosphatase was lower in fed and fasted NZO mice. These enzyme changes are consistent with a normal response to the hyperinsulinemia in NZO mice. In contrast, the activity of the third regulated gluconeogenic enzyme, fructose-1,6-bisphosphatase, was similar in fed and fasted NZO and NZC mice despite the higher insulin and glucose levels in the NZO mouse. This enzyme is primarily regulated by the powerful inhibitor fructose-2,6-bisphosphate. The levels of this metabolite were measured and found to be increased in both the fed and fasted states in the NZO mouse, suggesting that the activity of the bifunctional enzyme that regulates the level of inhibitor (6-phosphofructo-2-kinase/fructose-2,6- bisphosphatase) is normally regulated in the NZO mouse. We conclude that most insulin-responsive gluconeogenic and glycolytic enzymes are normally regulated in the NZO mouse, but an abnormality in the regulation of fructose-1,6-bisphosphatase may contribute to the increase hepatic glucose production in these mice.

Impaired suppression of gluconeogenesis induced by overexpression of a noninsulin-responsive phosphoenolpyruvate carboxykinase gene.

Despite detailed knowledge of the regulation of individual steps in the gluconeogenic pathway, the relative importance of each step to the overall control of gluconeogenesis by insulin is not known. The aim of this study was to determine the role of phosphoenolpyruvate carboxykinase (PEPCK) in the regulation of gluconeogenesis by insulin. Clones of the rat hepatoma cell line H4IIE-C3 were produced, overexpressing a PEPCK gene, driven by a promoter not responsive to insulin. In these cells basal gluconeogenesis from 2-[14C]pyruvate was increased 2.1-fold compared to controls (4.63 +/- 0.49 nmol/10(5) cells vs. 2.21 +/- 0.24 nmol/10(5) cells after 3 h, P < 0.05, n = 5). Increased gluconeogenesis was associated with an increase in basal PEPCK mRNA levels (1.9-fold) and enzyme activity (2.8-fold). Insulin (10(-7) M) suppressed basal gluconeogenesis, PEPCK mRNA levels, and enzyme activity in control cells, but no detectable decrease was observed in PEPCK-transfected cells. These experiments provide direct evidence in intact cells that PEPCK is the rate-limiting enzyme in gluconeogenesis from pyruvate and show that insulin's action to inhibit gluconeogenesis is predominantly on the inhibition of PEPCK transcription.

Identification of early metabolic defects in diabetes-prone Australian aborigines.

The aim of the present study was to identify in young, diabetes-prone subjects the early abnormalities which may predispose to the development of type 2 diabetes. We studied 10 full-blood Australian Aborigines all of whom had a family history of diabetes and who were from an urbanised community with a high prevalence of this disorder. They were compared to 10 age- and body-mass-index-matched Caucasian controls with no family history of diabetes. Glucose kinetics were measured basally and following an oral glucose load. Fasting plasma glucose was equal in the two groups, but 2 h following the 75 g glucose load, the Aboriginal subjects had higher glycaemia than the controls (P less than 0.01). Insulinaemia was higher in the Aborigines both basally and following the glucose drink (P less than 0.05). Despite the hyperinsulinaemia, hepatic glucose production was higher in the Aboriginal subjects (P less than 0.01), while metabolic clearance rate was lower. It is concluded that in young Australian Aborigines with a strong family history of type 2 diabetes, both hepatic and peripheral insulin resistance are early abnormalities.

Dexamethasone-induced increase in the rate of appearance in plasma of gut-derived glucose following an oral glucose load in rats.

Glucocorticoids are known to impair oral glucose tolerance and to induce insulin resistance. It has also been reported that glucocorticoids stimulate absorption of glucose, water, and electrolytes from the gut. The aim of the present study was to determine if dexamethasone treatment increased the rate of appearance in plasma of gut-derived glucose. Glucose turnover was measured following an oral glucose load in chronically catheterized, nonstressed rats treated for 96 hours with either normal saline (n = 14) or dexamethasone (5 micrograms twice daily intravenously [IV] (n = 10). Dexamethasone-treated rats had mild glucose intolerance and higher insulin levels than control rats. Total glucose turnover was increased at all time points following the glucose drink in the dexamethasone-treated rats, as was the rate of appearance of gut-derived glucose (154 +/- 25 v 321 +/- 62 mg/45 min; P = .018). It is concluded that in rats, dexamethasone treatment increases the rate of appearance in plasma of orally administered glucose.

Evolution of dexamethasone-induced insulin resistance in rats.

Glucocorticoids are known to cause insulin resistance and glucose intolerance. Although there have been many studies investigating the mechanism of this effect, several aspects remain to be clarified. The aim of this study was to investigate the evolution and sites of insulin resistance in dexamethasone-treated rats. To achieve this, chronically catheterized nonstressed rats had glucose kinetics measured during an oral glucose tolerance test by means of a double isotope technique. Studies were performed after 6, 48, or 96 h of dexamethasone administration (10 micrograms.rat-1.day-1) and were compared with control rats not treated with the steroid. Total hepatic glucose production (HGP) was increased in the 6-h (166 +/- 8.3, P less than 0.05) and 48-h (198 +/- 21, P less than 0.03) treated groups but not in the 96-h treated rats (140 +/- 8, P = 0.99) compared with the controls (141 +/- 8 mg/55 min). This increased HGP was despite the presence of higher insulin levels in the steroid-treated rats (1,220 +/- 115, P less than 0.09; 1,732 +/- 197, P less than 0.005; 1,567 +/- 107, P less than 0.001 in 6-, 48-, and 96-h treated rats, respectively, compared with 937 +/- 99 mU.l-1 x 55 min-1 in control rats). The metabolic clearance rate of glucose was higher in the dexamethasone-treated rats (200 +/- 14, P less than 0.07; 227 +/- 18, P less than 0.01; 227 +/- 17, P less than 0.01 in 6-, 48-, and 96-h groups, respectively, compared with 165 +/- 10 ml/55 min in control rats).(ABSTRACT TRUNCATED AT 250 WORDS)