Fasting protects against the side effects of irinotecan but preserves its anti-tumor effect in Apc15lox mutant mice

Irinotecan is a widely used topoisomerase-I-inhibitor with a very narrow therapeutic window because of its severe toxicity. In the current study we have examined the effects of fasting prior to irinotecan treatment on toxicity and anti-tumor activity. FabplCre;Apc^15lox/+ mice, which spontaneously develop intestinal tumors, of 27 weeks of age were randomized into 3-day fasted and ad libitum fed groups, followed by treatment with a flat-fixed high dose of irinotecan or vehicle. Side-effects were recorded until 11 days after the start of the experiment. Tumor size, and markers for cell-cycle activity, proliferation, angiogenesis, and senescence were measured. Fasted mice were protected against the side-effects of irinotecan treatment. Ad libitum fed mice developed visible signs of discomfort including weight loss, lower activity, ruffled coat, hunched-back posture, diarrhea, and leukopenia. Irinotecan reduced tumor size in fasted and ad libitum fed groups similarly compared to untreated controls (2.4 ± 0.67 mm and 2.4 ± 0.82 mm versus 3.0 ± 1.05 mm and 2.8 ± 1.08 mm respectively, P < 0.001). Immunohistochemical analysis showed reduced proliferation, a reduced number of vascular endothelial cells, and increased levels of senescence in tumors of both irinotecan treated groups. In conclusion, 3 days of fasting protects against the toxic side-effects of irinotecan in a clinically relevant mouse model of spontaneously developing colorectal cancer without affecting its anti-tumor activity. These results support fasting as a powerful way to improve treatment of colorectal carcinoma patients.     

MIDAS: software for analysis and visualisation of interallelic disequilibrium between multiallelic markers

Background  

Various software tools are available for the display of pairwise linkage disequilibrium across multiple single nucleotide polymorphisms. The HapMap project also presents these graphics within their website. However, these approaches are limited in their use of data from multiallelic markers and provide limited information in a graphical form.     

Restitution of defective glucose-stimulated insulin release of sulfonylurea type 1 receptor knockout mice by acetylcholine

Inhibition of ATP-sensitive K+ (K(ATP)) channels by an increase in the ATP/ADP ratio and the resultant membrane depolarization are considered essential in the process leading to insulin release (IR) from pancreatic beta-cells stimulated by glucose. It is therefore surprising that mice lacking the sulfonylurea type 1 receptor (SUR1-/-) in beta-cells remain euglycemic even though the knockout is expected to cause hypoglycemia. To complicate matters, isolated islets of SUR1-/- mice secrete little insulin in response to high glucose, which extrapolates to hyperglycemia in the intact animal. It remains thus unexplained how euglycemia is maintained. In recognition of the essential role of neural and endocrine regulation of IR, we evaluated the effects of acetylcholine (ACh) and glucagon-like peptide-1 (GLP-1) on IR and free intracellular Ca2+ concentration ([Ca2+]i) of freshly isolated or cultured islets of SUR1-/- mice and B6D2F1 controls (SUR1+/+). IBMX, a phosphodiesterase inhibitor, was also used to explore cAMP-dependent signaling in IR. Most striking, and in contrast to controls, SUR1-/-) islets are hypersensitive to ACh and IBMX, as demonstrated by a marked increase of IR even in the absence of glucose. The hypersensitivity to ACh was reproduced in control islets by depolarization with the SUR1 inhibitor glyburide. Pretreatment of perifused SUR1-/- islets with ACh or IBMX restored glucose stimulation of IR, an effect expectedly insensitive to diazoxide. The calcium channel blocker verapamil reduced but did not abolish ACh-stimulated IR, supporting a role for intracellular Ca2+ stores in stimulus-secretion coupling. The effect of ACh on IR was greatly potentiated by GLP-1 (10 nM). ACh caused a dose-dependent increase in [Ca2+]i at 0.1-1 microM or biphasic changes (an initial sharp increase in [Ca2+]i followed by a sustained phase of low [Ca2+]i) at 1-100 microM. The latter effects were observed in substrate-free medium or in the presence of 16.7 mM glucose. We conclude that SUR1 deletion depolarizes the beta-cells and markedly elevates basal [Ca2+]i. Elevated [Ca2+]i in turn sensitizes the beta-cells to the secretory effects of ACh and IBMX. Priming by the combination of high [Ca2+]i, ACh, and GLP-1 restores the defective glucose responsiveness, precluding the development of diabetes but not effectively enough to cause hyperinsulinemic hypoglycemia.     

A randomised sham controlled trial of vertebroplasty for painful acute osteoporotic vertebral fractures (VERTOS IV)

Background

Combination of erlotinib and bevacizumab is a promising regimen in advanced non-squamous non-small-cell lung cancer (NSCLC). We are conducting a single arm phase II trial which aims to evaluate the efficacy and safety of this regime as a second- or third-line chemotherapy.

Methods

Key eligibility criteria were histologically or cytologically confirmed non-squamous NSCLC, stage III/IV or recurrent NSCLC not indicated radical chemoradiation, prior one or two regimen of chemotherapy, age 20 years or more, and performance status of two or less. The primary endpoint is objective response rate. The secondary endpoints include overall survival, progression-free survival, disease control rate and incidence of adverse events. This trial plans to accrue 80 patients based on a two-stage design employing a binomial distribution with an alternative hypothesis response rate of 35% and a null hypothesis threshold response rate of 20%. A subset analysis according to EGFR mutation status is planned.

Discussion

We have presented the design of a single arm phase II trial to evaluate the efficacy and safety of combination of bevacizumab and erlotinib in advanced non-squamous NSCLC patients. In particular we are interested in determining the merit of further development of this regimen and whether prospective patient selection using EGFR gene is necessary in future trials.

Trial registration

This trial was registered at the UMIN Clinical Trials Registry as UMIN000004255 (http://www.umin.ac.jp/ctr/index.htm).     

Potential risk of biologic pollution associated to the introduction of <i>Pinus radiata</i> in grassland areas

Afforestation is a recommended practice to mitigate global warming. However, their implementation may generate undesirable impacts, mostly if exotic species are used. Plantations of Pinus radiata D Don in Ventania (Bs. As., Argentina) soils showed notorious increments of extractable P (Pe), which could affect the dynamic of this element as well as the degree of phosphorus saturation (GSP_Bray). The objectives of this study were: i) to quantify the GSP_Bray in Mollisols afforested with P. radiata comparing the results with those coming from adjacent, natural grassland areas (base line); ii) to evaluate the potential environmental risk induced by afforestation through the identification of a change point (PC) in the GSP_Bray indicative of a phosphate leaching increment. Treatments included mature stands of P. radiata (TB) and adjacent areas with natural grassland vegetation (TP). Samples were taken at 0-15; 15-30 and 30-45 cm soil depth, and texture, pH, total organic carbon (COT), Pe, soluble reactive phosphorus (PSR), phosphorus sorption index (ISP) and GSP_Bray were determined. The results showed a significant acidification in TB and an increase in the COT stock, indicating an additional atmospheric CO₂ sequestration by the trees. The Pe and PSR values were notoriously higher in TB, and they were reflected in a significant increment in the GSP_Bray with respect to TP. The detection of a significant PC in the GSP_Bray-PSR regression indicates higher chances of phosphate leaching in the forest stands, which could reach water courses, lakes and artificial reservoirs promoting their eutrophication. Because of the potential environmental pollution risk of biologic origin derived from the afforestation with P. radiata in Mollisols areas, their inclusion in clean development practices must be reconsidered.     

Rate of ATP synthesis in the perfused rat liver by 31P cryo-NMR

ATP concentrations in the perfused rat liver during normoxic perfusion, transient ischemia, and recovery from transient ischemia were measured using the modified 31P cryo-NMR method (Chance, B., Nakase, Y., Bond, M., Leigh, J. S., Jr., and McDonald, G. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 4925-4929). Transient ischemia was induced in the perfused livers of starved rats, and multiple freeze-trapped tissue samples were taken from each liver at short intervals (15-30 s) during ischemia or following reperfusion. The freeze-trapped tissue was pulverized together with an antifreezing agent and high energy metabolites were measured by 31P NMR at 243 K after thawing. By using the cryo-NMR technique, a biochemical time resolution of 2 s could be achieved. Absolute metabolite concentrations were calculated by comparing the peak areas with internal standards mixed into the samples. Good time resolution and reliable concentration measurements provided by the cryo-NMR method enable us to estimate the ATP synthesis rate in the perfused liver during reperfusion following transient ischemia. The rate of ATP synthesis in the normoxic perfusion was 1.95 mumol/min/g wet weight; the maximal ATP synthesis rate during the recovery phase from ischemia was 5.75 mumol/min/g wet weight.     

Glucokinase activation repairs defective bioenergetics of islets of Langerhans isolated from type 2 diabetics

It was reported previously that isolated human islets from individuals with type 2 diabetes mellitus (T2DM) show reduced glucose-stimulated insulin release. To assess the possibility that impaired bioenergetics may contribute to this defect, glucose-stimulated respiration (Vo(2)), glucose usage and oxidation, intracellular Ca(2+), and insulin secretion (IS) were measured in pancreatic islets isolated from three healthy and three type 2 diabetic organ donors. Isolated mouse and rat islets were studied for comparison. Islets were exposed to a "staircase" glucose stimulus, whereas IR and Vo(2) were measured. Vo(2) of human islets from normals and diabetics increased sigmoidally from equal baselines of 0.25 nmol/100 islets/min as a function of glucose concentration. Maximal Vo(2) of normal islets at 24 mM glucose was 0.40 ± 0.02 nmol·min(-1)·100 islets(-1), and the glucose S(0.5) was 4.39 ± 0.10 mM. The glucose stimulation of respiration of islets from diabetics was lower, V(max) of 0.32 ± 0.01 nmol·min(-1)·100 islets(-1), and the S(0.5) shifted to 5.43 ± 0.13 mM. Glucose-stimulated IS and the rise of intracellular Ca(2+) were also reduced in diabetic islets. A clinically effective glucokinase activator normalized the defective Vo(2), IR, and free calcium responses during glucose stimulation in islets from type 2 diabetics. The body of data shows that there is a clear relationship between the pancreatic islet energy (ATP) production rate and IS. This relationship was similar for normal human, mouse, and rat islets and the data for all species fitted a single sigmoidal curve. The shared threshold rate for IS was ～13 pmol·min(-1)·islet(-1). Exendin-4, a GLP-1 analog, shifted the ATP production-IS curve to the left and greatly potentiated IS with an ATP production rate threshold of ～10 pmol·min(-1)·islet(-1). Our data suggest that impaired β-cell bioenergetics resulting in greatly reduced ATP production is critical in the molecular pathogenesis of type 2 diabetes mellitus.     

Visibility of ATP and ADP in freeze-trapped tissue from perfused rat liver during normoxia and ischemia using 31P-cryo-NMR

The visibility of ATP and ADP to NMR was studied by comparing simultaneous measurements of freeze-trapped tissue sections from perfused rat liver under normoxia and ischemia using a modified 31P-cryo-NMR method and biochemical assay. The 31P-cryo-NMR method provides good time resolution and allows the quantitation of absolute metabolite concentrations. Prior to 31P-cryo-NMR measurements, freeze-trapped tissues were thawed in the presence of cryoprotectant and EDTA. With this sample preparation procedure, the integrity of the plasma and mitochondrial membranes was not maintained, inducing homogeneous microviscosity and chelation of intracellular divalent cations, thereby increasing the visibility of metabolites compared to the in vivo NMR measurement. With ischemic stress, total cellular ATP concentration decreased significantly (P less than 0.001). While ADP concentrations measured by cryo-NMR and biochemical analysis were consistent during normoxia and ischemia, ATP concentrations measured by cryo-NMR were significantly lower (P less than 0.05) than those obtained by biochemical analysis. The amount of invisible ATP (0.42 +/- 0.10 mumol/g wet weight: mean +/- S.E.) did not change after the induction of ischemia. The results of this study suggest that ATP invisibility to cryo-NMR is not due to compartmentation into regions of high paramagnetic ion concentrations or high microviscosity, but is influenced by other factors.     

Cholinergic regulation of fuel-induced hormone secretion and respiration of SUR1-/- mouse islets

Neural and endocrine factors (i.e., Ach and GLP-1) restore defective glucose-stimulated insulin release in pancreatic islets lacking sulfonylurea type 1 receptors (SUR1(-/-)) (Doliba NM, Qin W, Vatamaniuk MZ, Li C, Zelent D, Najafi H, Buettger CW, Collins HW, Carr RD, Magnuson MA, and Matschinsky FM. Am J Physiol Endocrinol Metab 286: E834-E843, 2004). The goal of the present study was to assess fuel-induced respiration in SUR1(-/-) islets and to correlate it with changes in intracellular Ca(2+), insulin, and glucagon secretion. By use of a method based on O(2) quenching of phosphorescence, the O(2) consumption rate (OCR) of isolated islets was measured online in a perifusion system. Basal insulin release (IR) was 7-10 times higher in SUR1(-/-) compared with control (CON) islets, but the OCR was comparable. The effect of high glucose (16.7 mM) on IR and OCR was markedly reduced in SUR1(-/-) islets compared with CON. Ach (0.5 microM) in the presence of 16.7 mM glucose caused a large burst of IR in CON and SUR1(-/-) islets with minor changes in OCR in both groups of islets. In SUR1(-/-) islets, high glucose failed to inhibit glucagon secretion during stimulation with amino acids or Ach. We conclude that 1) reduced glucose responsiveness of SUR1(-/-) islets may be in part due to impaired energetics, as evidenced by significant decrease in glucose-stimulated OCR; 2) elevated intracellular Ca(2+) levels may contribute to altered insulin and glucagon secretion in SUR1(-/-) islets; and 3) The amplitudes of the changes in OCR during glucose and Ach stimulation do not correlate with IR in normal and SUR1(-/-) islets suggesting that the energy requirements for exocytosis are minor compared with other ATP-consuming reactions.