Rapamycin does not adversely affect intrahepatic islet engraftment in mice and improves early islet engraftment in humans

Objective: In this study we examined the effect of rapamycin (RAPA), a key component of the immunosuppressive regimen in clinical islet transplantation, on islet engraftment and function in vivo.

Methods and results: Diabetic C57BL/6 or BALB/C recipient mice were transplanted with 350 syngeneic islets through the portal vein (PV-Tx; C57BL/6 n = 60; BALB/C n = 22) and treated with once-daily oral RAPA (1 mg/kg) or vehicle. No differences in post-transplant blood glucose concentrations and glucose tolerance were observed between RAPA- and vehicle-treated mice. The impact of RAPA on human islet engraftment was assessed in 10 patients with type 1 diabetes treated with

0.1 mg/kg/day rapamycin before islet transplantation. Compared to non pre-treated islet transplant recipents (n = 12), RAPA pre-treated patients had increased blood RAPA concentrations (p = 0.006) and fasting C-peptide concentrations (p = 0.005) in the two weeks post-transplant. RAPA pre-treatment was associated with a reduction in chemokines CCL2 and CCL3 concentrations pre-transplant (p < 0.01), and a dampened chemokine response (p = 0.005) post-transplant. Concordantly, in vitro RAPA inhibited the secretion of CCL2 and CCL3 by monocytes.

Conclusion: Rapamycin does not adversely affect intrahepatic islet engraftment in the mouse, and potentially improves islet engraftment in humans by an anti-inflammatory mechanism.     

Metformin does not adversely affect hormonal and symptomatic responses to recurrent hypoglycemia

Body weight gain and severe hypoglycemia are the major adverse effects of insulin therapy in type 2 diabetic patients. Metformin has been shown to prevent insulin therapy-induced body weight gain when used in combination with insulin. However, the effects of metformin on hormonal and symptomatic responses to hypoglycemia mediating hypoglycemia awareness have not been assessed to date. Fifteen young healthy men were treated with 850 mg metformin and placebo twice daily for a 16-d period in a double blind, cross-over design. On the last 2 d of the treatment period, the subjects underwent three hypoglycemic clamp experiments, with the first and the last performed with identical patterns of plasma glucose decrease. Differences between the effects of metformin and placebo (effect of metformin) as well as between first and last hypoglycemic clamps (effect of antecedent hypoglycemia) were assessed. Antecedent hypoglycemia significantly reduced epinephrine, ACTH, cortisol, glucagon, GH, and symptomatic responses to hypoglycemia (P < 0.05 for all variables). There was no detectable effect of metformin on epinephrine, norepinephrine, ACTH, cortisol, glucagon, or autonomic symptomatic response to hypoglycemia (P > 0.05 for all comparisons), except that metformin slightly increased the response of GH to hypoglycemia (P = 0.039). The latter finding may be due to an IGF-I-reducing effect of metformin, as after 14 d of metformin treatment baseline levels of IGF-I were significantly lower than in the placebo condition (236.9 +/- 13.9 vs. 263.2 +/- 14.4 microg/liter; P = 0.015). The data indicate that metformin does not adversely affect hormonal and symptomatic responses to hypoglycemia. This finding appears to be relevant with regard to the safety of the combination of metformin with insulin therapy.     

Overnight storage of autologous stem cell apheresis products before cryopreservation does not adversely impact early or long-term engraftment following transplantation

To reduce costs and avoid inconvenient overtime work, our institution changed policy in September 2000 so that autologous stem cell apheresis products were stored overnight before cryopreservation rather than immediately processed. This retrospective review was conducted to evaluate the possible impact of this policy change on hematopoietic engraftment following autologous stem cell transplantation (ASCT). In total, 229 consecutive lymphoma patients who underwent a single, unpurged ASCT in Calgary between January 1995 and November 2003 were evaluated. Of these patients, 131 patients' autografts underwent immediate processing and cryopreservation before September 2000, and 98 patients' autografts underwent next-day cryopreservation after overnight storage following this date. Results of univariate and multivariate analyses demonstrated no adverse effect of overnight storage before cryopreservation on the number of days to initial engraftment of platelets or neutrophils, on the proportion of patients with low blood counts 6 months post-ASCT, or on lymphoma relapse rates or overall survival post-ASCT. These data suggest that overnight storage of the autograft before cryopreservation does not adversely affect graft viability or influence long-term disease status, and support the continued use of overnight storage of stem cells before cryopreservation as a convenient, cost reduction measure.     

Short-term glucosamine infusion does not affect insulin sensitivity in humans

Overactivity of the hexosamine biosynthetic pathway may underlie hyperglycemia-associated insulin resistance, but to date human studies are lacking. Hexosamine pathway activation can be mimicked by glucosamine (GlcN). In the present placebo-controlled study we determined whether GlcN infusion affects insulin resistance in vivo. In 18 healthy subjects, we applied the double forearm balance technique (infused arm vs. control arm) combined with the euglycemic hyperinsulinemic clamp (60 mU/m(2).min insulin) for at least 300 min. During the clamp, subjects received infusions in the brachial artery of 4 micromol/dL.min GlcN from 90-240 min (n = 6) or from 0-300 min (n = 6) or saline (placebo; n = 6). We studied the effects of GlcN on forearm glucose uptake (FGU; infused arm vs. control arm, and vs. placebo experiments) and on whole body glucose uptake. GlcN infusion raised the plasma GlcN concentration in the infusion arms to 0.42 +/- 0.14 and 0.81 +/- 0.46 mmol/L; plasma GlcN remained very low (< 0.07 mmol/L) in the control arms and in the placebo group. GlcN infusion did not change forearm blood flow. During insulin, FGU increased more than 10-fold. At all time points, FGU was similar in the GlcN-infused arm compared with the control arm and was not different from FGU in the placebo experiments. Similar results were obtained for forearm arteriovenous glucose differences or extraction and for whole body glucose uptake. Thus, despite relevant GlcN concentrations for 5 h in the infused forearm, GlcN had no effect on insulin-induced glucose uptake. These results do not support involvement of the hexosamine pathway in the regulation of insulin sensitivity in humans, at least not in the short-term setting.     

Acute hyperglycemia does not affect the reactivity of coronary microcirculation in humans

OBJECTIVE: There is some evidence that acute hyperglycemia (H) may cause vascular dysfunction in normal subjects. This study investigates whether acute, short-term H affects coronary vasodilatory function in healthy subjects. DESIGN: Diastolic peak flow velocity in the left anterior descending coronary artery was measured at rest and after dipyridamole (0.56 mg/kg over 4 min) using transthoracic color Doppler echocardiography in 13 healthy men. Coronary flow reserve (CFR) was defined as the ratio of dipyridamole-induced coronary peak diastolic to resting peak diastolic flow velocity. CFR was measured both in euglycemia (E) and after 3 h H ( approximately 14 mmol/liter) by a variable infusion of glucose and octreotide (0.4 mg/h) to prevent increase in insulin concentration. RESULTS: Fasting plasma glucose increased to 14.3 +/- 0.33 mmol/liter during the study and maintained variability within less than 10%. Plasma insulin remained nearly stable during H. Resting diastolic flow velocity was 18.5 +/- 0.6 cm/sec in E and increased to 20.0 +/- 0.7 cm/sec during H (P < 0.005). Dipyridamole infusion produced a marked increase in coronary flow velocity, which reached values of 50.8 +/- 2.9 cm/sec in E and 51.8 +/- 2.1 cm/sec in H (P = not significant). CFR was 2.78 +/- 0.16 in E and 2.59 +/- 0.12 in H (P = not significant). CONCLUSION: Our study indicates that short-term hyperglycemia does not affect the vasodilatory response of coronary microcirculation in healthy subjects.     

Hypercholesterolemia in pregnant mice does not affect atherosclerosis in adult offspring

In humans, maternal hypercholesterolemia during pregnancy promotes microscopical fatty streaks in the children. The mechanism is unknown. Fatty streaks are clinically silent, and many of them regress and never develop into advanced atherosclerosis. The aim of this study was to investigate whether hypercholesterolemia in pregnant mice induced more advanced atherosclerosis in their adult progeny. Hypercholesterolemic (HC) apolipoprotein E knockout (apoE(-/-)) female mice were mated with normocholesterolemic (NC) wild-type (apoE(+/+)) males and vice versa. All parents were almost identical genetically except for apoE. Therefore, all progeny became genetically identical and heterozygous apoE(+/-). They were born of either HC (i.e. apoE(-/-)) or NC (i.e. apoE(+/+)) mothers. The progeny were killed 6 months after birth and the amount of atherosclerosis in the aortic root was assessed. Females developed more atherosclerosis than males (P<0.001) but, regardless of sex, maternal hypercholesterolemia during pregnancy had no influence on the amount of atherosclerosis in adult progeny. Males of HC mothers had lower plasma cholesterol levels than males of NC mothers. Thus, in mice, maternal hypercholesterolemia during pregnancy does not promote the development of advanced atherosclerosis in their adult progeny.     

Cytogenetic clonal evolution alone in CML relapse post-transplantation does not adversely affect response to imatinib mesylate treatment

Good prognosis after imatinib mesylate treatment has been reported if cytogenetic clonal evolution (CE) is the only criterion of accelerated phase (AP) chronic myelogenous leukemia (CML). To evaluate the impact of CE upon imatinib treatment in post-transplant settings, responses and toxicities in the relapsed AP-CE were analyzed in comparison with those in the relapsed chronic phase (CP). Both CP (n=7) and AP-CE patients (n=6) received imatinib mesylate in an oral dose of 400 mg/day. Complete cytogenetic responses were obtained in six patients of each group, CP (86%) and AC-CE (100%), while molecular remission was seen in 43 and 50%, respectively. Granulocytopenia or thrombocytopenia of grade III or more occurred in four (57%) and two (33%) patients with CP and AP-CE, respectively. Nonhematological adverse events were mild and tolerable in both groups and only one (7%) of the 13 patients experienced recurrent graft-versus-host disease after imatinib treatment. Although this is a relatively small group of patients, we suggest that imatinib mesylate should be considered as a front-line treatment for relapsed CML as it showed the high response rate and low toxicity. We also suggest that CE alone is not an important factor in the induction of cytogenetic and molecular remissions in post-transplant relapse.     

Fenofibrate administration does not affect muscle triglyceride concentration or insulin sensitivity in humans

Animal data suggest that males, in particular, rely on peroxisome proliferator activated receptor-α activity to maintain normal muscle triglyceride metabolism. We sought to examine whether this was also true in men vs women and its relationship to insulin sensitivity (Si). Normolipidemic obese men (n = 9) and women (n = 9) underwent an assessment of Si (intravenous glucose tolerance test) and intramuscular triglyceride (IMTG) metabolism (gas chromatography/mass spectrometry and gas chromatography-combustion isotope ratio mass spectrometry from plasma and muscle biopsies taken after infusion of [U-¹³C]palmitate) before and after 12 weeks of fenofibrate treatment. Women were more insulin sensitive (Si: 5.2 ± 0.7 vs 2.4 ± 0.4 ×10⁻⁴/ μU/mL, W vs M, P < .01) at baseline despite similar IMTG concentration (41.9 ± 15.5 vs 30.8 ± 5.1 μg/mg dry weight, W vs M, P = .43) and IMTG fractional synthesis rate (FSR) (0.27%/h ± 0.07%/h vs 0.35%/h ± 0.06%/h, W vs M, P = .41) as men. Fenofibrate enhanced FSR in men (0.35 ± 0.06 to 0.54 ± 0.06, P = .05), with no such change seen in women (0.27 ± 0.07 to 0.32 ± 0.13, P = .73) and no change in IMTG concentration in either group (23.0 ± 3.9 in M, P = .26 vs baseline; 36.3 ± 12.0 in W, P = .79 vs baseline). Insulin sensitivity was unaffected by fenofibrate (P ≥ .68). Lower percentage saturation of IMTG in women vs men before (29.1% ± 2.3% vs 35.2% ± 1.7%, P = .06) and after (27.3% ± 2.8% vs 35.1% ± 1.9%, P = .04) fenofibrate most closely related to their greater Si (R² = 0.34, P = .10) and was largely unchanged by the drug. Peroxisome proliferator activated receptor-α agonist therapy had little effect on IMTG metabolism in men or women. Intramuscular triglyceride saturation, rather than IMTG concentration or FSR, most closely (but not significantly) related to Si and was unchanged by fenofibrate administration.     

Trapidil does not affect serum levels and cardiotonic action of digoxin in healthy humans

The influence of trapidil on serum levels and cardiac effects of digoxin was investigated in 10 healthy men, intraindividually compared with placebo. Each subject took digoxin orally for 9 days (0.375 mg daily on days 1 to 8, 0.25 mg on day 9) either combined with trapidil (400 mg daily on days 1 to 8, 200 mg on day 9) or combined with placebo in a cross-over design. Trapidil failed to change significantly the steady-state serum digoxin concentrations on day 9 of treatment. It did also not alter significantly the following digoxin effects: shortening in QS2c time indicating the cardiotonic digoxin effect, shortening in QTc time, increase in PTQ index. But trapidil prevented the negative chronotropic digoxin effect.     

A probing dose of phenylacetate does not affect glucose production and gluconeogenesis in humans

Phenylacetate ingestion has been used to probe Krebs cycle metabolism and to augment waste nitrogen excretion in urea cycle disorders. Phenylalkanoic acids, including phenylacetate, have been proposed as potential therapeutic agents in the treatment of diabetes. They inhibit gluconeogenesis in the liver in vitro and reduce the blood glucose concentration in diabetic rats. The effect of sodium phenylacetate ingestion on blood glucose and the contribution of gluconeogenesis to glucose production have now been studied in 7 type 2 diabetic patients. The study was not designed to test whether the changes in glucose metabolism observed in the rat could be reproduced in humans. After an overnight fast, over a period of 1 hour, 4.8 g phenylacetate was ingested, which is the highest dose used to probe Krebs cycle metabolism. Glucose production was measured by tracer kinetics using [6,6-(2)H2]glucose and gluconeogenesis by the labeling of the hydrogens of blood glucose on (2)H20 ingestion. The concentration of phenylacetate in plasma peaked by 2 hours after its ingestion, and about 40% of the dose was excreted in 5 hours. The plasma glucose concentration and production, and the contribution of gluconeogenesis to glucose production, were unaffected by phenylacetate ingestion at the highest dose used to probe Krebs cycle metabolism.     

Duct ligation and pancreatic islet blood flow in rats: physiological growth of islets does not affect islet blood perfusion

OBJECTIVES: The aim of this study was to evaluate islet blood-flow changes during stimulated growth of the islet organ without any associated functional impairment of islet function. DESIGN: A duct ligation encompassing the distal two-thirds of the pancreas was performed in adult, male Sprague-Dawley rats. METHODS: Pancreatic islet blood flow was measured in duct-ligated and sham-operated rats 1, 2 or 4 weeks after surgery. In some animals studied 4 weeks after surgery, islet blood flow was also measured also during hyperglycaemic conditions. RESULTS: A marked atrophy of the exocrine pancreas was seen in all duct-ligated rats. Blood glucose and serum insulin concentrations were normal. An increased islet mass was only seen 4 weeks after surgery. No differences in islet blood perfusion were noted at any time point after duct ligation. In both sham-operated and duct-ligated rats islet blood flow was increased during hyperglycaemia; the response was, however, slightly more pronounced in the duct-ligated part of the gland. CONCLUSIONS: Normal, physiological islet growth does not cause any major changes in the islet blood perfusion or its regulation. This is in contrast to findings during increased functional demands on the islets or during deteriorated islet function, when increased islet blood flow is consistently seen.     

Aspirin does not adversely affect survival in patients with stable congestive heart failure treated with Angiotensin-converting enzyme inhibitors

BACKGROUND: Experimental studies and retrospective analyses of mortality trials with angiotensin-converting enzyme inhibitors (ACE-Is) have suggested that aspirin may reduce the beneficial effect of these drugs. The aim of this study was to assess a possible detrimental effect of aspirin on survival in stable patients with left ventricular systolic dysfunction who had congestive heart failure and had been treated with ACE-Is. METHODS AND RESULTS: We performed a retrospective analysis in 755 consecutive stable patients with left ventricular systolic dysfunction. A Cox regression model was used to select independent predictors of survival and to test for a possible interaction between aspirin and ACE-Is with an adjustment to differences in clinical characteristics in subgroups of patients. Of the 755 patients, 328 (43.4%) had proven ischemic cardiomyopathy, 693 patients (91.8%) were receiving ACE-Is, and 317 patients were receiving aspirin (mean [+/- SD] dose, 183 +/- 65 mg/d; 74% of the patients receiving < or = 200 mg/d). During a median follow-up period of 1,996 days, there were 273 cardiac-related deaths, 14 urgent transplantations, 71 nonurgent transplantations, and 46 noncardiac-related deaths, and 3 patients were lost to follow-up. The cardiovascular mortality rates were 11.5% and 19.0%, respectively, at 1 and 2 years. There were no interactions among aspirin, ACE-Is, and survival in the overall population (p = 0.21), or in subgroups of patients with ischemic cardiomyopathy (p = 0.41) or with nonischemic cardiomyopathy (p = 0.74). CONCLUSIONS: In this population of stable patients with left ventricular systolic dysfunction, our retrospective analysis did not demonstrate any interaction between the use of aspirin and survival in patients receiving ACE-Is.     

Conditional islet hypovascularisation does not preclude beta cell expansion during pregnancy in mice

Aims/hypothesis

Endothelial–endocrine cell interactions and vascular endothelial growth factor (VEGF)-A signalling are deemed essential for maternal islet vascularisation, glucose control and beta cell expansion during mouse pregnancy. The aim of this study was to assess whether pregnancy-associated beta cell expansion was affected under conditions of islet hypovascularisation.

Methods

Soluble fms-like tyrosine kinase 1 (sFLT1), a VEGF-A decoy receptor, was conditionally overexpressed in maternal mouse beta cells from 1.5 to 14.5 days post coitum. Islet vascularisation, glycaemic control, beta cell proliferation, individual beta cell size and total beta cell volume were assessed in both pregnant mice and non-pregnant littermates.

Results

Conditional overexpression of sFLT1 in beta cells resulted in islet hypovascularisation and glucose intolerance in both pregnant and non-pregnant mice. In contrast to non-pregnant littermates, glucose intolerance in pregnant mice was transient. sFLT1 overexpression did not affect pregnancy-associated changes in beta cell proliferation, individual beta cell size or total beta cell volume.

Conclusions/interpretation

Reduced intra-islet VEGF-A signalling results in maternal islet hypovascularisation and impaired glycaemic control but does not preclude beta cell expansion during mouse pregnancy.