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We examined whether exercising indoors vs. outdoors reduced the cardio-respiratory effects of outdoor air pollution. Adults ≥55 were randomly assigned to exercise indoors when the Air Quality Health Index was ≥5 and outdoors on other days (intervention group, n = 37), or outdoors everyday (control group, n = 35). Both groups completed cardio-respiratory measurements before and after exercise for up to 10 weeks. Data were analyzed using linear mixed effect regression models. In the control group, an interquartile range increase in fine particulate matter (PM 2.5 ) was associated with increases of 1.4% in heart rate (standard error (SE) = 0.7%) and 5.6% (SE = 2.6%) in malondialdehyde, and decreases of 5.6% (SE = 2.5%) to 16.5% (SE = 7.5%) in heart rate variability measures. While the hypothesized benefit of indoor vs. outdoor exercise could not be demonstrated due to an insufficient number of intervention days (n = 2), the study provides evidence of short-term effects of air pollution in older adults. ISRCTN #26552763.

Background : An oil refinery in Oakville, Canada, closed over 2004⁻2005, providing an opportunity for a natural experiment to examine the effects on oil refinery-related air pollution and residents' health. Methods : Environmental and health data were collected for the 16 years around the refinery closure. Toronto (2.5 million persons) and the Greater Toronto Area (GTA, 6.3 million persons) were used as control and reference populations, respectively, for Oakville (160,000 persons). We compared sulfur dioxide and age- and season-standardized hospitalizations, considering potential factors such as changes in demographics, socio-economics, drug prescriptions, and environmental variables. Results : The closure of the refinery eliminated 6000 tons/year of SO₂ emissions, with an observed reduction of 20% in wind direction-adjusted ambient concentrations in Oakville. After accounting for trends, a decrease in cold-season peak-centered respiratory hospitalizations was observed for Oakville (reduction of 2.2 cases/1000 persons per year, p = 0.0006 ) but not in Toronto (p = 0.856) and the GTA (p = 0.334). The reduction of respiratory hospitalizations in Oakville post closure appeared to have no observed link to known confounders or effect modifiers. Conclusion : The refinery closure allowed an assessment of the change in community health. This natural experiment provides evidence that a reduction in emissions was associated with improvements in population health. This study design addresses the impact of a removed source of air pollution.

Background: There is a paucity of mechanistic information that is central to the understanding of the adverse health effects of source emission exposures. To identify source emission-related effects, blood and saliva samples from healthy volunteers who spent five days near a steel plant (Bayview site, with and without a mask that filtered many criteria pollutants) and at a well-removed College site were tested for oxidative stress, inflammation and endothelial dysfunction markers., Methods: Biomarker analyses were done using multiplexed protein-array, HPLC-Fluorescence, EIA and ELISA methods. Mixed effects models were used to test for associations between exposure, biological markers and physiological outcomes. Heat map with hierarchical clustering and Ingenuity Pathway Analysis (IPA) were used for mechanistic analyses., Results: Mean CO, SO 2 and ultrafine particles (UFP) levels on the day of biological sampling were higher at the Bayview site compared to College site. Bayview site exposures "without" mask were associated with increased (p < 0.05) pro-inflammatory cytokines (e.g IL-4, IL-6) and endothelins (ETs) compared to College site. Plasma IL-1β, IL-2 were increased (p < 0.05) after Bayview site "without" compared to "with" mask exposures. Interquartile range (IQR) increases in CO, UFP and SO 2 were associated with increased (p < 0.05) plasma pro-inflammatory cytokines (e.g. IL-6, IL-8) and ET-1 (1-21) levels. Plasma/saliva BET-1 levels were positively associated (p < 0.05) with increased systolic BP. C-reactive protein (CRP) was positively associated (p < 0.05) with increased heart rate. Protein network analyses exhibited activation of distinct inflammatory mechanisms after "with" and "without" mask exposures at the Bayview site relative to College site exposures., Conclusions: These findings suggest that air pollutants in the proximity of steel mill site can influence inflammatory and vascular mechanisms. Use of mask and multiple biomarker data can be valuable in gaining insight into source emission-related health impacts.

Objective: The aim of this study was to assess cardiorespiratory effects of air pollution in older adults exercising outdoors in winter., Methods: Adults 55 years of age and older completed daily measurements of blood pressure, peak expiratory flow and oximetry, and weekly measurements of heart rate variability, endothelial function, spirometry, fraction of exhaled nitric oxide and urinary oxidative stress markers, before and after outdoor exercise, for 10 weeks. Data were analyzed using linear mixed effect models., Results: Pooled estimates combining 2014 (n = 36 participants) and 2015 (n = 34) indicated that an interquartile increase in the Air Quality Health Index was associated with a significant (P < 0.05) increase in heart rate (0.33%) and significant decreases in forced expiratory volume (0.30%), and systolic (0.28%) and diastolic blood pressure (0.39%)., Conclusion: Acute subclinical effects of air pollution were observed in older adults exercising outdoors in winter.

In developed countries, phthalate exposure is ubiquitous. Previous studies have shown an association between phthalate levels and health effects. To test associations between phthalate exposures, estimated from urinary phthalate metabolites, and insulin resistance, β-cell function and glucose control. Data were obtained from a cross-sectional, nationally representative study; the Canadian Health Measures Survey (CHMS, 2009-2011). Participants under the age of 12, those with diabetes, who were pregnant or who had not fasted overnight were excluded. Fasting blood glucose, insulin, and glycosylated hemoglobin (HbA 1C ) levels were measured in a subset of participants, and urine was collected for creatinine and phthalate metabolites. We tested associations between these variables using linear regression analysis. Of 4437 participants (12-79years old), 2119 had fasting glucose measurements and at least one phthalate metabolite above detection limits. MBzP, MCPP, MEHP, MEHHP, MiBP, and the sum of DEHP metabolites were positively associated with increased HbA 1C (p<0.05). DEHP metabolites were positively associated with increased fasting glucose, insulin, HOMA-IR and HOMA-β. An interquartile increase in the sum of log transformed DEHP metabolites was associated with increases in HOMA-IR and HOMA-β of 0.15 (95% CI 0.04, 0.26) and 10.24 (95% CI 3.71, 16.77) respectively. Increased concentrations of all measured phthalate metabolites were associated with reduced blood glucose control. DEHP metabolites were also associated with increased glucose concentrations, and indicators of β-cell function and insulin resistance. Our results suggest that exposure to phthalates may possibly impair control of blood glucose and thereby predispose to pre-diabetes., (Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.)

The effects of industrial air pollution on human health have not been as thoroughly investigated as those of urban air pollution which originates mostly from automotive transport. To better assess the health impacts of point sources of industrial air pollution, a randomized crossover exposure study was conducted. Sixty one young and healthy volunteers were randomly assigned to spend five consecutive eight-hour days near a steel mill or at a location five kilometres away. After a nine or sixteen-day washout period, volunteers spent another five consecutive days at the second site. Meteorological conditions and air pollutants were monitored at both exposure sites. On each exposure day, the first morning urine was collected along with a second urine sample obtained immediately before leaving the exposure site at the end of the day. Urinary levels of biomarkers of oxidative stress 8-hydroxy-2'-deoxyguanosine (8-OHdG, a biomarker of oxidative DNA damage), malondialdehyde (MDA, a biomarker of lipid peroxidation), 8-isoprostane (8-IsoP, a bioactive metabolite resulting from the peroxidation of arachidonic acid) and Vascular Endothelial Growth Factor (VEGF, involved in response to oxidative stress) were measured. According to mixed-effects linear regression models, intra-individual variations in 8-OHdG urinary levels were significantly associated with exposure site, but surprisingly, lower levels were observed at the steel mill site. Delayed, temporally-defined associations with specific air pollutants were observed for 8-OHdG, 8-IsoP and VEGF. However, these associations were subtle, presented complex patterns and their biological consequences remain unclear., (Crown Copyright © 2016. Published by Elsevier GmbH. All rights reserved.)

Background: Epidemiological studies have shown that as ambient air pollution (AP) increases the risk of cardiovascular mortality also increases. The mechanisms of this effect may be linked to alterations in autonomic nervous system function. We wished to examine the effects of industrial AP on heart rate variability (HRV), a measure of subtle changes in heart rate and rhythm representing autonomic input to the heart., Methods: Sixty healthy adults were randomized to spend five consecutive 8-h days outdoors in one of two locations: (1) adjacent to a steel plant in the Bayview neighbourhood in Sault Ste Marie Ontario or (2) at a College campus, several kilometers from the plant. Following a 9-16 day washout period, participants spent five consecutive days at the other site. Ambient AP levels and ambulatory electrocardiogram recordings were collected daily. HRV analysis was undertaken on a segment of the ambulatory ECG recording during a 15 min rest period, near the end of the 8-h on-site day. Standard HRV parameters from both time and frequency domains were measured. Ambient AP was measured with fixed site monitors at both sites. Statistical analysis was completed using mixed-effects models., Results: Compared to the College site, HRV was statistically significantly reduced at the Bayview site by 13% (95%CI 3.6,19.2) for the standard deviation of normal to normal, 8% (95%CI 0.1, 4.9) for the percent normal to normal intervals differing by more than 50 ms, and 15% (95%CI 74.9, 571.2) for low frequency power. Levels of carbon monoxide, sulphur dioxide, nitrogen dioxide, and fine and ultrafine particulates were slightly, but statistically significantly, elevated at Bayview when compared to College. Interquartile range changes in individual air pollutants were significantly associated with reductions in HRV measured on the same day. The patterns of effect showed a high degree of consistency, with nearly all pollutants significantly inversely associated with at least one measure of HRV., Conclusions: The significant associations between AP and changes in HRV suggest that ambient AP near a steel plant may impact autonomic nervous system control of the heart.

Exposure to residential volatile organic compounds (VOCs) is ubiquitous in homes, and may influence respiratory health with important public health implications. To investigate the association between VOCs measured in residential indoor air and lung function in the Canadian population Cycle 2 of the Canadian Health Measures Survey - a population based cross-sectional survey designed to be representative of the Canadian population - was carried out between 2009 and 2011. Of the 84 VOCs measured, 47 were detectable in at least 50% of homes and ten were negatively associated with lung function: decanal, 2-furancarboxaldehyde, hexanal, nonanal, octanal, benzene, styrene, α-pinene, 2-methyl-1,2-butadiene and naphthalene. Differences were observed between males and females, as well as by age, and significant associations were most frequent in those under 17 years. These results provide evidence that some VOCs measured indoors are negatively associated with lung function in the Canadian population., (Crown Copyright © 2014. Published by Elsevier Ltd. All rights reserved.)

Objective: The acute cardiorespiratory effects of air quality among children living in areas with considerable heavy industry have not been well investigated. We conducted a panel study of children with asthma living in proximity to an industrial complex housing two refineries in Montreal, Quebec, in order to assess associations between their personal daily exposure to air pollutants and changes in pulmonary function and selected indicators of cardiovascular health., Methods: Seventy-two children with asthma age 7-12 years in 2009-2010 participated in this panel study for a period of 10 consecutive days. They carried a small backpack for personal monitoring of sulphur dioxide (SO2), benzene, fine particles (PM2.5), nitrogen dioxide (NO2) and polycyclic aromatic hydrocarbons (PAHs) and underwent daily spirometry and cardiovascular testing (blood pressure, pulse rate and oxygen saturation). To estimate these associations, we used mixed regression models, adjusting for within-subject serial correlation, and for the effects of a number of personal and environmental variables (e.g., medication use, ethnicity, temperature)., Results: Children with asthma involved in the study had relatively good pulmonary function test results (mean FEV1 compared to standard values: 89.8%, mean FVC: 97.6%, mean FEF25-75: 76.3%). Median diastolic, systolic blood pressures and oxygen saturation were 60/94 mmHg and 99%, respectively. Median personal concentrations of pollutants were NO2, 5.5 ppb; benzene, 2.1 µg/m(3); PM2.5, 5.7 µg/m(3); and total PAH, 130 µg/m(3). Most personal concentrations of SO2 were below the level of detection. No consistent associations were observed between cardio-pulmonary indices and personal exposure to PM2.5, NO2 and benzene, although there was a suggestion for a small decrease in respiratory function with total concentrations of PAHs (e.g., adjusted association with FVC: -9.9 ml per interquartile range 95%CI: -23.4, 3.7)., Conclusions: This study suggests that at low daily average levels of exposure to industrial emissions, effects on pulmonary and cardiovascular functions in children with asthma may be difficult to detect over 10 consecutive days., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: Studying the physiologic effects of components of fine particulate mass (PM2.5) could contribute to a better understanding of the nature of toxicity of air pollution., Objectives: We examined the relation between acute changes in cardiovascular and respiratory function, and PM2.5-associated-metals., Methods: Using generalized linear mixed models, daily changes in ambient PM2.5-associated metals were compared to daily changes in physiologic measures in 59 healthy subjects who spent 5-days near a steel plant and 5-days on a college campus., Results: Interquartile increases in calcium, cadmium, lead, strontium, tin, vanadium and zinc were associated with statistically significant increases in heart rate of 1-3 beats per minute, increases of 1-3 mmHg in blood pressure and/or lung function decreases of up to 4% for total lung capacity., Conclusion: Metals contained in PM2.5 were found to be associated with acute changes in cardiovascular and respiratory physiology., (Crown Copyright © 2014. Published by Elsevier Ltd. All rights reserved.)

Background: Iron and steel industry is an important source of air pollution emissions. Few studies have investigated cardiovascular effects of air pollutants emitted from steel plants., Objective: We examined the influence of outdoor air pollution in the vicinity of a steel plant on cardiovascular physiology in Sault Ste. Marie, Canada., Methods: Sixty-one healthy, non-smoking subjects (females/males=33/28, median age 22 years) spent 5 consecutive 8-hour days outdoors in a residential area neighbouring a steel plant, or on a college campus approximately 5 kilometres away from the plant, and then crossed over to the other site with a 9-day washout. Mid day, subjects underwent daily 30-minute moderate intensity exercise. Blood pressure (BP) and pulse rate were determined daily and post exercise at both sites. Flow-mediated vasodilation (FMD) was determined at the site near the plant. Air pollution was monitored at both sites. Mixed-effects regressions were run for statistical associations, adjusting for weather variables., Results: Concentrations of ultrafine particles, sulphur dioxide (SO2), nitrogen dioxide (NO2) and carbon monoxide (CO) were 50-100% higher at the site near the plant than at the college site, with minor differences in temperature, humidity, and concentrations of particulate matter ≤2.5 μm in size (PM2.5) and ozone (O3). Resting pulse rate [mean (95% confidence interval)] was moderately higher near the steel plant [+1.53 bpm (0.31, 2.78)] than at the college site, male subjects having the highest pulse rate elevation [+2.77 bpm (0.78, 4.76)]. Resting systolic and diastolic BP and pulse pressure, and post-exercise BP and pulse rate were not significantly different between two sites. Interquartile range concentrations of SO2 (2.9 ppb), NO2 (5.0 ppb) and CO (0.2 ppm) were associated with increased pulse rate [0.19 bpm (-0.00, 0.38), 0.86 bpm (0.03, 1.68), and 0.11 bpm (0.00, 0.22), respectively], ultrafine particles (10,256 count/cm(3)) associated with increased pulse pressure [0.85 mmHg (0.23, 1.48)], and NO2 and CO inversely associated with FMD [-0.14% (-0.31, 0.02), -0.02% (-0.03, -0.00), respectively]. SO2 during exercise was associated with increased pulse rate [0.26 bpm (0.01, 0.51)]., Conclusion: Air quality in residential areas near steel plants may influence cardiovascular physiology., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

Background: Steel production is a major industry worldwide yet there is relatively little information on the pulmonary effects of air quality near steel manufacturing plants., Objectives: The aim of this study was to examine how lung function changes acutely when healthy subjects are situated near a steel plant which is adjacent to a residential area., Methods: Sixty-one subjects were randomly assigned to spend 5 consecutive, 8-hour days in a residential neighborhood approximately 0.9km from a steel plant, or approximately 4.5km away at a college campus. Subjects crossed-over between sites after a nine-day washout period. Lung function was measured daily at both sites along with air pollutants including SO2, NO2, O3, PM2.5, and ultrafine particles. Diffusion capacity and pulse oximetry were also examined., Results: Compared with the college site, the forced expiratory volume in 1-second/forced vital capacity, forced expiratory flow between 25% and 75% of the FVC, total lung capacity, functional residual capacity, and residual volume were lower near the steel plant by 0.67% (95% CI: 0.28, 1.06),1.62% (95% CI: 0.50, 2.75), 1.54% (95% CI: 0.68, 2.39), 3.54% (95% CI: 1.95, 5.13) and 11.3% (95% CI: 4.92, 17.75), respectively. Diffusion capacity, forced expiratory volume in 1s, and pulse oximetry were also lower near the plant but these effects were not statistically significant. Sulfur dioxide, ultrafine particulates, and oxides of nitrogen were greater near the steel plant site compared to the college site., Conclusions: Spending short periods of time near a steel plant is associated with a decrease in lung function., (Crown Copyright © 2013. Published by Elsevier Ltd. All rights reserved.)

Type 1 diabetes is inhibited in diabetes-prone BioBreeding (BBdp) rats fed a low-antigen hydrolyzed casein (HC) diet. In cereal-fed BBdp rats, islet expansion is defective accompanied by a futile upregulation of islet neogenesis without increased islet mass, due to a subtle blockage in islet cell cycle. We hypothesized that islet growth is enhanced before insulitis in HC-fed young BBdp rats and that islet neogenesis could be stimulated by a trophic factor, islet neogenesis-associated protein (INGAP). beta-Cell homeostasis was analyzed using immunohistochemistry, morphometry, laser capture microdissection and RT-PCR in BBdp rats fed HC or cereal diets. beta-cell proliferation in small and medium islets, and the number and area fraction of medium and large islets were increased in HC-fed animals. In situ islet cell cycle analysis revealed an increased proportion of proliferating S + G2 cells in medium and large islets of 25-45 day HC-fed rats. Expression of the cell cycle inhibitor, p16(INK4a) correlated with islet size and the percentage of p16(INK4a+) beta-cells increased in HC-fed BBdp rats, likely reflecting an increase in large islet area fraction. In HC-fed rats, extra-islet insulin(+) clusters (EIC), insulin(+) duct cells, large islet area fraction, and beta-cell mass were increased. Neurogenin-3 and Pdx-1, markers of beta-cell progenitors, were increased in EIC of weanling HC-fed rats. Daily injection of INGAP (30-45 days) increased the number of small islets, total islets, and insulin(+) cells in small ducts. Thus, in BBdp rats fed a protective HC diet, beta-cell expansion is enhanced through increased beta-cell proliferation and stimulation of islet neogenesis.

Prior to its application for in vitro toxicological assays, thorough characterization of a cell line is essential. The present study uses global transcriptional profiling to characterize a lung epithelial cell line (FE1) derived from MutaMouse [White, P.A., Douglas, G.R., Gingerich, J., Parfett, C., Shwed, P., Seligy, V., Soper, L., Berndt, L., Bayley, J., Wagner, S., Pound, K., Blakey, D., 2003. Development and characterization of a stable epithelial cell line from Muta Mouse lung. Environmental and Molecular Mutagenesis 42, 166-184]. Results presented here demonstrate the origin of the FE1 lung cell line as epithelial, presenting both type I and type II alveolar phenotype. An assessment of toxicologically-relevant genes, including those involved in the response to stress and stimuli, DNA repair, cellular metabolism, and programmed cell death, revealed changes in expression of 22-27% of genes in one or more culture type (proliferating and static FE1 cultures, primary epithelial cultures) compared with whole lung isolates. Gene expression analysis at 4 and 24h following benzo(a)pyrene exposure revealed the induction of cyp1a1, cyp1a2, and cyp1b1 in FE1 cells and lung isolates. The use of DNA microarrays for gene expression profiling allows an improved understanding of global, coordinated cellular events arising in cells under different physiological conditions. Taken together, these data indicate that the FE1 cell line is derived from a cell type relevant to toxic responses in vivo, and shows some similarity in response to chemical insult as the original tissue.

We reported previously that young BioBreeding diabetes-prone (BBdp) rats display increased neogenic extra-islet insulin+ clusters (EICs, <4 insulin+ cells) without an increase in beta-cell mass. Therefore, we investigated the possibility that abnormal islet expansion occurs in BBdp rats before the appearance of islet inflammation. Islet expansion was analyzed in pancreata from 14 to 45 day BBdp and control (BioBreeding control, BBc) rats using immunohistochemistry, morphometry, laser capture microdissection and reverse transcriptase-PCR. mRNA expression for Neurogenin-3, a developmental marker of endocrine progenitors, was three-fold greater in EIC of weanling BBdp and BBc rats compared with islet cells. With increasing age (14-30 days), Neurogenin-3 expression decreased in EIC and increased in islets. In BBdp rats, EIC number and beta-cell proliferation within EIC was greater compared with BBc animals; apoptosis did not differ. The area of small and medium islets in BBdp rats was greater than BBc rats between 14 and 30 days, but this did not result in increased total islet area or beta-cell mass. In addition, the number and area of very large islets was low at 45 days. The frequency of proliferating beta-cells decreased with increasing islet size in BBdp but was constant in BBc rats. Cell cycle analysis of islets revealed more G1 cells and fewer G2 cells in BBdp rats. The ratio of cyclinD2/Cdkn1a, genes that respectively promote or inhibit cell cycle progression, was decreased in BBdp islets. These results suggest that despite increased islet neogenesis, the capacity for islet expansion in diabetes-prone rats is compromised possibly due to decreased proliferative capacity with increasing islet size associated with a partial block at the G1/S cell cycle boundary in islet cells.

C57BL/6 (B6) mice develop glucose intolerance with age, whereas C3H/He (C3H) mice do not. In this study, we examined whether this differential glucose homeostasis was associated with differences of proteolytic activation of pancreatic prohormones. Radioimmunoassays showed comparable levels of fasting plasma insulin between the two strains but a significantly lower glucagon level in B6 mice. Pulse-chase analysis of glucagon biosynthesis in isolated pancreatic islets revealed that proglucagon was less efficiently processed in B6 mice. Because proprotein convertase (PC)2 and its 7B2 helper protein are required for this processing, we quantified islet mRNA levels by RT-PCR and protein levels by immunoblotting. The levels of proPC2 mRNA were similar between the two strains, but B6 protein extracts contained less of the mature PC2. In contrast, 7B2 mRNA and protein levels were both significantly lower in B6 pancreas. Sequencing of the 7B2 gene promoter and cDNA in the two strains revealed seven single nucleotide polymorphisms and one dinucleotide insertion/deletion in the cDNA as well as a single nucleotide polymorphism and two insertions/deletions in the promoter. Differential expression of 7B2 may contribute to the difference between B6 and C3H mice not only in glucagon production and secretion but also in glucose tolerance.

Glucose induces complex patterns of oscillations in intracellular Ca2+ concentration ([Ca2+]i), metabolism and secretion in islets of Langerhans including "slow" and "fast" pulses with period of 2-5 min and 10-20 s respectively. In an effort to elucidate the origin of slow oscillations, individual mouse islets were exposed to different fuels including glyceraldehyde, pyruvate, methyl pyruvate and alpha-ketoisocaproate (KIC), all of which bypass key steps of glycolytic metabolism, while monitoring [Ca2+]i, oxygen consumption and secretion. Glyceraldehyde gave rise to slow oscillations only when substimulatory glucose was also added to the media. Glucosamine, an inhibitor of glucokinase, blocked these slow oscillations. KIC, pyruvate, and methyl pyruvate did not give rise to slow oscillations alone or with glucose present. The addition of glucose to islets bathed in nutrient-rich cell culture media accelerated metabolism and initiated slow oscillations while glyceraldehyde did not. It is concluded that glucose has a special role in accelerating metabolism and generating slow oscillations in isolated islets of Langerhans from mice. Combined with previous observations of Ca2+ dependency for all oscillations in islets, we propose that interactions between Ca2+ influx and glycolysis are responsible for the slow oscillations. In contrast, fast oscillations can occur independent of glycolytic flux.

Mice with deletion of insulin receptor substrate (IRS)-1 (IRS-1 knockout [KO] mice) show mild insulin resistance and defective glucose-stimulated insulin secretion and reduced insulin synthesis. To further define the role of IRS-1 in islet function, we examined the insulin secretory defect in the knockouts using freshly isolated islets and primary beta-cells. IRS-1 KO beta-cells exhibited a significantly shorter increase in intracellular free Ca(2+) concentration ([Ca(2+)](i)) than controls when briefly stimulated with glucose or glyceraldehyde and when l-arginine was used to potentiate the stimulatory effect of glucose. These changes were paralleled by a lower number of exocytotic events in the KO beta-cells in response to the same secretagogues, indicating reduced insulin secretion. Furthermore, the normal oscillations in intracellular Ca(2+) and O(2) consumption after glucose stimulation were dampened in freshly isolated KO islets. Semiquantitative RT-PCR showed a dramatically reduced islet expression of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)-2b and -3 in the mutants. These data provide evidence that IRS-1 modulation of insulin secretion is associated with Ca(2+) signaling and expression of SERCA-2b and -3 genes in pancreatic islets and provides a direct link between insulin resistance and defective insulin secretion.

Glucagon-like peptide-1 (GLP-1) is the most insulinogenic of the glucagon-like peptides secreted mainly by L cells in the small and large intestine in response to the ingestion of nutrients. It binds to a specific GLP-1 receptor (GLP-1R) on beta-cells and can increase islet neogenesis and beta-cell mass. It is not clear whether the transmission of information from the gut to islet beta-cells by messengers such as GLP-1 is different in individuals who develop autoimmune diabetes. In the present study the expression of bioactive GLP-1 protein in the gut and its receptor in the pancreas was examined in diabetes-prone BioBreeding (BBdp) rats in the period before overt diabetes and in age-matched control, non-diabetes-prone BB (BBc) rats. An N-terminal directed antibody specific for the bioactive forms of GLP-1 (GLP-1(7-37) and GLP-1(7-36amide)) was used to mea-sure GLP-1 by radioimmunoassay in proximal, median, and distal gut. Pancreas GLP-1R area fraction, GLP-1R gene expression, and insulin content were analyzed, as were plasma GLP-1, glucose, and insulin. The concentration of GLP-1 protein in the jejunum and ileum of BBdp rats was lower than in BBc rats. Although these animals maintained normal blood glucose, there was impaired pancreatic endocrine function, characterized by low baseline insulin concentration in plasma and pancreas. GLP-1R mRNA expression was threefold less in islets isolated from BBdp rats, and GLP-1R+ islet area fraction in pancreas sections was decreased. When injected iv with GLP-1, BBdp rats displayed lower second-phase insulin response (and insulin/glucose ratios) compared with BBc rats. Thus, young BBdp rats displayed decreased concentrations of bioactive GLP-1 in jejunum and ileum, reduced GLP-1R in islets, and lower second-phase insulin response to iv GLP-1 than controls. The decrease in insulinogenic and islet beta-cell mass-promoting signal from GLP-1 in BBdp rats may contribute to impaired glucoregulation and ineffective maintenance of normal islet mass that shifts islet homeostasis in favor of development of diabetes.

A microscale method for purines involved in intracellular signaling and energy metabolism, including ADP, ATP, cyclic-AMP, NADH and GTP, was developed. The analytes were separated on a fused-silica capillary liquid chromatography column (50 microm inner diameter by 25 cm long) packed with 7 microm reversed-phase particles and detected with a carbon fiber cylinder microelectrode at +1.50 V versus Ag/AgCl reference electrode. With an acetonitrile gradient, the separation was carried out within 15 min. With a 100 nl injection volume, the detection limits varied from 0.9 to 8 fmol depending upon the analyte. The low detection limits make the method suitable for analysis of small tissue samples. As a demonstration of the method, islets of Langerhans were analyzed for their adenosine-related messenger content.

A glucose oxidase-based glucose microsensor (<10 micro m tip diameter) was used to measure the glucose concentration within single islets under static conditions and during step changes in glucose level. The sensors had response times of 4.1+/-0.5s (n=7) and sensitivities of 8.7+/-1.8 pA/mM (n=11). The sensors performed independent of oxygen up to 15 mM glucose as long as the oxygen level was > 70 mm Hg. Spatially resolved glucose measurements revealed a glucose gradient around and inside single islets. From measurement of the glucose gradient, a glucose consumption rate of 0.48+/-0.14 pmol/nL islet/min (n=6) and an intraislet glucose diffusion coefficient of 3.8 x 10(-7)cm(2)/s were determined. The measurement of the gradient demonstrates that not all cells within an islet in culture are exposed to the same glucose concentration. The sensor was also used to measure the time required for intraislet glucose concentration to reach steady state following a step increase in glucose concentration from 3 to 10 mM at the islet surface. At a depth of 70 micro m inside an islet, glucose reached steady state in 180+/-7s (n=7) for islets with a diameter of 180-220 micro m (smaller islets reach steady-state faster). In the presence of 10 mM mannoheptulose, an inhibitor of glucokinase, the equilibration time was reduced to 122+/-11s (n=6), indicating that glucose utilization by glycolysis limited the time required for glucose to diffuse into the islets. The long times to reach steady state and presence of glucose gradients are important in interpreting data from experiments involving islets in culture.

Whereas the mechanisms underlying oscillatory insulin secretion remain unknown, several models have been advanced to explain if they involve generation of metabolic oscillations in beta-cells. Evidence, including measurements of oxygen consumption, glucose consumption, NADH, and ATP/ADP ratio, has accumulated to support the hypothesis that energy metabolism in beta-cells can oscillate. Where simultaneous measurements have been made, these oscillations are well correlated with oscillations in intracellular [Ca(2+)] and insulin secretion. Considerable evidence has been accumulated to suggest that entry of Ca(2+) into cells can modulate metabolism both positively and negatively. The main positive effect of Ca(2+) is an increase in oxygen consumption, believed to involve activation of mitochondrial dehydrogenases. Negative feedback by Ca(2+) includes decreases in glucose consumption and decreases in the mitochondrial membrane potential. Ca(2+) also provides negative feedback by increasing consumption of ATP. The negative feedback provided by Ca(2+) provides a mechanism for generating oscillations based on a model in which glucose stimulates a rise in ATP/ADP ratio that closes ATP-sensitive K(+) (K(ATP)) channels, thus depolarizing the cell membrane and allowing Ca(2+) entry through voltage-sensitive channels. Ca(2+) entry reduces the ATP/ADP ratio and allows reopening of the K(ATP) channel.

Micron-sized sensors were used to monitor glucose and oxygen levels in the extracellular space of single islets of Langerhans in real-time. At 10 mM glucose, oscillations in intraislet glucose concentration were readily detected. Changes in glucose level correspond to changes in glucose consumption by glycolysis balanced by mass transport into the islet. Oscillations had a period of 3.1 +/- 0.2 min and amplitude of 0.8 +/- 0.1 mM glucose (n = 21). Superimposed on these oscillations were faster fluctuations in glucose level during the periods of low glucose consumption. Oxygen level oscillations that were out of phase with the glucose oscillations were also detected. Oscillations in both oxygen and glucose consumption were strongly dependent upon extracellular Ca(2+) and sensitive to nifedipine. Simultaneous measurements of glucose with intracellular Ca(2+) ([Ca(2+)](i)) revealed that decreases in [Ca(2+)](i) preceded increases in glucose consumption by 7.4 +/- 2.1 s during an oscillation (n = 9). Conversely, increases in [Ca(2+)](i) preceded increases in oxygen consumption by 1.5 +/- 0.2 s (n = 4). These results suggest that during oscillations, bursts of glycolysis begin after Ca(2+) has stopped entering the cell. Glycolysis stimulates further Ca(2+) entry, which in turn stimulates increases in respiration. The data during oscillation are in contrast to the time course of events during initial exposure to glucose. Under these conditions, a burst of oxygen consumption precedes the initial rise in [Ca(2+)](i). A model to explain these results is described.

An oxygen microsensor with a < 3-micron tip diameter was developed for monitoring oxygen levels at single cells and mouse pancreatic islets. The sensor was fabricated by electrochemically recessing an etched Pt wire inside a pulled glass micropipet and then coating with cellulose acetate. This fabrication process was found to be simpler than previous oxygen electrode designs of comparable size. The microsensors had a average sensitivity of 0.59 +/- 0.29 pA/mmHg (mean +/- SD, n = 42), signals that were minimally perturbed by convection, and response times of < 1 s. The electrode was used to measure the oxygen gradient around and inside single mouse islets. The measurements demonstrate that oxygen levels within even the largest islets at maximal glucose stimulation are 67 +/- 1.6 mmHg (mean +/- SD, n = 5), indicating that islets have adequate oxygen supplies by diffusion under tissue culture conditions to support insulin secretion. The electrode was also used to record the dynamics of oxygen level at single islets as a function of glucose concentration. As glucose level was changed from 3 to 10 mM, oxygen level decreased by 15.8 +/- 2.3 mmHg (mean +/- SEM, n = 6) and oscillations with a period of 3.3 +/- 0.6 min (mean +/- SEM, n = 6) appeared in the oxygen level. In islets bathed in quiescent solutions containing 10 mM glucose, similar oscillations could be observed. In addition, in the quiet solutions it was possible to detect faster oscillations with a period of 12.1 +/- 1.7 s (mean +/- SEM, n = 6) superimposed on the slower oscillations. Oxygen consumption could also be observed at single insulinoma cells using the electrode. Individual cells also showed oscillations in oxygen consumption with a period of a few seconds. The results demonstrate that the electrode can be used for dynamic oxygen level recordings in biological microenvironments.