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This systematic review aims to summarize and critically evaluate the current evidence regarding the impact of hypoglycaemia in children and adolescents with type 1 diabetes on parental quality of life. MEDLINE, PsycINFO, CINAHL, and the Cochrane Library were searched. Inclusion criteria were: 1) quantitative design, 2) included parents of children or adolescents with type 1 diabetes, 3) assessment of hypoglycemia in children/adolescents with type 1 diabetes, 4) assessment of parent quality of life (or related domains of life), and 5) analysis of the relationship(s) between the child's hypoglycaemia and parents' quality of life. The data were summarised in accordance with Synthesis Without Meta-Analysis Guidelines. Twelve studies were included, reporting data from 1895 parents across six countries. Ten studies were cross-sectional; two included prospective data. Evidence suggested that greater frequency and severity of hypoglycemia was associated with greater parental fear of hypoglycemia, emotional distress and family burden. Children's hypoglycaemia has a negative impact on the well-being of parents, but there is an absence of evidence regarding the impact on their overall quality of life. Research into the hypoglycaemia-specific quality of life of parents is needed to explore the impact on various areas, such as social and physical dimensions., (© 2022 John Wiley & Sons A/S . Published by John Wiley & Sons Ltd.)

Introduction: Fertility treatment with frozen thawed embryo transfer (FET) is widely used. Women treated in artificial cycles (AC-FET) receive high doses of estrogen in contrast to natural cycles (NC-FET), where no estrogen is administered. Estrogen substitution may be associated with increased risk of thromboembolism. Our aim is therefore to characterize changes in blood coagulation parameters defined as surrogate thrombotic risk markers in women undergoing estrogen substitution during AC-FET., Materials: In our prospective cohort study, we enrolled 34 women in either: AC-FET (n = 19) or NC-FET (n = 15). Women were recruited at the Department of Obstetrics and Gynaecology, Horsens Fertility Clinic, Denmark, from August 2019 - November 2020. Blood samples were obtained at four timepoints. Thrombin generation, platelet aggregation and fibrinolysis were evaluated as thrombotic risk markers., Results: Within the AC-FET group, we found a significantly shorter lagtime (p < 0.05) and time to peak (TTP) (p < 0.001) after hormone substitution compared to baseline. Furthermore, a significantly higher mean peak (p < 0.0001) and larger endogenous thrombin potential (ETP) (p < 0.0001) was observed. When compared to the NC-FET group, women receiving AC-FET had a significantly shorter mean TTP (p < 0.005), higher mean peak (p < 0.0001) and larger ETP (p < 0.05). Additionally, we demonstrated a significantly prolonged lysis time within the AC-FET group (p < 0.001)., Conclusion: Our results indicate that women receiving AC-FET have a significantly increased thrombin generation which may increase the thromboembolic risk in women being estrogen substituted., (Copyright © 2021. Published by Elsevier Ltd.)

Aim: To summarize and critically appraise the recent qualitative evidence regarding the impact of hypoglycaemia on the quality of life of family members of adults with type 1 or type 2 diabetes., Methods: Four databases were searched systematically (MEDLINE, PsycINFO, CINAHL and Cochrane Library), and results were screened for eligibility. Article quality was assessed using the Joanna Briggs Institute Critical Appraisal Checklist for Qualitative Research. Data were extracted, coded and analysed using thematic analysis. The systematic review was conducted in accordance with Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) and Enhancing transparency in reporting of qualitative research (ENTREQ) guidelines., Results: Eight qualitative studies were included in the final review. The majority of participants were partners or spouses of adults with type 1 diabetes. Thematic synthesis resulted in six analytical themes: 'Hypoglycaemia alters everyday life, reducing freedoms and increasing disruptions', 'Hypoglycaemia has an adverse impact on sleep', 'Hypoglycaemia negatively changes the relationship with the person with diabetes', 'Hypoglycaemia negatively impacts emotional well-being', 'The detection, prevention, and treatment of hypoglycaemia consumes time and energy' and 'Family members have unmet needs for informational and emotional support regarding hypoglycaemia'. Across the six analytical themes, family members described how hypoglycaemia has a severe negative impact on different aspects of their lives, including daily living, personal relationships and emotional well-being., Conclusions: Family members experience the impact of hypoglycaemia as a major recurrent challenge in their lives. The unmet needs of family members need further attention in research and clinical practice., (© 2021 Diabetes UK.)

Metabolic fuels regulate insulin secretion by generating second messengers that drive insulin granule exocytosis, but the biochemical pathways involved are incompletely understood. Here we demonstrate that stimulation of rat insulinoma cells or primary rat islets with glucose or glutamine + 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (Gln + BCH) induces reductive, "counter-clockwise" tricarboxylic acid (TCA) cycle flux of glutamine to citrate. Molecular or pharmacologic suppression of isocitrate dehydrogenase-2 (IDH2), which catalyzes reductive carboxylation of 2-ketoglutarate to isocitrate, results in impairment of glucose- and Gln + BCH-stimulated reductive TCA cycle flux, lowering of NADPH levels, and inhibition of insulin secretion. Pharmacologic suppression of IDH2 also inhibits insulin secretion in living mice. Reductive TCA cycle flux has been proposed as a mechanism for generation of biomass in cancer cells. Here we demonstrate that reductive TCA cycle flux also produces stimulus-secretion coupling factors that regulate insulin secretion, including in non-dividing cells., Competing Interests: Declaration of interests The authors declare no conflicts of interest in conduct of this research. C.B.N. is a paid consultant for Eli Lilly, Axcella Health, Boehringer Ingelheim, and Sigilon. Whereas all of these companies have interests in diabetes therapy, they have no involvement or competing interests in the research described in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Importance: As survival rates from cancer have improved dramatically over the last decades, there is a need to explore the long-term consequences. Adolescents and young adults with cancer are at risk for several therapy-related late effects; however, these have not been studied extensively., Objective: To investigate the lifetime risks of endocrine late effects of cancer and cancer treatment in adolescent and young adult cancer survivors., Design, Setting, and Participants: This Danish, nationwide, population-based cohort study was conducted from January 1, 1976, through December 31, 2009, and included follow-up from January 1, 1977, through December 31, 2010. A total of 32 548 one-year cancer survivors diagnosed at ages 15 to 39 years were identified using the Danish Cancer Registry and 188 728 cancer-free comparison participants matched by year of birth and sex were randomly chosen from the Danish Civil Registration system. Analyses were performed from July 3, 2015, to February 27, 2018., Exposures: Individuals in the survivor cohort were diagnosed with a first primary cancer at ages 15 to 39 years and received treatment according to recommendations and guidelines at time of diagnosis., Main Outcomes and Measures: By linkage to the National Patient Register, all hospital contacts for endocrine diseases were identified, and standardized hospitalization rate ratios (RRs) and absolute excess risks (AERs) were calculated., Results: A total of 32 548 adolescent and young adult 1-year cancer survivors (14 021 [43.1%] male) in the Danish Patient Registry were followed up for 379 157 person-years (median [range]: 10 [0-34] years) and 188 728 cancer-free participants (82 669 [43.8%] male) for comparison were followed up for 2 958 994 person-years (median [range]: 15 [0-34] years). A total of 2129 survivors (6.5%) had at least 1 hospital contact for an endocrine disease, while 1232.0 (3.8%) were expected, yielding a statistically significant increased RR of 1.73 (95% CI, 1.65-1.81). The RRs were highest for testicular hypofunction (75.12; 95% CI, 45.99-122.70), ovarian hypofunction (14.65; 95% CI, 8.29-25.86), and pituitary hypofunction (11.14; 95% CI, 8.09-15.34). The leading reasons for hospital contacts were thyroid disease (38.0% of total AER), testicular dysfunction (17.1% of total AER), and diabetes (14.4% of total AER). Leukemia survivors were at a high risk for any endocrine disease (RR, 3.97; 95% CI, 3.10-5.09), while Hodgkin lymphoma survivors (RR, 3.06; 95% CI, 2.62-3.57) had the highest disease-specific excess risk for hypothyroidism (AER, 362 per 100 000 person-years; 95% CI, 280-443 per 100 000 person-years)., Conclusions and Relevance: The increased risk for endocrine diseases in adolescent and young adult cancer survivors indicates the need for counseling and follow-up, and could guide future preventive measures and surveillance strategies. Additional studies are required to determine exact associations between treatment regimens and endocrine diseases.

After multiple decades of investigation, the precise mechanisms involved in fuel-stimulated insulin secretion are still being revealed. One avenue for gaining deeper knowledge is to apply emergent tools of "metabolomics," involving mass spectrometry and nuclear magnetic resonance-based profiling of islet cells in their fuel-stimulated compared with basal states. The current article summarizes recent insights gained from application of metabolomics tools to the specific process of glucose-stimulated insulin secretion, revealing 2 new mechanisms that may provide targets for improving insulin secretion in diabetes., (© 2017 John Wiley & Sons Ltd.)

Proteins in the serine esterase family are widely distributed in bacterial phyla and display activity against a range of biologically produced and chemically synthesized esters. A serine esterase from the psychrophilic bacterium Pseudoalteromonas arctica with a C-terminal OsmC-like domain was recently characterized; here we report on the identification and characterization of further putative esterases with OsmC-like domains constituting a new esterase family that is found in a variety of bacterial species from different environmental niches. All of these proteins contained the Ser-Asp-His motif common to serine esterases and a highly conserved pentapeptide nucleophilic elbow motif. We produced these proteins heterologously in Escherichia coli and demonstrated their activity against a range of esterase substrates. Two of the esterases characterized have activity of over two orders of magnitude higher than other members of the family, and are active over a wide temperature range. We determined the crystal structure of the esterase domain of the protein from Rhodothermus marinus and show that it conforms to the classical α/β hydrolase fold with an extended 'lid' region, which occludes the active site of the protein in the crystal. The expansion of characterized members of the esterase family and demonstration of activity over a wide-range of temperatures could be of use in biotechnological applications such as the pharmaceutical, detergent, bioremediation and dairy industries., Competing Interests: The authors have declared that no competing interests exist.

Osteocytes are suggested to play a central role in bone remodeling. Evaluation of iliac crest biopsies is a standard procedure for evaluating bone conditions in the clinical setting. Despite the widespread use of such biopsies, little is known about the population of osteocytes in the iliac crest from normal individuals. Contradicting results have been reported on osteocyte lacunar properties in human bone. Hence, a solid understanding of the osteocyte population in healthy bone and the effect of age and sex is needed as good reference data are lacking. Furthermore, the role of cortical bone in bone quality has recently been suggested to be more important than previously realized. Therefore, the present study assesses osteocyte lacunar properties and cortical microstructure of the iliac crest as a function of age and sex. A total of 88 iliac crest bone samples from healthy individuals (46 women, aged 18.5-96.4years and 42 men, aged 22.6-94.6years) with an even age-distribution were examined using synchrotron radiation μCT and in house μCT, with >5×10(6) osteocyte lacunae measured and analyzed. The study revealed that osteocyte lacunar volumes were unaffected by both age and sex. Osteocyte lacunar density did not differ between women and men, and only showed a significant decrease with age when pooling data from both sexes. Cortical porosity and Haversian canal density increased while cortical thickness decreased with age, with cortical thinning dominating the age-related cortical bone loss. None of the cortical microstructural parameters showed any sex dependency. Only weak links between osteocyte lacunar properties and cortical microstructural properties in iliac crest bone were found. Interestingly, the Haversian canal diameters were significantly but weakly negatively correlated with osteocyte lacunar volumes., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Metabolomics, the characterization of the set of small molecules in a biological system, is advancing research in multiple areas of islet biology. Measuring a breadth of metabolites simultaneously provides a broad perspective on metabolic changes as the islets respond dynamically to metabolic fuels, hormones, or environmental stressors. As a result, metabolomics has the potential to provide new mechanistic insights into islet physiology and pathophysiology. Here we summarize advances in our understanding of islet physiology and the etiologies of type-1 and type-2 diabetes gained from metabolomics studies., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Pancreatic islet failure, involving loss of glucose-stimulated insulin secretion (GSIS) from islet β cells, heralds the onset of type 2 diabetes (T2D). To search for mediators of GSIS, we performed metabolomics profiling of the insulinoma cell line 832/13 and uncovered significant glucose-induced changes in purine pathway intermediates, including a decrease in inosine monophosphate (IMP) and an increase in adenylosuccinate (S-AMP), suggesting a regulatory role for the enzyme that links the two metabolites, adenylosuccinate synthase (ADSS). Inhibition of ADSS or a more proximal enzyme in the S-AMP biosynthesis pathway, adenylosuccinate lyase, lowers S-AMP levels and impairs GSIS. Addition of S-AMP to the interior of patch-clamped human β cells amplifies exocytosis, an effect dependent upon expression of sentrin/SUMO-specific protease 1 (SENP1). S-AMP also overcomes the defect in glucose-induced exocytosis in β cells from a human donor with T2D. S-AMP is, thus, an insulin secretagogue capable of reversing β cell dysfunction in T2D., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Insulin secretion from β cells of the pancreatic islets of Langerhans controls metabolic homeostasis and is impaired in individuals with type 2 diabetes (T2D). Increases in blood glucose trigger insulin release by closing ATP-sensitive K+ channels, depolarizing β cells, and opening voltage-dependent Ca2+ channels to elicit insulin exocytosis. However, one or more additional pathway(s) amplify the secretory response, likely at the distal exocytotic site. The mitochondrial export of isocitrate and engagement with cytosolic isocitrate dehydrogenase (ICDc) may be one key pathway, but the mechanism linking this to insulin secretion and its role in T2D have not been defined. Here, we show that the ICDc-dependent generation of NADPH and subsequent glutathione (GSH) reduction contribute to the amplification of insulin exocytosis via sentrin/SUMO-specific protease-1 (SENP1). In human T2D and an in vitro model of human islet dysfunction, the glucose-dependent amplification of exocytosis was impaired and could be rescued by introduction of signaling intermediates from this pathway. Moreover, islet-specific Senp1 deletion in mice caused impaired glucose tolerance by reducing the amplification of insulin exocytosis. Together, our results identify a pathway that links glucose metabolism to the amplification of insulin secretion and demonstrate that restoration of this axis rescues β cell function in T2D.

The aim of this study was to determine the time course of physiological adaptations and their relationship with performance improvements during 2 weeks of heat acclimatization. Nine trained cyclists completed 2 weeks of training in naturally hot environment (34 ± 3 °C; 18 ± 5% relative humidity). On days 1, 6, and 13, they performed standardized heat response tests (HRT-1, 2, 3), and 43.4-km time trials in the heat (TTH-1, 2, 3) were completed on days 2, 7, and 14. Within the first 5-6 days, sweat sodium concentration decreased from 75 ± 22 mmol/L to 52 ± 24 mmol/L, sweat rate increased (+20 ± 15%), and resting hematocrit decreased (-5.6 ± 5.4%), with no further changes during the remaining period. In contrast, power output during TTHs gradually improved from TTH-1 to TTH-2 (+11 ± 8%), and from TTH-2 to TTH-3 (+5 ± 4%). Individual improvements in performance from TTH-1 to TTH-2 correlated with individual changes in hematocrit (assessed after the corresponding HRT; r = -0.79, P < 0.05), however, were not related to changes in performance from TTH-2 to TTH-3. In trained athletes, sudomotor and hematological adaptations occurred within 5-6 days of training, whereas the additional improvement in performance after the entire acclimatization period did not relate to changes in these parameters., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

This study investigated if well-trained cyclists improve V ˙ O 2 m a x and performance in cool conditions following heat acclimatization through natural outdoor training in hot conditions. Eighteen trained male cyclists were tested for physiological adaptations, V ˙ O 2 m a x , peak aerobic power output, exercise efficiency, and outdoor time trial (TT) performance (43.4 km in cool environment, ∼5-13 °C) before and after 2 weeks of training in a cool (CON, n = 9) or hot (∼35 °C, HA, n = 9) environment. After heat acclimatization, TT performance in the heat was improved by 16%; however, there was no change in the HA group in V ˙ O 2 m a x (4.79 ± 0.21 L/min vs 4.82 ± 0.35 L/min), peak aerobic power output (417 ± 16 W vs 422 ± 17 W), and outdoor TT performance in cool conditions (300 ± 14 W/69 ± 3 min vs 302 ± 9 W/69 ± 4 min). The present study shows that 2 weeks of heat acclimatization was associated with marked improvements in TT performance in the heat. However, for the well-trained endurance athletes, this did not transfer to an improved aerobic exercise capacity or outdoor TT performance in cool conditions., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Immobilization causes rapid and massive bone loss. By comparing Botulinum Toxin A (BTX)-induced bone loss in mouse strains with different genetic backgrounds we investigated whether the genetic background had an influence on the severity of the osteopenia. Secondly, we investigated whether BTX had systemic effects on bone. Female mice from four inbred mouse strains (BALB/cJ, C57BL/6 J, DBA/2 J, and C3H/HeN) were injected unilaterally with BTX (n = 10/group) or unilaterally with saline (n = 10/group). Mice were euthanized after 21 days, and the bone properties evaluated using μCT, DXA, bone histomorphometry, and mechanical testing. BTX resulted in substantially lower trabecular bone volume fraction (BV/TV) and trabecular thickness in all mouse strains. The deterioration of BV/TV was significantly greater in C57BL/6 J (- 57%) and DBA/2 J (- 60%) than in BALB/cJ (- 45%) and C3H/HeN (- 34%) mice. The loss of femoral neck fracture strength was significantly greater in C57BL/6 J (- 47%) and DBA/2 J (- 45%) than in C3H (- 25%) mice and likewise the loss of mid-femoral fracture strength was greater in C57BL/6 J (- 17%), DBA/2 J (- 12%), and BALB/cJ (- 9%) than in C3H/HeN (- 1%) mice, which were unaffected. Using high resolution μCT we found no evidence of a systemic effect on any of the microstructural parameters of the contralateral limb. Likewise, there was no evidence of a systemic effect on the bone strength in any mouse strain. We did, however, find a small systemic effect on aBMD in DBA/2 J and C3H/HeN mice. The present study shows that BTX-induced immobilization causes the greatest loss of cortical and trabecular bone in C57BL/6 J and DBA/2 J mice. A smaller loss of bone microstructure and fracture strength was seen in BALB/cJ mice, while the bone microstructure and fracture strength of C3H/HeN mice were markedly less affected. This indicates that BTX-induced loss of bone is mouse strain dependent. We found only minimal systemic effects of BTX.

Unlabelled: Age-related changes of vertebra and iliac crest 3D microstructure were investigated, and we showed that they were in general similar. The 95th percentile of vertebral trabecular thickness distribution increased with age for women. Surprisingly, vertebral and iliac crest bone microstructure was only weakly correlated (r = 0.38 to 0.75), despite the overall similar age-related changes., Introduction: The purposes of the study were to determine the age-related changes in iliac and vertebral bone microstructure for women and men over a large age range and to investigate the relationship between the bone microstructure at these skeletal sites., Methods: Matched sets of transiliac crest bone biopsies and lumbar vertebral body (L2) specimens from 41 women (19-96 years) and 39 men (23-95 years) were micro-computed tomography (μCT) scanned, and the 3D microstructure was quantified., Results: For both women and men, bone volume per total volume (BV/TV), connectivity density (CD), and trabecular number (Tb.N) decreased significantly, while structure model index (SMI) and trabecular separation (Tb.Sp) increased significantly with age at either skeletal site. Vertebral trabecular thickness (Tb.Th) was independent of age for both women and men, while iliac Tb.Th decreased significantly with age for men, but not for women. In general, the vertebral and iliac age-related changes were similar. The 95th percentile of the Tb.Th distribution increased significantly with age for women but was independent of age for men at the vertebral body, while it was independent of age for either sex at the iliac crest. The Tb.Th probability density functions at the two skeletal sites became significantly more similar with age for women, but not for men. The microstructural parameters at the iliac crest and the vertebral bodies were only moderately correlated from r = 0.38 for SMI in women to r = 0.75 for Tb.Sp in men., Conclusion: Age-related changes in vertebral and iliac bone microstructure were in general similar. The iliac and vertebral Tb.Th distributions became more similar with age for women. Despite the overall similar age-related changes in trabecular bone microstructure, the vertebral and iliac bone microstructural measures were only weakly correlated (r = 0.38 to 0.75).

Loss of functional β-cell mass is a hallmark of type 1 and type 2 diabetes, and methods for restoring these cells are needed. We have previously reported that overexpression of the homeodomain transcription factor NK6 homeobox 1 (Nkx6.1) in rat pancreatic islets induces β-cell proliferation and enhances glucose-stimulated insulin secretion, but the pathway by which Nkx6.1 activates β-cell expansion has not been defined. Here, we demonstrate that Nkx6.1 induces expression of the nuclear receptor subfamily 4, group A, members 1 and 3 (Nr4a1 and Nr4a3) orphan nuclear receptors, and that these factors are both necessary and sufficient for Nkx6.1-mediated β-cell proliferation. Consistent with this finding, global knockout of Nr4a1 results in a decrease in β-cell area in neonatal and young mice. Overexpression of Nkx6.1 and the Nr4a receptors results in increased expression of key cell cycle inducers E2F transcription factor 1 and cyclin E1. Furthermore, Nkx6.1 and Nr4a receptors induce components of the anaphase-promoting complex, including ubiquitin-conjugating enzyme E2C, resulting in degradation of the cell cycle inhibitor p21. These studies identify a unique bipartite pathway for activation of β-cell proliferation, suggesting several unique targets for expansion of functional β-cell mass.

Background: Most likely because of low statistical power, no previous studies have shown any significant association between long-term survival and anastomotic leakage in patients who have undergone gastroesophageal cancer resection., Material and Methods: The present study included, prospectively and consecutively, nationwide collected patients who underwent gastroesophageal cancer resection between 2003 and 2011 in Denmark. The operation was carried out as an Ivor Lewis procedure. Only patients with intrathoracic anastomosis were included in the analysis., Results: From 2003 to 2011, 1,296 patients underwent gastroesophageal resection, and 128 (9.9 %) of these experienced anastomotic leakage. The overall 5-year survival rates in patients with and without anastomotic leakage were 20 and 35 % (P < 0.0001), respectively. After exclusion of 4 weeks mortality, the 5-year survival rate in patients with leakage was 22 % compared to 36 % in patients without anastomotic leakage (P < 0.001). After exclusion of 8 weeks mortality, the 5-year survival rate was 23 % in patients with leakage and 36 % in those without (P = 0.009). The corresponding median time of survival was 74 versus 128, 87 versus 138, and 95 versus 138 weeks, respectively. The overall hazard ratios of death after anastomotic leakage, unadjusted, and after adjusting for potentially confounding factors, were 1.59 (1.27-1.99) and 1.45 (1.14-1.84). The unadjusted and adjusted odds ratios after exclusion of 4 weeks mortality were 1.51 (1.19-1.90) and 1.41 (1.10-1.81). After exclusion of 8 weeks mortality the odds ratios were 1.38 (1.08-1.77) and 1.32 (1.02-1.71)., Conclusions: This nationwide study confirms that patients experiencing anastomotic leakage after gastroesophageal cancer resection have a significantly lower long-term survival, even following full recovery after the leakage.

Recent studies have shown that the pyruvate-isocitrate cycling pathway, involving the mitochondrial citrate/isocitrate carrier and the cytosolic NADP-dependent isocitrate dehydrogenase (ICDc), is involved in control of glucose-stimulated insulin secretion (GSIS). Here we demonstrate that pyruvate-isocitrate cycling regulates expression of the voltage-gated potassium channel family member Kv2.2 in islet β-cells. siRNA-mediated suppression of ICDc, citrate/isocitrate carrier, or Kv2.2 expression impaired GSIS, and the effect of ICDc knockdown was rescued by re-expression of Kv2.2. Moreover, chronic exposure of β-cells to elevated fatty acids, which impairs GSIS, resulted in decreased expression of Kv2.2. Surprisingly, knockdown of ICDc or Kv2.2 increased rather than decreased outward K(+) current in the 832/13 β-cell line. Immunoprecipitation studies demonstrated interaction of Kv2.1 and Kv2.2, and co-overexpression of the two channels reduced outward K(+) current compared with overexpression of Kv2.1 alone. Also, siRNA-mediated knockdown of ICDc enhanced the suppressive effect of the Kv2.1-selective inhibitor stromatoxin1 on K(+) currents. Our data support a model in which a key function of the pyruvate-isocitrate cycle is to maintain levels of Kv2.2 expression sufficient to allow it to serve as a negative regulator of Kv channel activity.

The voltage-gated potassium channels Kv2.1 and Kv2.2 are highly expressed in pancreatic islets, yet their contribution to islet hormone secretion is not fully understood. Here we investigate the role of Kv2 channels in pancreatic islets using a combination of genetic and pharmacologic approaches. Pancreatic β-cells from Kv2.1(-/-) mice possess reduced Kv current and display greater glucose-stimulated insulin secretion (GSIS) relative to WT β-cells. Inhibition of Kv2.x channels with selective peptidyl [guangxitoxin-1E (GxTX-1E)] or small molecule (RY796) inhibitors enhances GSIS in isolated wild-type (WT) mouse and human islets, but not in islets from Kv2.1(-/-) mice. However, in WT mice neither inhibitor improved glucose tolerance in vivo. GxTX-1E and RY796 enhanced somatostatin release in isolated human and mouse islets and in situ perfused pancreata from WT and Kv2.1(-/-) mice. Kv2.2 silencing in mouse islets by adenovirus-small hairpin RNA (shRNA) specifically enhanced islet somatostatin, but not insulin, secretion. In mice lacking somatostatin receptor 5, GxTX-1E stimulated insulin secretion and improved glucose tolerance. Collectively, these data show that Kv2.1 regulates insulin secretion in β-cells and Kv2.2 modulates somatostatin release in δ-cells. Development of selective Kv2.1 inhibitors without cross inhibition of Kv2.2 may provide new avenues to promote GSIS for the treatment of type 2 diabetes.

Glucose-stimulated insulin secretion from pancreatic islet beta-cells is dependent in part on pyruvate cycling through the pyruvate/isocitrate pathway, which generates cytosolic alpha-ketoglutarate, also known as 2-oxoglutarate (2OG). Here, we have investigated if mitochondrial transport of 2OG through the 2-oxoglutarate carrier (OGC) participates in control of nutrient-stimulated insulin secretion. Suppression of OGC in clonal pancreatic beta-cells (832/13 cells) and isolated rat islets by adenovirus-mediated delivery of small interfering RNA significantly decreased glucose-stimulated insulin secretion. OGC suppression also reduced insulin secretion in response to glutamine plus the glutamate dehydrogenase activator 2-amino-2-norbornane carboxylic acid. Nutrient-stimulated increases in glucose usage, glucose oxidation, glutamine oxidation, or ATP:ADP ratio were not affected by OGC knockdown, whereas suppression of OGC resulted in a significant decrease in the NADPH:NADP(+) ratio during stimulation with glucose but not glutamine + 2-amino-2-norbornane carboxylic acid. Finally, OGC suppression reduced insulin secretion in response to a membrane-permeant 2OG analog, dimethyl-2OG. These data reveal that the OGC is part of a mechanism of fuel-stimulated insulin secretion that is common to glucose, amino acid, and organic acid secretagogues, involving flux through the pyruvate/isocitrate cycling pathway. Although the components of this pathway must remain intact for appropriate stimulus-secretion coupling, production of NADPH does not appear to be the universal second messenger signal generated by these reactions.

Glucose-stimulated insulin secretion (GSIS) is central to normal control of metabolic fuel homeostasis, and its impairment is a key element of beta-cell failure in type 2 diabetes. Glucose exerts its effects on insulin secretion via its metabolism in beta-cells to generate stimulus/secretion coupling factors, including a rise in the ATP/ADP ratio, which serves to suppress ATP-sensitive K(+) (K(ATP)) channels and activate voltage-gated Ca(2+) channels, leading to stimulation of insulin granule exocytosis. Whereas this K(ATP) channel-dependent mechanism of GSIS has been broadly accepted for more than 30 years, it has become increasingly apparent that it does not fully describe the effects of glucose on insulin secretion. More recent studies have demonstrated an important role for cyclic pathways of pyruvate metabolism in control of insulin secretion. Three cycles occur in islet beta-cells: the pyruvate/malate, pyruvate/citrate, and pyruvate/isocitrate cycles. This review discusses recent work on the role of each of these pathways in control of insulin secretion and builds a case for the particular relevance of byproducts of the pyruvate/isocitrate cycle, NADPH and alpha-ketoglutarate, in control of GSIS.

We have previously demonstrated a role for pyruvate cycling in glucose-stimulated insulin secretion (GSIS). Some of the possible pyruvate cycling pathways are completed by conversion of malate to pyruvate by malic enzyme. Using INS-1-derived 832/13 cells, it has recently been shown by other laboratories that NADP-dependent cytosolic malic enzyme (MEc), but not NAD-dependent mitochondrial malic enzyme (MEm), regulates GSIS. In the current study, we show that small interfering RNA-mediated suppression of either MEm or MEc results in decreased GSIS in both 832/13 cells and a new and more glucose- and incretin-responsive INS-1-derived cell line, 832/3. The effect of MEm to suppress GSIS in these cell lines was linked to a substantial decrease in cell growth, whereas MEc suppression resulted in decreased NADPH, shown previously to be correlated with GSIS. However, adenovirus-mediated delivery of small interfering RNAs specific to MEc and MEm to isolated rat islets, while leading to effective suppression of the targets transcripts, had no effect on GSIS. Furthermore, islets isolated from MEc-null MOD1(-/-) mice exhibit normal glucose- and potassium-stimulated insulin secretion. These results indicate that pyruvate-malate cycling does not control GSIS in primary rodent islets.

Glucose-stimulated insulin secretion (GSIS) is mediated in part by glucose metabolism-driven increases in ATP/ADP ratio, but by-products of mitochondrial glucose metabolism also play an important role. Here we investigate the role of the mitochondrial citrate/isocitrate carrier (CIC) in regulation of GSIS. Inhibition of CIC activity in INS-1-derived 832/13 cells or primary rat islets by the substrate analogue 1,2,3-benzenetricarboxylate (BTC) resulted in potent inhibition of GSIS, involving both first and second phase secretion. A recombinant adenovirus containing a CIC-specific siRNA (Ad-siCIC) dose-dependently reduced CIC expression in 832/13 cells and caused parallel inhibitory effects on citrate accumulation in the cytosol. Ad-siCIC treatment did not affect glucose utilization, glucose oxidation, or ATP/ADP ratio but did inhibit glucose incorporation into fatty acids and glucose-induced increases in NADPH/NADP+ ratio relative to cells treated with a control siRNA virus (Ad-siControl). Ad-siCIC also inhibited GSIS in 832/13 cells, whereas overexpression of CIC enhanced GSIS and raised cytosolic citrate levels. In normal rat islets, Ad-siCIC treatment also suppressed CIC mRNA levels and inhibited GSIS. We conclude that export of citrate and/or isocitrate from the mitochondria to the cytosol is an important step in control of GSIS.

Glucose-stimulated insulin secretion (GSIS) from pancreatic islet beta-cells is central to control of mammalian fuel homeostasis. Glucose metabolism mediates GSIS in part via ATP-regulated K+ (KATP) channels, but multiple lines of evidence suggest participation of other signals. Here we investigated the role of cytosolic NADP-dependent isocitrate dehydrogenase (ICDc) in control of GSIS in beta-cells. Delivery of small interfering RNAs specific for ICDc caused impairment of GSIS in two independent robustly glucose-responsive rat insulinoma (INS-1-derived) cell lines and in primary rat islets. Suppression of ICDc also attenuated the glucose-induced increments in pyruvate cycling activity and in NADPH levels, a predicted by-product of pyruvate cycling pathways, as well as the total cellular NADP(H) content. Metabolic profiling of eight organic acids in cell extracts revealed that suppression of ICDc caused increases in lactate production in both INS-1-derived cell lines and primary islets, consistent with the attenuation of pyruvate cycling, with no significant changes in other intermediates. Based on these studies, we propose that a pyruvate cycling pathway involving ICDc plays an important role in control of GSIS.

We have previously reported that glucose-stimulated insulin secretion (GSIS) is tightly correlated with pyruvate carboxylase (PC)-catalyzed anaplerotic flux into the tricarboxylic acid cycle and stimulation of pyruvate cycling activity. To further evaluate the role of PC in beta-cell function, we constructed a recombinant adenovirus containing a small interfering RNA (siRNA) specific to PC (Ad-siPC). Ad-siPC reduced PC mRNA levels by 83 and 64% and PC protein by 56 and 35% in INS-1-derived 832/13 cells and primary rat islets, respectively. Surprisingly, this manipulation did not impair GSIS in rat islets. In Ad-siPC-treated 832/13 cells, GSIS was slightly increased, whereas glycolytic rate and glucose oxidation were unaffected. Flux through PC at high glucose was decreased by only 20%, suggesting an increase in PC-specific activity. Acetyl carnitine, a surrogate for acetyl-CoA, an allosteric activator of PC, was increased by 36% in Ad-siPC-treated cells, suggesting a mechanism by which PC enzymatic activity is maintained with suppressed PC protein levels. In addition, the NADPH:NADP ratio, a proposed coupling factor for GSIS, was unaffected in Ad-siPC-treated cells. We conclude that beta-cells activate compensatory mechanisms in response to suppression of PC expression that prevent impairment of anaplerosis, pyruvate cycling, NAPDH production, and GSIS.

Hyperlipidemia appears to play an integral role in loss of glucose-stimulated insulin secretion (GSIS) in type 2 diabetes. This impairment can be simulated in vitro by chronic culture of 832/13 insulinoma cells with high concentrations of free fatty acids, or by study of lipid-laden islets from Zucker diabetic fatty rats. Here we show that impaired GSIS is not a simple result of saturation of lipid storage pathways, as adenovirus-mediated overexpression of a cytosolically localized variant of malonyl-CoA decarboxylase in either cellular model results in dramatic lowering of cellular triglyceride stores but no improvement in GSIS. Instead, the glucose-induced increment in "pyruvate cycling" activity (pyruvate exchange with tricarboxylic acid cycle intermediates measured by (13)C NMR), previously shown to play an important role in GSIS, is completely ablated in concert with profound suppression of GSIS in lipid-cultured 832/13 cells, whereas glucose oxidation is unaffected. Moreover, GSIS is partially restored in both lipid-cultured 832/13 cells and islets from Zucker diabetic fatty rats by addition of a membrane permeant ester of a pyruvate cycling intermediate (dimethyl malate). We conclude that chronic exposure of islet beta-cells to fatty acids grossly alters a mitochondrial pathway of pyruvate metabolism that is important for normal GSIS.

Type 1 and type 2 diabetes are both diseases of insulin insufficiency, although they develop by distinct pathways. The recent surge in the incidence of type 2 diabetes and the chronic ailments confronted by patients with either form of the disease highlight the need for better understanding of beta-cell biology. In this review, we present recent work focused on this goal. Our hope is that basic research being conducted in this and other laboratories will ultimately contribute to the development of methods for enhancing beta-cell function and survival in the context of both major forms of diabetes. Our strategy for understanding the beta-cell involves a multidisciplinary approach in which tools from the traditional fields of biochemistry, enzymology, and physiology are teamed with newer technologies from the fields of molecular biology, gene discovery, cell and developmental biology, and biophysical chemistry. We have focused on two important aspects of beta-cell biology in our studies: beta-cell function, specifically the metabolic regulatory mechanisms involved in glucose-stimulated insulin secretion, and beta-cell resistance to immune attack, with emphasis on resistance to inflammatory cytokines and reactive oxygen species.

There is a growing appreciation for the complexity of the pathways involved in glucose-stimulated insulin secretion (GSIS) from pancreatic islet beta-cells. In our laboratory, this has stimulated the development of an interdisciplinary approach to the problem. In this study, we review recent studies combining the tools of recombinant adenovirus for gene delivery, the development of novel cell lines that exhibit either robust or weak GSIS, and nuclear magnetic resonance imaging for metabolic fingerprinting of glucose-stimulated cells. Using these tools, we demonstrate a potentially important role for pyruvate carboxylase-mediated pyruvate cycling pathways in the control of GSIS, and discuss potential coupling factors produced by such pathways.

Thiazolidinediones (TZDs) are effective therapies for type 2 diabetes, which has reached epidemic proportions in industrialized societies. TZD treatment reduces circulating free fatty acids (FFAs), which oppose insulin actions in skeletal muscle and other insulin target tissues. Here we report that TZDs, acting as ligands for the nuclear receptor peroxisome proliferator-activated receptor (PPAR)-gamma, markedly induce adipocyte glycerol kinase (GyK) gene expression. This is surprising, as standard textbooks indicate that adipocytes lack GyK and thereby avoid futile cycles of triglyceride breakdown and resynthesis from glycerol and FFAs. By inducing GyK, TZDs markedly stimulate glycerol incorporation into triglyceride and reduce FFA secretion from adipocytes. The 'futile' fuel cycle resulting from expression of GyK in adipocytes is thus a novel mechanism contributing to reduced FFA levels and perhaps insulin sensitization by antidiabetic therapies.

Cellular metabolism of glucose is required for stimulation of insulin secretion from pancreatic beta cells, but the precise metabolic coupling factors involved in this process are not known. In an effort to better understand mechanisms of fuel-mediated insulin secretion, we have adapted 13C NMR and isotopomer methods to measure influx of metabolic fuels into the tricarboxylic acid (TCA) cycle in insulinoma cells. Mitochondrial metabolism of [U-13C3]pyruvate, derived from [U-13C6]glucose, was compared in four clonal rat insulinoma cell 1-derived cell lines with varying degrees of glucose responsiveness. A 13C isotopomer analysis of glutamate isolated from these cells showed that the fraction of acetyl-CoA derived from [U-13C6]glucose was the same in all four cell lines (44 +/- 5%, 70 +/- 3%, and 84 +/- 4% with 3, 6, or 12 mM glucose, respectively). The 13C NMR spectra also demonstrated the existence of two compartmental pools of pyruvate, one that exchanges with TCA cycle intermediates and a second pool derived from [U-13C6]glucose that feeds acetyl-CoA into the TCA cycle. The 13C NMR spectra were consistent with a metabolic model where the two pyruvate pools do not randomly mix. Flux between the mitochondrial intermediates and the first pool of pyruvate (pyruvate cycling) varied in proportion to glucose responsiveness in the four cell lines. Furthermore, stimulation of pyruvate cycling with dimethylmalate or its inhibition with phenylacetic acid led to proportional changes in insulin secretion. These findings indicate that exchange of pyruvate with TCA cycle intermediates, rather than oxidation of pyruvate via acetyl-CoA, correlates with glucose-stimulated insulin secretion.

Introduction: In 1996 the Danish Parliament passed a new Act on industrial injuries, including the so-called sudden lifting injuries. This type of lifting injury had been experienced in some industries in connection with lifting situations causing sudden pain in the back. Less frequently, other regions of the body may be affected. This study is a distribution of a sample of reported industrial injuries of this type in groups according to types of lifts. The study also includes a comparison with parameters from the Danish Work Environment Cohort Study., Material and Methods: The sample consisted of a total of 482 cases of back injuries. Of these, 346 cases were distributed according to six categories of manual lifting work and to age and gender. The rest, 136 cases, were excluded for technical reasons., Results: In the distribution, 8% of the cases were in jobs with many heavy lifts, 66% of the cases were in jobs with moderate daily lifted weights or in jobs with only few or light lifts. Approximately 25% of cases concerned employees performing patient transfers. The distribution contained more men than the Danish Work Environment Cohort Study, and the persons in the cases were slightly older than this reference population., Discussion: Altogether, this study points towards an occurrence of sudden lifting injuries among employees performing certain types of manual lifting work. The industrial injuries cases may have been subject to some selection.

Long-chain acyl-CoA esters (LCA) act both as substrates and intermediates in metabolism and as regulators of various intracellular functions. Acyl-CoA binding protein (ACBP) binds LCA with high affinity and is believed to play an important role in intracellular acyl-CoA transport and pool formation and therefore also for the function of LCA as metabolites and regulators of cellular functions . The free concentration of cytosolic LCA is efficiently buffered to low nanomole concentration by ACBP and fatty acid binding protein (FABP). An additional important factor is the activity of acyl-CoA hydrolases. The estimated cellular free LCA concentration is two to four orders of magnitude lower than the concentrations reported to be necessary to regulate most LCA-affected cellular functions. Preliminary evidence indicates that the regulatory effect of LCA might be mediated by the LCA/ACBP complex.

Long chain acylCoA esters (LCAs) act both as substrates and intermediates in intermediary metabolism and as regulators in various intracellular functions. AcylCoA binding protein (ACBP) binds LCAs with high affinity and is believed to play an important role in intracellular acylCoA transport and pool formation and therefore also for the function of LCAs as metabolites and regulators of cellular functions [1]. The major factors controlling the free concentration of cytosol long chain acylCoA ester (LCA) include ACBP [2], sterol carrier protein 2 (SCP2) [3] and fatty acid binding protein (FABP) [4]. Additional factors affecting the concentration of free LCA include feed back inhibition of the acylCoA synthetase [5], binding to acylCoA receptors (LCA-regulated molecules and enzymes), binding to membranes and the activity of acylCoA hydrolases [6].

The aim of this study was to estimate the risk of being hospitalized due to prolapsed cervical interverteral disc among male professional drivers in Denmark. A cohort consisting of all economically active persons in Denmark, identified January 1981, gives information on the most important occupation during 1980. The cohort was followed for ten years. Additional data on occupational exposures were extracted from a national survey. Occupational groups such as bus and taxi-cab drivers were found to have an increased risk, of cervical prolapse as did the group of professional drivers as a whole.

Study Design: This study of professional drivers is a part of a longitudinal record linkage study of all economically active men in Denmark, identified on January 1, 1981. Information about the main occupation was identified in 1980. The cohort was followed for first hospitalization with prolapsed cervical intervertebral disc until December 31, 1990., Objectives: To examine the risk of prolapsed cervical intervertebral disc in all Danish professional drivers, and to analyze exposures of the male drivers in a sample of all Danish male drivers., Summary of Background Data: Only a few studies on occupation and prolapsed cervical intervertebral disc have been published. These studies suggest that professional driving may be a risk factor for development of prolapsed cervical intervertebral disc. Drivers are exposed to whole-body vibrations, heavy lifting, and a sedentary position. Other potential exposures are accelerations and decelerations and whiplash accidents. Such exposure may be involved in the causation of prolapsed cervical intervertebral disc., Methods: A standardized hospitalization ratio was calculated for each subgroup of drivers using all economically active people as the standard. Additional exposure information was extracted from a national survey on work environment., Results: Almost all men in occupations involving professional driving had a statistically significant elevated risk of being hospitalized with prolapsed cervical intervertebral disc., Conclusions: Professional driving is a risk factor for prolapsed cervical intervertebral disc.

This study validated hypertension (ICD-8: 401.99) in The National Inpatient Register with reference to the use of the diagnosis in the Occupational Hospitalization Register. A university hospital and a regional hospital were chosen for the evaluation. A sample of case records with the discharge diagnosis essential hypertension and additional case records with other diagnoses were re-coded blindly and independently by two doctors. Cause of admission or admission diagnosis was recorded for essential hypertension cases. The agreement with The National Inpatient Register ranged from 60 to 40%. About half of the cases with the discharge diagnosis "essential hypertension" were admitted to hospital due to hypertension, about a quarter due to diagnoses within "other arteriosclerotic diseases" (ICD-8: 400, 402-440). The misclassification may lead to an underestimation of risks of hypertension in various occupations in the Occupational Hospitalization Register or bias the occupational risk pattern of essential hypertension to become more alike that of arteriosclerotic diseases.

All Danish occupational groups were screened for an increased risk of hospitalization due to a prolapsed lumbar intervertebral disc (PLID) (ICD-8: 725.11). A cohort of all gainfully employed Danes aged 20 to 59 years in 1981 was followed-up for 10 years for first hospitalization with PLID. A Standardized Hospitalization Ratio was calculated using all economically active persons as the reference group. Male groups with an elevated risk were found in building and construction, the iron and metal industry, in the food and nutrition sector and in occupational driving. Almost all groups of professional drivers had an elevated risk. Female groups with an elevated risk were mainly found in the same industries, but home helps, service workers in the private sector and sewing machine operators also had an elevated risk. We conclude that there are significant and systematic differences between occupational groups as concerns the risk of hospital admission due to PLID.