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Background: In rare diseases, limited access to services and rare disease experts may force families to act as medical advocates for their child; they can volunteer to support clinician-initiated research or initiate and lead research themselves. Ketotic Hypoglycemia International (KHI) is a new, global organization for families affected by idiopathic ketotic hypoglycemia (IKH) and is run solely by volunteers. Doing research together, families and international experts in a collaborative process such as at KHI, also referred to as patient and public involvement and engagement (PPIE) or extreme citizen science, is often praised for its positive effects on the research and the stakeholders involved., Methods: We used auto-ethnographic narratives from parents and medical professionals in KHI to report on their experiences with co-produced health research. All co-authors wrote down their experiences in relation to three topics: time invested, work invested and power dynamics., Results: Whilst the parents and health care professionals felt a new hope for (their) children with IKH, they also felt pressure to contribute time or to be flexible in how and when they dedicated time towards the organization. The power dynamics were characterised by a change in the relationship between the parents and medical experts; the parent being taught by the expert shifted to the expert learning from the lived experience of the parent. Both parents and medical experts struggled with maintaining boundaries and safeguarding their mental health., Conclusion: Our findings call for the need to secure and prioritize funding for patient organizations, to enable them to create the sustainable architecture required for meaningful PPIE within these organizations. The morals and often deeply personal reasons for engaging with voluntary work in health research, can lead to overstepping of boundaries. As a result of our research, we call for the development of ethics of care guidelines within collaborative health research., (© 2024. The Author(s).)

We introduce techniques for probing the dynamics of triplet states. We employ these tools, along with conventional techniques, to develop a detailed understanding of a complex chemical system: a negative-tone, radical photoresist for multiphoton absorption polymerization in which isopropylthioxanthone (ITX) is the photoinitiator. This work reveals that the same color of light used for the 2-photon excitation of ITX, leading to population of the triplet manifold through intersystem crossing, also depletes this triplet population via linear absorption followed by reverse intersystem crossing (RISC). Using spectroscopic tools and kinetic modeling, we identify the reactive triplet state and a non-reactive reservoir triplet state. We present compelling evidence that the deactivation channel involves RISC from an excited triplet state to a highly vibrationally excited level of the electronic ground state. The work described here offers the enticing possibility of understanding, and ultimately controlling, the photochemistry and photophysics of a broad range of triplet processes., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

Background: Ketotic hypoglycemia (KH) without an identifiable underlying metabolic or hormonal disease is historically named idiopathic KH. The prevalence is unknown, but idiopathic KH is considered the most frequent cause of hypoglycemia beyond the neonatal period. KH in Down syndrome (DS) has not been reported., Methods: We conducted a web-based survey on KH in DS through the non-profit patient organization Ketotic Hypoglycemia International. The responses were evaluated for consistency with KH by two authors. Two DS patient histories with documented KH were shared in more details., Results: Survey data on 139 DS patients were obtained. After validation, 10 patients (7.2%) had reported episodes of documented hypoglycemia, ketosis, and/or symptoms compatible with KH beyond the neonatal period. Glucose concentrations ranged 1.2-2.9 mmol/L; betahydroxybutyrate was up to 5.5 mmol/L during hypoglycemia. One girl had trisomy 21 with no response to i.m. glucagon also had a heterozygous Xp22.23 deletion including GYG2 , which protein, glycogenin 2, is a substrate for glycogen synthase. Treatment with extended release cornstarch was effective., Conclusion: This is the first demonstration of a possible high prevalence of KH in DS. Even though this finding needs to be confirmed in other research settings, identification of KH in DS could have a dramatic impact, as simple treatments with cornstarch, protein and frequent meals may prevent KH attacks and, analogous to other conditions with KH, improve growth, stamina and prevent overeating and obesity. GYG2 deletion may contribute to KH in DS, resembling glycogen storage disease type 0., Competing Interests: The authors declare that they have no competing interests., (© 2021 The Authors. JIMD Reports published by John Wiley & Sons Ltd on behalf of SSIEM.)

Background: Idiopathic Ketotic hypoglycemia (IKH) is a diagnosis of exclusion. Although considered as the most frequent cause of hypoglycemia in childhood, little progress has been made to advance the understanding of IKH since the medical term was coined in 1964. We aimed to review the literature on ketotic hypoglycemia (KH) and introduce a novel patient organization, Ketotic Hypoglycemia International (KHI)., Results: IKH may be diagnosed after the exclusion of various metabolic and hormonal diseases with KH. Although often mild and self-limiting, more severe and long-lasting IKH occurs. We therefore divide IKH in physiological KH and pathological KH, the latter defined as recurrent symptomatic, or occasionally symptomatic, episodes with beta-hydroxybutyrate ≥ 1.0 mmol/L and blood glucose < 70 mg/dL (3.9 mol/L), in the absence of prolonged fasting, acute infections and chronic diseases known to cause KH. Pathological KH may represent undiscovered diseases, e.g. glycogen storage disease IXa, Silver-Russel syndrome, and ketone transporter defects, or suggested novel disease entities identified by exome sequencing. The management of KH aims to prevent hypoglycemia, fatty acid oxidation and protein deficiency by supplying adequate amounts of carbohydrates and protein, including nutritional therapy, uncooked cornstarch, and sometimes continuous tube feeding by night. Still, intravenous dextrose may be needed in acute KH episodes. Failure to acknowledge that IKH can be more than normal variation may lead to under-treatment. KHI is a non-profit, patient-centric, global organization established in 2020. The organization was created by adult IKH patients, patient family members, and volunteers. The mission of KHI is to enhance the understanding of IKH while advocating for patients, their families and the continued research into KH., Conclusion: IKH is a heterogeneous disorder including physiological KH and pathological KH. IKH may represent missed diagnoses or novel disease entities, but shares common management principles to prevent fatty acid oxygenation. KHI, a novel patient organization, aims to enhance the understanding of IKH by supporting IKH families and research into IKH.

Recent articles have highlighted the lack of reproducibility of data from scientific publications. Here we would argue that a better way to describe and also tackle this matter is to use the term "lack of robustness," since it points toward potential solutions. Presenting several case reports, we highlight examples with common underlying issues from Novo Nordisk's experience: animal model variability, reagent quality, and inter-lab variability. We discuss means to prevent these issues and argue for increased collaborative work and transparent manuscript revision procedures. Collectively, we believe these measures will help promote a more rapid and efficient self-corrective process in diabetes drug target research., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Purpose: The aim of this study was to determine whether first trimester ultrasound measurements of crown rump length (CRL) and gestational sac diameter (GSD) differ depending on the method of conception among infertile women., Method: Infertile women, ages 21-50 years old, who conceived viable, singleton pregnancies via fresh embryo transfer (ET), frozen ET, non-in vitro fertilization (IVF) fertility treatment, or spontaneously were included in this observational cohort study at an academic fertility practice. Embryonic growth trajectories defined by the CRL and GSD at 6 and 8 weeks' gestation were analyzed and compared among the methods of conception., Results: Crown rump length at 6 weeks' gestation was smaller for conceptions achieved via fresh ET compared with frozen ET in a natural cycle (1.50 vs. 2.50 mm, p = 0.017). Crown rump length was smaller at 8 weeks' gestation in conceptions achieved via fresh ET compared to frozen ET in a programmed cycle (16.13 vs. 17.02 mm, p = 0.039)., Conclusion: Among infertile women, embryo growth may differ between fresh and frozen ET as early as 6 and 8 weeks' gestation.

Roux-en-Y Gastric bypass surgery (RYGB) is emerging as a powerful tool for treatment of obesity and may also cause remission of type 2 diabetes. However, the molecular mechanism of RYGB leading to diabetes remission independent of weight loss remains elusive. In this study, we profiled plasma metabolites and proteins of 10 normal glucose-tolerant obese (NO) and 9 diabetic obese (DO) patients before and 1-week, 3-months, 1-year after RYGB. 146 proteins and 128 metabolites from both NO and DO groups at all four stages were selected for further analysis. By analyzing a set of bi-molecular associations among the corresponding network of the subjects with our newly developed computational method, we defined the represented physiological states (called the edge-states that reflect the interactions among the bio-molecules), and the related molecular networks of NO and DO patients, respectively. The principal component analyses (PCA) revealed that the edge states of the post-RYGB NO subjects were significantly different from those of the post-RYGB DO patients. Particularly, the time-dependent changes of the molecular hub-networks differed between DO and NO groups after RYGB. In conclusion, by developing molecular network-based systems signatures, we for the first time reveal that RYGB generates a unique path for diabetes remission independent of weight loss., (Copyright © 2018. Published by Elsevier B.V.)

SLE is a complex autoimmune inflammatory disease characterized by pathogenic autoantibody production as a consequence of uncontrolled T-B cell activity and immune-complex deposition in various organs, including kidney, leading to tissue damage and function loss. There is a high unmet need for better treatment options other than corticosteroids and immunosuppressants. Phosphoinositol-3 kinase δ (PI3Kδ) is a promising target in this respect as it is essential in mediating B- and T-cell function in mouse and human. We report the identification of selective PI3Kδ inhibitors that blocked B-, T-, and plasmacytoid dendritic cell activities in human peripheral blood and in primary cell co-cultures (BioMAP(®)) without detecting signs of undesired toxicity. In an IFNα-accelerated mouse SLE model, our PI3Kδ inhibitors blocked nephritis development, whether administered at the onset of autoantibody appearance or the onset of proteinuria. Disease amelioration correlated with normalized immune cell numbers in the spleen, reduced immune-complex deposition as well as reduced inflammation, fibrosis, and tissue damage in the kidney. Improvements were similar to those achieved with a frequently prescribed drug for lupus nephritis, the potent immunosuppressant mycophenolate mofetil. Finally, we established a pharmacodynamics/pharmacokinetic/efficacy model that revealed that a sustained PI3Kδ inhibition of 50% is sufficient to achieve full efficacy in our disease model. These data demonstrate the therapeutic potential of PI3Kδ inhibitors in SLE and lupus nephritis.

The purpose of the present study is to explore the progression of type 1 diabetes (T1D) in Danish children 12 months after diagnosis using Latent Factor Modelling. We include three data blocks of dynamic paraclinical biomarkers, baseline clinical characteristics and genetic profiles of diabetes related SNPs in the analyses. This method identified a model explaining 21.6% of the total variation in the data set. The model consists of two components: (1) A pattern of declining residual β-cell function positively associated with young age, presence of diabetic ketoacidosis and long duration of disease symptoms (P = 0.0004), and with risk alleles of WFS1, CDKN2A/2B and RNLS (P = 0.006). (2) A second pattern of high ZnT8 autoantibody levels and low postprandial glucagon levels associated with risk alleles of IFIH1, TCF2, TAF5L, IL2RA and PTPN2 and protective alleles of ERBB3 gene (P = 0.0005). These results demonstrate that Latent Factor Modelling can identify associating patterns in clinical prospective data--future functional studies will be needed to clarify the relevance of these patterns.

Aims/hypothesis: Recent studies suggest that proton pump inhibitor treatment may increase insulin secretion and improve glucose metabolism in type 2 diabetes. In a randomised double-blind prospective placebo-controlled 2 × 2 factorial study, we examined the effect of esomeprazole on insulin secretion, HbA(1c) and cardiovascular risk factors in type 2 diabetes., Methods: Forty-one patients with type 2 diabetes using dietary control or oral glucose-lowering treatment were randomised to receive add-on esomeprazole 40 mg (n = 20) or placebo (n = 21) for 12 weeks. Randomisation was carried out prior to inclusion on the basis of a computer-generated random-number list. The allocation sequence was concealed in sealed envelopes from the researcher enrolling and assessing participants. The study was undertaken at Steno Diabetes Center, Gentofte, Denmark. The primary outcome was change in AUC for insulin levels during a meal test. Secondary outcomes were the levels of HbA(1c) and biochemical markers of cardiovascular risk, including lipids, coagulation factors, inflammation markers, markers of endothelial function and 24 h ambulatory BP measurements., Results: Forty-one participants were analysed. In the esomeprazole-treated group the AUC for insulin did not change (before vs after treatment: 28,049 ± 17,659 vs 27,270 ± 32,004 pmol/l × min (p = 0.838). In the placebo group AUC for insulin decreased from 27,392 ± 14,348 pmol/l × min to 22,938 ± 11,936 pmol/l × min (p = 0.002). Esomeprazole treatment (n = 20) caused a ninefold increase in the AUC for gastrin. HbA(1c) increased from 7.0 ± 0.6% (53 ± 5 mmol/mol) to 7.3 ± 0.8% (56 ± 6 mmol/mol) in the esomeprazole-treated group and from 7.0 ± 0.6% (53 ± 5 mmol/mol) to 7.4 ± 0.8% (57 ± 6 mmol/mol) in the placebo group (n = 21) (p for difference in change >0.05). Except for BP, there were no differences between the groups in the markers of cardiovascular risk (p > 0.05). Monitoring of 24 h ambulatory BP showed a significant decrease in daytime systolic BP, daytime diastolic BP and 24 h diastolic BP in the placebo group (p < 0.05). No change in BP was seen in the patients treated with esomeprazole., Conclusions/interpretation: Treatment with esomeprazole over 12 weeks did not improve insulin secretion, glycaemic control or cardiovascular disease biomarkers in patients with type 2 diabetes.

Aims/hypothesis: Gastrin has been implicated in islet growth/neogenesis, and proton pump inhibitors (PPIs) have been shown to increase endogenous gastrin levels in animals and humans. Therefore, we investigated the effect of PPIs in a model of type 2 diabetes, Psammomys obesus., Methods: P. obesus (morning blood glucose [mBG] 16.9 +/- 0.6 mmol/l) were treated with vehicle or different doses (1-15 mg/kg) of lansoprazole for 17 days., Results: Treatment with lansoprazole resulted in up to ninefold dose-dependent increases in endogenous gastrin levels (p < 0.05 for 10 mg/kg lansoprazole vs vehicle). There was a significant reduction in mBG levels in all animals in the high-dose lansoprazole groups during the 17 day treatment period, whereas there was no significant improvement in mBG in animals in the vehicle groups. The mBG at end of study was 18.2 +/- 2.1, 8.7 +/- 2.2 (p < 0.01), and 6.1 +/- 2.3 (p < 0.001) mmol/l for vehicle and lansoprazole 10 and 15 mg/kg, respectively. The animals treated with 15 mg/kg lansoprazole, compared with vehicle, had a 2.3-fold increase in the intensity of insulin staining in beta cells (p=0.0002) and 50% higher beta cell mass (p=0.04)., Conclusions/interpretations: The PPI lansoprazole had significant glucose-lowering effects in an animal model of type 2 diabetes, an effect that is most likely mediated through an increase in endogenous gastrin levels.

Metabolic syndrome and obesity-related diseases are affecting more and more people in the Western world. The basis for an effective treatment of these patients is a better understanding of the underlying pathophysiology. Here, we characterize fructose- and fat-fed rats (FFFRs) as a new animal model of metabolic syndrome. Sprague-Dawley rats were fed a 60 kcal/100 kcal fat diet with 10% fructose in the drinking water. After 6, 12, 18, 24, 36, and 48 wk of feeding, blood pressure, glucose tolerance, plasma insulin, glucose, and lipid levels were measured. Cardiac function was examined by in vivo pressure volume measurements, and intramyocardial lipid accumulation was analyzed by confocal microscopy. Cardiac AMP-activated kinase (AMPK) and hepatic phosphoenolpyruvate carboxykinase (PEPCK) levels were measured by Western blotting. Finally, an ischemia-reperfusion study was performed after 56 wk of feeding. FFFRs developed severe obesity, decreased glucose tolerance, increased serum insulin and triglyceride levels, and an initial increased fasting glucose, which returned to control levels after 24 wk of feeding. The diet had no effect on blood pressure but decreased hepatic PEPCK levels. FFFRs showed significant intramyocardial lipid accumulation, and cardiac hypertrophy became pronounced between 24 and 36 wk of feeding. FFFRs showed no signs of cardiac dysfunction during unstressed conditions, but their hearts were much more vulnerable to ischemia-reperfusion and had a decreased level of phosphorylated AMPK at 6 wk of feeding. This study characterizes a new animal model of the metabolic syndrome that could be beneficial in future studies of metabolic syndrome and cardiac complications.

Introduction: High cholesterol-fructose (HCF) fed rats have previously been described as an animal model of impaired cardiac insulin signaling and decreased contractile performance. In this study, we evaluated the metabolic and cardiac effects of a HCF diet in rats., Methods: Male Sprague-Dawley rats received a HCF diet for 16 to 17weeks. Body weight was measured weekly and mean arterial blood pressure, fasting blood glucose, fasting plasma insulin, glucose tolerance, and blood lipid levels were measured following 15weeks of feeding. One to 2weeks later, while still on the HCF diet, cardiac function was examined by in vivo pressure-volume measurements in the left ventricle. Finally, protein and glucose content in the urine was measured and all organs were weighed at the end of the study., Results: Rats fed a HCF diet showed increased cholesterol and decreased high-density lipoprotein (HDL) levels in serum compared to control fed rats and they had more than a twofold increase in liver weight. However, in contrast to what has previously been reported, HCF diet had no effect on body weight, blood pressure, fasting blood glucose, fasting plasma insulin, glucose tolerance, or cardiac function during unstressed conditions., Discussion: We were unable to reproduce previous findings that a HCF diet causes changes in glucose tolerance and cardiac contractile performance. Therefore, further studies are warranted to evaluate specific interactions between genetic, environmental, and dietary factors on metabolic and cardiovascular disease progression associated with intake of a westernized diet., (2010 Elsevier Inc. All rights reserved.)

Aims: Rotigaptide is proposed to exert its anti-arrhythmic effects by improving myocardial gap-junction communication. To directly investigate the mechanisms of rotigaptide action, we treated cultured neonatal murine ventricular cardiomyocytes with clinical pharmacological doses of rotigaptide and directly determined its effects on gap-junctional currents., Methods and Results: Neonatal murine ventricular cardiomyocytes were enzymatically isolated and cultured for 1-4 days. Primary culture cell pairs were subjected to dual whole cell patch-clamp procedures to directly measure gap-junctional currents (I(j)) and voltage (V(j)). Rotigaptide (0-350 nM) was applied overnight or acutely perfused into 35 mm culture dishes. Rotigaptide (35-100 nM) acutely and chronically increased the resting gap-junction conductance (g(j)), and normalized steady-state minimum g(j) (G(min)) by 5-20%. Higher concentrations produced a diminishing response, which mimics the observed therapeutic efficacy of the drug. The inactivation kinetics was similarly slowed in a therapeutic concentration-dependent manner without affecting the V(j) dependence of inactivation or recovery. The effects of 0-100 nM rotigaptide on ventricular g(j) during cardiac action potential propagation were accurately modelled by computer simulations which demonstrate that clinically effective concentrations of rotigaptide can partially reverse conduction slowing due to decreases in g(j) and inactivation., Conclusion: These results demonstrate that therapeutic concentrations of rotigaptide increase the resting gap-junction conductance and reduce the magnitude and kinetics of steady-state inactivation in a concentration-dependent manner. Rotigaptide may be effective in treating re-entrant forms of cardiac arrhythmias by improving conduction and preventing the formation of re-entrant circuits in partially uncoupled myocardium.

Human extracellular superoxide dismutase (EC-SOD) is a tetrameric glycoprotein responsible for the removal of superoxide generated in the extracellular space. Two different folding variants of EC-SOD exist based on the disulfide bridge connectivity, resulting in enzymatically active (aEC-SOD) and inactive (iEC-SOD) subunits. As a consequence of this, the assembly of the EC-SOD tetramers produces molecules with variable activity and may represent a way to regulate the antioxidant level in the extracellular space. To determine whether the formation of these two folding variants is an intra- or extracellular event, we analyzed the biosynthesis in human embryonic kidney 293 cells expressing wild-type EC-SOD. These analyses revealed that both folding variants were present in the intra- and extracellular spaces, suggesting that the formation is an intracellular event. To further analyze the biosynthesis, we constructed mutants with the capacity to generate only aEC-SOD (C195S) or iEC-SOD (C45S). The expression of these suggested that the cellular biosynthetic machinery supported the secretion of aEC-SOD but not iEC-SOD. The coexpression of these two mutants did not affect the expression pattern. This study shows that generation of the EC-SOD folding variants is an intracellular event that depends on a free cysteine residue not involved in disulfide bonding.

Background and Purpose: ZP120 (Ac-RYYRWKKKKKKK-NH(2)), is a new partial nociceptin/orphanin FQ (NOP) receptor agonist with sodium-potassium sparing aquaretic effects. The mechanisms of vasodilatation of ZP120 were examined in rat mesenteric resistance arteries., Experimental Approach: Arterial segments (internal diameters 206+/-4 microm, n=224) were mounted in microvascular myographs for isometric tension recordings and electrical field stimulation (EFS)., Key Results: ZP120 and the endogenous NOP receptor ligand, N/OFQ, did not relax arteries contracted with noradrenaline or adenosine-triphosphate. EFS-evoked contractions were inhibited by a purinoceptor antagonist, suramin, and the alpha(1)-adrenoceptor antagonist prazosin. N/OFQ inhibited, concentration-dependently, EFS-evoked contractions with a maximal effect of 52+/-3% (n=8) at 1 microM. The maximal effect of 1 microM ZP120 was lower (27+/-5%, P<0.05, n=9) than for N/OFQ. Endothelial removal or pretreatment with capsaicin did not influence the vasodilator effects of ZP120 and N/OFQ. ZP120 and N/OFQ responses were preserved in the presence of suramin. The alpha(2)-adrenoceptor antagonist, rauwolscine, antagonized the effect of clonidine and brimonidine, but ZP120 and N/OFQ inhibition of EFS-evoked contraction was unaltered. The competitive NOP receptor antagonist, UFP-101 (10 microM), prevented the inhibitory effect of N/OFQ, but not ZP120 suggesting that N/OFQ and ZP120 have distinct modes of interaction with the NOP receptor., Conclusions and Implications: Our findings suggest that the vasodilator effect of ZP120 and N/OFQ in rat mesenteric resistance arteries is mediated by prejunctional inhibition of adrenergic neurotransmission. These properties, that promote diuresis and attenuate the cardiovascular consequences of increased sympathetic nerve activity, make ZP120 a promising drug candidate.

The peptide hormone glucagon-like peptide (GLP)-1 has important actions resulting in glucose lowering along with weight loss in patients with type 2 diabetes. As a peptide hormone, GLP-1 has to be administered by injection. Only a few small-molecule agonists to peptide hormone receptors have been described and none in the B family of the G protein coupled receptors to which the GLP-1 receptor belongs. We have discovered a series of small molecules known as ago-allosteric modulators selective for the human GLP-1 receptor. These compounds act as both allosteric activators of the receptor and independent agonists. Potency of GLP-1 was not changed by the allosteric agonists, but affinity of GLP-1 for the receptor was increased. The most potent compound identified stimulates glucose-dependent insulin release from normal mouse islets but, importantly, not from GLP-1 receptor knockout mice. Also, the compound stimulates insulin release from perfused rat pancreas in a manner additive with GLP-1 itself. These compounds may lead to the identification or design of orally active GLP-1 agonists.

We have previously shown that systemic treatment with the somatostatin analog octreotide has marked beneficial effects on renal function in rats with liver cirrhosis induced by common bile duct ligation (CBL; Jonassen TEN, Christensen S, Sørensen AM, Marcussen N, Flyvbjerg A, Andreasen F, and Petersen JS. Hepatology 29: 1387-1395, 1999). In the present study, we tested the hypothesis that octreotide has a direct effect on renal tubular function. Rats (CBL or Sham-CBL) were intrarenally treated with low-dose octreotide in a long-acting release formulation, which had no systemic actions (100 microg/kg body wt as a single dose). Rats receiving low-dose octreotide (sc) were used as controls. The rats were chronically instrumented, and renal function was examined 4 wk after CBL or Sham-CBL. Intrarenal octreotide administration (IROA) prevented sodium retention in CBL rats without changes in renal plasma flow, glomerular filtration rate, or circulating levels of aldosterone and vasopressin. Renal clearance studies revealed that IROA normalized the increased natriuretic efficacy of furosemide found in CBL rats. Furthermore, IROA protected against the development of hypertrophy of the inner stripe of the outer medulla and thereby the increased the volume of thick ascending limb of Henle's loop (TAL) epithelium found in CBL rats. Finally, Western blot analyses of outer medullary homogenates showed increased abundance of the furosemide-sensitive Na-K-2Cl (NKCC2) cotransporter. IROA did not affect the abundance of NCKK2 within the outer medulla. Together with the histological findings, these results indicate that IROA reduces the total number of NKCC2 within the outer medulla. In conclusion, the results indicate a direct intrarenal effect of octreotide on TAL function and morphology in cirrhotic rats.

We investigated the effects of rotigaptide (ZP123), a stable hexapeptide with antiarrhythmic properties, on gap junction mediated intercellular communication in contracting rat neonatal cardiac myocytes, HL-1 cells derived from cardiac atrium and in HeLa cells transfected with cDNA encoding Cx43-GFP, Cx32-GFP, Cx26-GFP, wild-type Cx43 or wild-type Cx26. Intercellular communication was monitored before and after treatment with rotigaptide following microinjection of small fluorescent dyes (MW<1 kDa). The communication-modifying effect of rotigaptide was confined to cells expressing Cx43 since the peptide had no effect on dye transfer in HeLa cells expressing Cx32-GFP, Cx26-GFP or wild-type Cx26. In contrast, HeLa cells expressing Cx43-GFP exposed to 50 nM rotigaptide for 5 h showed a 40% increase in gap junction mediated communication. Rotigaptide (50 nM) increased intercellular dye transfer in myocytes and atrial HL-1 cells, where Cx43 is the dominant connexin. However, it caused no change in cell beating rates of cardiac myocytes. Western blot analysis showed that rotigaptide did not modify the overall level of Cx43 expression and changes in the phosphorylation status of the protein were not observed.We conclude that the effects of rotigaptide were confined to cells expressing Cx43.

Treatment with non-selective drugs (eg, long-chain alcohols, halothane) that reduce gap junction intercellular communication (GJIC) is associated with reduced infarct size after myocardial infarction (MI). Therefore, it has been suggested that gap junction intercellular communication stimulating compounds may increase infarct size. The antiarrhythmic peptide analogue rotigaptide (ZP123) increases cardiac gap junction intercellular communication and the purpose of the present study was to examine the effects of rotigaptide treatment on infarct size. Myocardial infarction was induced in male rats by ligation of the left anterior descending artery (LAD). Rats (n = 156) were treated with rotigaptide at three dose levels or vehicle from the onset of ischemia and for 3 weeks following LAD occlusion. Infarct size was determined using histomorphometry after 3 weeks treatment. Rotigaptide treatment producing steady state plasma levels of 0.8 +/- 0.1, 5.5 +/- 0.5, and 86 +/- 8 nmol/L had no effect on mortality, but reduced infarct size to 90 +/- 10% (P = 0.41), 67 +/- 7% (P = 0.005), and 82 +/- 7% (P = 0.13), respectively relative to vehicle-treated myocardial infarction rats (100 +/- 12%). In contrast to what was predicted, our data demonstrates that rotigaptide treatment was associated with a significant infarct size reduction. We conclude that whereas treatment with non-selective inhibitors of gap junction intercellular communication cause a reduction in infarct size, this information cannot be extrapolated to the effects of compounds that selectively increase gap junction intercellular communication.

Gap junctions play an important role in bone development and function, but the lack of pharmacological tools has hampered the gap junction research. The antiarrhythmic peptides stimulate gap junction communication between cardiomyocytes, but effects in noncardiac tissue are unknown. The purpose of this study was to examine whether antiarrhythmic peptides, which are small peptides increasing gap junctional conductivity, show specific binding to osteoblasts and investigate the effect of the stable analog rotigaptide (ZP123) on gap junctional intercellular communication in vitro and on bone mass and strength in vivo. Cell coupling and calcium signaling were assessed in vitro on human, primary, osteoblastic cells. In vivo effects of rotigaptide on bone strength and density were determined 4 wk after ovariectomy in rats treated with either vehicle, sc injection twice daily (300 nmol per kilogram body weight) or by continuous ip infusion (158 nmol per kilogram body weight per day). During metabolic stress, a high affinity-binding site (KD=0.1 nM) with low density (15 fmol/mg protein) for [125I]di-I-AAP10 was demonstrated. During physiological conditions, specific binding sites for [125I]AAP10 could not be shown. Studies of the effects of rotigaptide on propagation of intercellular calcium waves and cell-to-cell coupling demonstrated that 10 nM rotigaptide produced a small increase in intercellular communication during physiological conditions (+4.5+/-1.6% vs. vehicle; P<0.05). During conditions with metabolic stress, 10 nM rotigaptide produced an increase in coupling measured by both methods. Four weeks after ovariectomy, bone strength of the femoral head was reduced by 20% in vehicle-treated ovariectomized rats, which was completely prevented in both rotigaptide-treated groups. Rotigaptide also prevented decreases in bone mineral. We conclude that the stable analog rotigaptide increases gap junctional communication in osteoblasts in vitro and preferably during conditions with metabolic stress. Rotigaptide further prevents ovariectomy-induced bone loss in vivo. Thus, gap junction modulation may be a promising new target for osteoporosis therapy.

The role of gap junction intercellular communication (GJIC) in ischemia-induced focal ventricular tachycardia (VT) is unknown. We have developed a new, stable antiarrhythmic peptide analog named ZP123 that selectively increases GJIC and prevents reentrant VT. Our aim in this study was to use ZP123 as a tool to assess the role of GJIC on occurrence of ischemia-induced focal VT and triggered activity (TA) due to delayed afterdepolarizations (DADs). Focal VT was induced by programmed stimulation in alpha-chloralose-anesthetized, open-chest dogs 1-4 h after coronary artery occlusion. Three-dimensional activation mapping was done using 6 bipolar electrograms on each of 23 multipolar needles in the risk zone. Dogs were randomly assigned to receive either saline or ZP123 cumulatively at three dose levels (an intravenous bolus followed by a 30-min infusion per dose). Attempts to induce VT were repeated in each dose. Mass spectrometry was used to measure plasma ZP123 concentrations. Standard microelectrode techniques were used for in vitro study of DADs and TA. Twenty-six dogs with focal VT were included. ZP123 did not affect the inducibility of focal VT at any plasma concentrations vs. saline (0.8 +/- 0.1 nM, 77 vs. 75%; 7.8 +/- 0.4 nM, 86 vs. 77%; and 78.8 +/- 5.0 nM, 77 vs. 91%). In vitro, ZP123 did not affect the induction of DADs (12/12) and TAs (10/10) in ischemic tissues or tissue removed from the origin of focal VT (DADs, 8/8; TAs, 4/4). Therefore, although indirect, the data with the doses and concentrations used suggest that GJIC may not play a major role in the genesis of focal activity in the ischemic models studied.

Nociceptin, the endogenous ligand of the inhibitory G protein-coupled opioid receptor-like 1 receptor, produces aquaresis (i.e., increases the excretion of solute-free urine) in rats. However, the mechanism underlying this effect has not yet been explained. Using immunohistochemistry, we found the opioid receptor-like 1 receptor in the rat kidney colocalized with the vasopressin-regulated water channel aquaporin-2 in inner medullary collecting ducts. We investigated the aquaretic effect of opioid receptor-like 1 receptor stimulation by infusing the selective nociceptin analog ZP120C; volume depletion was prevented by computer-driven, servo-controlled intravenous volume replacement with 50 mM glucose. ZP120C induced a marked and sustained aquaresis in normal and congestive heart failure rats in the absence of changes in vasopressin plasma concentrations. The ZP120C-induced aquaresis was associated with downregulation of the aquaporin-2 protein level in both rat groups, suggesting that opioid receptor-like 1 receptor stimulation produces aquaresis by inhibiting the vasopressin type-2 receptor-mediated stimulation on collecting duct water reabsorption. However, substantial amounts of PKA-mediated serine 256 phosphorylated aquaporin-2 were still present after 4 h of ZP120C treatment. Furthermore, neither preincubation with nociceptin nor ZP120C inhibited vasopressin-mediated cAMP accumulation in isolated collecting ducts. We conclude that renal opioid receptor-like 1 receptor stimulation in normal and congestive heart failure rats produces aquaresis by a direct renal effect, via aquaporin-2 downregulation, through a mechanism not involving inhibition of vasopressin type-2 receptor-mediated cAMP production.

Non-pharmacological in vivo models of atrial fibrillation (AF) have been developed in large animals only. We aimed to develop and characterize a new small animal non-pharmacological in vivo model of AF. AF was induced by transesophageal atrial burst pacing during 35 seconds periods of asphyxia in anesthetized male Sprague-Dawley rats. AF was reproducibly induced in 81% of the rats. The presence of AF was associated with an increased heart rate, and a decreased blood pressure. Treatment with amiodarone, D,L-sotalol, flecainide, and propranolol all reduced duration of AF, whereas verapamil treatment was associated with a marked profibrillatory effect. Increasing gap junction intracellular communication using the antiarrhythmic peptide analogue AAP10 did not affect AF duration. Basal plasma level of epinephrine and norepinephrine were increased 5- to 20-fold relative to values reported by others, but unchanged following 35 seconds of asphyxia. The results from our study demonstrate that the rat model shares several clinical key characteristics with human AF: (1) hemodynamic response to AF; (2) increased autonomic tone; (3) antiarrhythmic effects of clinically used drugs; (4) profibrillatory effect of verapamil. Relative to existing models of AF in larger animals, this model offers rapid, predictive, and inexpensive testing of antiarrhythmic/profibrillatory effects of new drugs.

Our earlier investigations have demonstrated a critical difference in the efficacy of orally administered porcine compared to human or mouse insulin (no effect) in preventing type I diabetes in two distinct experimental models. Based on these findings one has to assume that certain insulins might not be suitable for the induction of oral 'tolerance'/bystander suppression, which might be one cause for recent failures in human oral antigen trials. Here we demonstrate that coupling to the non-toxic subunit of cholera toxin (CTB) can abolish these differences in efficacy between human and porcine insulin. As expected, an added benefit was the much smaller oral antigen dose required to induce CD4+ insulin-B specific regulatory cells that bystander-suppress autoaggressive responses. Mechanistically we found that uptake or transport of insulin-CTB conjugates in the gut occurs at least partially via binding to GM-1, which would explain the enhanced clinical efficacy. Both B chains bound well to major histocompatibility complex (MHC) class II, indicating comparable immunological potential once uptake and processing has occurred. Thus, our findings delineate a pathway to overcome issues in oral antigen choice for prevention of type I diabetes.

During ischemia, cardiac gap junctions close and neighboring cells uncouple. This leads to slow conduction, increased dispersion of APD90 (duration from action potential beginning to 90% of repolarization), nonuniform anisotropy, and unidirectional conduction block, all of which favor the induction of reentry arrhythmias. It has been suggested that anti-arrhythmic peptides increase gap junction conductance during states of reduced coupling. The aim of this study was to test the effect of the anti-arrhythmic peptide N-3-(4-hydroxyphenyl)propionyl Pro-Hyp-Gly-Ala-Gly-OH (HP-5) (10(-10) ) on dispersion of epicardial APD90 during both normokalemic and hypokalemic ischemia/reperfusion in isolated perfused rabbit hearts. HP-5 did not affect average APD90, heart rate, left ventricular contractility (LVP dP/dtmax), or mean coronary flow. HP-5 significantly reduced the epicardial APD dispersion during hypokalemic ischemia (HP-5 treated: 24.1 +/- 3.4 ms, untreated: 33.9 +/- 3.1 ms, p < 0.05 versus untreated) and during normokalemic reperfusion but not during normokalemic ischemia or control conditions. In addition, among untreated hearts subjected to hypokalemic ischemia/reperfusion, seven of 10 developed ventricular fibrillation, whereas only three of nine hearts perfused with HP-5 developed ventricular fibrillation. These results show that HP-5 is able to reduce APD90 dispersion during hypokalemic ischemia in rabbit hearts.

1 This study reports on the pharmacological characterization of ZP120, a novel ligand of the nociceptin/orphanin FQ (N/OFQ) peptide receptor, NOP. ZP120 is a structure inducing probes modified NOP ligand: Zealand Pharma proprietary SIP technology was used to increase the enzymatic stability and half-life of peptide. 2 In vitro, ZP120 mimicked the inhibitory effects of N/OFQ in the electrically stimulated mouse vas deferens, showing however higher potency (pEC(50) 8.88 vs 7.74), lower maximal effects (E(max) 69+/-5% vs 91+/-2%), and slower onset of action. Like N/OFQ, the effects of ZP120 were not modified by 1 micro M naloxone, but they were antagonized by the NOP receptor selective antagonist J-113397 (pA(2) 7.80 vs ZP120, 7.81 vs N/OFQ). 3 In vivo, ZP120 mimicked the effects of N/OFQ, producing pronociceptive effects in the tail withdrawal assay and decreased locomotor activity after i.c.v., but not after i.v. administration in mice. ZP120 elicited similar maximal effects as N/OFQ, but it was about 10 fold more potent and its effects lasted longer. 4 In conclusion, the novel NOP receptor ligand ZP120 is a highly potent and selective partial agonist of the NOP receptor with prolonged effects in vivo.

The pentameric B-subunit of cholera toxin (CTB) can be used as an efficient mucosal carrier of either immunogenic or tolerogenic T-cell epitopes. In this study a series of fusions was constructed between the genes encoding CTB and the B-chain of human insulin (InsB). The resulting fusion proteins were expressed in Escherichia coli and isolated as cytoplasmic inclusion bodies that were then dissolved and assembled in vitro. GM1 enzyme-linked immunosorbent assay (ELISA), sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analyses showed that the protein construct in which InsB was fused to the C-terminus of a CTB monomer (CI) assembled into structures that both bound to the receptor GM1 ganglioside and reacted with monoclonal antibodies to CTB and insulin. Fusion of InsB to the N-terminus of CTB resulted in protein that could not assemble into pentameric CTB. In vitro assays showed that the CI fusion protein was 300-fold more potent than native insulin at inducing interleukin-2 (IL-2) production by an insulin-specific T-cell hybridoma. When administered orally, the CI fusion protein induced efficient immunological suppression of ovalbumin-specific T-cell responses in mice co-immunized parenterally with insulin and ovalbumin. These results demonstrate the stability, GM1 receptor-binding activity and antigenic authenticity of the CI fusion protein as well as its ability to elicit insulin-specific T-cell responses in vitro. In addition, we demonstrate that the CI fusion protein induces efficient immunosuppression after oral administration, raising the possibility of using such constructs in the treatment of type-1 diabetes.

Simultaneous blockade of systemic AT1 and AT2 receptors or converting enzyme inhibition (CEI) attenuates the hypoglycemia-induced reflex increase of epinephrine (Epi). To examine the role of brain AT1 and AT2 receptors in the reflex regulation of Epi release, we measured catecholamines, hemodynamics, and renin during insulin-induced hypoglycemia in conscious rats pretreated intracerebroventricularly with losartan, PD-123319, losartan and PD-123319, or vehicle. Epi and norepinephrine (NE) increased 60-and 3-fold, respectively. However, the gain of the reflex increase in plasma Epi (Deltaplasma Epi/Deltaplasma glucose) and the overall Epi and NE responses were similar in all groups. The ensuing blood pressure response was similar between groups, but the corresponding bradycardia was augmented after PD-123319 (P < 0.05 vs. vehicle) or combined losartan and PD-123319 (P < 0.01 vs. vehicle). The findings indicate 1) brain angiotensin receptors are not essential for the reflex regulation of Epi release during hypoglycemia and 2) the gain of baroreceptor-mediated bradycardia is increased by blockade of brain AT2 receptors in this model.

Previous studies have suggested that mineralocorticoids are needed for a normal action of vasopressin on collecting duct osmotic water permeability. However, the mechanisms behind this are unknown. To investigate if aldosterone-receptor blockade influences vasopressin type 2 receptor (V(2))-mediated renal water reabsorption and the renal expression of the vasopressin-regulated water channel aquaporin-2 (AQP2), rats were treated with the aldosterone-receptor antagonist canrenoate (20 mg/day iv) for 4 wk. Daily urine flow was increased significantly by 44%, and urine osmolality was decreased by 27% in canrenoate-treated rats. Acute V(2)-receptor blockade (OPC-31260, 800 microgram. kg(-1). h(-1)) was performed under conditions in which volume depletion was prevented. In control rats, OPC-31260 induced a significant increase in urine flow rate (V, +25%) and free water clearance (C(H(2)O), -29%). In canrenoate-treated rats, the effect of OPC-31260 was significantly reduced, and semiquantiative immunoblotting demonstrated a significant reduction (45%) in AQP2 expression. Because rats with common bile duct ligation (CBL) have a reduced vasopressin-mediated water reabsorption compared with normal rats (V: -24%; C(H(2)O): -28%, and 86% downregulation of AQP2), the effect of canrenoate combined with OPC-31260 was tested. Canrenoate treatment of CBL rats significantly increased daily urine flow, decreased urine osmolality, and impaired the aquaretic response to OPC-31260 (V: -23%; C(H(2)O): -31%) with maintained suppression of the renal AQP2 expression. Thus canrenoate treatment of normal and CBL rats showed 1) increased urine production, 2) reduced aquaretic effect of acute V(2)-receptor blockade, and 3) a marked reduction in AQP2 expression. This strongly supports the view that aldosterone plays a significant role for vasopressin-mediated water reabsorption.

Glutamic acid decarboxylase (GAD) is an important autoantigen in insulin-dependent diabetes mellitus (IDDM), but little is known about its regulation and function in islet cells. We investigated the effects of the GABA-transaminase inhibitor gamma-vinyl-GABA (GVG) on GAD expression in rat islets and brain in vitro and in vivo. In islets incubated in high glucose culture medium there was an increase in GAD activity, GAD65 and GAD67 protein levels compared to low-glucose conditions; however, even in high glucose, GVG still significantly suppressed GAD activity and GAD67 expression. Our observations suggest that glucose and GVG act on GAD in islets through different mechanisms. Quantitative immunohistochemistry of pancreatic sections from rats treated with GVG in vivo using novel monoclonal antibodies specific for GAD65 and GAD67, showed a decrease in GAD67 expression (p < 0.005) relative to untreated rats. The effects of GVG on rat pancreatic islets were very similar to those observed in brain of rats treated with GVG in vivo. In homogenates of cerebral tissue from GVG treated rats containing both membrane-bound and soluble protein GAD67 levels were significantly decreased while GAD65 levels were not significantly changed compared to untreated rats. In contrast, in homogenates of cerebral tissues containing only soluble cytosolic protein, GVG-treatment was also significantly found to decrease GAD65 levels. Taken together, these results suggest that GVG potentially could be of use to decrease GAD expression in islet cells and consequently to deviate/inhibit the autoimmune response against the beta cells seen in IDDM.

An unselected population of 755 siblings of children with insulin-dependent diabetes mellitus (IDDM) was studied to evaluate the predictive characteristics of islet cell antibodies (ICA), antibodies to the IA-2 protein (IA-2A), antibodies to the 65-kD isoform of glutamic acid decarboxylase (GADA), insulin autoantibodies (IAA), and combinations of these markers. We also evaluated whether the histochemical ICA test could be replaced by the combined detection of other markers. 32 siblings progressed to IDDM within 7.7 yr of the initial sample taken at or close to the diagnosis of the index case (median follow-up, 9.1 yr). The positive predictive values of ICA, IA-2A, GADA, and IAA were 43, 55, 42, and 29%, and their sensitivities 81, 69, 69, and 25%, respectively. In contrast to the other three antibody specificities, GADA levels were not related to the risk for IDDM. The risk for IDDM in siblings with four, three, two, one, or no antibodies was 40, 70, 25, 2, and 0.8%, respectively. Combined screening for IA-2A and GADA identified 70% of all ICA-positive siblings, and all of the ICA-positive progressors were also positive for at least one of the three other markers. The sensitivity of the combined analysis of IA-2A and GADA was 81%, and the positive predictive value was 41%. In conclusion, combined screening for IA-2A and GADA may replace the ICA assay, giving comparable sensitivity, specificity, and positive predictive value. Accurate assessment of the risk for IDDM in siblings is complicated, as not even all those with four antibody specificities contract the disease, and some with only one or no antibodies initially will progress to IDDM.

Angiotensin II facilitates epinephrine release during insulin-induced hypoglycemia, and this effect appears to be independent of type 1 angiotensin II (AT1) receptors in man. In the present study, we hypothesized that the action of angiotensin II on adrenomedullary epinephrine release is mediated by an AT2 receptor-dependent mechanism. In conscious chronically instrumented rats, we measured plasma concentrations of catecholamines during acute insulin-induced hypoglycemia in groups of rats pretreated with the AT1 receptor antagonist losartan (10 mg/kg i.v.), the AT2 receptor antagonist PD123319 (30 mg/kg i.v.), combined losartan + PD123319, the converting enzyme inhibitor enalapril (1 mg/kg i.v.), or vehicle. In vehicle-treated rats, the area under the curve for changes in plasma epinephrine concentration [AUC(plasma epinephrine)] during insulin-induced hypoglycemia was 111+/-8 nmolXh/L (+/-SEM). Pretreatment with losartan alone did not affect AUC(plasma epinephrine) (113+/-17 nmol x h/L), while pretreatment with PD123319 tended to reduce the response (87+/-10 nmol x h/L; P=.08 versus vehicle). However, AUC(plasma epinephrine) was significantly reduced in rats that were pretreated with combined losartan + PD123319 (68+/-5 nmol x h/L; P<.001 versus vehicle) or enalapril: 86+/-10 nmol x h/L (P<.05 versus vehicle). Thus, combined treatment with losartan + PD 123319 proved more effective in attenuating the reflex increase in plasma epinephrine concentration during hypoglycemia than either of the two AT receptor antagonists given alone. We speculate that angiotensin II through binding to both receptor subtypes facilitates the sympathoadrenal reflex response by actions at several anatomical levels of the neural pathways involved in the sympathoadrenal reflex response elicited during insulin-induced hypoglycemia.

Objective: To study the comparative importance of environment and genes in the development of islet cell autoimmunity associated with insulin dependent diabetes mellitus., Design: Population based study of diabetic twins., Setting: Danish population., Subjects: 18 monozygotic and 36 dizygotic twin pairs with one or both partners having insulin dependent diabetes., Main Outcome Measures: Presence of islet cell antibodies, insulin autoantibodies, and autoantibodies to glutamic acid decarboxylase (GAD65) in serum samples from twin pairs 10 years (range 0-30 years) and 9.5 years (2-30 years) after onset of disease., Results: In those with diabetes the prevalence of islet cell antibodies, insulin autoantibodies, and autoantibodies to glutamic acid decarboxylase in the 26 monozygotic twins was 38%, 85%, and 92%, respectively, and in the dizygotic twins was 57%, 70%, and 57%, respectively. In those without diabetes the proportions were 20%, 50%, and 40% in the 10 monozygotic twins and 26%, 49%, and 40% in the 35 dizygotic twins., Conclusion: There is no difference between the prevalence of islet cell autoantibodies in dizygotic and monozygotic twins without diabetes, suggesting that islet cell autoimmunity is environmentally rather than genetically determined. Furthermore, the prevalence of islet cell antibodies was higher in the non-diabetic twins than in other first degree relatives of patients with insulin dependent diabetes. This implies that the prenatal or early postnatal period during which twins are exposed to the same environment, in contrast with that experienced by first degree relatives, is of aetiological importance.

We tested whether severe congestive heart failure (CHF), a condition associated with excess free-water retention, is accompanied by altered regulation of the vasopressin-regulated water channel, aquaporin-2 (AQP2), in the renal collecting duct. CHF was induced by left coronary artery ligation. Compared with sham-operated animals, rats with CHF had severe heart failure with elevated left ventricular end-diastolic pressures (LVEDP): 26.9 +/- 3.4 vs. 4.1 +/- 0.3 mmHg, and reduced plasma sodium concentrations (142.2 +/- 1. 6 vs. 149.1 +/- 1.1 mEq/liter). Quantitative immunoblotting of total kidney membrane fractions revealed a significant increase in AQP2 expression in animals with CHF (267 +/- 53%, n = 12) relative to sham-operated controls (100 +/- 13%, n = 14). In contrast, immunoblotting demonstrated a lack of an increase in expression of AQP1 and AQP3 water channel expression, indicating that the effect on AQP2 was selective. Furthermore, postinfarction animals without LVEDP elevation or plasma Na reduction showed no increase in AQP2 expression (121 +/- 28% of sham levels, n = 6). Immunocytochemistry and immunoelectron microscopy demonstrated very abundant labeling of the apical plasma membrane and relatively little labeling of intracellular vesicles in collecting duct cells from rats with severe CHF, consistent with enhanced trafficking of AQP2 to the apical plasma membrane. The selective increase in AQP2 expression and enhanced plasma membrane targeting provide an explanation for the development of water retention and hyponatremia in severe CHF.

Intravenous administration of the antihyperglycemic agent metformin decreases arterial pressure and sympathetic nerve activity (SNA). To test the hypothesis that metformin inhibits SNA by interrupting ganglionic neurotransmission, we compared the actions of intravenous administration of metformin and the ganglionic blocker trimethaphan on postganglionic renal and preganglionic adrenal sympathetic nerves in pentobarbital-anesthetized male Sprague-Dawley rats. Intravenous metformin elicited dose-dependent decreases in postganglionic renal SNA (1 mg/kg: 0 +/- 0%; 10 mg/kg: -20 +/- 4%; 100 mg/kg: -92 +/- 3%; n = 7). Conversely, only the maximal dose of metformin affected preganglionic adrenal SNA (100 mg/kg: delta adrenal SNA = -14 +/- 6%; n = 8). Ganglionic blockade with intravenous trimethaphan (5 mg/kg) produced a differential sympathoinhibitory response similar to the response observed after high-dose metformin (delta renal SNA = -100 +/- 3%; delta adrenal SNA = -17 +/- 7%; P < .001). Preganglionic renal neurons were electrically stimulated in the spinal cord, before and during the peak of the sympathoinhibitory response to intravenous metformin, and the magnitude of the stimulus-evoked increases in postganglionic renal SNA were compared. Metformin dose-dependently attenuated the magnitude of the increase in postganglionic renal SNA elicited by stimulation of the spinal cord (30 mg/kg: -23 +/- 8%; 90 mg/kg: -65 +/- 11%; 270 mg/kg: -91 +/- 8%; n = 6 per dose). We conclude that high-dose intravenous metformin interrupts ganglionic neurotransmission in renal nerves.

We have studied the presence of GAD65 autoantibodies in women with insulin-dependent diabetes mellitus (IDDM) (n = 28) or gestational diabetes (GDM) (n = 139) diagnosed during pregnancy and investigated the temporal relationship between these autoantibodies and the subsequent recurrence or development of IDDM. Among the GDM patients, 4.3% (6 of 139) developed true IDDM during a median follow-up period of 6.3 years (range 4.0-11.0). Of these, 2.2% (3 of 139) were positive for GAD65 autoantibodies at diagnosis of GDM compared to 0% (0 of 27) of healthy pregnant women. All 3 GAD65 autoantibody positive GDM patients subsequently developed IDDM after a median of 14 months (range 4-34). GAD65 autoantibodies were present in 50% (14 of 28) of sera from women with IDDM diagnosed during pregnancy. The non-insulin-requiring remission period was significantly shorter in GAD65 autoantibody positive patients (median 0.5 years [range 0-6.0 years]) than in GAD65 antibody negative patients (median 2.6 years; range 0-9.7 years; p < 0.05). The results suggest that screening for GAD65 autoantibodies in women with GDM or IDDM diagnosed during pregnancy may be useful for predicting the clinical course of the disease.

Chronic treatment with the antihyperglycemic agent metformin prevents hypertension in spontaneously hypertensive rats. This effect has been ascribed to normalization of plasma insulin levels. However, whether metformin affects arterial pressure via changes in sympathetic nerve activity is unknown. Therefore, the objective of this study was to examine whether acute administration of metformin produces changes in mean arterial pressure, heart rate, or efferent renal sympathetic nerve activity in spontaneously hypertensive rats. Rats were anesthetized with alphaxalone-alphadolone (Saffan), paralyzed with pancuronium, and artificially ventilated. Intravenous administration of metformin (0, 1, 10, 100 mg/kg) produced dose-dependent reversible decreases in mean arterial pressure, heart rate, and efferent renal sympathetic nerve activity that were not affected by arterial or cardiopulmonary baroreceptor denervation, nitric oxide synthase inhibition by N(omega)-nitro-L-arginine methyl ester, or cyclooxygenase inhibition by indomethacin. Metformin given into the lateral cerebral ventricle (250, 500, 1000 microg) produced dose-dependent decreases in mean arterial pressure, heart rate, and efferent renal sympathetic nerve activity in doses that caused no changes when given intravenously. The sympathoinhibitory response to intracerebroventricular administration of metformin was not affected by alpha2-adrenoceptor blockade by intracerebroventricular yohimbine. We conclude that metformin has acute sympathoinhibitory effects (decreased arterial pressure, heart rate, and efferent renal sympathetic nerve activity) that are produced by a direct central nervous system site of action.

The mouse homeodomain protein insulin promoter factor-1 (IPF-1) and the rat homologue somatostatin transactivating factor-1 (STF-1) are involved in early pancreatic development and have been implicated in the cell-specific regulation of insulin- and somatostatin-gene expression in mature islet beta- and delta-cells. The cell specificity of IPF-1/STF-1 expression in mature islets is, however, still unclear. Using antisera against recombinant IPF-1 and STF-1 in combination with antisera against islet hormones we find that all beta-cells in monolayers of newborn rat islet cells express STF-1, as do a fraction of the delta-cells. In adult rat and mouse pancreas we find a similar distribution. IPF-1/STF-1 expression was not detected in glucagon-producing alpha-cells. In islet cell tumour models we found that a glucagon/islet amyloid polypeptide (IAPP)-producing pluripotent rat islet cell line (NHI-6F-GLU) expresses STF-1 in all cells prior to insulin gene activation induced by in vivo culture. In contrast, a mouse alpha-cell line (alpha TC1) exclusively expressed IPF-1 in a small subset of insulin-producing cells while an insulin-negative subclone (alpha TC1.9) was negative for IPF-1. In transfection experiments using alpha TC1.9 cells STF-1 activated a rat insulin 1 reporter gene dependent not only on both STF-1-binding sites, but also on the E1-binding site for the helix-loop-helix factor IEF-1. However, the endogenous mouse insulin genes remained inactive in these cells. These results suggest that the insulin promoter acquires its very high, yet cell-specific, activity at least partly through the action of IPF-1/STF-1. This action is dependent on helix-loop-helix factors bound to the E1 element.

To examine whether furosemide elicits a cardiorenal reflex response via stimulation of cardiac afferents, furosemide was administered intrapericardially in sinoaortic denervated rats. The role of vagal afferents was evaluated by intrapericardial (IPC) administration of furosemide before and after bilateral vagotomy. The role of cardiac sympathetic afferents was examined by IPC administration of furosemide before and after IPC lidocaine blockade in rats with bilateral vagotomy. Low-dose furosemide (10 micrograms) elicited renal sympathoinhibition, whereas high-dose furosemide (1000 micrograms) produced a rapid and transient change in efferent renal sympathetic nerve activity of either inhibitory (19/49, or 39%) or excitatory (30/49, or 61%) nature. The responses were not affected by vagotomy but were abolished by IPC lidocaine blockade. In rats with a renal sympathoinhibitory response to IPC administration of 1000 micrograms furosemide, both the hypotensive and sympathoinhibitory responses were inhibited by indomethacin, whereas indomethacin did not affect reflex responses in rats showing a renal sympathoexcitatory response to IPC injection of 1000 micrograms furosemide. We conclude that furosemide elicits a nonuniform reflex response mediated via cardiac sympathetic afferents of which the renal sympathoinhibitory response is dependent on intact cyclooxygenase function.

Impaired heparan sulphate biosynthesis through diabetes-induced inhibition of glucosaminyl N-deacetylase may have a central role in the development of diabetic nephropathy, and genetic differences in the vulnerability of the N-deacetylase could influence the risk of developing nephropathy. We studied N-deacetylase activity in fibroblast cultures from Type 1 (insulin-dependent) diabetic patients with (n = 14) or without (n = 13) diabetic nephropathy, together with non-diabetic control subjects (n = 7). No difference in N-deacetylase activity was found (p = 0.13), and no inhibition of N-deacetylase was found in cultures grown at 25 mmol/l glucose. N-deacetylase activity was inversely correlated to growth rate (r = -0.59, p = 0.0008), and in patients with nephropathy a negative correlation between HbA1C and fibroblast N-deacetylase activity (r = -0.72, p = 0.012) was found. Cell-cycle analysis revealed an increased fraction of S-phase cells in patients with nephropathy (28%(21-52%)) compared to healthy control subjects (17% (9-24%)), p = 0.0008, but not between patients with and without nephropathy (latter group 26%(11-43%)), p = 0.43. Forskolin, an activator of protein kinase A, specifically decreased N-deacetylase activity, whereas activation of protein kinase C produced a combined reduction in N-deacetylase activity and total protein synthesis. In conclusion, no constitutive defects in N-deacetylase activity were found in fibroblasts from these patients. Further studies should consider possible associations between fibroblast characteristics and pre-biopsy environmental parameters related to cellular memory phenomena. Finally, activation of protein kinase A provides a potential general pathway for regulating N-deacetylase activity.

The vagal-mediated reflex responses elicited by the selective serotonin type 3 receptor agonist 2-methyl-serotonin were examined by administration (6.25, 12.5, 25, and 50 micrograms/kg i.v.) of 2-methylserotonin to sinoaortic-denervated rats with either intact or sectioned vagi. To study the influence of dietary sodium intake on 2-methylserotonin-induced vagal reflex responses, we performed experiments in rats fed either a high or low sodium diet. Left ventricular end-diastolic pressure was significantly higher in animals on high than low salt diet. However, mean arterial pressure and heart rate were similar in high and low salt groups. In rats with intact vagi, 2-methylserotonin produced a dose-dependent increase in afferent vagal nerve activity and a dose-dependent decrease in efferent renal sympathetic nerve activity, mean arterial pressure, and heart rate. The sympathoinhibitory responses of decreased efferent renal sympathetic nerve activity, mean arterial pressure, and heart rate were abolished by vagotomy and were not affected by changes in dietary sodium intake. We conclude that the sympathoinhibitory effect of 2-methylserotonin is due to stimulation of vagal afferents with inhibitory action on peripheral sympathetic nerve activity and that the sympathoinhibitory responses are unaffected by changes in dietary sodium intake.

Pancreatic beta-cell destruction and development of Type 1 (insulin-dependent) diabetes mellitus are associated with circulating islet cell antibodies. Mice with severe combined immunodeficiency (SCID mice) were reconstituted with peripheral blood mononuclear cells from Type 1 diabetic patients, one who was antibody positive and one antibody negative, and from healthy individuals. Reconstituted mice were subsequently immunized with rat islets in incomplete Freunds adjuvant or adjuvant alone. Seventeen mice received peripheral blood mononuclear cells obtained at three different time points from the islet cell antibody positive patient. Before immunization with rat islets two mice developed antibodies to glutamic acid decarboxylase, a major target for antibodies in Type 1 diabetes, whereas none were positive for cytoplasmic islet cell antibodies. Following immunization with rat islets, glutamic acid decarboxylase antibodies were detected by immunoprecipitation in three additional mice, two of which also became positive for cytoplasmic islet cell antibodies. Of 22 mice which received peripheral blood mononuclear cells from either the islet cell antibody negative patient (n = 5) or from two healthy individuals (n = 17), none were positive for islet cell autoantibodies before or after immunization. None of the islet cell antibody positive mice became hyperglycaemic, showed impaired glucose tolerance or islet cell damage when studied 40 days after immunization (i.e. 100 days after reconstitution). In conclusion these results show that human B lymphocytes producing diabetes-associated autoantibodies can be transferred to SCID mice and remain antigen sensitive, but also that autoantibodies alone are not sufficient to induce beta-cell destruction.

At and before onset, most insulin-dependent diabetics (IDDM) have islet GAD65 autoantibodies (GAD65Ab). Since IDDM also occurs in older patients where non-insulin-dependent diabetes is common, we studied GAD65Ab at onset to classify diabetes type. Our quantitative immunoprecipitation assay uses recombinant human islet GAD65 stably expressed in hamster fibroblasts. Electrophoretic mobility was identical to native islet GAD65. Like native antigen, recombinant GAD65 migrated as two bands during electrophoresis, but converted to one under stronger reduction. Immunoprecipitation was linear with respect to antibody or antigen concentration. In 120 population-based diabetic patients of all ages grouped by treatment at onset and after 18 mo, GAD65Ab were present in 70% on insulin (n = 37), 10% on oral agent (n = 62, P < 0.0001), 69% changing from oral agent to insulin (n = 16, P < 0.001), and 1 of 33 controls. 65% with GAD65Ab, versus 8% without, changed from oral agent to insulin (P < 0.01). The GAD65Ab quantitative index was remarkably stable, and only 2 of 32 patients changed antibody status during follow-up. Concordance between GAD65Ab and islet cell antibodies was 93%. Quantitative correlation was approximate but significant. This highly sensitive, quantitative, high capacity assay for GAD65Ab reveals treatment requirements better than clinical criteria, perhaps guiding immunomodulatory therapy.