Towards enhanced understanding of idiopathic ketotic hypoglycemia: a literature review and introduction of the patient organization, Ketotic Hypoglycemia International.

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.

Systems Signatures Reveal Unique Remission-path of Type 2 Diabetes Following Roux-en-Y Gastric Bypass Surgery.

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.

Partial remission definition: validation based on the insulin dose-adjusted HbA1c (IDAA1C) in 129 Danish children with new-onset type 1 diabetes.

OBJECTIVE: To validate the partial remission (PR) definition based on insulin dose-adjusted HbA1c (IDAA1c). SUBJECTS AND METHODS: The IDAA1c was developed using data in 251 children from the European Hvidoere cohort. For validation, 129 children from a Danish cohort were followed from the onset of type 1 diabetes (T1D). Receiver operating characteristic curve (ROC) analysis was used to evaluate the predictive value of IDAA1c and age on partial C-peptide remission (stimulated C-peptide, SCP > 300 pmol/L). RESULTS: PR (IDAA1c ≤ 9) in the Danish and Hvidoere cohorts occurred in 62 vs. 61% (3 months, p = 0.80), 47 vs. 44% (6 months, p = 0.57), 26 vs. 32% (9 months, p = 0.32) and 19 vs. 18% (12 months, p = 0.69). The effect of age on SCP was significantly higher in the Danish cohort compared with the Hvidoere cohort (p < 0.0001), likely due to higher attained Boost SCP, so the sensitivity and specificity of those in PR by IDAA1c ≤ 9, SCP > 300 pmol/L was 0.85 and 0.62 at 6 months and 0.62 vs. 0.38 at 12 months, respectively. IDAA1c with age significantly improved the ROC analyses and the AUC reached 0.89 ± 0.04 (age) vs. 0.94 ± 0.02 (age + IDAA1c) at 6 months (p < 0.0004) and 0.76 ± 0.04 (age) vs. 0.90 ± 0.03 (age + IDAA1c) at 12 months (p < 0.0001). CONCLUSIONS: The diagnostic and prognostic power of the IDAA1c measure is kept but due to the higher Boost stimulation in the Danish cohort, the specificity of the formula is lower with the chosen limits for SCP (300 pmol/L) and IDAA1c ≤9, respectively.

Disease progression among 446 children with newly diagnosed type 1 diabetes located in Scandinavia, Europe, and North America during the last 27 yr.

OBJECTIVE: To clarify whether the rate of decline in stimulated C-peptide (SCP) from 2 to 15 months after diagnosis has changed over an interval of 27 yr. RESEARCH DESIGN AND METHODS: The rate of decline in SCP levels at 1, 2, 3, 6, 9, 12, and 15 months after diagnosis was compared in four paediatric cohorts from Scandinavian and European countries including 446 children with new onset type 1 diabetes (T1D, 1982-2004). Findings were evaluated against 78 children (2004-2009) from the TrialNet studies. RESULTS: The mean rate of decline [%/month (±SEM)] in SCP for a 10-yr-old child was 7.7%/month (±1.5) in the 1982-1985 Cohort, 6.3%/month (±1.7) in the 1995-1998 Cohort, 7.8%/month (±0.7) in the 1999-2000 Cohort, and 10.7%/month (±0.9) in the latest 2004-2005 Cohort (p = 0.05). Including the TrialNet Cohort with a rate of decline in SCP of 10.0%/month (±0.9) the differences between the cohorts are still significant (p = 0.039). The rate of decline in SCP was negatively associated with age (p < 0.0001), insulin antibodies (IA) (p = 0.003), and glutamic acid decarboxylase-65 (GAD65A) (p = 0.03) initially with no statistically significant effect of body mass index (BMI) Z-score at 3 months. Also, at 3 months the time around partial remission, the effect of age on SCP was significantly greater in children ≤5 yr compared with older children (p ≤ 0.0001). CONCLUSIONS: During the past 27 yr, initial C-peptide as well as the rate of C-peptide decline seem to have increased. The rate of decline was affected significantly by age, GAD65A, and IA, but not BMI Z-score or initial C-peptide.

Treatment with a proton pump inhibitor improves glycaemic control in type 2 diabetic patients - a retrospective analysis.

We retrospectively studied whether treatment with esomeprazole improved HbA1(c) levels in type 2 diabetic patients. We selected 21 patients who had been treated with esomeprazole for 11 ± 3 months and 21 controls. HbA1(c) levels decreased in the esomeprazole-treated group. Our data indicate that proton pump inhibitors may improve glycaemic control in type 2 diabetic patients.

Small-molecule agonists for the glucagon-like peptide 1 receptor.

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.

Stimulation of pancreatic beta-cell replication by incretins involves transcriptional induction of cyclin D1 via multiple signalling pathways.

The incretin hormones, glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP), have been suggested to act as beta-cell growth factors and may therefore be of critical importance for the maintenance of a proper beta-cell mass. We have investigated the molecular mechanism of incretin-induced beta-cell replication in primary monolayer cultures of newborn rat islet cells. GLP-1, GIP and the long-acting GLP-1 derivative, liraglutide, increased beta-cell replication 50-80% at 10-100 nM upon a 24 h stimulus, whereas glucagon at a similar concentration had no significant effect. The stimulatory effect of GLP-1 and GIP was efficiently mimicked by the adenylate cyclase activator, forskolin, at 10 nM (approximately 90% increase) and was additive (approximately 170-250% increase) with the growth response to human growth hormone (hGH), indicating the use of distinct intracellular signalling pathways leading to mitosis by incretins and cytokines, respectively. The response to both GLP-1 and GIP was completely blocked by the protein kinase A (PKA) inhibitor, H89. In addition, the phosphoinositol 3-kinase (PI3K) inhibitor wortmannin and the mitogen-activated protein kinase kinase (MEK) inhibitor PD98059, both inhibited GLP-1- and GIP-stimulated proliferation. The p38 mitogen-activated protein kinase (MAPK) inhibitor, SB203580, had no inhibitory effect on either GLP-1 or GIP stimulated proliferation. Cyclin Ds act as molecular switches for the G0/G1-S phase transition in many cell types and we have previously demonstrated hGH-induced cyclin D2 expression in the insulinoma cell line, INS-1. GLP-1 time-dependently induced the cyclin D1 mRNA and protein levels in INS-1E, whereas the cyclin D2 levels were unaffected. However, minor effect of GLP-1 stimulation was observed on the cyclin D3 mRNA levels. Transient transfection of a cyclin D1 promoter-luciferase reporter construct into islet monolayer cells or INS-1 cells revealed approximately a 2-3 fold increase of transcriptional activity in response to GLP-1 and GIP, and a 4-7 fold increase in response to forskolin. However, treatment of either cell type with hGH had no effect on cyclin D1 promoter activity. The stimulation of the cyclin D1 promoter by GLP-1 was inhibited by H89, wortmannin, and PD98059. We conclude that incretin-induced beta-cell replication is dependent on cAMP/PKA, p42 MAPK and PI3K activities, which may involve transcriptional induction of cyclin D1. GLP-1, GIP and liraglutide may have the potential to increase beta-cell replication in humans which would have significant impact on long-term diabetes treatment.

The long-acting glucagon-like peptide-1 analogue, liraglutide, inhibits beta-cell apoptosis in vitro.

We here show that GLP-1 and the long-acting GLP-1 analogue, liraglutide, interfere with diabetes-associated apoptotic processes in the beta-cell. Studies using primary neonatal rat islets showed that native GLP-1 and liraglutide inhibited both cytokine- and free fatty acid-induced apoptosis in a dose-dependent manner. The anti-apoptotic effect of liraglutide was mediated by the GLP-1 receptor as the specific GLP-1 receptor antagonist, exendin(9-39), blocked the effects. The adenylate cyclase activator, forskolin, had an anti-apoptotic effect similar to those of GLP-1 and liraglutide indicating that the effect was cAMP-mediated. Blocking the PI3 kinase pathway using wortmannin but not the MAP kinase pathways by PD98059 inhibited the effects of liraglutide. In conclusion, GLP-1 receptor activation has anti-apoptotic effect on both cytokine, and free fatty acid-induced apoptosis in primary islet-cells, thus suggesting that the long-acting GLP-1 analogue, liraglutide, may be useful for retaining beta-cell mass in both type 1 and type 2 diabetic patients.

The imidazoline NNC77-0020 affects glucose-dependent insulin, glucagon and somatostatin secretion in mouse pancreatic islets.

The effect of the novel imidazoline compound 2-[2-(4,5-dihydro-1H-imidazol-2-yl)-1-(5-methyl-2,3-dihydrobenzofuran-7-yl)-ethyl]-pyridine (NNC77-0020) on stimulus-secretion coupling and hormone secretion was investigated in mouse pancreatic islets and isolated alpha- and beta-cells. In the presence of elevated glucose concentrations NNC77-0020 stimulated insulin secretion concentration dependently (EC(50) 64 nM) by 200% without affecting the whole-cell K(+) current or cytoplasmic Ca(2+) levels. Capacitance measurements in single mouse beta-cells showed that intracellular application of NNC77-0020 via the recording pipette enhanced Ca(2+)-dependent exocytosis. This action was dependent on protein kinase C (PKC) and cytoplasmic phospholipase A(2) (cPLA(2)) activity and required functional granular ClC-3 Cl(-) channels. In intact islets NNC77-0020 stimulated glucose-dependent somatostatin secretion, an effect that was also dependent on PKC and cPLA(2) activity. NNC77-0020 also inhibited glucagon secretion. In single mouse alpha-cells this action was not associated with a change in spontaneous electrical activity and resulted from a reduction in the rate of Ca(2+)-dependent exocytosis. Inhibition of exocytosis by NNC77-0020 was pertussis toxin sensitive and mediated by activation of the protein phosphatase calcineurin. In conclusion, our data suggest that the imidazoline compound NNC77-0020 modulates pancreatic hormone secretion in a complex fashion, comprising glucose-dependent stimulation of insulin and somatostatin secretion and inhibition of glucagon release. These mechanisms of action constitute an ideal basis for the development of novel imidazoline-containing anti-diabetic compounds.

Imidazoline NNC77-0074 stimulates Ca2+-evoked exocytosis in INS-1E cells by a phospholipase A2-dependent mechanism.

We have previously demonstrated that the novel imidazoline compound (+)-2-(2-(4,5-dihydro-1H-imidazol-2-yl)-thiopene-2-yl-ethyl)-pyridine (NNC77-0074) increases insulin secretion from pancreatic beta-cells by stimulation of Ca(2+)-dependent exocytosis. Using capacitance measurements, we now show that NNC77-0074 stimulates exocytosis in clonal INS-1E cells. NNC77-0074-stimulated exocytosis was antagonised by the cytoplasmic phospholipase A(2) (cPLA(2)) inhibitors ACA and AACOCF(3) and in cells treated with antisense oligonucleotide against cPLA(2)alpha. NNC77-0074-evoked insulin secretion was likewise inhibited by ACA, AACOCF(3), and cPLA(2)alpha antisense oligonucleotide treatment. In pancreatic islets NNC77-0074 stimulated PLA(2) activity. We propose that cPLA(2)alpha plays an important role in the regulation of NNC77-0074-evoked exocytosis in insulin secreting beta-cells.

Imidazoline NNC77-0074 stimulates insulin secretion and inhibits glucagon release by control of Ca(2+)-dependent exocytosis in pancreatic alpha- and beta-cells.

We have investigated the effects of the novel imidazoline compound (+)-2-(2-(4,5-dihydro-1H-imidazol-2-yl)-thiopene-2-yl-ethyl)-pyridine (NNC77-0074) on stimulus-secretion coupling in isolated pancreatic alpha- and beta-cells. NNC77-0074 stimulated glucose-dependent insulin secretion in intact mouse pancreatic islets. No effect was observed at

Genetic fusion of human insulin B-chain to the B-subunit of cholera toxin enhances in vitro antigen presentation and induction of bystander suppression 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.

A new model for analyzing immunomodulation of T cell responses induced by oral administration of islet cell antigens.

Based on the coimmunization of BALB/c mice with insulin and ovalbumin, we here describe a feasible efficacy model for analyzing the tolerogenic effect of the oral administration of sequestered antigens-for example, islet-specific proteins such as insulin and GAD65.