A 1-year pilot study of intralymphatic injections of GAD-alum in individuals with latent autoimmune diabetes in adults (LADA) with signs of high immunity: No safety concerns and resemblance to juvenile type 1 diabetes.

AIMS: To test, for the first time in latent autoimmune diabetes in adults (LADA), the effects of autoantigen-specific immunotherapy by intralymphatic administration of aluminium-formulated recombinant human glutamic acid decarboxylase 65 (GAD-alum); specifically, to test if this treatment is safe, to test whether it induces a strong immunological response akin to a similar protocol in type 1 diabetes and to look for associations with preserved beta-cell function. MATERIALS AND METHODS: Three GAD-alum injections, 4 µg each, were administered 1 month apart into an inguinal lymph node in 14 people with newly diagnosed LADA (age 30-62 years) presenting with high levels of antibodies against glutamic acid decarboxylase (GADA). Adverse effects, immunological variables and beta-cell function were monitored, with detailed measurements at 5 and 12 months from baseline. RESULTS: Clinical adverse effects were minor and transient and measured laboratory variables were unaffected. All participants completed the study. Treatment raised levels of GADA, elicited strong effects on reactivity of peripheral blood mononuclear cells to GAD and raised cytokine/chemokine levels. Beta-cell function appeared stable preferentially in the seven participants carrying human leukocyte antigen (HLA) haplotypes DR3DQ2, as assessed by C-peptide glucagon tests (P < 0.05 vs. seven non-carriers). CONCLUSION: Intralymphatic treatment with GAD-alum in LADA is without clinical or other safety concerns over a 12-month period. As in a similar protocol used in type 1 diabetes, treatment exerts a strong immunological impact and is compatible with protection of beta-cell function preferentially in HLA-DR3DQ2 LADA patients. These findings pave the way for a randomized controlled trial in this important subgroup of LADA patients.

Latent Autoimmune Diabetes in Adults: Background, Safety and Feasibility of an Ongoing Pilot Study With Intra-Lymphatic Injections of GAD-Alum and Oral Vitamin D.

Background: Latent Autoimmune Diabetes in Adults (LADA) constitutes around 10% of all diabetes. Many LADA patients gradually lose their insulin secretion and progress to insulin dependency. In a recent trial BALAD (Behandling Av LADa) early insulin treatment compared with sitagliptin failed to preserve insulin secretion, which deteriorated in individuals displaying high levels of antibodies to GAD (GADA). These findings prompted us to evaluate a treatment that directly affects autoimmunity. Intra-lymphatic GAD-alum treatment has shown encouraging results in Type 1 diabetes patients. We therefore tested the feasibility of such therapy in LADA-patients (the GADinLADA pilot study). Material and Methods: Fourteen GADA-positive (>190 RU/ml), insulin-independent patients 30-70 years old, with LADA diagnosed within < 36 months were included in an open-label feasibility trial. They received an intra-nodal injection of 4 µg GAD-alum at Day 1, 30 and 60 plus oral Vitamin D 2000 U/d from screening 30 days before (Day -30) for 4 months if the vitamin D serum levels were below 100 nmol/L (40 ng/ml). Primary objective is to evaluate safety and feasibility. Mixed Meal Tolerance Test and i.v. Glucagon Stimulation Test at baseline and after 5 and 12 months are used for estimation of beta cell function. Results will be compared with those of the recent BALAD study with comparable patient population. Immunological response is followed. Results: Preliminary results show feasibility and safety, with almost stable beta cell function and metabolic control during follow-up so far (5 months). Conclusions: Intra-lymphatic GAD-alum treatment is an option to preserve beta cell function in LADA-patients. An ongoing trial in 14 LADA-patients show feasibility and safety. Clinical and immunological responses will determine how to proceed with future trials.

Time-dependent effects on circulating cytokines in patients with LADA: A decrease in IL1-ra and IL-1 beta is associated with progressive disease.

BACKGROUND: Cytokines and chemokines participate in autoimmune processes at cellular targets which include insulin-producing beta cells. To which extent such participation is reflected in the circulation has not been conclusively resolved. AIM: We compared the time course of cytokines/chemokines in Latent Autoimmune Diabetes in Adults (LADA) patients heterogeneous for high or low autoimmune activity as determined by levels of antibodies against glutamic acid decarboxylase (GADA). METHODS: Serum samples to be measured were from a two-armed randomized controlled trial (RCT) in 68 LADA patients. The study encompassed 21 months with C-peptide as primary endpoint. We measured 27 immune mediators at baseline, at 9 and at 21 months (end of study). Results of measurements were analyzed by multiple linear regression. RESULTS: At baseline, a high body mass index (BMI) (>26 kg/m2) was associated with elevated levels of the interleukins (IL) IL-1 beta, IL-1ra, IL-2, IL-5, IL-6 and IL-13. Treatment during RCT (sitagliptin vs. insulin) did not affect the time course (21 months) of levels of cytokines/chemokines (by univariate analyses). However, levels of the cytokines IL-1ra and IL-1 beta decreased significantly (p < 0.04 or less) in patients with high vs. low GADA when adjusted for BMI, age, gender (male/female), treatment (insulin/sitagliptin) and study site (Norwegian/Swedish). CONCLUSIONS: In LADA, high levels of GADA, a proxy for high autoimmune activity and linked to a decline in C-peptide, was associated with a decrease of selected cytokines over time. This implies that the decline of IL-1ra and IL-1 beta in the circulation reflects autoimmune activity and beta cell demise in LADA.

Investigating optimal ß-cell-preserving treatment in latent autoimmune diabetes in adults: Results from a 21-month randomized trial.

AIMS: To compare outcomes of glucagon-stimulated C-peptide tests (GSCTs) in people with latent autoimmune diabetes in adults (LADA) after a 21-month intervention with either insulin or the dipeptidyl peptidase-4 inhibitor sitagliptin. RESEARCH DESIGN AND METHODS: We included 64 glutamic acid decarboxylase (GAD) antibody-positive individuals, who were diagnosed with diabetes <3 years before the study, aged 30 to 70 years, and without clinical need for insulin treatment. We stratified participants by age and body mass index (BMI) and evaluated ß-cell function by GSCT after a 48-hour temporary withdrawal of study medication. RESULTS: Age at randomization (mean 53 years), BMI (mean 27 kg/m2 ) and metabolic markers were similar between treatment arms. Glycated haemoglobin concentrations during intervention did not differ between arms. Fasting C-peptide concentrations after the intervention were similar, as were stimulated C-peptide levels (0.82 ± 0.63 nmol/L after insulin, 0.82 ± 0.46 nmol/L after sitagliptin; nonsignificant). Autoimmunity in the study population (estimated from GAD antibody titres and positivity/no positivity for zinc transporter 8 and islet antigen 2 antibodies) affected the evolution of the GSCT results significantly, which deteriorated in participants with high but not in those with low autoimmunity. Adjustment using analysis of covariance for the degree of autoimmunity did not alter the findings of no difference between treatment arms. CONCLUSIONS: ß-cell function after intervention was similar in patients with insulin- and sitagliptin-treated LADA, regardless of the strength of autoimmunity. Further, participants with low levels of GAD antibodies did not experience progressive deterioration of ß-cell function over a 21-month period. Taken together, these findings could be useful for clinicians' choices of treatment in people with LADA.

Treatment of Latent Autoimmune Diabetes in Adults: What is Best?

Latent Autoimmune Diabetes in Adults (LADA), although formally classified as Type 1 Diabetes (T1D), very often (at least in Western countries) appear clinically with Type 2 Diabetes (T2D)-like features as overweight and insulin resistance. LADA patients do not need exogenous insulin at the time they are diagnosed with diabetes, but a large percentage will within a few years develop need for such treatment. The decline in beta cell function progresses much faster in LADA than in T2D, presumably because of the ongoing autoimmune assault in LADA, and therefore necessitates insulin therapy much earlier in LADA than in T2D. Despite high prevalence of LADA (about 10% of the total diabetic population in many countries), the treatment of LADA patients is far less elucidated than is the case for T1D and T2D. Finding a treatment strategy for LADA from the time of diagnosis, that can reduce the decline of beta cell function, ensure adequate metabolic control and thereby reduce the risk of diabetic complications is thus an important clinical challenge. Conclusions from the randomized treatment studies so far do not indicate an optimal treatment strategy in LADA. This review aims to give an overview of current practices for the medical treatment of LADA as well as an update on results from recent studies on the treatment of the disease.

Metabolic Outcomes in Adults Born Preterm With Very Low Birthweight or Small for Gestational Age at Term: A Cohort Study.

Context and Objectives: Low birthweight (LBW) has emerged as a risk factor of metabolic syndrome (MetS). Whether adults with very low birthweight (VLBW) born preterm are at higher risk than individuals who were term-born small for gestational age (tb-SGA) is not established. We assessed metabolic outcomes, including relation with skeletal parameters, in these two LBW categories. Design, Participants, and Outcomes: This follow-up cohort study included 189 individuals (females 51%), aged 25 to 28 years; 55 were preterm VLBW (≤1500 g), 59 were tb-SGA (<10th percentile), and 75 were controls (≥10th percentile). Outcomes were indices of MetS: blood pressure (BP), waist circumference, fasting glucose, lipid profile, and association between calculated MetS score and bone mineral density (BMD) and trabecular bone score (TBS), a measure of bone quality. Results: Compared with controls, individuals with VLBW displayed higher systolic [mean (SD), 126 (13.3) vs 119 (12.3) mm Hg; 95% CI, 1.27 to 11.48 mm Hg] and diastolic [71.9 (7.6) vs 68.6 (7.1) mm Hg; 95% CI, 0.3 to 6.2 mm Hg] BP, higher glycated hemoglobin, higher C-peptide, increased insulin resistance (Homeostatic Model Assessment 2), and lower high-density lipoprotein cholesterol [1.34 (0.3) vs 1.50 (0.4); 95% CI, 0.32 to 0.01]. Substantial differences were mainly seen between control females and females with VLBW. The adults who were tb-SGA had higher waist circumference and higher total and low-density lipoprotein cholesterol compared with controls. In males, MetS score correlated positively with BMD and inversely with TBS. Conclusions: The LBW groups and preferentially females in the VLBW group displayed a less favorable metabolic profile than did controls. The inverse association between MetS score and bone quality suggests enhanced future fracture risk.

Culture at low glucose up-regulates mitochondrial function in pancreatic ß cells with accompanying effects on viability.

We tested whether exposure of ß cells at reduced glucose leads to mitochondrial adaptions and whether such adaptions modulate effects of hypoxia. Rat islets, human islets and INS-1 832/13 cells were pre-cultured short term at half standard glucose concentrations (5.5 mM for rat islets and cells, 2.75 mM for human islets) without overtly negative effects on subsequently measured function (insulin secretion and cellular insulin contents) or on viability. Culture at half standard glucose upregulated complex I and tended to upregulate complex II in islets and INS-1 cells alike. An increased release of lactate dehydrogenase that followed exposure to hypoxia was attenuated in rat islets which had been pre-cultured at half standard glucose. In INS-1 cells exposure to half standard glucose attenuated hypoxia-induced effects on several viability parameters (MTT, cell number and incremental apoptotic DNA). Thus culture at reduced glucose of pancreatic islets and clonal ß cells leads to mitochondrial adaptions which possibly lessen the negative impact of hypoxia on ß cell viability. These findings appear relevant in the search for optimization of pre-transplant conditions in a clinical setting.

Mitochondrial Respiration in Insulin-Producing ß-Cells: General Characteristics and Adaptive Effects of Hypoxia.

OBJECTIVE: To provide novel insights on mitochondrial respiration in ß-cells and the adaptive effects of hypoxia. METHODS AND DESIGN: Insulin-producing INS-1 832/13 cells were exposed to 18 hours of hypoxia followed by 20-22 hours re-oxygenation. Mitochondrial respiration was measured by high-resolution respirometry in both intact and permeabilized cells, in the latter after establishing three functional substrate-uncoupler-inhibitor titration (SUIT) protocols. Concomitant measurements included proteins of mitochondrial complexes (Western blotting), ATP and insulin secretion. RESULTS: Intact cells exhibited a high degree of intrinsic uncoupling, comprising about 50% of oxygen consumption in the basal respiratory state. Hypoxia followed by re-oxygenation increased maximal overall respiration. Exploratory experiments in peremabilized cells could not show induction of respiration by malate or pyruvate as reducing substrates, thus glutamate and succinate were used as mitochondrial substrates in SUIT protocols. Permeabilized cells displayed a high capacity for oxidative phosphorylation for both complex I- and II-linked substrates in relation to maximum capacity of electron transfer. Previous hypoxia decreased phosphorylation control of complex I-linked respiration, but not in complex II-linked respiration. Coupling control ratios showed increased coupling efficiency for both complex I- and II-linked substrates in hypoxia-exposed cells. Respiratory rates overall were increased. Also previous hypoxia increased proteins of mitochondrial complexes I and II (Western blotting) in INS-1 cells as well as in rat and human islets. Mitochondrial effects were accompanied by unchanged levels of ATP, increased basal and preserved glucose-induced insulin secretion. CONCLUSIONS: Exposure of INS-1 832/13 cells to hypoxia, followed by a re-oxygenation period increases substrate-stimulated respiratory capacity and coupling efficiency. Such effects are accompanied by up-regulation of mitochondrial complexes also in pancreatic islets, highlighting adaptive capacities of possible importance in an islet transplantation setting. Results also indicate idiosyncrasies of ß-cells that do not respire in response to a standard inclusion of malate in SUIT protocols.

Marked over expression of uncoupling protein-2 in beta cells exerts minor effects on mitochondrial metabolism.

Evidence is conflicting as to the impact of elevated levels of uncoupling protein-2 (UCP-2) on insulin-producing beta cells. Here we investigated effects of a fourfold induction of UCP-2 protein primarily on mitochondrial parameters and tested for replication of positive findings at a lower level of induction. We transfected INS-1 cells to obtain a tet-on inducible cell line. A 48 h exposure to 1 µg/ml of doxycycline (dox) induced UCP-2 fourfold (424 ± 113%, mean±SEM) and 0.1 µg/ml twofold (178 ± 29%, n=3). Fourfold induced cells displayed normal viability (MTT, apoptosis), normal cellular insulin contents and, glucose-induced insulin secretion (+27 ± 11%) as well as D-[U-(14)C]-glucose oxidation (+5 ± 9% at 11 mM glucose). Oxidation of [1-(14)C]-oleate was increased from 4088 to 5797 fmol/µg prot/2h at 3.3mM glucose, p<0.03. Oxidation of L-[(14)C(U)]-glutamine was unaffected. Induction of UCP-2 did not significantly affect measures of mitochondrial membrane potential (Rhodamine 123) or mitochondrial mass (Mitotracker Green) and did not affect ATP levels. Oligomycin-inhibited oxygen consumption (a measure of mitochondrial uncoupling) was marginally increased, the effect being significant in comparison with dox-only treated cells, p<0.05. Oxygen radicals, assessed by dichlorofluorescin diacetate, were decreased by 30%, p<0.025. Testing for the lower level of UCP-2 induction did not reproduce any of the positive findings. A fourfold induction of UCP-2 was required to exert minor metabolic effects. These findings question an impact of moderately elevated UCP-2 levels in beta cells as seen in diabetes.