Henrik U. Andersen

RNA modifications by oxidation: a novel disease mechanism?

The past decade has provided exciting insights into a novel class of central (small) RNA molecules intimately involved in gene regulation. Only a small percentage of our DNA is translated into proteins by mRNA, yet 80% or more of the DNA is transcribed into RNA, and this RNA has been found to encompass various classes of novel regulatory RNAs, including, e.g., microRNAs. It is well known that DNA is constantly oxidized and repaired by complex genome maintenance mechanisms. Analogously, RNA also undergoes significant oxidation, and there are now convincing data suggesting that oxidation, and the consequent loss of integrity of RNA, is a mechanism for disease development. Oxidized RNA is found in a large variety of diseases, and interest has been especially devoted to degenerative brain diseases such as Alzheimer disease, in which up to 50-70% of specific mRNA molecules are reported oxidized, whereas other RNA molecules show virtually no oxidation. The iron-storage disease hemochromatosis exhibits the most prominent general increase in RNA oxidation ever observed. Oxidation of RNA primarily leads to strand breaks and to oxidative base modifications. Oxidized mRNA is recognized by the ribosomes, but the oxidation results in ribosomal stalling and dysfunction, followed by decreased levels of functional protein as well as the production of truncated proteins that do not undergo proper folding and may result in protein aggregation within the cell. Ribosomal dysfunction may also signal apoptosis by p53-independent pathways. There are very few reports on interventions that reduce RNA oxidation, one interesting observation being a reduction in RNA oxidation by ingestion of raw olive oil. High urinary excretion of 8-oxo-guanosine, a biomarker for RNA oxidation, is highly predictive of death in newly diagnosed type 2 diabetics; this demonstrates the clinical relevance of RNA oxidation. Taken collectively the available data suggest that RNA oxidation is a contributing factor in several diseases such as diabetes, hemochromatosis, heart failure, and β-cell destruction. The mechanism involves free iron and hydrogen peroxide from mitochondrial dysfunction that together lead to RNA oxidation that in turn gives rise to truncated proteins that may cause aggregation. Thus RNA oxidation may well be an important novel contributing mechanism for several diseases.

The Effect of Real-Time Continuous Glucose Monitoring in Pregnant Women With Diabetes: A randomized controlled trial

OBJECTIVE To assess whether intermittent real-time continuous glucose monitoring (CGM) improves glycemic control and pregnancy outcome in unselected women with pregestational diabetes. RESEARCH DESIGN AND METHODS A total of 123 women with type 1 diabetes and 31 women with type 2 diabetes were randomized to use real-time CGM for 6 days at 8, 12, 21, 27, and 33 weeks in addition to routine care, including self-monitored plasma glucose seven times daily, or routine care only. To optimize glycemic control, real-time CGM readings were evaluated by a diabetes caregiver. HbA1c, self-monitored plasma glucose, severe hypoglycemia, and pregnancy outcomes were recorded, with large-for-gestational-age infants as the primary outcome. RESULTS Women assigned to real-time CGM (n = 79) had baseline HbA1c similar to that of women in the control arm (n = 75) (median 6.6 [range 5.3–10.0] vs. 6.8% [5.3–10.7]; P = 0.67) (49 [34–86] vs. 51 mmol/mol [34–93]). Forty-nine (64%) women used real-time CGM per protocol. At 33 weeks, HbA1c (6.1 [5.1–7.8] vs. 6.1% [4.8–8.2]; P = 0.39) (43 [32–62] vs. 43 mmol/mol [29–66]) and self-monitored plasma glucose (6.2 [4.7–7.9] vs. 6.2 mmol/L [4.9–7.9]; P = 0.64) were comparable regardless of real-time CGM use, and a similar fraction of women had experienced severe hypoglycemia (16 vs. 16%; P = 0.91). The prevalence of large-for-gestational-age infants (45 vs. 34%; P = 0.19) and other perinatal outcomes were comparable between the arms. CONCLUSIONS In this randomized trial, intermittent use of real-time CGM in pregnancy, in addition to self-monitored plasma glucose seven times daily, did not improve glycemic control or pregnancy outcome in women with pregestational diabetes.

Nicotinamide Prevents Interleukin-1 Effects on Accumulated Insulin Release and Nitric Oxide Production in Rat Islets of Langerhans

Nicotinamide (NA) prevents macrophage- and interleukin-1 (IL-1)-mediated β-cell damage in vitro as well as diabetes development in animal models of insulin-dependent diabetes mellitus (IDDM). IL-1 β-mediated inhibition of insulin release and damage to β-cells are associated with intracellular production of nitric oxide (NO) radicals. Therefore, we studied whether NA prevented IL-1 β-induced islet NO production, measured as nitrite release from isolated rat islets, and, if so, whether this action was associated with prevention of IL-1 β-mediated inhibition of insulin release. NA dose- and time-dependently inhibited and delayed IL-1 β-induced islet NO production. Light microscopy detected that 25 mM of NA protected against IL-1 β-induced islet damage. Five to 50 mM of NA dose-dependently reduced inhibition of accumulated islet insulin release induced by 150 pg/ml of IL-1 β. NA was not able to reverse the reduced ability of IL-1 β-treated islets to respond to an acute glucose challenge. NO or nitrite did not interact directly with NA, because NA did not reduce sodium nitroprusside-generated nitrite. No-synthase inhibition with L-arginine depletion abolished NO production but only partially reduced IL-1 β-induced inhibition of accumulated insulin release. Complete inhibition of IL-1 β effects could not be obtained by adding L-arginine analogues to L-arginine-depleted medium, indicating that an NO-independent action of IL-1 β on islet insulin release may exist. These results suggest a novel mechanism for NA-mediated protection of IL-1–induced P-cell damage via an inhibitory effect of NA on the formation of NO.

Cystic Fibrosis–Related Diabetes The presence of microvascular diabetes complications

OBJECTIVE—Cystic fibrosis (CF)-related diabetes has been regarded as a mild form of diabetes with a low risk of severe diabetes complications. The prevalence of CF-related diabetes increases with age, resulting in a 50% prevalence of diabetes at age 30 years. We sought to investigate whether microvascular complications in CF-related diabetes appear with a relevant frequency. RESEARCH DESIGN AND METHODS—Thirty-eight patients aged 30 (range 18–55) years with CF-related diabetes for 20 (0–31) years were screened for diabetes complications. Because of chronic pulmonary infections, the majority of patients were regularly treated with aminoglycoside and cyclosporine given frequently. RESULTS—Since the pharmacological treatment of lung transplant patients could influence metabolical regulation and renal function, the results are given separately for nontransplanted (n = 29) and transplanted (n = 9) CF patients. Nine patients (27%) had retinopathy, two of which had proliferative retinopathy and needed laser treatment. Lung transplantation did not affect the prevalence of retinopathy. In nontransplanted patients, nine had hypertension, three microalbuminuria, and one elevated creatinine. None had macroalbuminuria. In transplanted patients, eight of nine had hypertension, two had microalbuminuria, and none had macroalbuminuria. Seven of nine lung transplant patients had elevated plasma creatinine, and severely reduced glomerular filtration rate was significantly more frequent. CONCLUSIONS—A high frequency of diabetic retinopathy was found in patients with insulin-treated CF-related diabetes, stressing the need for a regular screening program as in type 1 diabetes. Severely impaired kidney function was common in lung transplant patients, probably secondary to cyclosporine treatment.

Two-Dimensional Gel Electrophoresis of Rat Islet Proteins: Interleukin 1β-Induced Changes in Protein Expression are Reduced by L-Arginine Depletion and Nicotinamide

Interleukin (IL)-1 β-mediated damage to β-cells in isolated islets of Langerhans depends upon de novo synthesis of proteins that have not been fully identified. Further, IL-1 β-induced and tumor necrosis factor alpha-induced islet damage partly depends on the intracellular production of the nitric oxide (NO) radical. IL-1 β has also been reported to induce the synthesis of cellular defense proteins, e.g., heme-oxygenase and heat shock proteins 70 and 90. Nicotinamide, while in itself inactive, inhibited IL-1 β-induced NO production in a time- and dose-dependent manner. To enable the identification of IL-1 β-induced proteins with possible protective and deleterious effects, we characterized the effects of IL-1 β, nicotinamide, and NO synthesis inhibition by L-arginine depletion on rat islet protein expression detected by high-resolution two-dimensional gel electrophoresis. More than 1,600 proteins were reproducibly detected in control rat islets. Incubation with IL-1 β–, nicotinamide-, or L-arginine-depleted control medium upregulated 29, 3, and 1 protein, respectively, and downregulated 4, 0, and 1 protein, respectively. Addition of nicotinamide and L-arginine depletion reduced the upregulation of 16 and 20 IL-1 β-induced proteins, respectively. The identity of these proteins is under study. The demonstrated changes in protein expression caused by IL-1 β ± nicotinamide and L-arginine depletion may form the basis for identification of proteins with possible protective and deleterious roles in the initial β-cell destruction in insulin-dependent diabetes mellitus.

Cloning and Expression of Cytokine-Inducible Nitric Oxide Synthase cDNA From Rat Islets of Langerhans

An inducible nitric oxide (NO) synthase isoform (iNOS) is specifically induced in the β-cells of interleuldn (IL)-1β–exposed rat islets, suggesting a role for NO in the pathogenesis of type I diabetes. The aim of this study was to clone and characterize iNOS cDNA from cytokineexposed islets. Neither NO production nor iNOS transcription could be detected in rat islets or in rat insulinoma RIN-5AH β-cells cultured in the absence of cytokines. Addition of IL-1α alone or in combination with tumor necrosis factor-α induced a concentration- and time-dependent expression of the iNOS gene and associated NO production (measured as nitrite) from both islets and RIN cells. iNOS transcripts were cloned by reverse transcriptase-polymerase chain reaction from the cytokine-exposed rat islets and RIN cells, and DNA sequence analysis revealed a near 100% identity to the recently published iNOS cDNA cloned from cytokineexposed rat hepatocytes and smooth muscle cells. Recombinant rat islet iNOS was transiently and stably expressed in human kidney 293 fibroblasts, and the high enzymatic activity was inhibited by addition of the Larginine analogs, Nω-nitro-L-arginine methyl ester and aminoguanidine. Two-dimensional gel electrophoresis revealed the recombinant iNOS as a series of spots with the expected molecular mass of 131 kDa and pi values in the range of 6.8 to 7.0. In conclusion, the IL-1β-induced iNOS cloned and expressed from rat islets and RIN cells is encoded by the same transcript as the iNOS induced in other cell types.

Twelve-Week Treatment With Liraglutide as Add-on to Insulin in Normal-Weight Patients With Poorly Controlled Type 1 Diabetes: A Randomized, Placebo-Controlled, Double-Blind Parallel Study

OBJECTIVE This study investigated the efficacy and safety of once-daily liraglutide 1.2 mg versus placebo as add-on to insulin treatment in normal-weight patients with poorly controlled type 1 diabetes. RESEARCH DESIGN AND METHODS In a randomized (1:1), double-blind, placebo-controlled design, 40 patients with type 1 diabetes (HbA1c ≥8% [64 mmol/mol]) received once-daily liraglutide 1.2 mg or placebo for 12 weeks. Continuous glucose monitoring was performed before and at the end of treatment. The primary end point was change in HbA1c. Secondary end points included change in insulin dose, weight, glycemic excursions, heart rate, and blood pressure. RESULTS Baseline HbA1c was similar in the liraglutide and placebo group (8.8 ± 0.2 and 8.7 ± 0.1% [72.5 ± 2.2 and 71.8 ± 1.5 mmol/mol]). Change in HbA1c from baseline was −0.6 ± 0.2% (−6.22 ± 1.71 mmol/mol) with liraglutide and −0.5 ± 0.2% (−5.56 ± 1.67 mmol/mol) with placebo (P = 0.62). Variation in glycemic excursions did not change in either group. Change in body weight was −3.13 ± 0.58 and +1.12 ± 0.42 kg (P < 0.0001) with liraglutide and placebo, respectively. The bolus insulin dose decreased in liraglutide-treated patients and did not change with placebo treatment (4.0 ± 1.3 vs. 0.0 ± 1.0 IU, P = 0.02). Heart rate increased within the liraglutide group (P = 0.04) but not compared with placebo, whereas mean systolic blood pressure decreased compared with placebo (between-group difference 3.21 mmHg [95% CI −8.31 to 1.90], P = 0.04). Liraglutide was more frequently associated with gastrointestinal adverse effects. The incidence of hypoglycemia did not differ between groups. CONCLUSIONS Liraglutide significantly reduces body weight and insulin requirements but has no additional effect on HbA1c in normal-weight patients with type 1 diabetes inadequately controlled on insulin alone.

Lack of Predictive Value of Islet Cell Antibodies, Insulin Antibodies, and HLA-DR Phenotype for Remission in Cyclosporin-Treated IDDM Patients

The effect of immunosuppression on the humoral immune response to islet autoantigens and exogenously administered insulin and the predictive value of islet cell cytoplasmic antibodies (ICAs), insulin antibodies (IAs), and HLA-DR phenotype for remission during immunosuppression were studied in a prospective randomized double-blind trial of cyclosporin administration in 98 newly diagnosed insulin-dependent diabetes mellitus (IDDM) patients. HLA-DR phenotype and glycosylated hemoglobin were determined at study entry, and insulin requirement, glucagon-stimulated C-peptide, ICAs, and IAs were measured at entry and after 1, 3, 6, 9, and 12 mo of follow-up. Cyclosporin therapy caused significant suppression of the prevalence and serum concentrations of ICAs and lAs. Cyclosporin-treated IDDM patients ICA+ at study entry had higher levels of stimulated C-peptide after 1 mo of study, but the increased (β-cell function was not associated with a higher frequency of insulin-free remission at 1 mo. ICA and IA status at entry did not predict cyclosporininduced remission as assessed by the prevalence of insulin-free remission or β-cell function at 3–12 mo of study, and significant decrements in the titers or total disappearance of ICAs were not associated with an increased prevalence or duration of non-insulinrequiring remission or higher stimulated C-peptide values. There was no correlation between the serum levels of ICAs and lAs at entry and β-cell function at 12 mo of follow-up. Although patients who were HLADR3/X seemed to respond better to cyclosporin therapy at 6 mo than patients who were HLA-DR3/4 or -DR4/X when analyzed cross-sectionally, a split-plot analysis of variance with time as the split-plot factor showed that the HLA-DR phenotype failed to identify patients with an overall better response to cyclosporin therapy. These data indicate that, although cyclosporin therapy inhibits the humoral immune response to islet components and exogenous insulin, significant decrements in ICA titers are not useful in monitoring efficacy of immunosuppression with cyclosporin, and the determination of ICA and IA status and immune response phenotype at diagnosis is of no predictive value for remission in selecting recent-onset IDDM patients for cyclosporin immunointervention.

Changes in basal rates and bolus calculator settings in insulin pumps during pregnancy in women with type 1 diabetes

Abstract Objective: To explore insulin pump settings in a cohort of pregnant women with type 1 diabetes on insulin pump therapy with a bolus calculator. Methods: Twenty-seven women with type 1 diabetes on insulin pump therapy were included in this study. At 8, 12, 21, 27 and 33 weeks, insulin pump settings and HbA1c were recorded. Results were compared with 96 women with type 1 diabetes on multiple daily injection therapy. Results: Throughout pregnancy, the carbohydrate-to-insulin ratio decreased at all three main meals. The most pronounced decrease was observed at breakfast, where the carbohydrate-to-insulin ratio was reduced, from median 12 (range 4–20) in early pregnancy to 3 (2–10) g carbohydrate per unit insulin in late pregnancy. Basal insulin delivery increased by ∼50%, i.e. from 0.8 (0.5–2.2) to 1.2 (0.6–2.5) IU/h at 5 a.m. and from 1.0 (0.6–1.5) to 1.3 (0.2–2.3) IU/h at 5 p.m. during pregnancy. HbA1c levels during pregnancy, the occurrence of severe hypoglycemia and pregnancy outcomes were similar in the two groups. Conclusions: In women with type 1 diabetes on insulin pump therapy with a bolus calculator, the carbohydrate-to-insulin ratio declined 4-fold from early to late pregnancy, whereas changes in basal insulin delivery were smaller.

Total Mortality by Elevated Transferrin Saturation in Patients With Diabetes

OBJECTIVE It is not known to what extent iron overload predicts prognosis in patients with diabetes after diagnosis or whether iron overload is a risk factor independent of the HFE genotype. We investigated total and cause-specific mortality according to increased transferrin saturation (≥50 vs. <50%), whether mortality is driven by the HFE genotype, and whether early measurement of transferrin saturation helps to predict mortality outcome. RESEARCH DESIGN AND METHODS Cohort 1 included patients with late-onset type 1 diabetes (n = 716) with a cross-sectional measurement of transferrin saturation and HFE genotype. Cohort 2 included consecutively recruited patients with any diabetes (n = 6,120), transferrin saturation measurement at referral, and HFE genotype if transferrin saturation was above 50%. RESULTS In cohort 1, the hazard ratio for total mortality was 2.3 (95% CI 1.3–3.9; P = 0.002) and for cause-specific mortality by neoplasms was 5.8 (2.4–14; P = 0.00007) in patients with transferrin saturation ≥50 vs. <50%. Excluding genotypes C282Y/C282Y and C282Y/H63D gave similar results. The hazard ratio for total mortality was 4.0 (1.2–13; P = 0.01) and for cause-specific mortality by neoplasms was 13 (3.6–49; P = 0.0001) in patients with C282Y/C282Y versus wild type. In cohort 2, total mortality was not different in patients with transferrin saturation ≥50 vs. <50%. In patients with late-onset type 1 diabetes and transferrin saturation ≥50%, the hazard ratio for total mortality was 0.4 (0.2–0.9; P = 0.03) in cohort 2 versus cohort 1. CONCLUSIONS Increased transferrin saturation and HFE genotype C282Y/C282Y predict total mortality in patients with late-onset type 1 diabetes, and increased transferrin saturation after diagnosis is an independent risk factor. Early measurement of transferrin saturation in these patients leading to early intervention improves life expectancy.