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Aims/hypothesisEmerging evidence suggests that in addition to hyperglycaemia, dyslipidaemia could represent a contributing pathogenetic factor to diabetic neuropathy, while obesity and insulin resistance play a role in the development of diabetic cardiac autonomic neuropathy (CAN) characterised by reduced heart rate variability (HRV), particularly in type 2 diabetes. We hypothesised that distinct lipid metabolites are associated with diminished HRV in recent-onset type 2 diabetes rather than type 1 diabetes.MethodsWe analysed 127 plasma lipid metabolites (11 acylcarnitines, 39 NEFA, 12 sphingomyelins (SMs), 56 phosphatidylcholines and nine lysophosphatidylcholines) using MS in participants from the German Diabetes Study baseline cohort recently diagnosed with type 1 (n = 100) and type 2 diabetes (n = 206). Four time-domain HRV indices (number of normal-to-normal (NN) intervals >50 ms divided by the number of all NN intervals [pNN50]; root mean square of successive differences [RMSSD]; SD of NN intervals [SDNN]; and SD of differences between adjacent NN intervals) and three frequency-domain HRV indices (very-low-frequency [VLF], low-frequency [LF] and high-frequency [HF] power spectrum) were computed from NN intervals recorded during a 3 h hyperinsulinaemic–euglycaemic clamp at baseline and in subsets of participants with type 1 (n = 60) and type 2 diabetes (n = 95) after 5 years.ResultsIn participants with type 2 diabetes, after Bonferroni correction and rigorous adjustment, SDNN was inversely associated with higher levels of diacyl-phosphatidylcholine (PCaa) C32:0, PCaa C34:1, acyl-alkyl-phosphatidylcholine (PCae) C36:0, SM C16:0 and SM C16:1. SD of differences between NN intervals was inversely associated with PCaa C32:0, PCaa C34:1, PCaa C34:2, PCae C36:0 and SM C16:1, and RMSSD with PCae C36:0. For VLF power, inverse associations were found with PCaa C30:0, PCaa C32:0, PCaa C32:1, PCaa C34:2 and SM C16:1, and for LF power inverse associations were found with PCaa C32:0 and SM C16:1 (r = −0.242 to r = −0.349; p ≤ 0.0005 for all correlations). In contrast, no associations of lipid metabolites with measures of cardiac autonomic function were noted in participants recently diagnosed with type 1 diabetes. After 5 years, HRV declined due to ageing rather than diabetes, whereby prediction analyses for lipid metabolites were hampered.Conclusions/interpretationHigher plasma levels of specific lipid metabolites are closely linked to cardiac autonomic dysfunction in recent-onset type 2 diabetes but not type 1 diabetes, suggesting a role for perturbed lipid metabolism in the early development of CAN in type 2 diabetes.

The pathogenesis of fatty liver is not understood in detail, but lipid overflow as well as de novo lipogenesis (DNL) seem to be the key points of hepatocyte accumulation of lipids. One key transcription factor in DNL is sterol regulatory element-binding protein (SREBP)-1c. We generated mice with liver-specific over-expression of mature human SREBP-1c under control of the albumin promoter and a liver-specific enhancer (alb-SREBP-1c) to analyze systemic perturbations caused by this distinct alteration. SREBP-1c targets specific genes and causes key enzymes in DNL and lipid metabolism to be up-regulated. The alb-SREBP-1c mice developed hepatic lipid accumulation featuring a fatty liver by the age of 24 weeks under normocaloric nutrition. On a molecular level, clinical parameters and lipid-profiles varied according to the fatty liver phenotype. The desaturation index was increased compared to wild type mice. In liver, fatty acids (FA) were increased by 50% (p<0.01) and lipid composition was shifted to mono unsaturated FA, whereas lipid profile in adipose tissue or serum was not altered. Serum analyses revealed a ∼2-fold (p<0.01) increase in triglycerides and free fatty acids, and a ∼3-fold (p<0.01) increase in insulin levels, indicating insulin resistance; however, no significant cytokine profile alterations have been determined. Interestingly and unexpectedly, mice also developed adipositas with considerably increased visceral adipose tissue, although calorie intake was not different compared to control mice. In conclusion, the alb-SREBP-1c mouse model allowed the elucidation of the systemic impact of SREBP-1c as a central regulator of lipid metabolism in vivo and also demonstrated that the liver is a more active player in metabolic diseases such as visceral obesity and insulin resistance.

Although fibrosis depicts a reparative mechanism, maladaptation of the heart due to excessive production of extracellular matrix accelerates cardiac dysfunction. The anthraquinone Rhein was examined for its anti-fibrotic potency to mitigate cardiac fibroblast-to-myofibroblast transition (FMT). Primary human ventricular cardiac fibroblasts were subjected to hypoxia and characterized with proteomics, transcriptomics and cell functional techniques. Knowledge based analyses of the omics data revealed a modulation of fibrosis-associated pathways and cell cycle due to Rhein administration during hypoxia, whereas p53 and p21 were identified as upstream regulators involved in the manifestation of cardiac fibroblast phenotypes. Mechanistically, Rhein acts inhibitory on HDAC classes I/II as enzymatic inhibitor. Rhein-mediated cellular effects were linked to the histone deacetylase (HDAC)-dependent protein stabilization of p53 under normoxic but not hypoxic conditions. Functionally, Rhein inhibited collagen contraction, indicating anti-fibrotic property in cardiac remodeling. This was accompanied by increased abundance of SMAD7, but not SMAD2/3, and consistently SMAD-specific E3 ubiquitin ligase SMURF2. In conclusion, this study identifies Rhein as a novel potent direct HDAC inhibitor that may contribute to the treatment of cardiac fibrosis as anti-fibrotic agent. As readily available drug with approved safety, Rhein constitutes a promising potential therapeutic approach in the supplemental and protective intervention of cardiac fibrosis.

Cytokine-induced signal transduction is executed by natural biological switches, which among many others control immune-related processes. Here, we show that synthetic cytokine receptors (SyCyRs) can induce cytokine signaling using non-physiological ligands. High-affinity GFP- and mCherry-nanobodies were fused to transmembrane and intracellular domains of the IL-6/IL-11 and IL-23 cytokine receptors gp130 and IL-12Rβ1/IL-23R, respectively. Homo- and heterodimeric GFP:mCherry fusion proteins as synthetic cytokine-like ligands were able to induce canonical signaling in vitro and in vivo. Using SyCyR ligands, we show that IL-23 receptor homodimerization results in its activation and IL-23-like signal transduction. Moreover, trimeric receptor assembly induces trans-phosphorylation among cytokine receptors with associated Janus kinases. The SyCyR technology allows biochemical analyses of transmembrane receptor signaling in vitro and in vivo, cell-specific activation through SyCyR ligands using transgenic animals and possible therapeutic regimes involving non-physiological targets during immunotherapy. Cytokine-induced signaling acts as an ON/OFF switch dependent on the presence of ligands. Here the authors construct synthetic cytokine receptors responsive to synthetic ligands able to activate canonical signaling pathways.

Aims/hypothesisThe aim of this study was to investigate the modifying effect of the glucose transporter (GLUT2) gene SLC2A2 (rs8192675) variant on the glycaemic response to metformin in individuals recently diagnosed with type 2 diabetes.MethodsIndividuals with type 2 diabetes (n = 508) from the prospective German Diabetes Study (age [mean ± SD] 53 ± 10 years; 65% male; BMI 32 ± 6 kg/m2, metformin use 57%) underwent detailed metabolic characterisation (hyperinsulinaemic–euglycaemic clamp, IVGTT) during the first year after diagnosis. Participants provided self-reported data from the time of diagnosis. The change in fasting glucose was assessed in relation to SLC2A2 genotype and glucose-lowering treatment using two-way ANCOVA with gene×treatment interactions adjusted for age, sex, BMI and diabetes duration.ResultsThe C variant allele of rs8192675 was associated with a higher prevalence of diabetes symptoms at diabetes diagnosis. In the metformin monotherapy group only, patients with a C allele showed a larger adjusted blood glucose reduction during the first year after diabetes diagnosis than patients with the TT genotype (6.3 mmol/l vs 3.9 mmol/l; genotype difference 2.4 mmol/l, p = 0.02; p value for genotype interaction [metformin monotherapy vs non-pharmacological therapy] <0.01). The greater decline in fasting glucose (CC/CT vs TT) in metformin monotherapy persisted after further adjusting for glucose values at diagnosis (genotype difference 1.0 mmol/l, p = 0.01; genotype×treatment interaction p = 0.06).Conclusions/interpretationThe variant rs8192675 in the SLC2A2 gene (C allele) is associated with an improved glucose response to metformin monotherapy during the first year after diagnosis in type 2 diabetes.Trial registrationClinicalTrials.gov NCT01055093

IntroductionShunting of glycolytic intermediates into the pentose phosphate pathway via transketolase activation by benfotiamine has been suggested to protect from hyperglycemia-induced microvascular damage, but the long-term effects of benfotiamine on diabetic sensorimotor polyneuropathy (DSPN) remain unclear.Research design and methodsThis 1:1 randomized double-blind, placebo-controlled parallel group monocentric phase II trial compared the efficacy and safety of 1-year treatment with benfotiamine 300 mg two times per day versus placebo over 12 months in participants with type 2 diabetes and mild-to-moderate symptomatic DSPN. The primary endpoint was the change in corneal nerve fiber length (CNFL) assessed by corneal confocal microscopy (CCM) from baseline to 12 months. Secondary endpoints included three other CCM parameters, skin biopsy (four parameters), nerve conduction studies (13 measures), quantitative sensory testing (six parameters), cardiovascular autonomic function tests (17 indices), sudomotor function tests (five parameters), 15 clinical scores and scales for neuropathic symptoms and signs and 13 health-related quality of life and depression instruments. Pharmacokinetics included measurement of six thiamine analytes in blood.ResultsA total of 57 participants underwent randomization. The changes from baseline to 12 months in CNFL did not differ between the two groups. The corresponding changes in the secondary morphometric, functional and clinical neuropathic outcomes as well as quality of life were also similar in the two groups. Only the Neuropathy Symptom Score tended to improve after benfotiamine treatment (p=0.098 vs placebo). Benfotiamine treatment increased the concentrations of all six thiamine analytes studied (p≤0.003 vs placebo). Safety analysis showed no relevant differences between the groups in the rates of adverse events.ConclusionsIn type 2 diabetes individuals with mild-to-moderate symptomatic DSPN, treatment with benfotiamine for 12 months was well tolerated, but had no significant effects on multiple morphometric, neurophysiological and clinical measures of neuropathy.Trial registration numberEuropean Clinical Trials Database (EudraCT) 2017-003054-16 registered on April 10 (https://eudract.ema.europa.eu/), 2018 and German Register for Clinical Trials DRKS00014832 registered on August 3, 2018 (https://drks.de/search/de).

Type 2 diabetes (T2D) has a strong genetic component. Most of the gene variants driving the pathogenesis of T2D seem to target pancreatic β-cell function. To identify novel gene variants acting at early stage of the disease, we analyzed whole transcriptome data to identify differential expression (DE) and alternative exon splicing (AS) transcripts in pancreatic islets collected from two metabolically diverse mouse strains at 6 weeks of age after three weeks of high-fat-diet intervention. Our analysis revealed 1218 DE and 436 AS genes in islets from NZO/Hl vs C3HeB/FeJ. Whereas some of the revealed genes present well-established markers for β-cell failure, such as Cd36 or Aldh1a3 , we identified numerous DE/AS genes that have not been described in context with β-cell function before. The gene Lgals2 , previously associated with human T2D development, was DE as well as AS and localizes in a quantitative trait locus (QTL) for blood glucose on Chr.15 that we reported recently in our N 2 (NZOxC3H) population. In addition, pathway enrichment analysis of DE and AS genes showed an overlap of only half of the revealed pathways, indicating that DE and AS in large parts influence different pathways in T2D development. PPARG and adipogenesis pathways, two well-established metabolic pathways, were overrepresented for both DE and AS genes, probably as an adaptive mechanism to cope for increased cellular stress. Our results provide guidance for the identification of novel T2D candidate genes and demonstrate the presence of numerous AS transcripts possibly involved in islet function and maintenance of glucose homeostasis.

IntroductionDiabetic sensorimotor polyneuropathy (DSPN) affects approximately 30% of people with diabetes, while around half of cases are symptomatic. Currently, there are only few pathogenetically oriented pharmacotherapies for DSPN, one of which is benfotiamine, a prodrug of thiamine with a high bioavailability and favourable safety profile. While benfotiamine has shown positive effects in preclinical and short-term clinical studies, no long-term clinical trials are available to demonstrate disease-modifying effects on DSPN using a comprehensive set of disease-related endpoints.Methods and analysisThe benfotiamine on morphometric, neurophysiological and clinical measures in patients with type 2 diabetes trial is a randomised double-blind, placebo-controlled parallel group monocentric phase II clinical trial to assess the effects of treatment with benfotiamine compared with placebo in participants with type 2 diabetes and mild to moderate symptomatic DSPN. Sixty participants will be 1:1 randomised to treatment with benfotiamine 300 mg or placebo two times a day over 12 months. The primary endpoint will be the change in corneal nerve fibre length assessed by corneal confocal microscopy (CCM) after 12 months of benfotiamine treatment compared with placebo. Secondary endpoints will include other CCM measures, skin biopsy and function indices, variables from somatic and autonomic nerve function tests, clinical examination and questionnaires, general health, health-related quality of life, cost, safety and blood tests.Ethics and disseminationThe trial was approved by the competent authority and the local independent ethics committee. Trial results will be published in peer-reviewed journals, conference abstracts, and via online and print media.Trial registration numberDRKS00014832.

The transcription factor sterol regulatory element binding protein (SREBP)-1a plays a pivotal role in lipid metabolism. Using the SREBP-1a expressing human hepatoma cell line HepG2 we have shown previously that human SREBP-1a is phosphorylated at serine 117 by ERK-mitogen-activated protein kinases (MAPK). Using a combination of cell biology and protein chemistry approach we show that SREBP-1a is also target of other MAPK-families, i.e. c-JUN N-terminal protein kinases (JNK) or p38 stress activated MAP kinases. Serine 117 is also the major phosphorylation site in SREBP-1a for JNK. In contrast to that the major phosphorylation sites of p38 MAPK family are serine 63 and threonine 426. Functional analyses reveal that phosphorylation of SREBP-1a does not alter protein/DNA interaction. The identified phosphorylation sites are specific for both kinase families also in cellular context. To provide direct evidence that phosphorylation of SREBP-1a is a regulatory principle of biological and clinical relevance, we generated transgenic mice expressing mature transcriptionally active N-terminal domain of human SREBP-1a variant lacking all identified phosphorylaton sites designed as alb-SREBP-1aΔP and wild type SREBP-1a designed as alb-SREBP-1a liver specific under control of the albumin promoter and a liver specific enhancer. In contrast to alb-SREBP-1a mice the phosphorylation-deficient mice develop no enlarged fatty livers under normocaloric conditions. Phenotypical examination reveales a massive accumulation of adipose tissue in alb-SREBP-1a but not in the phosphorylation deficient alb-SREBP-1aΔP mice. Moreover, preventing phosphorylation of SREBP-1a protects mice also from dyslipidemia. In conclusion, phosphorylation of SREBP-1a by ERK, JNK and p38 MAPK-families resembles a biological principle and plays a significant role, in vivo.

In non-alcoholic fatty liver disease (NAFLD) caused by ectopic lipid accumulation, lipotoxicity is a crucial molecular risk factor. Mechanisms to eliminate lipid overflow can prevent the liver from functional complications. This may involve increased secretion of lipids or metabolic adaptation to ß-oxidation in lipid-degrading organelles such as mitochondria and peroxisomes. In addition to dietary factors, increased plasma fatty acid levels may be due to increased triglyceride synthesis, lipolysis, as well as de novo lipid synthesis (DNL) in the liver. In the present study, we investigated the impact of fatty liver caused by elevated DNL, in a transgenic mouse model with liver-specific overexpression of human sterol regulatory element-binding protein-1c (alb-SREBP-1c), on hepatic gene expression, on plasma lipids and especially on the proteome of peroxisomes by omics analyses, and we interpreted the results with knowledge-based analyses. In summary, the increased hepatic DNL is accompanied by marginal gene expression changes but massive changes in peroxisomal proteome. Furthermore, plasma phosphatidylcholine (PC) as well as lysoPC species were altered. Based on these observations, it can be speculated that the plasticity of organelles and their functionality may be directly affected by lipid overflow.