Additive efficacy of a bispecific anti-TNF/IL-6 nanobody compound in translational models of rheumatoid arthritis.

Rheumatoid arthritis (RA) is one of the most common autoimmune diseases affecting primarily the joints. Despite successful therapies including antibodies against tumor necrosis factor (TNF) and interleukin-6 (IL-6) receptor, only 20 to 30% of patients experience remission. We studied whether inhibiting both TNF and IL-6 would result in improved efficacy. Using backtranslation from single-cell RNA sequencing (scRNA-seq) data from individuals with RA, we hypothesized that TNF and IL-6 act synergistically on fibroblast-like synoviocytes (FLS) and T cells. Coculture of FLS from individuals with RA and T cells supported this hypothesis, revealing effects on both disease-driving pathways and biomarkers. Combining anti-TNF and anti-IL-6 antibodies in collagen-induced arthritis (CIA) mouse models resulted in sustained long-term remission, improved histology, and effects on bone remodeling pathways. These promising data initiated the development of an anti-TNF/IL-6 bispecific nanobody compound 1, with similar potencies against TNF and IL-6. We observed additive efficacy of compound 1 in a FLS/T cell coculture affecting arthritis and T helper 17 (TH17) pathways. This nanobody compound transcript signature inversely overlapped with described RA endotypes, indicating a potential efficacy in a broader patient population. In summary, we showed superiority of a bispecific anti-TNF/IL-6 nanobody compound or combination treatment over monospecific treatments in both in vitro and in vivo models. We anticipate improved efficacy in upcoming clinical studies.

Liver-Specific Knockdown of Class IIa HDACs Has Limited Efficacy on Glucose Metabolism but Entails Severe Organ Side Effects in Mice.

Histone deacetylases (HDACs) are important regulators of epigenetic gene modification that are involved in the transcriptional control of metabolism. In particular class IIa HDACs have been shown to affect hepatic gluconeogenesis and previous approaches revealed that their inhibition reduces blood glucose in type 2 diabetic mice. In the present study, we aimed to evaluate the potential of class IIa HDAC inhibition as a therapeutic opportunity for the treatment +of metabolic diseases. For that, siRNAs selectively targeting HDAC4, 5 and 7 were selected and used to achieve a combinatorial knockdown of these three class IIa HDAC isoforms. Subsequently, the hepatocellular effects as well as the impact on glucose and lipid metabolism were analyzed in vitro and in vivo. The triple knockdown resulted in a statistically significant decrease of gluconeogenic gene expression in murine and human hepatocyte cell models. A similar HDAC-induced downregulation of hepatic gluconeogenesis genes could be achieved in mice using a liver-specific lipid nanoparticle siRNA formulation. However, the efficacy on whole body glucose metabolism assessed by pyruvate-tolerance tests were only limited and did not outweigh the safety findings observed by histopathological analysis in spleen and kidney. Mechanistically, Affymetrix gene expression studies provide evidence that class IIa HDACs directly target other key factors beyond the described forkhead box (FOXP) transcription regulators, such as hepatocyte nuclear factor 4 alpha (HNF4a). Downstream of these factors several additional pathways were regulated not merely including glucose and lipid metabolism and transport. In conclusion, the liver-directed combinatorial knockdown of HDAC4, 5 and 7 by therapeutic siRNAs affected multiple pathways in vitro, leading in vivo to the downregulation of genes involved in gluconeogenesis. However, the effects on gene expression level were not paralleled by a significant reduction of gluconeogenesis in mice. Combined knockdown of HDAC isoforms was associated with severe adverse effects in vivo, challenging this approach as a treatment option for chronic metabolic disorders like type 2 diabetes.

AMPKß1 and AMPKß2 define an isoform-specific gene signature in human pluripotent stem cells, differentially mediating cardiac lineage specification.

AMP-activated protein kinase (AMPK) is a key regulator of energy metabolism that phosphorylates a wide range of proteins to maintain cellular homeostasis. AMPK consists of three subunits: α, ß, and γ. AMPKα and ß are encoded by two genes, the γ subunit by three genes, all of which are expressed in a tissue-specific manner. It is not fully understood, whether individual isoforms have different functions. Using RNA-Seq technology, we provide evidence that the loss of AMPKß1 and AMPKß2 lead to different gene expression profiles in human induced pluripotent stem cells (hiPSCs), indicating isoform-specific function. The knockout of AMPKß2 was associated with a higher number of differentially regulated genes than the deletion of AMPKß1, suggesting that AMPKß2 has a more comprehensive impact on the transcriptome. Bioinformatics analysis identified cell differentiation as one biological function being specifically associated with AMPKß2. Correspondingly, the two isoforms differentially affected lineage decision toward a cardiac cell fate. Although the lack of PRKAB1 impacted differentiation into cardiomyocytes only at late stages of cardiac maturation, the availability of PRKAB2 was indispensable for mesoderm specification as shown by gene expression analysis and histochemical staining for cardiac lineage markers such as cTnT, GATA4, and NKX2.5. Ultimately, the lack of AMPKß1 impairs, whereas deficiency of AMPKß2 abrogates differentiation into cardiomyocytes. Finally, we demonstrate that AMPK affects cellular physiology by engaging in the regulation of hiPSC transcription in an isoform-specific manner, providing the basis for further investigations elucidating the role of dedicated AMPK subunits in the modulation of gene expression.

Hepatic transcriptomic signatures of statin treatment are associated with impaired glucose homeostasis in severely obese patients.

BACKGROUND: Clinical data identified an association between the use of HMG-CoA reductase inhibitors (statins) and incident diabetes in patients with underlying diabetes risk factors such as obesity, hypertension and dyslipidemia. The molecular mechanisms however are unknown. METHODS: An observational cross-sectional study included 910 severely obese patients, mean (SD) body mass index (BMI) 46.7 (8.7), treated with or without statins (ABOS cohort: a biological atlas of severe obesity). Data and sample collection took place in France between 2006 and 2016. Transcriptomic signatures of statin treatment in human liver obtained from genome-wide transcriptomic profiling of five different statin drugs using microarrays were correlated to clinico-biological phenotypes and also assigned to biological pathways and mechanisms. Patients from the non-statin-users group were matched to patients in the statin users group by propensity score analysis to minimize confounding effects from age, gender, parental familial history of diabetes, BMI, waist circumference, systolic and diastolic blood pressure and use of anti-hypertensive drugs as pre-specified covariates. RESULTS: We determined the hepatic, statin-related gene signature from genome-wide transcriptomic profiling in severely obese patients with varying degrees of glucose tolerance and cardio-metabolic comorbidities. One hundred and fifty seven patients on statin treatment in the matched cohort showed higher diabetes prevalence (OR = 2.67; 95%CI, 1.60-4.45; P = 0.0002) and impairment of glucose homeostasis. This phenotype was associated with molecular signatures of increased hepatic de novo lipogenesis (DNL) via activation of sterol regulatory element-binding protein 1 (SREBP1) and concomitant upregulation of the expression of key genes in both fatty acid and triglyceride metabolism. CONCLUSIONS: A DNL gene activation profile in response to statins is associated with insulin resistance and the diabetic status of the patients. Identified molecular signatures thus suggest that statin treatment increases the risk for diabetes in humans at least in part via induction of DNL. TRIAL REGISTRATION: NCT01129297 . Registered May 242,010 (retrospectively registered).

Genetic Nicotinamide N-Methyltransferase (Nnmt) Deficiency in Male Mice Improves Insulin Sensitivity in Diet-Induced Obesity but Does Not Affect Glucose Tolerance.

Antisense oligonucleotide knockdown (ASO-KD) of nicotinamide N-methyltransferase (NNMT) in high-fat diet (HFD)-fed mice has been reported to reduce weight gain and plasma insulin levels and to improve glucose tolerance. Using NNMT-ASO-KD or NNMT knockout mice (NNMT-/-), we tested the hypothesis that Nnmt deletion protects against diet-induced obesity and its metabolic consequences in males and females on obesity-inducing diets. We also examined samples from a human weight reduction (WR) study for adipose NNMT (aNNMT) expression and plasma 1-methylnicotinamide (MNAM) levels. In Western diet (WD)-fed female mice, NNMT-ASO-KD reduced body weight, fat mass, and insulin level and improved glucose tolerance. Although NNMT-/- mice fed a standard diet had no obvious phenotype, NNMT-/- males fed an HFD showed strongly improved insulin sensitivity (IS). Furthermore, NNMT-/- females fed a WD showed reduced weight gain, less fat, and lower insulin levels. However, no improved glucose tolerance was observed in NNMT-/- mice. Although NNMT expression in human fat biopsy samples increased during WR, corresponding plasma MNAM levels significantly declined, suggesting that other mechanisms besides aNNMT expression modulate circulating MNAM levels during WR. In summary, upon NNMT deletion or knockdown in males and females fed different obesity-inducing diets, we observed sex- and diet-specific differences in body composition, weight, and glucose tolerance and estimates of IS.

Systems biology of the IMIDIA biobank from organ donors and pancreatectomised patients defines a novel transcriptomic signature of islets from individuals with type 2 diabetes.

AIMS/HYPOTHESIS: Pancreatic islet beta cell failure causes type 2 diabetes in humans. To identify transcriptomic changes in type 2 diabetic islets, the Innovative Medicines Initiative for Diabetes: Improving beta-cell function and identification of diagnostic biomarkers for treatment monitoring in Diabetes (IMIDIA) consortium ( www.imidia.org ) established a comprehensive, unique multicentre biobank of human islets and pancreas tissues from organ donors and metabolically phenotyped pancreatectomised patients (PPP). METHODS: Affymetrix microarrays were used to assess the islet transcriptome of islets isolated either by enzymatic digestion from 103 organ donors (OD), including 84 non-diabetic and 19 type 2 diabetic individuals, or by laser capture microdissection (LCM) from surgical specimens of 103 PPP, including 32 non-diabetic, 36 with type 2 diabetes, 15 with impaired glucose tolerance (IGT) and 20 with recent-onset diabetes (<1 year), conceivably secondary to the pancreatic disorder leading to surgery (type 3c diabetes). Bioinformatics tools were used to (1) compare the islet transcriptome of type 2 diabetic vs non-diabetic OD and PPP as well as vs IGT and type 3c diabetes within the PPP group; and (2) identify transcription factors driving gene co-expression modules correlated with insulin secretion ex vivo and glucose tolerance in vivo. Selected genes of interest were validated for their expression and function in beta cells. RESULTS: Comparative transcriptomic analysis identified 19 genes differentially expressed (false discovery rate ≤0.05, fold change ≥1.5) in type 2 diabetic vs non-diabetic islets from OD and PPP. Nine out of these 19 dysregulated genes were not previously reported to be dysregulated in type 2 diabetic islets. Signature genes included TMEM37, which inhibited Ca2+-influx and insulin secretion in beta cells, and ARG2 and PPP1R1A, which promoted insulin secretion. Systems biology approaches identified HNF1A, PDX1 and REST as drivers of gene co-expression modules correlated with impaired insulin secretion or glucose tolerance, and 14 out of 19 differentially expressed type 2 diabetic islet signature genes were enriched in these modules. None of these signature genes was significantly dysregulated in islets of PPP with impaired glucose tolerance or type 3c diabetes. CONCLUSIONS/INTERPRETATION: These studies enabled the stringent definition of a novel transcriptomic signature of type 2 diabetic islets, regardless of islet source and isolation procedure. Lack of this signature in islets from PPP with IGT or type 3c diabetes indicates differences possibly due to peculiarities of these hyperglycaemic conditions and/or a role for duration and severity of hyperglycaemia. Alternatively, these transcriptomic changes capture, but may not precede, beta cell failure.

Correction: Chronic Activation of Hepatic Nrf2 Has No Major Effect on Fatty Acid and Glucose Metabolism in Adult Mice.

[This corrects the article DOI: 10.1371/journal.pone.0166110.].

Inhibition of citrate cotransporter Slc13a5/mINDY by RNAi improves hepatic insulin sensitivity and prevents diet-induced non-alcoholic fatty liver disease in mice.

OBJECTIVE: Non-alcoholic fatty liver disease is a world-wide health concern and risk factor for cardio-metabolic diseases. Citrate uptake modifies intracellular hepatic energy metabolism and is controlled by the conserved sodium-dicarboxylate cotransporter solute carrier family 13 member 5 (SLC13A5, mammalian homolog of INDY: mINDY). In Drosophila melanogaster and Caenorhabditis elegans INDY reduction decreased whole-body lipid accumulation. Genetic deletion of Slc13a5 in mice protected from diet-induced adiposity and insulin resistance. We hypothesized that inducible hepatic mINDY inhibition should prevent the development of fatty liver and hepatic insulin resistance. METHODS: Adult C57BL/6J mice were fed a Western diet (60% kcal from fat, 21% kcal from carbohydrate) ad libitum. Knockdown of mINDY was induced by weekly injection of a chemically modified, liver-selective siRNA for 8 weeks. Mice were metabolically characterized and the effect of mINDY suppression on glucose tolerance as well as insulin sensitivity was assessed with an ipGTT and a hyperinsulinemic-euglycemic clamp. Hepatic lipid accumulation was determined by biochemical measurements and histochemistry. RESULTS: Within the 8 week intervention, hepatic mINDY expression was suppressed by a liver-selective siRNA by over 60%. mINDY knockdown improved hepatic insulin sensitivity (i.e. insulin-induced suppression of endogenous glucose production) of C57BL/6J mice in the hyperinsulinemic-euglycemic clamp. Moreover, the siRNA-mediated mINDY inhibition prevented neutral lipid storage and triglyceride accumulation in the liver, while we found no effect on body weight. CONCLUSIONS: We show that inducible mINDY inhibition improved hepatic insulin sensitivity and prevented diet-induced non-alcoholic fatty liver disease in adult C57BL6/J mice. These effects did not depend on changes of body weight or body composition.

Chronic Activation of Hepatic Nrf2 Has No Major Effect on Fatty Acid and Glucose Metabolism in Adult Mice.

The transcription factor NF-E2-related factor 2 (Nrf2) induces cytoprotective genes, but has also been linked to the regulation of hepatic energy metabolism. In order to assess the pharmacological potential of hepatic Nrf2 activation in metabolic disease, Nrf2 was activated over 7 weeks in mice on Western diet using two different siRNAs against kelch-like ECH-associated protein 1 (Keap1), the inhibitory protein of Nrf2. Whole genome expression analysis followed by pathway analysis demonstrated successful knock-down of Keap1 expression and induction of Nrf2-dependent genes involved in anti-oxidative stress defense and biotransformation, proving the activation of Nrf2 by the siRNAs against Keap1. Neither the expression of fatty acid- nor carbohydrate-handling proteins was regulated by Keap1 knock-down. Metabolic profiling of the animals did also not show effects on plasma and hepatic lipids, energy expenditure or glucose tolerance. The data indicate that hepatic Keap1/Nrf2 is not a major regulator of glucose or lipid metabolism in mice.

Characterization of RA839, a Noncovalent Small Molecule Binder to Keap1 and Selective Activator of Nrf2 Signaling.

The activation of the transcription factor NF-E2-related factor 2 (Nrf2) maintains cellular homeostasis in response to oxidative stress by the regulation of multiple cytoprotective genes. Without stressors, the activity of Nrf2 is inhibited by its interaction with the Keap1 (kelch-like ECH-associated protein 1). Here, we describe (3S)-1-[4-[(2,3,5,6-tetramethylphenyl) sulfonylamino]-1-naphthyl]pyrrolidine-3-carboxylic acid (RA839), a small molecule that binds noncovalently to the Nrf2-interacting kelch domain of Keap1 with a Kd of ∼6 µM, as demonstrated by x-ray co-crystallization and isothermal titration calorimetry. Whole genome DNA arrays showed that at 10 µM RA839 significantly regulated 105 probe sets in bone marrow-derived macrophages. Canonical pathway mapping of these probe sets revealed an activation of pathways linked with Nrf2 signaling. These pathways were also activated after the activation of Nrf2 by the silencing of Keap1 expression. RA839 regulated only two genes in Nrf2 knock-out macrophages. Similar to the activation of Nrf2 by either silencing of Keap1 expression or by the reactive compound 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid methyl ester (CDDO-Me), RA839 prevented the induction of both inducible nitric-oxide synthase expression and nitric oxide release in response to lipopolysaccharides in macrophages. In mice, RA839 acutely induced Nrf2 target gene expression in liver. RA839 is a selective inhibitor of the Keap1/Nrf2 interaction and a useful tool compound to study the biology of Nrf2.

Minimal modeling of insulin secretion in the perfused rat pancreas: a drug effect case study.

The experimental protocol of the perfused rat pancreas is commonly used to evaluate ß-cell function. In this context, mathematical models become useful tools through the determination of indexes that allow the assessment of ß-cell function in different experimental groups and the quantification of the effects of antidiabetic drugs, secretagogues, or treatments. However, a minimal model applicable to the isolated perfused rat pancreas has so far been unavailable. In this work, we adapt the C-peptide minimal model applied previously to the intravenous glucose tolerance test to obtain a specific model for the experimental settings of the perfused pancreas. Using the model, it is possible to estimate indexes describing ß-cell responsivity for first (ΦD) and second phase (ΦS, T) of insulin secretion. The model was initially applied to untreated pancreata and afterward used for the assessment of pharmacologically relevant agents (the gut hormone GLP-1, the potent GLP-1 receptor agonist lixisenatide, and a GPR40/FFAR1 agonist, SAR1) to quantify and differentiate their effect on insulin secretion. Model fit was satisfactory, and parameters were estimated with good precision for both untreated and treated pancreata. Model application showed that lixisenatide reaches improvement of ß-cell function similarly to GLP-1 (11.7- vs. 13.1-fold increase in ΦD and 2.3- vs. 2.8-fold increase in ΦS) and demonstrated that SAR1 leads to an additional improvement of ß-cell function in the presence of postprandial GLP-1 levels.

Global gene expression in human myocardium-oligonucleotide microarray analysis of regional diversity and transcriptional regulation in heart failure.

To obtain region- and disease-specific transcription profiles of human myocardial tissue, we explored mRNA expression from all four chambers of eight explanted failing [idiopathic dilated cardiomyopathy (DCM), n=5; ischemic cardiomyopathy (ICM), n=3], and five non-failing hearts using high-density oligonucleotide arrays (Affymetrix U95Av2). We performed pair-wise comparisons of gene expression in the categories (1) atria versus ventricles, (2) disease-regulated genes in atria and (3) disease-regulated genes in ventricles. In the 51 heart samples examined, 549 genes showed divergent distribution between atria and ventricles (272 genes with higher expression in atria, 277 genes with higher expression in ventricles). Two hundred and eighty-eight genes were differentially expressed in failing myocardium compared to non-failing hearts (19 genes regulated in atria and ventricles, 172 regulated in atria only, 97 genes regulated in ventricles only). For disease-regulated genes, down-regulation was 4.5-times more common than up-regulation. Functional classification according to Gene Ontology identified specific biological patterns for differentially expressed genes. Eleven genes were validated by RT-PCR showing a good correlation with the microarray data. Our goal was to determine a gene expression fingerprint of the heart, accounting for region- and disease-specific aspects. Recognizing common gene expression patterns in heart failure will significantly contribute to the understanding of heart failure and may eventually lead to the development of pathway-specific therapies.