The effect of GLP-1 receptor agonist lixisenatide on experimental diabetic retinopathy.

AIMS: Glucagon-like peptide-1 receptor agonists are effective treatments for type 2 diabetes, effectively lowering glucose without weight gain and with low risk for hypoglycemia. However, their influence on the retinal neurovascular unit remains unclear. In this study, we analyzed the effects of the GLP-1 RA lixisenatide on diabetic retinopathy. METHODS: Vasculo- and neuroprotective effects were assessed in experimental diabetic retinopathy and high glucose-cultivated C. elegans, respectively. In STZ-diabetic Wistar rats, acellular capillaries and pericytes (quantitative retinal morphometry), neuroretinal function (mfERG), macroglia (GFAP western blot) and microglia (immunohistochemistry) quantification, methylglyoxal (LC-MS/MS) and retinal gene expressions (RNA-sequencing) were determined. The antioxidant properties of lixisenatide were tested in C. elegans. RESULTS: Lixisenatide had no effect on glucose metabolism. Lixisenatide preserved the retinal vasculature and neuroretinal function. The macro- and microglial activation was mitigated. Lixisenatide normalized some gene expression changes in diabetic animals to control levels. Ets2 was identified as a regulator of inflammatory genes. In C. elegans, lixisenatide showed the antioxidative property. CONCLUSIONS: Our data suggest that lixisenatide has a protective effect on the diabetic retina, most likely due to a combination of neuroprotective, anti-inflammatory and antioxidative effects of lixisenatide on the neurovascular unit.

Effects on weight loss and glycemic control with SAR441255, a potent unimolecular peptide GLP-1/GIP/GCG receptor triagonist.

Unimolecular triple incretins, combining the activity of glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP), and glucagon (GCG), have demonstrated reduction in body weight and improved glucose control in rodent models. We developed SAR441255, a synthetic peptide agonist of the GLP-1, GCG, and GIP receptors, structurally based on the exendin-4 sequence. SAR441255 displays high potency with balanced activation of all three target receptors. In animal models, metabolic outcomes were superior to results with a dual GLP-1/GCG receptor agonist. Preclinical in vivo positron emission tomography imaging demonstrated SAR441255 binding to GLP-1 and GCG receptors. In healthy subjects, SAR441255 improved glycemic control during a mixed-meal tolerance test and impacted biomarkers for GCG and GIP receptor activation. Single doses of SAR441255 were well tolerated. The results demonstrate that integrating GIP activity into dual GLP-1 and GCG receptor agonism provides improved effects on weight loss and glycemic control while buffering the diabetogenic risk of chronic GCG receptor agonism.

Spectrum of mutations in monogenic diabetes genes identified from high-throughput DNA sequencing of 6888 individuals.

BACKGROUND: Diagnosis of monogenic as well as atypical forms of diabetes mellitus has important clinical implications for their specific diagnosis, prognosis, and targeted treatment. Single gene mutations that affect beta-cell function represent 1-2% of all cases of diabetes. However, phenotypic heterogeneity and lack of family history of diabetes can limit the diagnosis of monogenic forms of diabetes. Next-generation sequencing technologies provide an excellent opportunity to screen large numbers of individuals with a diagnosis of diabetes for mutations in disease-associated genes. METHODS: We utilized a targeted sequencing approach using the Illumina HiSeq to perform a case-control sequencing study of 22 monogenic diabetes genes in 4016 individuals with type 2 diabetes (including 1346 individuals diagnosed before the age of 40 years) and 2872 controls. We analyzed protein-coding variants identified from the sequence data and compared the frequencies of pathogenic variants (protein-truncating variants and missense variants) between the cases and controls. RESULTS: A total of 40 individuals with diabetes (1.8% of early onset sub-group and 0.6% of adult onset sub-group) were carriers of known pathogenic missense variants in the GCK, HNF1A, HNF4A, ABCC8, and INS genes. In addition, heterozygous protein truncating mutations were detected in the GCK, HNF1A, and HNF1B genes in seven individuals with diabetes. Rare missense mutations in the GCK gene were significantly over-represented in individuals with diabetes (0.5% carrier frequency) compared to controls (0.035%). One individual with early onset diabetes was homozygous for a rare pathogenic missense variant in the WFS1 gene but did not have the additional phenotypes associated with Wolfram syndrome. CONCLUSION: Targeted sequencing of genes linked with monogenic diabetes can identify disease-relevant mutations in individuals diagnosed with type 2 diabetes not suspected of having monogenic forms of the disease. Our data suggests that GCK-MODY frequently masquerades as classical type 2 diabetes. The results confirm that MODY is under-diagnosed, particularly in individuals presenting with early onset diabetes and clinically labeled as type 2 diabetes; thus, sequencing of all monogenic diabetes genes should be routinely considered in such individuals. Genetic information can provide a specific diagnosis, inform disease prognosis and may help to better stratify treatment plans.

Proteomic Profiling of Cardiomyocyte-Specific Cathepsin A Overexpression Links Cathepsin A to the Oxidative Stress Response.

Cathepsin A (CTSA) is a lysosomal carboxypeptidase present at the cell surface and secreted outside the cell. Additionally, CTSA binds to ß-galactosidase and neuraminidase 1 to protect them from degradation. CTSA has gained attention as a drug target for the treatment of cardiac hypertrophy and heart failure. Here, we investigated the impact of CTSA on the murine cardiac proteome in a mouse model of cardiomyocyte-specific human CTSA overexpression using liquid chromatography-tandem mass spectrometry in conjunction with an isotopic dimethyl labeling strategy. We identified up to 2000 proteins in each of three biological replicates. Statistical analysis by linear models for microarray data (limma) found >300 significantly affected proteins (moderated p-value ≤0.01), thus establishing CTSA as a key modulator of the cardiac proteome. CTSA strongly impaired the balance of the proteolytic system by upregulating several proteases such as cathepsin B, cathepsin D, and cathepsin Z while down-regulating numerous protease inhibitors. Moreover, cardiomyocyte-specific human CTSA overexpression strongly reduced the levels of numerous antioxidative stress proteins, i.e., peroxiredoxins and protein deglycase DJ-1. In vitro, using cultured rat cardiomyocytes, ectopic overexpression of CTSA resulted in accumulation of reactive oxygen species. Collectively, our proteomic and functional data strengthen an association of CTSA with the cellular oxidative stress response.

Cathepsin A inhibition attenuates myocardial infarction-induced heart failure on the functional and proteomic levels.

BACKGROUND: Myocardial infarction (MI) is a major cause of heart failure. The carboxypeptidase cathepsin A is a novel target in the treatment of cardiac failure. We aim to show that recently developed inhibitors of the protease cathepsin A attenuate post-MI heart failure. METHODS: Mice were subjected to permanent left anterior descending artery (LAD) ligation or sham operation. 24 h post-surgery, LAD-ligated animals were treated with daily doses of the cathepsin A inhibitor SAR1 or placebo. After 4 weeks, the three groups (sham, MI-placebo, MI-SAR1) were evaluated. RESULTS: Compared to sham-operated animals, placebo-treated mice showed significantly impaired cardiac function and increased plasma BNP levels. Cathepsin A inhibition prevented the increase of plasma BNP levels and displayed a trend towards improved cardiac functionality. Proteomic profiling was performed for the three groups (sham, MI-placebo, MI-SAR1). More than 100 proteins were significantly altered in placebo-treated LAD ligation compared to the sham operation, including known markers of cardiac failure as well as extracellular/matricellular proteins. This ensemble constitutes a proteome fingerprint of myocardial infarction induced by LAD ligation in mice. Cathepsin A inhibitor treatment normalized the marked increase of the muscle stress marker CA3 as well as of Igγ 2b and fatty acid synthase. For numerous further proteins, cathepsin A inhibition partially dampened the LAD ligation-induced proteome alterations. CONCLUSIONS: Our proteomic and functional data suggest that cathepsin A inhibition has cardioprotective properties and support a beneficial effect of cathepsin A inhibition in the treatment of heart failure after myocardial infarction.

Identification of a novel AS160 splice variant that regulates GLUT4 translocation and glucose-uptake in rat muscle cells.

AS160 (AKT substrate of 160 kDa) is an important mediator of GLUT4 (glucose transporter 4) translocation and glucose-uptake in adipocytes and muscle cells. In our study we have identified a novel splice variant of AS160 (variant 2 of AS160, AS160_v2) that lacks exon 11 and 12. The protein is phosphorylated in response to insulin via the PI3K/AKT pathway. Expression of this splice variant in human tissues from different donors was examined with quantitative RT-PCR. Our data reveal a tissue specific distribution pattern of both isoforms with highest overall expression of AS160_v2. To investigate the function of the novel splice variant we established the doxycycline-inducible expression of the protein in a rat myoblast cell line co-expressing GLUT4-myc. In contrast to data reported for the full-length AS160 protein, over expression and activation of transcript variant 2 in this cell line increased GLUT4 translocation and glucose-uptake rates in response to insulin and IGF-1 but not in response to AICAR or metformin. Immunofluorescence based studies indicated a direct association of AS160_v2 with GLUT4 under basal but not under insulin-stimulated conditions. Additionally, over expression of AS160_v2 slightly improved glucose-uptake rates in a model of insulin resistance but was not able to fully prevent induction of insulin resistance. This was accompanied with decreased phosphorylation of AS160_v2 and AKT. Taken together, our data suggest a tissue specific distribution of full-length AS160 and the novel AS160 splice variant (AS160_v2) indicating different functions. In contrast to full-length AS160, transcript variant 2 of AS160 seems to be a novel regulator of glucose transport that positively influences glucose-uptake rates.