Effect of vitamin B12 supplementation on retinal lesions in diabetic rats.

Purpose: Diabetic retinopathy (DR) is the most common complication of diabetes involving microvasculature and neuronal alterations in the retina. Previously, we reported that vitamin B12 deficiency could be an independent risk factor for DR in humans. However, the effect of vitamin B12 supplementation in experimental DR is unknown. Thus, in this study, we investigated the impact of dietary supplementation of vitamin B12 on retinal changes in diabetic rats. Methods: Diabetes was induced in 2-month-old Sprague-Dawley rats and maintained for 4 months. One group of diabetic rats were fed normal levels of vitamin B12, and one group double the quantity of vitamin B12 (50 µg/kg diet). Vitamin B12 and homocysteine levels in the plasma were analyzed with radioimmunoassay (RIA) and high-performance liquid chromatography (HPLC), respectively. At the end of 4 months of experimentation, the eyeballs were collected. Retinal changes were analyzed with hematoxylin and eosin (H&E) staining, immunoblotting, and immunofluorescence methods. Results: Dietary supplementation of vitamin B12 had no effect on food intake, bodyweight, fasting blood glucose, and plasma homocysteine levels in the diabetic rats. However, vitamin B12 supplementation prevented loss of rhodopsin, and overexpression of VEGF, and completely prevented overexpression of HIF1α, GFAP, and endoplasmic reticulum (ER) stress markers (GRP78, ATF6α, XBP1, CHOP, and caspase 12) in the diabetic rat retina. Further, vitamin B12 ameliorated apoptosis in the retina as shown with terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) and prevented retinal thinning. Conclusions: Vitamin B12 supplementation of diabetic rats appeared to be beneficial by circumventing retinal hypoxia, VEGF overexpression, and ER stress-mediated cell death in the retina. The present study adds another potential therapeutic strategy of vitamin B12 in diabetes.

Circulating proteolytic signatures of chemotherapy-induced cell death in humans discovered by N-terminal labeling.

It is known that many chemotherapeutics induce cellular apoptosis over hours to days. During apoptosis, numerous cellular proteases are activated, most canonically the caspases. We speculated that detection of proteolytic fragments released from apoptotic cells into the peripheral blood may serve as a unique indicator of chemotherapy-induced cell death. Here we used an enzymatic labeling process to positively enrich free peptide α-amines in the plasma of hematologic malignancy patients soon after beginning treatment. This N-terminomic approach largely avoids interference by high-abundance proteins that complicate traditional plasma proteomic analyses. Significantly, by mass spectrometry methods, we found strong biological signatures of apoptosis directly in the postchemotherapy plasma, including numerous caspase-cleaved peptides as well as relevant peptides from apoptotic and cell-stress proteins second mitochondria-derived activator of caspases, HtrA serine peptidase 2, and activating transcription factor 6. We also treated hematologic cancer cell lines with clinically relevant chemotherapeutics and monitored proteolytic fragments released into the media. Remarkably, many of these peptides coincided with those found in patient samples. Overall, we identified 153 proteolytic peptides in postchemotherapy patient plasma as potential indicators of cellular apoptosis. Through targeted quantitative proteomics, we verified that many of these peptides were indeed increased post- vs. prechemotherapy in additional patients. Our findings reveal that numerous proteolytic fragments are released from dying tumor cells. Monitoring posttreatment proteolysis may lead to a novel class of inexpensive, rapid biomarkers of cell death.

The ATF6-Met[67]Val substitution is associated with increased plasma cholesterol levels.

OBJECTIVE: Activating transcription factor 6 (ATF6) is a sensor of the endoplasmic reticulum stress response and regulates expression of several key lipogenic genes. We used a 2-stage design to investigate whether ATF6 polymorphisms are associated with lipids in subjects at increased risk for cardiovascular disease (CVD). METHODS AND RESULTS: In stage 1, 13 tag-SNPs were tested for association in Dutch samples ascertained for familial combined hyperlipidemia (FCHL) or increased risk for CVD (CVR). In stage 2, we further investigated the SNP with the strongest association from stage 1, a Methionine/Valine substitution at amino-acid 67, in Finnish FCHL families and in subjects with CVR from METSIM, a Finnish population-based cohort. The combined analysis of both stages reached region-wide significance (P=9 x 10(-4)), but this association was not seen in the entire METSIM cohort. Our functional analysis demonstrated that Valine at position 67 augments ATF6 protein and its targets Grp78 and Grp94 as well as increases luciferase expression through Grp78 promoter. CONCLUSIONS: A common nonsynonymous variant in ATF6 increases ATF6 protein levels and is associated with cholesterol levels in subjects at increased risk for CVD, but this association was not seen in a population-based cohort. Further replication is needed to confirm the role of this variant in lipids.