Caloric restriction, among other behavioral interventions, has demonstrated benefits on improving glycemic control in obesity-associated diabetic subjects. However, an acute and severe intervention without proper maintenance could reverse the initial benefits, with additional metabolic derangements. To assess the effects of an acute caloric restriction in a metabolic syndrome model, a cohort of 15-week old Long Evans Tokushima Otsuka (LETO) and Otsuka Long Evans Tokushima Fatty (OLETF) rats were calorie restricted (CR: 50% × 10 days) with or without a 10-day body mass (BM) recovery period, along with their respective ad libitum controls. An oral glucose tolerance test (oGTT) was performed after CR and BM recovery. Both strains had higher rates of mass gain during recovery vs. ad lib controls; however, the regain was partial (ca. 50% of ad lib controls) over the measurement period. Retroperitoneal and epididymal adipose masses decreased 30% (8.8 g, P < 0.001) in OLETF; however, this loss only accounted for 11.5% of the total BM loss. CR decreased blood glucose AUC 16% in LETO and 19% in OLETF, without significant decreases in insulin. Following CR, hepatic expression of the gluconeogenic enzyme, PEPCK, was reduced 55% in OLETF compared to LETO, and plasma triglycerides (TG) decreased 86%. Acute CR induced improvements in glucose tolerance and TG suggestive of improvements in metabolism; however, partial recovery of BM following CR abolished the improvement in glucose tolerance. The present study highlights the importance of proper maintenance of BM after CR as only partial recovery of the lost BM reversed benefits of the initial mass loss.

Disordered activation of the Renin‐Angiotensin‐Aldosterone‐System (RAAS) is associated with hypertension and insulin resistance. The hormones Angiotensin II (Ang II) and Aldosterone (Aldo) are two of its principal effector molecules that have been shown to regulate sodium and volume homeostasis leading to blood pressure control. Ang II has been shown to activate endothelial cells and increase the secretion of extracellular protein disulfide isomerase (PDI). PDI is a multifunctional protein critical to thrombus formation and regulation of reactive oxygen species among other functions. Our group has recently reported that caloric restriction (CR) in rodents leads to increases in Aldo secretion from adrenal zona glomerulosa cells. However, the effects of CR on PDI are unknown. We hypothesize that CR, due to its association with activating RAAS, will lead to increases in the circulating PDI activity. To test our hypothesis, we propose to study the in vitro effects of AngII and Aldo on PDI activity. We will study the in vivo effects of CR on the circulating activity of PDI in Long‐Evans Tokushima Fatty (LETO) and Otsuka Long‐Evans Tokushima Fatty (OLETF). We use two assays to measure PDI activity. In one approach we will use insulin degradation assays. The measurement of circulating PDI activity will be further validated with a fluorescent of Di‐E‐GSSG assay. Initial studies have shown specificity of our assay using the insulin degradation assay and recombinant PDI. Controls of our fluorescent assay are currently underway. We expect that CR will be associated with increased PDI activity.Support or Funding InformationK.V. is supported by an NIH MARC grant R25HL121029 and by the Division of Medical Sciences.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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Activation of the Renin-Angiotensin-Aldosterone-System (RAAS) is proposed to play a role in the development of insulin resistance and type II diabetes. Angiotensin II (AngII) is a principal effector molecule that binds AT1 receptors (AT1R) in various tissues. Recent data shows that activated endothelial cells secrete protein disulfide isomerase (PDI), a multifunctional protein that has been shown to be critical to the initiation and regulation of thrombus formation. However, the effects of RAAS on PDI regulation are unknown. We hypothesized that RAAS activation would lead to increased PDI levels thus contributing to inflammation. First, we studied the in vitro effects of AngII on EA.hy926 human endothelial cells and measured PDI activity. Our results show that AngII increased PDI activity; an event that was blocked by preincubation with 0.5 M losartan, an AT1R antagonist (ARB) (P<0.05, n=6). We then studied the in vivo effects of exogenous AngII infusion in Sprague–Dawley rats under the following conditions: (1) control; (2) AngII infused; (3) AngII + ARB. In these rats, AngII infusion led to significant increases in plasma PDI levels that were partially prevented by ARB treatment (P<0.05, n>5). We then studied the obese Otsuka Long Evans Tokushima Fatty (OLETF) (n = 6/group) rats, a model of naturally increased AngII and RAAS mediated insulin resistance and hypertension. OLETF and their lean strain controls were randomly assigned to the following groups: (1) untreated Long Evans Tokushima Otsuka (lean, control); (2) untreated OLETF; (3) OLETF + ARB. Our results show that OLETF rats had increased insulin resistance and significantly greater circulating PDI activity than control rats (P<0.05) that was likewise blocked by ARB treatment (P<0.05). To assess the relevance to humans of these findings we measured circulating PDI levels in patients with type 2 diabetes. In our cohort, PDI activity was significantly greater in patients with type 2 diabetes than non diabetics (P<0.001, n=134). Our data suggest that RAAS activation represents a novel mechanism for PDI secretion. Thus we posit that PDI may contribute to the deleterious effects of RAAS-mediated vascular disease.

Protein disulfide isomerase (PDI), a multifunctional oxidoreductase that regulates thrombus formation, leukocyte adherence to the endothelium and nitric oxide delivery, is present at high levels and regulates KCNN4 (Gardos Channel) activity in erythrocytes from humans with Sickle Cell Disease (SCD) and sickle mouse models. We reported evidence of elevated erythrocyte-surface associated and circulating PDI activity in humans and mouse models of SCD when compared to either wild-type mice, transgenic mice expressing normal human globins or otherwise healthy individuals; results that suggest a novel role for PDI in the pathophysiology of SCD through unclear mechanisms. We studied the effects of deoxygenation (5% O2) on PDI activity in endothelial cells and sickle erythrocytes from humans with SCD and two sickle transgenic knockout mouse models expressing human sickle hemoglobin, BERK and βSAntilles. Our data shows that deoxygenation of human and mouse sickle erythrocytes increased surface-associated reductive capacity that was sensitive to monoclonal antibodies against PDI (mAb PDI [RL-90]). We then studied sickle human erythrocytes and showed that PDI inhibitors (quercentin-3-rutinoside [Q3R] and mAb PDI) significantly reduced deoxy-stimulated sickle cell dehydration and Gardos channel activity (n=5; P <0.03). We characterized erythrocyte density with a phthalate density-distribution assay, generated density distribution curves and calculated the D50. Both mAb PDI and Q3R significantly reduced D50 when compared to vehicle (1.113±0.002 to 1.102±0.001 [P <0.0001]; and 1.101±0.002 [P <0.0001]; respectively). In contrast, incubation with exogenous PDI (3 nM) increased cellular dehydration (from D50= 1.110 ± 0.001 to D50= 1.115 ±0.001, P <0.01). We also measured the effect of hypoxia and endothelin-1 (ET-1; 10nM) in the human vascular endothelial cell line, EA.hy926. We observed that hypoxia induced PDI secretion that was further enhanced by co-incubation with ET-1 (10nM; n=3; P <0.05). The selective inhibitor of ET-1 subtype B receptor antagonist, BQ788 blocked ET-1 stimulated PDI increases in these cells as was previously reported in red blood cells. Consistent with these results hypoxia was associated with increased mRNA expression of MCP-1, VEGF-a but not ICAM by qRT-PCR and Taqman probes (n=3; P <0.05). Of importance we observed that early cultures of mouse aortic endothelial cells from BERK mice showed similar results. We then evaluated the effects of PDI on erythrocyte hemolysis by exposing cells from patients with SCD to 20 mM 2-2'-azo-bis- (2-amidinopropane) dihydrochloride (AAPH) with or without Q3R or mAb PDI. AAPH-induced hemolysis was dose-dependently blocked by Q3R (IC50: 4.1nM; n=6, P <0.0001 compared to vehicle) or mAb PDI (IC50: 11nM; n=3). Irrelevant IgG did not affect hemolysis under these conditions. Experiments performed in blood from BERK or βSAntilles mice showed similar results. We then studied sickle mice that express varying levels of HbF; BERK (<1% HbF), BERKγM (25% HbF), and BERKγH (45% HbF). BERKγH had the lowest circulating and cell associated PDI activity among the three mouse types that was associated with lower circulating ET-1 levels (n=2; P <0.05). Consistent with these results we observed an inverse correlation between levels of HbF and PDI activity in cells from humans with SCD (n=4). Thus we posit that in SCD elevated erythrocyte PDI activity is important for cell survival and stability and that its inhibition may represent a novel therapeutic approach for improving both the hematological and vascular complications of SCD as it may not only increase sickle erythrocyte survival but may likewise interfere with cellular adhesion leading to reduced vaso-occlusive episodes. [Supported by NIH: HL090632 (AR) and HL096518 (JRR)] Disclosures No relevant conflicts of interest to declare.