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Bariatric surgery is emerging as an effective treatment for obesity and the metabolic syndrome. Recently, we demonstrated that Roux-en-Y gastric bypass (RYGB), but not vertical sleeve gastrectomy (VSG), resulted in improvements to white adipose physiology and enhanced brown adipose functioning. Since beneficial alterations to liver health are also expected after bariatric surgery, comparing the post-operative effects of RYGB and VSG on liver physiology is essential to their application in the treatment of non-alcoholic fatty liver disease (NAFLD).The effects of RYGB and VSG on liver physiology were compared using diet induced mouse model of obesity. High-fat diet (HFD) was administered for 12 weeks after surgery and alterations to liver physiology were assessed.Both RYGB and VSG showed decreased liver weight as well as reductions to hepatic cholesterol and triglyceride levels. There were demonstrable improvements to NAFLD activity score (NAS) and fibrosis stage scoring after both surgeries. In RYGB, these beneficial changes to liver function resulted from the downregulation of pro-fibrotic and upregulation anti-fibrotic genes, as well as increased fatty acid oxidation and bile acid flux. For VSG, though similar alterations were observed, they were less potent. However, VSG did significantly downregulate pro-fibrotic genes and showed increased glycogen content paralleled by decreased glycogenolysis which may have contributed to the resolution of NAFLD.RYGB and VSG improve liver physiology and function, but RYGB is more efficacious. Resolutions of NAFLD in RYGB and VSG are achieved through different processes, independent of weight loss.

The obesity epidemic is associated with an increased prevalence and severity of type 2 diabetes, cardiovascular disease, and nonalcoholic fatty liver disease (NAFLD) . Bariatric surgery has become a widely accepted treatment for individuals with severe obesity and obesity related health problems. Previous studies from our lab have implicated the role of lipocalin-type prostaglandin D2 synthase (L-PGDS) in adipose tissue dysfunction and NAFLD. Our lab has also shown the effect of Roux-en Y gastric bypass (RYGB) on body weight changes, improvement to insulin sensitivity and adipose tissue physiology. In the current study, we examined the effects of L-PGDS using both diet-induced obesity (DIO) and RYGB models. We found reduced expression of L-PGDS in liver of DIO mice after 14 weeks on high fat diet. We then performed RYGB surgery in DIO mice and compared the effects of diets (normal chow and HFD for 6 weeks) on post-operative outcomes of RYGB. Our results show that both surgery and diet contribute to the substantial improvement in liver weight and HOMA-IR. H&E images of liver sections also demonstrate that both diet and surgery play significant roles in reversal of NAFLD. Interestingly, we only found significant increase to liver L-PGDS expression after RYGB when HFD is administered. We conclude that both surgery and diet plays a significant role in modulating the expression of L-PGDS in liver of obese mice. Thus, our study shows that L-PGDS may serve as a novel therapeutic target in treatment of obesity and NAFLD. Disclosure A.Srivastava: None. M.Stevenson: None. T.Palaia: None. C.Hall: None. J.Lee: None. L.Ragolia: None. Funding American Heart Association: (#15GRNT22420001) and The George Link Foundation

Understanding the effects of Roux-en-Y gastric bypass (RYGB) and vertical sleeve gastrectomy (VSG) on adipose tissue physiology is important for the treatment of obesity-related metabolic disorders. By using robust mouse models of bariatric surgery that closely resemble those performed in humans, we can compare the effects of RYGB and VSG on adipose physiology in the absence of post-operative confounds such as diet and lifestyle changes. RYGB and VSG were compared using a diet-induced mouse model of obesity. High-fat diet (HFD) was administered post-operatively and changes to white and brown adipose tissue were evaluated, along with alterations to weight, glucose homeostasis, dyslipidemia, and insulin sensitivity. After prolonged exposure to high-fat diet post-operatively, RYGB was effective in achieving sustained weight loss, while VSG unexpectedly accelerated weight gain rates. The resolution of obesity-related comorbidities such as glucose and insulin intolerance, dyslipidemia, and insulin sensitivity was improved after RYGB, but not for VSG. In RYGB, there were improvements to the function and health of white adipose tissue, enhanced brown adipose metabolism, and the browning of subcutaneous white adipose tissue, with no comparable changes seen for these factors after VSG. Some markers of systemic inflammation improved after both RYGB and VSG. There are significantly different effects between RYGB and VSG when HFD is administered post-operatively and robust mouse models of bariatric surgery are used. RYGB resulted in lasting physiological and metabolic changes but VSG showed little difference from that of its sham-operated, DIO counterpart.

Although coronavirus disease 2019 (COVID-19) is transmitted via respiratory droplets, there are multiple gastrointestinal and hepatic manifestations of the disease, including abnormal liver-associated enzymes. However, there are not many published articles on the pathological findings in the liver of patients with COVID-19. We collected the clinical data from 17 autopsy cases of patients with COVID-19 including age, sex, Body mass index (BMI), liver function test (alanine aminotransaminase (ALT), aspartate aminotransaminase (AST), alkaline phosphatase (ALP), direct bilirubin, and total bilirubin), D-dimer, and anticoagulation treatment. We examined histopathologic findings in postmortem hepatic tissue, immunohistochemical (IHC) staining with antibody against COVID-19 spike protein, CD68 and CD61, and electron microscopy. We counted the number of megakaryocytes in liver sections from these COVID-19-positive cases. Abnormal liver-associated enzymes were observed in 12 of 17 cases of COVID-19 infection. With the exception of three cases that had not been tested for D-dimer, all 14 patients' D-dimer levels were increased, including the cases that received varied doses of anticoagulation treatment. Microscopically, the major findings were widespread platelet-fibrin microthrombi, steatosis, histiocytic hyperplasia in the portal tract, mild lobular inflammation, ischemic-type hepatic necrosis, and zone 3 hemorrhage. Rare megakaryocytes were found in sinusoids. COVID-19 IHC demonstrates positive staining of the histiocytes in the portal tract. Under electron microscopy, histiocyte proliferation is present in the portal tract containing lipid droplets, lysosomes, dilated ribosomal endoplasmic reticulum, microvesicular bodies, and coronavirus. The characteristic findings in the liver of patients with COVID-19 include numerous amounts of platelet-fibrin microthrombi, as well as various degrees of steatosis and histiocytic hyperplasia in the portal tract. Possible mechanisms are also discussed.

Cerebral cavernous malformations (CCMs) are enlarged leaking vascular lesions in the brain caused by loss-of-function mutations in CCM1/2/3 genes or loss of expression. Although we previously showed that Nogo-B receptor (NgBR) knockout in endothelial cells (ECs) results in CCMs-like cerebrovascular lesions in the mouse embryo, the molecular mechanism by which NgBR regulates CCM1/2 expression has not been elucidated. Here, we show that temporal genetic depletion of NgBR in ECs at both the postnatal and adult stages results in CCM1/2 expression deficiency and consequently CCMs-like lesions such as enlarged vessels, blood-brain barrier (BBB) hyperpermeability, and intracerebral hemorrhage. These cerebrovascular defects in the brain of NgBR endothelial-specific knockout (ecKO) mice can be rescued by adeno-associated virus (AAV)-mediated overexpression of CCM1 and CCM2 genes. To reveal the molecular mechanism, we used RNA-seq analysis to examine changes in the transcriptome. Surprisingly, we found that acetyltransferase HBO1 was downregulated in NgBR deficient ECs. The mechanistic study elucidated that NgBR is required for maintaining the expression of CCM1/2 in ECs via HBO1-mediated histone acetylation. ChIP-qPCR data further demonstrated that loss of NgBR impairs the binding of both the HBO1 and acetylated H4K5/K12 at the promoter of CCM1 and CCM2 genes. Similarly, AAV-mediated overexpression of HBO1 restores the acetylation of H4K5/K12 and rescues the CCMs-like cerebrovascular defects in the brain of NgBR ecKO mice. Our findings on epigenetic regulation of CCM1 and CCM2 provide a perspective that NgBR and HBO1-mediated histone H4 acetylation may be targeted for preventing the onset of CCMs-like cerebrovascular disease.

Cerebral cavernous malformations (CCMs) are enlarged leaking vascular lesions in the brain caused by loss-of-function mutations in CCM1/2/3 genes or loss of expression. Although we previously showed that Nogo-B receptor (NgBR) knockout in endothelial cells (ECs) results in CCMs-like cerebrovascular lesions in the mouse embryo, the molecular mechanism by which NgBR regulates CCM1/2 expression has not been elucidated. Here, we show that temporal genetic depletion of NgBR in ECs at both the postnatal and adult stages results in CCM1/2 expression deficiency and consequently CCMs-like lesions such as enlarged vessels, blood-brain barrier (BBB) hyperpermeability, and cerebral hemorrhage. These cerebrovascular defects in the brain of NgBR endothelial-specific knockout (ecKO) mice can be rescued by adeno-associated virus (AAV)-mediated overexpression of CCM1 and CCM2 genes. To reveal the molecular mechanism, we used RNA-seq analysis to examine changes in the transcriptome. Surprisingly, we found that acetyltransferase HBO1 was downregulated in NgBR deficient ECs. The mechanistic study elucidated that NgBR is required for maintaining the expression of CCM1/2 in ECs via HBO1-mediated histone acetylation. ChIP-qPCR data further demonstrated that loss of NgBR impairs the binding of both the HBO1 and acetylated H4K5/K12 at the promoter of CCM1 and CCM2 genes. Similarly, AAV-mediated overexpression of HBO1 restores the acetylation of H4K5/K12 and rescues the CCMs-like cerebrovascular defects in the brain of NgBR ecKO mice. Our findings on epigenetic regulation of CCM1 and CCM2 provide a perspective that NgBR and HBO1-mediated histone H4 acetylation may be targeted for preventing the onset of CCMs-like cerebrovascular disease.

Introduction: Lipases constitute an important class of water-soluble enzymes that catalyze the hydrolysis of hydrophobic triacylglycerol (TAG). Their activity is usually measured after isolation and quantification of the hydrolyzed free fatty acids. We have used NBD-labeled lipids previously to develop TAG, cholesteryl ester, and phospholipid transfer assays for microsomal triglyceride transfer protein. To date, however, NBD-labeled TAG (NBD-TAG) has not been used to assess the enzymatic activities of different lipases. Methods: We developed methods to measure lipase activities using adipose triglyceride lipase (ATGL), lipoprotein lipase (LpL), and pancreatic triglyceride lipase (PNLIP) as model lipases. In these assays, we incubated a source of ATGL, LpL, or PNLIP with substrate emulsions containing nitrobenzoxadiazole (NBD)-labeled TAG in phosphatidylcholine (PC) and phosphatidylinositol (PI), measured increases in NBD fluorescence with time, and calculated enzyme activities. Results: Incorporation of NBD-TAG into PC vesicles resulted in some hydrolysis; but required product isolation step to measure NBD fluorescence. However, incorporation of PI into these NBD-TAG/PC vesicles significantly increased substrate hydrolysis in time, protein, and substrate concentration dependent manner negating the need to isolate products to measure lipase activities. Further, increasing the ratio of NBD-TAG to PC enhanced substrate hydrolysis. Next, we tested specific lipase inhibitors and found that orlistat inhibits all three enzymes indicating that it is a pan-lipase inhibitor. Conclusions: We describe a simple, rapid fluorescence-based TAG hydrolysis assay to assess three major TAG hydrolases: ATGL localized intracellularly, LpL localized at the extracellular endothelium, and PNLIP produced and secreted from the pancreas into the intestinal lumen. The major advantages of this method are its speed, simplicity, and avoidance of a product isolation step. This assay is potentially applicable to a wide range of lipases and is amenable to high-throughput screening to discover novel modulators of triacylglycerol hydrolases, and in the diagnosis of diseases associated with increases in plasma lipase activities.

Adipose tissue dysfunction is the primary defect in obesity which contributes to the development of dyslipidemia, insulin resistance, cardiovascular diseases, type 2 diabetes, nonalcoholic fatty liver disease and some cancers. Previous study from our lab implicated the role of lipocalin-type prostaglandin D2 synthase (L-PGDS) in glucose intolerance and atherosclerosis. Our lab also showed the development of NAFLD in L-PGDS knockout mice on both low fat and high fat diet. In the present study, we examined the role of L-PGDS in adipose tissue in response to high fat diet. We found decreased expression of L-PGDS in visceral and subcutaneous white adipose tissue (WAT) of C57BL/6 mice after 12 weeks on high fat diet (HFD). Our study showed increased fat content in L-PGDS KO mice as compared to control C57BL/6 mice after 14 weeks on HFD. Histological analysis showed hypertrophied adipocytes in visceral WAT of L-PGDS KO mice as compared to control mice. Furthermore, we found increased expression of the lipogenesis genes LXRα, SREBP-1c and FAS in visceral WAT of L-PGDS KO mice after 14 weeks on HFD. L-PGDS KO mice showed reduced gene expression of adiponectin and PPARγ in visceral WAT on HFD. The plasma adiponectin level was also decreased in L-PGDS KO mice. We conclude that absence of L-PGDS has a deleterious effect on adipose tissue functioning which further reduces insulin sensitivity in adipose tissue. Thus, our study suggests L-PGDS as a novel therapeutic target for the treatment of obesity-associated metabolic syndrome. Disclosure A. Srivastava: None. T. Palaia: None. C. Hall: None. J. Lee: None. M. Stevenson: None. L. Ragolia: None. Funding American Heart Association (15GRNT22420001); George Link, Jr. Foundation Inc. (3500-7294)

The loss function of cerebral cavernous malformation (CCM) genes leads to most CCM lesions characterized by enlarged leaking vascular lesions in the brain. Although we previously showed that NOGOB receptor (NGBR) knockout in endothelial cells (ECs) results in cerebrovascular lesions in the mouse embryo, the molecular mechanism by which NGBR regulates CCM1/2 expression has not been elucidated. Here, we show that genetic depletion of Ngbr in ECs at both postnatal and adult stages results in CCM1/2 expression deficiency and cerebrovascular lesions such as enlarged vessels, blood-brain-barrier hyperpermeability, and cerebral hemorrhage. To reveal the molecular mechanism, we used RNA-sequencing analysis to examine changes in the transcriptome. Surprisingly, we found that the acetyltransferase HBO1 and histone acetylation were downregulated in NGBR-deficient ECs. The mechanistic studies elucidated that NGBR is required for maintaining the expression of CCM1/2 in ECs via HBO1-mediated histone acetylation. ChIP-qPCR data further demonstrated that loss of NGBR impairs the binding of HBO1 and acetylated histone H4K5 and H4K12 on the promotor of the CCM1 and CCM2 genes. Our findings on epigenetic regulation of CCM1 and CCM2 that is modulated by NGBR and HBO1-mediated histone H4 acetylation provide a perspective on the pathogenesis of sporadic CCMs.

The prevalence of obesity has increased steadily over the last few decades and is a major risk factor for type 2 diabetes and cardiovascular disease. Obesity is associated with alterations in adipose tissue functioning leading to metabolic dysfunction. In previous studies, we have reported the role of lipocalin-type prostaglandin D2 synthase (L-PGDS) in glucose intolerance and atherosclerosis. More recently, we demonstrated that L-PGDS KO mice develop NAFLD even on a low fat diet. Our present study explored the role of L-PGDS in adipose tissue. We found decreased expression of L-PGDS in insulin resistant 3T3-L1 adipocytes. Enzymatic inhibition of L-PGDS in 3T3-L1 adipocytes using AT56 impaired insulin signaling by reducing the phosphorylation of AKT protein and the expression and translocation of GLUT4 to plasma membrane. Furthermore, we found that inhibition of L-PGDS reduces the expression of adiponectin and AMPK phosphorylation in 3T3-L1 adipocytes. Rosiglitazone treatment in insulin resistant 3T3-L1 adipocytes significantly increased L-PGDS expression. Treatment with rosiglitazone restored the expression of adiponectin and phosphorylation of AMPK in AT56 treated 3T3-L1 adipocytes. Correspondingly, we found decreased L-PGDS expression in visceral fat of obese humans. Cumulatively, these data confirm that the absence of L-PGDS results in decreased insulin sensitivity via impaired insulin signaling and reduced adiponectin levels in 3T3-L1 adipocytes. Thus, our findings suggest that L-PGDS plays a significant role in maintaining the insulin sensitivity in 3T3-L1 adipocytes and provides a novel opportunity for therapeutic intervention of obesity-associated metabolic syndrome. Disclosure A. Srivastava: None. T. Palaia: None. J. Lee: None. C. Hall: None. M. Stevenson: None. A.G. Fernando: None. L. Ragolia: None. Funding American Heart Association (15GRNT22420001); George Link, Jr. Foundation, Inc.

Background Glucagon-like peptide-1 (GLP-1) level was significantly increased post Vertical Sleeve Gastrectomy (VSG), an effect believed to contribute to its beneficial cardiometabolic effects. Objective To validate the beneficial GLP-1 mediated cardiometabolic effects post VSG using GLP-1 antagonist (exendin 9-39) in Zucker diabetic fatty rats. Methods Animals were divided into three (n = 5) groups: (i) sham, (ii) VSG, and (iii) VSG received exendin 9–39 (GLP-1 receptor antagonist). The study was performed over 12 weeks and parameters were measured 12 weeks post-surgery. Results and discussion As expected, fasting blood glucose and insulin levels were improved post VSG due to enhanced GLP-1 secretion. However, both fasting glucose and insulin levels were impaired in the presence of GLP-1 antagonist. Baseline total cholesterol level pre-surgery was 100±1 mg/dl which remained unchanged in the VSG group but significantly increased to 140±8 mg/dl in the presence of antagonist. Interestingly, post-surgery there was a nearly 70% reduction in triglyceride level in the VSG group compared to sham which was overcome in the presence of antagonist. Myographic studies using aortic rings showed no significant change between groups. Additionally, blood pressure and heart rate also remained unchanged in all groups. Serum bile acid and L-PGDS levels increased post VSG but significantly decreased in the presence of antagonist, suggesting a strong association with GLP-1 and a novel mechanism of action. Conclusion Enhanced GLP-1 secretion post VSG imparted beneficial cardiometabolic effects on blood glucose, insulin, total cholesterol, triglyceride, bile acids and L-PGDS levels which were abated in the presence of GLP-1 antagonist., Highlights • GLP-1 increases post-VSG 30 min after glucose load. • Post-VSG GLP-1 secretion is associatged with lower cholesterol and triglycerides. • Bile acids and L-PGDS increase post-VSG and are inhibited in the presence of GLP-1 antagonist. • Heart rate, blood pressure and myograph profile remain unchanged.

Non-alcoholic fatty liver disease (NAFLD) is an emerging risk factor for type 2 diabetes mellitus, cardiovascular disease, and all-cause mortality. Previously, we demonstrated that lipocalin-type prostaglandin D

Nonalcoholic fatty liver disease (NAFLD) is an emerging risk factor for type 2 diabetes mellitus (T2DM), cardiovascular disease, and all-cause mortality. Obesity is a major determinant of the high prevalence of NAFLD and T2DM worldwide. Previously, we demonstrated that lipocalin-type prostaglandin D2 synthase (L-PGDS) knockout mice show increased glucose intolerance and accelerated atherosclerosis. In the present study, we investigated the role of L-PGDS in causing NAFLD in L-PGDS knockout (KO) and control C57BL/6 mice (n=6/group) on both low fat and high fat diets for 14 weeks. We observed that L-PGDS KO mice remain slightly lighter in weight compared to control mice, yet develop NAFLD faster, even on the low fat diet. In addition, we found elevated fasting glucose and insulin levels in L-PGDS KO mice with increased lipid accumulation in the liver with time on both diets as compared to controls. We also found total cholesterol and LDL levels were significantly elevated in L-PGDS KO mice. Finally, lipogenesis marker proteins such as SREBP-1c and LXRα, and CD36, a fatty acid transporter protein, were also found to be increased in L-PGDS KO mice on both diets as compared to control mice. These data confirm that absence of L-PGDS results in hyperinsulinemia and dyslipidemia. Therefore, we conclude that L-PGDS plays a significant role in developing NAFLD independent of obesity and may serve as a novel therapeutic target for the treatment of NAFLD. Disclosure A. Srivastava: None. T. Palaia: None. C. Hall: None. J. Lee: None. M. Stevenson: None. L. Ragolia: None. Funding George Link, Jr. Foundation, Inc.; American Heart Association

In Latin, placebo translates to “I shall please” and was used in the eighth century as part of a psalm sung at funerals. The term was initially used in medicine in the late eighteenth century to indicate a commonplace method or medicine. By the early nineteenth century, it was used to refer to any medicine adapted more to please than to benefit the patient. Modern usage typically refers to a drug or therapy that simulates medical treatment, but has no specific action on the condition being treated. It is important to note, however, that placebo can also refer to the response experienced by the patient and not only the word for a seemingly inert stimulus. Beecher quantified the placebo effect at 35% based on 15 uncontrolled observational studies. While later studies have estimated this number as both higher and lower, the Beecher placebo effect size is the most commonly cited number.

Background Vertical sleeve gastrectomy (VSG) ameliorates metabolic complications in obese and diabetic patients through unknown mechanisms. Objective The objective of this study was to investigate the role of lipocalin-type prostaglandin D 2 synthase (L-PGDS) in glucose regulation in response to VSG using L-PGDS knock-out (KO), knock-in (KI), and C57BL/6 (wild type) mice. Setting Winthrop University Hospital Research Institute. Methods Animals were divided into 6 groups: L-PGDS KO sham/VSG (n = 5), L-PGDS KI sham/VSG (n = 5), and C57BL/6 (wild type) sham/VSG (n = 5). Related parameters were measured in fasting animals after 10 weeks. Results Our intraperitoneal glucose tolerance tests and homeostatic model assessment insulin resistance results showed significant glycemic improvement 10 weeks post-VSG in both C57BL/6 and KI groups compared with the sham group. In contrast, the KO group developed glucose intolerance and insulin resistance similar to or greater than the sham group 10 weeks post-VSG. Interestingly, weight gain was insignificant 10 weeks post-VSG in all the groups and even trended higher in the KO group compared with sham. Peptide YY levels in the KO group post-VSG were slightly increased but significantly less than other groups. Similarly, the KO group showed significantly less leptin sensitivity in response to VSG compared with the KI group. Total cholesterol level remained unchanged in all groups irrespective of sham or surgery but interestingly, the KO group had significantly higher cholesterol levels. In parallel, adipocyte size was also found to be significantly increased in the KO group post-VSG compared with the sham group. Conclusion Our findings propose that L-PGDS plays an important role in the beneficial metabolic effects observed after VSG.

Non-alcoholic fatty liver disease (NAFLD) is an emerging risk factor for type 2 diabetes mellitus, cardiovascular disease, and all-cause mortality. Previously, we demonstrated that lipocalin-type prostaglandin D2 synthase (L-PGDS) knockout mice show increased glucose intolerance and accelerated atherosclerosis. In the present study, we investigated the role of L-PGDS in mediating NAFLD utilizing L-PGDS knockout (KO) and control C57BL/6 mice fed either low fat (LFD) or high fat diet (HFD) for 14 weeks. Our present study demonstrates that L-PGDS KO mice remain slightly lighter in weight compared to control mice, yet develop NAFLD faster and eventually progress to the more severe non-alcoholic steatohepatitis (NASH). We found increased lipid accumulation in the liver of KO mice over time on both diets, as compared to control mice. The L-PGDS KO mice showed elevated fasting glucose and insulin levels and developed insulin resistance on both LFD and HFD. Lipogenesis marker proteins such as SREBP-1c and LXRα were increased in L-PGDS KO mice after 14 weeks on both diets, when compared to control mice. We replicated our in vivo findings in vitro using HepG2 cells treated with a combination of free fatty acids (oleic and palmitic acid) and exposure to a L-PGDS inhibitor and prostaglandin D2 receptor (DP1) antagonists. We conclude that the absence or inhibition of L-PGDS results in dyslipidemia, altered expression of lipogenesis genes and the acceleration of NAFLD to NASH, independent of diet and obesity. We propose L-PGDS KO mice as a useful model to explore the pathogenesis of NAFLD and NASH, and L-PGDS as a potential therapeutic target for treatment.

Hepatosteatosis is strongly associated with hyperinsulinaemia and obesity. Previously, we demonstrated accelerated atherosclerosis and impaired glucose tolerance in L-PGDS knockout (KO) mice. Interestingly, L-PGDS KO mice also exhibit hyperinsulinemia on high fat diet in a time-dependent manner. In this study, we investigated the role of L-PGDS on liver pathophysiology using L-PGDS KO mice compared to control C57BL/6 mice) (n=6/group). Mice were kept on high fat diet (60% fat) for 14 weeks and all parameters were measured initially and at 4-, 8- and 14-weeks post high fat diet. Data were analyzed by unpaired t-test or one-way ANOVA where appropriate with p Disclosure S. Kumar: None. T. Palaia: None. C. Hall: None. J. Lee: None. M. Stevenson: None. L. Ragolia: None.

Background: Lipocalin prostaglandin D2 synthase (LPGDS) is a bile acid binding protein that has been found to have a significant role in glucose metabolism and appetite. In performing sleeve gastrectomy (SG) and gastric bypass in rodent models, we have also demonstrated LPGDS appears to have an important role in the beneficial metabolic effects seen after surgery. We have sought to correlate if LPGDS has a significant clinical effect in humans for surgical weight loss. Methods: Serum LPGDS levels were obtained in patients undergoing SG before, and then one month following surgery. Serum LPGDS was also measured in subjects undergoing a very low calorie diet (VLCD) program before, and then one month following the start of the program. LPGDS levels were measured by ELISA and analyzed. Results: Subjects that underwent surgical loss went from 304.45 +/-74 pounds before surgery to 271.4 +/- 70 pounds one month following surgery. Subjects that underwent VLCD went from 273.1 +/- 65 pounds before the program to 255 +/-61 pounds one month after starting the program. LPGDS levels for surgical weight loss increased from 579.9 +/-161 ng/mL before the program to 694.6 +/- 448 ng/mL one month after surgery. LPGDS for VLCD was 583.1 +/- 187 ng/mL before the program and 598.4 +/- 165 ng/mL one month after starting the program. Conclusion: LPGDS levels appear to increase after sleeve gastrectomy but not VLCD, although this trend is not statistically significant. Further study is needed to confirm this trend. Disclosure R.G. Lau: None. S. Kumar: None. J. Lee: None. T. Palaia: None. S. Islam: None. C. Brathwaite: None. L. Ragolia: None.

Diabetes is associated with disturbances in the normal levels of both insulin and glucagon, both of which play critical roles in the regulation of glycemia. Recent studies have found lipocalin-type prostaglandin D2 synthase (l-PGDS) to be an emerging target involved in the pathogenesis of type-2 diabetes. This study focused on the effect of l-PGDS on glucagon secretion from cultured pancreatic Alpha TC-1 Clone 6 cells. When cells were treated with various concentrations of l-PGDS (0, 10, 50, and 100 ug/ml) for 2 h in 1 mM glucose; glucagon secretion decreased to 670±45, 838±38, 479±11, and 437±45 pg/ml, respectively. In addition, pancreatic islets were isolated from C57BL/6 mice and stained for prostaglandin D2 receptors, DP1 and DP2, using immunohistochemistry. Our results showed that these islets express only the DP1 receptor. Pancreatic islets were then stained for alpha and beta cells, as well as DP1, to find the primary location of the receptor within the islets using immunofluorescence. Interestingly, DP1 receptor density was found primarily in alpha cells rather than in beta cells. Our study is the first to report a correlation between l-PGDS and glucagon secretion in alpha cells. Based on our obtained results, it can be concluded that higher concentrations of l-PGDS significantly reduced the secretion of glucagon in alpha cells, which may contribute to the pathogenesis of diabetes as well as offer a novel therapeutic site for the treatment of diabetes., Highlights • Alpha TC1 Clone 6) derived from C57BL/6 mice express DP1 receptor. • High [l-PGDS] significantly reduce the secretion of glucagon in alpha cells. • DP1 receptor density is found primarily in alpha cells rather than beta cells.

Inflammation plays a crucial role in neurodegenerative diseases, but the irritants responsible for this response remain largely unknown. This report addressed the hypothesis that hypochlorous acid reacts with dopamine to produce melanic precipitates that promote cerebral inflammation. Spectrophotometric studies demonstrated that nM amounts of HOCl and dopamine react within seconds. A second-order rate constant for the reaction of HOCl and dopamine of 2.5 × 10(4)M(-1)s(-1) was obtained by measuring loss of dopaminergic fluorescence due to HOCl. Gravimetric measurements, electron microscopy, elemental analysis, and a novel use of flow cytometry confirmed that the major product of this reaction is a precipitate with an average diameter of 1.5 μm. Flow cytometry was also used to demonstrate the preferential reaction of HOCl with dopamine rather than albumin. Engulfment of the chlorodopamine particulates by phagocytes in vitro caused these cells to release TNFα and die. Intrastriatal administration of 10(6) particles also increased the content of TNFα in the brain and led to a 50% loss of the dopaminergic neurons in the nigra. These studies indicate that HOCl and dopamine react quickly and preferentially with each other to produce particles that promote inflammation and neuronal death in the brain.

The objective of the study was to investigate the role of prostaglandin D2 during pregnancy and its mediator Lipocalin-type prostaglandin D2 synthase (L-PGDS) as a predictor of preterm birth (PTB). Transgenic L-PGDS (+/+), L-PGDS (-/-) and C57BL/6 control pregnant mice models were used to determine the effect of DP1 and DP2 receptor antagonists in lipopolysaccharide (LPS)-induced PTB mice. In addition, L-PGDS levels were measured in the cervicovaginal secretions (CVS) of 370 pregnant women using ELISA and further processed for isoform detection using 2-D gel electrophoresis. Our results found that C57BL/6 control mice (n = 26), transgenic L-PGDS (+/+) (n = 26), demonstrated an 89% and 100% preterm birth in LPS (intraperitoneal injection, 20mg/kg) induced mice model respectively. Interestingly, the incidence of PTB was significantly reduced to 40% in L-PGDS (-/-) knockout mice (n = 26). DP1 and DP2 receptor antagonists (0.264 μg/day, dose of 0.1 μg/μl with the flow of 0.11 μl/h for 28 day using Alzet pumps) were used to investigate the effect in LPS-induced PTB in C57BL/6 mice and found 3.3-fold increase in viable pups after LPS-induction. In addition, L-PGDS levels were measured in CVS samples and found that PTB women (n = 296) had two-fold higher levels compared to full term births (n = 74) and established a significant inverse correlation between levels of L-PGDS and days to expected delivery by using 370 preterm birth CVS samples. Elevated L-PGDS levels in the CVS of women may be considered as a potential biomarker for PTB in future. Secondly, the use of DP1 and DP2 receptor antagonists may represent novel tocolytic agents for the treatment of PTB.

Summary Background/objective Lipocalin Prostaglandin D2 synthase (LPGDS) contributes to the production of PGD2, which has been associated with adipogenesis. In this study, we aimed to investigate the role of PGD2 on obesity through its DP1 and DP2 receptor signaling using intraperitoneal injection of their respective agonists and antagonists. Methods ApoE−/− mice were divided into five groups: vehicle control (n = 5), DP1 receptor agonist (n = 5), DP1 receptor antagonist (n = 5), DP2 receptor agonist (n = 5), and DP2 receptor antagonist (n = 5), and the study was carried out for 10 weeks. Results Despite being on high fat diet, mice receiving DP1 receptor agonist sustained a significant inhibition of weight gain throughout the study gaining only 11.4% body weight compared to the controls gaining 61% body weight. Interestingly, parallel to the body weight, the DP1 receptor agonist group showed a significant reduction in food intake throughout the study. Consistently, fasting leptin, insulin and bile acids levels were elevated in the DP1 receptor agonist group compared to controls. As expected, there was a significant reduction in fasting glucose level in DP1 receptor agonist group. At last, as a result of weight gain inhibition, DP1 receptor agonist also imparted cardiovascular benefits showing significant reduction in aortic wall thickness, intima, adventia and lumen size. Conclusion Based on the obtained results, we believe DP1 receptor agonism inhibited diet induced weight gain possibly through controlling appetite which consequently imparted beneficial cardiometabolic effects. DP1 receptor agonism may represent a novel therapeutic target for the management of obesity.

Vertical sleeve gastrectomy (VSG) ameliorates metabolic complications in obese and diabetic patients through unknown mechanisms.The objective of this study was to investigate the role of lipocalin-type prostaglandin DWinthrop University Hospital Research Institute.Animals were divided into 6 groups: L-PGDS KO sham/VSG (n = 5), L-PGDS KI sham/VSG (n = 5), and C57BL/6 (wild type) sham/VSG (n = 5). Related parameters were measured in fasting animals after 10 weeks.Our intraperitoneal glucose tolerance tests and homeostatic model assessment insulin resistance results showed significant glycemic improvement 10 weeks post-VSG in both C57BL/6 and KI groups compared with the sham group. In contrast, the KO group developed glucose intolerance and insulin resistance similar to or greater than the sham group 10 weeks post-VSG. Interestingly, weight gain was insignificant 10 weeks post-VSG in all the groups and even trended higher in the KO group compared with sham. Peptide YY levels in the KO group post-VSG were slightly increased but significantly less than other groups. Similarly, the KO group showed significantly less leptin sensitivity in response to VSG compared with the KI group. Total cholesterol level remained unchanged in all groups irrespective of sham or surgery but interestingly, the KO group had significantly higher cholesterol levels. In parallel, adipocyte size was also found to be significantly increased in the KO group post-VSG compared with the sham group.Our findings propose that L-PGDS plays an important role in the beneficial metabolic effects observed after VSG.

Background. Endothelial Microparticles (EMPs) are small vesicles shed from activated or apoptotic endothelial cells and involved in cellular cross-talk. Whether EMP immunophenotypes vary according to stimulus in Diabetes Mellitus (DM) is not known. We studied the cellular adhesion molecule (CAM) profile of circulating EMPs in patients with and without Diabetes Mellitus type 2, who were undergoing elective cardiac catheterization.Methods and Results. EMPs were analyzed by flow cytometry. The absolute median number of EMPs (EMPs/L) specific for CD31, CD105, and CD106 was significantly increased in the DM population. The ratio of CD62E/CD31 EMP populations reflected an apoptotic process.Conclusion. Circulating CD31+, CD105+, and CD106+ EMPs were significantly elevated in patients with DM. EMPs were the only independent predictors of DM in our study cohort. In addition, the EMP immunophenotype reflected an apoptotic process. Circulating EMPs may provide new options for risk assessment.

CASE PRESENTATION A 76-year-old woman, who sustained an accidental fracture of the femoral neck, developed hyponatremia after admission. The diagnosis of the syndrome of inappropriate secretion of antidiuretic hormone (SIADH) was made by the consulting internist and fluid restriction of 700 cc/day was instituted. Her serum sodium (Na) fluctuated between 122 and 129mmol/l and a renal consultation was requested on the eighth day after undergoing a hemiarthroplasty of the hip when she was ambulating and on no pain medication. Her blood pressure was 100/60 mm Hg, pulse 92 beats/min and there was poor tissue turgor, flat jugular veins, and dry mucous membranes. There was no edema, third spacing, postural hypotension or neurological abnormalities. Her medications included docusate sodium, milk of magnesia, simvastatin, and warfarin. She was not on drugs such as atorvastatin, fenofibrate, or losartan which reduce serum urate by increasing renal urate excretion. 1-3 A low baseline urine Na (UNa) concentration of 6 mmol/l suggested a hypovolemic condition with normal renal function 4 that should result in a prerenal state characterized by low UNa, azotemia, and hyperuricemia. The baseline serum urate of 3.4 mg/dl, however, was inconsistent with prerenal azotemia and its coexistence with hyponatremia was suggestive of SIADH or renal salt wasting. 4-6 The patient was, therefore, included in an IRB-approved protocol to differentiate SIADH from renal salt wasting. After obtaining an informed consent, a blood volume study was performed, using a radioisotope dilution technique.7 With a hematocrit of 31%, the red cell volume was decreased by 28% and the plasma volume increased by 4.7%, resulting in a net blood volume reduction of 7.1%. The pertinent baseline laboratory data are summarized in Table 1. The baseline plasma renin, aldosterone, antidiuretic hormone (ADH), and fractional urate excretion were elevated at 8.63ng/ml/h, 16.5ng/dl, 2.5pg/ml, and 29.6% (normal 5-10%), respectively. Plasma osmolality was decreased at 260 mosm/kgH 2 O, atrial natriuretic peptide was in the low normal range at 35 pg/ml, serum phosphorus and urate were decreased at 2.4 and 3.4 mg/dl, respectively. Renal, adrenal, and thyroid functions were normal. The chest X-ray and urinalysis were normal. Saline was then infused at 125 ml/h for 48 h. Urine osmolality of all spontaneously voided urine was immediately determined and when her urine osmolality decreased to 152 mosM/kgH 2 O at 13.3h, a repeat plasma ADH was undetectable when the serum osmolality was 268 mosm/kgH 2 O. At this time, she had received 1660 ml of saline overnight and excreted 955 ml of urine. The plasma renin and aldosterone decreased to 2.95 ng/ml/h and 7.6ng/dl, respectively, and plasma atrial natriuretic peptide increased to a high normal 65 pg/ml. The serum Na remained unchanged at 129mmol/l but the serum urate decreased from 3.4 to 1.7 mg/dl. It should be noted that the baseline urine osmolality of 321 mosm/kgH 2 O increased to 587 mosm/kgH 2 O and the UNa only increased from a baseline of 6 to 18 mmol/l in the first urine passed 4 h after initiation of saline infusion (Figure 1, Table 2). The serum Na gradually increased from 129 to 138 mmol/l at 45.8 h (Figure 2), as serum urate decreased to 1.0 mg/dl and fractional urate excretion remained elevated (Figure 2). Her UNa increased to a peak of 82 mmol/l at 8.1 h (Table 2).

Lipocalin-type prostaglandin D(2) synthase (L-PGDS) is a highly glycosylated protein found in several body fluids. Elevated L-PGDS levels have been observed in the serum of patients with renal impairment, diabetes mellitus, and hypertension. Recently, we demonstrated the ability of L-PGDS to induce apoptosis in a variety of cell types including epithelial cells, neuronal cells, and vascular smooth muscle cells (VSMCs). The aim of this study was to investigate the effect several site-directed mutations had on L-PGDS-induced apoptosis in order to identify potential sites of regulation. Point mutations created in a glycosylation site (Asn51), a protein kinase C phosphorylation site (Ser106), and the enzymatic active site (Cys65) all inhibited L-PGDS-induced apoptosis as determined by both terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) and caspase3 activity. We also compared the L-PGDS isoforms present in GK rat serum to WKY control serum using two-dimensional gel electrophoresis and observed distinct differences which vanished after PNGase F glycolytic digestion. We conclude that post-translational modification of L-PGDS, by either glycosylation or phosphorylation, enhances its apoptotic activity and inhibits VSMC hyperproliferation and postulate that this process is altered in type 2 diabetes.

Background Roux-en-Y gastric bypass (RYGB) may improve cardiometabolic risk through alteration of bile acids and L-PGDS levels. Objective The objective of this study was to investigate the effect of RYGB on aortic wall thickness, in relation to bile acid and L-PGDS metabolism. Methods Zucker diabetic fatty (ZDF) rats were divided into two groups, ad lib (n = 4), and RYGB (n = 6). Bile acid and L-PGDS were measured presurgery and fourteen weeks post-surgery. Results Elevation of bile acid levels following RYGB in Zucker Diabetic Fatty (ZDF) rodents was observed, as compared to ad lib . RYGB in ZDF rodents led to a significantly decreased aortic wall thickness (25%) as compared to ad lib control. Although bile acid metabolism is implicated in these alterations, other mediators are likely involved. Our laboratory has demonstrated lipocalin prostaglandin D2 synthase (L-PGDS) is a kno n cardiometabolic modulator that also functions as a bile acid binding protein. Therefore, L-PGDS levels were measured and a significant elevation was observed with RYGB compared to ad lib control. Conclusion Based on these findings, RYGB showed beneficial effect on aortic wall thickness, possibly through bile acids and L-PGDS elevation in a severely obese and diabetic rodent model.

Lipocalin-type prostaglandin D2 synthase (L-PGDS) has recently been linked to a variety of pathophysiological cardiovascular conditions including hypertension and diabetes. In this study, we report on the 50% increase in L-PGDS protein expression observed in vascular smooth muscle cells (VSMC) isolated from spontaneously hypertensive rats (SHR). L-PGDS expression also increased 50% upon the differentiation of normotensive control cells (WKY, from Wistar-Kyoto rats). In addition, we demonstrate differential effects of L-PGDS treatment on cell proliferation and apoptosis in VSMCs isolated from SHR versus WKY controls. L-PGDS (50 microg/ml) was able to significantly inhibit VSMC proliferation and DNA synthesis and induce the apoptotic genes bax, bcl-x, and ei24 in SHR but had no effect on WKY cells. Hyperglycemic conditions also had opposite effects, in which increased glucose concentrations (20 mm) resulted in decreased L-PGDS expression in control cells but actually stimulated L-PGDS expression in SHR. Furthermore, we examined the effect of L-PGDS incubation on insulin-stimulated Akt, glycogen synthase kinase-3beta (GSK-3beta), and ERK phosphorylation. Unexpectedly, we found that when WKY cells were pretreated with L-PGDS, insulin could actually induce apoptosis and failed to stimulate Akt/GSK-3beta phosphorylation. Insulin-stimulated ERK phosphorylation was unaffected by L-PGDS pretreatment in both cell lines. We propose that L-PGDS is involved in the balance of VSMC proliferation and apoptosis and in the increased expression observed in the hypertensive state is an attempt to maintain a proper equilibrium between the two processes via the induction of apoptosis and inhibition of cell proliferation.

Background Roux-en-Y gastric bypass (RYGB) ameliorates type 2 diabetes (T2DM) and obesity through alteration in gastrointestinal (GI) hormones. Objective The objective of this study was to investigate the effect of RYGB on GI hormones and cardiometabolic parameters in Zucker diabetic fatty (ZDF) rodents. Setting Winthrop University Hospital, Research and Academic Center Methods Animals were divided into 3 groups, pair-fed (n = 4), ad lib (n = 4), and RYGB (n = 5). This study was carried out for 4 weeks and all related parameters were measured pre- and postsurgery in fasted obese diabetic Zucker rodents. Results Postoperatively, RYGB significantly decreased fasting blood glucose by 32% compared with ad lib. Plasma insulin and leptin levels were also found to be significantly decreased, by 66% and 38%, respectively, after surgery. Moreover, both glucose-dependent insulinotropic polypeptide (GIP) and peptide tyrosine-tyrosine (PYY) were significantly increased after RYGB—by 300% and 51%, respectively. Glucagon-like peptide-1 (GLP-1) levels were also increased, but the increase was not statistically significant. Total cholesterol levels of the RYGB group remained unchanged for 4 weeks. However, total cholesterol in the ad lib and pair-fed groups increased by 25% and 34%, respectively, compared with initial levels. The cholesterol/high-density lipoprotein (HDL) ratio was decreased in the RYGB group by 14% and 30% compared with the ad lib and pair-fed group, respectively. The RYGB group had a significant decrease in aortic wall thickness of 25% compared with the ad lib and pair-fed groups. Similarly, the RYGB group had a 20-unit (mm Hg) decrease in systolic blood pressure compared with the presurgical value. Conclusion RYGB has beneficial cardiometabolic effects through alterations in GI hormones in a severely obese and diabetic rodent model.

Objective: Atherosclerosis contributes to 30% of deaths in systemic lupus erythematosus (SLE) and cardiovascular mortality is 50% higher in rheumatoid arthritis (RA) than in the general population....

Prostaglandin D 2 synthase induces apoptosis in pig kidney LLC-PK1 cells. Background Prostaglandin D 2 synthase (PGD 2 S), a unique member of the lipocalin family, is found at elevated levels in the serum of patients with renal impairment and has recently been implicated as a new biochemical marker of renal insufficiency. The aim of this study was to investigate the apoptotic effects of PGD 2 S on a pig kidney epithelial cell line (LLC-PK1) and to investigate the effects of prostaglandins and growth factors on this process. Methods Apoptosis was detected by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL), annexin V staining, and electron microscopy. Results A four- to fivefold increase in apoptosis was observed in PGD 2 S-treated cells as compared with controls and the apoptosis appeared to act via caspase-3. A cyclooxygenase-2 inhibitor, anti-PGD 2 S antibody, and selenium all significantly inhibited the apoptosis induced by PGD 2 S; however, none had any effect on the apoptosis induced by the known apoptotic inducer camptothecin. Furthermore, prostaglandins E 1 and E 2 , known to induce mitogen-activated protein (MAP) kinase phosphorylation and exhibit cytoprotective effects, both inhibited PGD 2 S-induced apoptosis, while prostaglandin H 2 had no significant effect. Growth factors such as insulin, insulin-like growth factor-1, and platelet-derived growth factor also decreased PGD 2 S-induced apoptosis. In addition, PGD 2 S isolated from human serum seemed slightly more effective at inducing apoptosis than recombinantly expressed protein. Conclusions We report on the induction of apoptosis by PGD 2 S in LLC-PK1 pig kidney epithelial cells, and speculate that the accumulation of PGD 2 S in the serum of kidney failure patients may further exacerbate renal problems and is most likely regulated by other prostaglandins and growth factors.

We have previously demonstrated that a plasma natriuretic factor is present in Alzheimer’s disease (AD), but not in multi-infarct dementia (MID) or normal controls (C). We postulated that the natriuretic factor might induce the increased cytosolic calcium reported in AD by inhibiting the sodium-calcium antiporter, thereby activating the apoptotic pathway. To test for a factor in AD plasma that induces apoptosis, we exposed nonconfluent cultured LLC-PK1cells to plasma from AD, MID, and C for 2 h and performed a terminal transferase-dUTP-nick-end labeling (TUNEL) assay. The plasma from AD increased apoptosis nearly fourfold compared with MID and C. The effect was dose dependent and the peak effect was attained after a 2-h exposure. Additionally, apoptotic morphology was detected by electron microscopy, and internucleosomal DNA cleavage was found. We inhibited apoptosis by removing calcium from the medium, inhibiting protein synthesis with cycloheximide, alternately boiling or freezing and thawing the plasma, and digesting a partially purified fraction with trypsin. Heating AD plasma to 56°C did not deactivate the apoptotic factor. These results demonstrate the presence of an apoptotic factor in the plasma of patients with AD.

A rabbit model of TNBS-colitis was used to study the effect of intestinal inflammation on epithelial cell function. Epithelial cells were isolated using a non-enzymatic isolation method without any apparent contamination with infiltrating immune cells. The isolated cells were found to be viable using dye exclusion studies, unidirectional Na+-fluxes, proliferation assays and morphological studies. The cells, however, showed morphological changes that suggested the presence of increased number of secretory vesicles. This increase correlated well with the increase observed in ion and water secretion as measured by the short-circuit current. Finally, in the colitic tissue the number of PGE2 receptors was greatly reduced with no changes observed in the affinity of PGE2 to its receptor. The reduced number of PGE2 receptors might be due to sensitization of the receptor. In conclusion, we have demonstrated that morphologically and functionally normal epithelial cells can be isolated from the rabbit inflamed distal colon.

In intestinal inflammation, inflammatory cells infiltrate the submucosa and are found juxtaposed to intestinal epithelial cell (IEC) basolateral membranes and may directly regulate IEC function. In this study we determined whether macrophage (M phi), P388D1 and J774A.1, are coupled by gap junctions to IEC lines, Mode-K and IEC6. Using flow cytometric analysis, we show bi-directional transfer of the fluorescent dye, calcein (700 Da) between IEC and M phi resulting in a 3.5-20-fold increase in recipient cell fluorescence. Homocellular and heterocellular dye transfer between M phi and/or IEC was detected in cocultures of P388D1, J774A.1, Mode-K, IEC6 and CMT93. However, transfer between P388D1 and Mode-K was asymmetrical in that transfer from P388D1 to Mode-K was always more efficient than transfer from Mode-K to P388D1. Dye transfer was strictly dependent on IEC-M phi adhesion which in turn was dependent on the polarity of IEC adhesion molecule expression. Both calcein dye transfer and adhesion were inhibited by the addition of heptanol to cocultures. Furthermore we demonstrate both IEC homocellular, and M phi-IEC heterocellular propagation of calcium waves in response to mechanical stimulation, typical of gap junctional communication. Finally, areas of close membrane apposition were seen in electron micrographs of IEC-M phi cocultures, suggestive of gap junction formation. These data indicate that IEC and M phi are coupled by gap junctions suggesting that gap junctional communication may provide a means by which inflammatory cells might regulate IEC function.

Apoptosis is a mode of cell death currently thought to occur in the absence of inflammation. In contrast, inflammation follows unscheduled events such as acute tissue injury which results in necrosis, not apoptosis. We examined the relevance of this paradigm in three distinct models of acute lung injury; hyperoxia, oleic acid, and bacterial pneumonia. In every case, it was found that apoptosis is actually a prominent component of the acute and inflammatory phase of injury. Moreover, using strains of mice that are differentially sensitive to hyperoxic lung injury we observed that the percent of apoptotic cells was well correlated with the severity of lung injury. These observations suggest that apoptosis may be one of the biological consequences during acute injury and the failure to remove these apoptotic cells may also contribute to the inflammatory response.

All forms of aerobic life are faced with the threat of oxidation from molecular oxygen (O2) and have evolved antioxidant defenses to cope with this potential problem. However, cellular antioxidants can become overwhelmed by oxidative insults, including supraphysiologic concentrations of O2 (hyperoxia). Oxidative cell injury involves the modification of cellular macromolecules by reactive oxygen intermediates (ROI), often leading to cell death. O2 therapy, which is a widely used component of life-saving intensive care, can cause lung injury. It is generally thought that hyperoxia injures cells by virtue of the accumulation of toxic levels of ROI, including H2O2 and the superoxide anion (O−2), which are not adequately scavenged by endogenous antioxidant defenses. These oxidants are cytotoxic and have been shown to kill cells via apoptosis, or programmed cell death. If hyperoxia-induced cell death is a result of increased ROI, then O2 toxicity should kill cells via apoptosis. We studied cultured epithelial cells in 95% O2 and assayed apoptosis using a DNA-binding fluorescent dye, in situ end-labeling of DNA, and electron microscopy. Using all approaches we found that hyperoxia kills cells via necrosis, not apoptosis. In contrast, lethal concentrations of either H2O2 or O−2 cause apoptosis. Paradoxically, apoptosis is a prominent event in the lungs of animals injured by breathing 100% O2. These data indicate that O2 toxicity is somewhat distinct from other forms of oxidative injury and suggest that apoptosis in vivo is not a direct effect of O2.

Insulin resistance associated with Type 2 diabetes contributes to impaired vasorelaxation and therefore contributes to the enhanced incidence of hypertension observed in diabetes. In this study, we examined the role of insulin on the association of the myosin-binding subunit of myosin phosphatase (MYPT1) to myosin phosphatase Rho-interacting protein (MRIP), a relatively novel member of the myosin phosphatase complex that directly binds RhoA in vascular smooth muscle cells (VSMCs). Through a series of molecular and cellular studies, we investigated whether insulin stimulates the binding of MRIP to MYPT1 and compared the results generated from VSMCs isolated from both Wistar-Kyoto (WKY) control and Goto-Kakizaki (GK) diabetic rats. We demonstrate for the first time that insulin stimulates the binding of MRIP to MYPT1 in a dose- and time-dependent manner, as determined by immunoprecipitation, implying a regulatory role for MRIP in insulin-induced vasodilation signaling via MYPT1 interaction. VSMCs from GK model of Type 2 diabetes had impaired insulin-induced MRIP/MYPT1 binding as well as reduced MRIP expression. Adenovirus-mediated overexpression of MRIP in GK VSMCs led to significantly improved insulin-stimulated MRIP/MYPT1 binding. Finally, insulin-stimulated MRIP translocation out of stress fibers, which was observed in control VSMCs, was impaired in GK VSMCs. We believe the impaired expression of MRIP, and therefore decreased insulin-stimulated MRIP/MYPT1 association, in the GK diabetic model may contribute to the impaired insulin-mediated vasodilation observed in the diabetic vasculature and provides a novel therapeutic strategy for the treatment of Type 2 diabetes.

Previously, we demonstrated that lipocalin-type prostaglandin D(2) synthase (L-PGDS) induces apoptosis and prevents cell cycle progression in several cell types. In this study we determined the expression of L-PGDS in a variety of human lung tumor types. While L-PGDS expression was evident in the surrounding margins, we observed significantly decreased protein and gene expression in the tumor tissue. Using RT-PCR we demonstrated that L-PGDS gene expression decreased proportionately with tumor progression. In addition, we demonstrated that exogenously added L-PGDS could suppress the hyperproliferation and PDGF-stimulated migration of A549 cells, a cultured carcinomic human alveolar basal epithelial cell line. We conclude that L-PGDS may play a key role in modulating lung cancer growth and may offer a novel diagnostic and therapeutic approach for treatment.

Insulin resistance associated with Type 2 diabetes contributes to impaired vasorelaxation. Previously, we showed the phosphorylation of myosin-bound phosphatase substrate MYPT1, a marker of the vascular smooth muscle cell (VSMC) contraction, was negatively regulated by Akt (protein kinase B) phosphorylation in response to insulin stimulation. In this study we examined the role of Akt phosphorylation on impaired insulin-induced vasodilation in the Goto-Kakizaki (GK) rat model of Type 2 diabetes. GK VSMCs had impaired basal and insulin-induced Akt phosphorylation as well as increases in basal MYPT1 phosphorylation, inducible nitric oxide synthase (iNOS) expression, and nitrite/nitrate production compared with Wistar-Kyoto controls. Both iNOS expression and the inhibition of angiotensin (ANG) II-induced MYPT1 phosphorylation were resistant to the effects of insulin in diabetic GK VSMC. We also measured the isometric tension of intact and denuded GK aorta using a myograph and observed significantly impaired insulin-induced vasodilation. Adenovirus-mediated overexpression of constitutively active Akt in GK VSMC led to significantly improved insulin sensitivity in terms of counteracting ANG II-induced contractile signaling via MYPT1, myosin light chain dephosphorylation, and reduced iNOS expression, S-nitrosylation and survivin expression. We demonstrated for the first time the presence of Akt-independent iNOS expression in the GK diabetic model and that the defective insulin-induced vasodilation observed in the diabetic vasculature can be restored by the overexpression of active Akt, which advocates a novel therapeutic strategy for treating diabetes.

Previously, we demonstrated that lipocalin-type prostaglandin D2 synthase (L-PGDS) knockout mice become glucose intolerant and display signs of diabetic nephropathy and accelerated atherosclerosis. In the current study we sought to explain the link between L-PGDS and glucose tolerance. Using the insulin-sensitive rat skeletal muscle cell line, L6, we showed that L-PGDS could stimulate glucose transport approximately 2-fold as well as enhance insulin-stimulated glucose transport, as measured by 2-deoxy-[3H]-glucose uptake. The increased glucose transport was not attributed to increased GLUT4 production but rather the stimulation of GLUT4 translocation to the plasma membrane, a phenomenon that was lost when cells were cultured under hyperglycemic (20 mM) conditions or pretreated with wortmannin. There was however, an increase in GLUT1 expression as well as a 3-fold increase in hexokinase III expression, which was increased to nearly 5-fold in the presence of insulin, in response to L-PGDS at 20 mM glucose. In addition, adipocytes isolated from L-PGDS knockout mice were significantly less sensitive to insulin-stimulated glucose transport than wild-type. We conclude that L-PGDS, via production of prostaglandin D2, is an important mediator of muscle and adipose glucose transport which is modulated by glycemic conditions and plays a significant role in the glucose intolerance associated with type 2 diabetes.