Louis Ragolia

Accelerated Glucose Intolerance, Nephropathy, and Atherosclerosis in Prostaglandin D2 Synthase Knock-out Mice

Type 2 diabetics have an increased risk of developing atherosclerosis, suggesting the mechanisms that cause this disease are enhanced by insulin resistance. In this study we examined the effects of gene knock-out (KO) of lipocalin-type prostaglandin D2 synthase (L-PGDS), a protein found at elevated levels in type 2 diabetics, on diet-induced glucose tolerance and atherosclerosis. Our results show that L-PGDS KO mice become glucose-in-tolerant and insulin-resistant at an accelerated rate when compared with the C57BL/6 control strain. Adipocytes were significantly larger in the L-PGDS KO mice compared with controls on the same diets. Cell culture data revealed significant differences between insulin-stimulated mitogen-activated protein kinase phosphatase-2, protein-tyrosine phosphatase-1D, and phosphorylated focal adhesion kinase expression levels in L-PGDS KO vascular smooth muscle cells and controls. In addition, only the L-PGDS KO mice developed nephropathy and an aortic thickening reminiscent to the early stages of atherosclerosis when fed a “diabetogenic” high fat diet. We conclude that L-PGDS plays an important role regulating insulin sensitivity and atherosclerosis in type 2 diabetes and may represent a novel model of insulin resistance, atherosclerosis, and diabetic nephropathy.

Ang-II-induced Ca2+ influx is mediated by the 1/4/5 subgroup of the transient receptor potential proteins in cultured aortic smooth muscle cells from diabetic Goto-Kakizaki rats

Angiotensin-II (Ang-II) exerts many of its vascular effects, including the pathophysiological changes associated with type 2 diabetes, through changes in intracellular calcium concentration [Ca(2+)](i). We sought to clarify the mechanism responsible for Ang-II-induced Ca(2+) influx in cultured aortic VSMC using the Goto-Kakizaki (GK) rat model of type 2 diabetes. Ang-II-induced Ca(2+) influx was blocked by neither VDCC nor c-src inhibition but was sensitive to inositol 1,4,5-trisphosphate receptor inhibition, lanthanide and the diacylglycerol analogue, oleoyl-2-acetyl-sn-glycerol. Since transient receptor potential canonical (TRPC)-3 gene expression was undetectable in both WKY and GK VSMCs and TRPC6 gene and protein expression were significantly down-regulated in GK, we believe the 1/4/5 subgroup of TRPC proteins plays a significant role. Furthermore, in GK VSMC the elevated calcium influx observed was not attributable to increased TRPC expression, but rather an alteration of TRPC activity.

Lipocalin-type prostaglandin D2 synthase stimulates glucose transport via enhanced GLUT4 translocation

Previously, we demonstrated that lipocalin-type prostaglandin D(2) 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-[(3)H]-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 D(2), 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.

Diminished lipocalin-type prostaglandin D2 synthase expression in human lung tumors

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.

Functional melanocortin-2 receptors are expressed by mouse aorta-derived mesenchymal progenitor cells

A local melanocortin system is active during tissue injury and inflammation. Thus far this system has been described as autocrine in nature where local production of pro-opiomelanocortin (POMC) peptides by leukocytes feeds back on melanocortin receptor (MC-R) expressing immune cells to quell inflammatory cytokine production. Here we present evidence that POMC peptides may generate extracellular matrix (ECM) changes by inducing matrix production by cells of the mesenchymal lineage through activation of the MC2-R. Using immunoblot, we determined that mouse aorta-derived mesenchymal progenitor cells express both MC2-R and MC3-R. These progenitors respond to treatment with ACTH by increasing collagen matrix synthesis as assessed by picrosirius red stain and (3)H-proline incorporation. ACTH also induces transient increases in intracellular calcium ([Ca(2+)](i)) as assessed using the fluorescent Ca(2+) indicator, fura-2. The ACTH-induced changes in [Ca(2+)](i) are consistent with MC2-R signaling and consist of both an intracellular release and an extracellular influx of Ca(2+). Both mouse aortic mesenchymal progenitors and mouse macrophage cells express POMC and the prohormone convertase 1/3 (PC1/3) indicating they have the potential to contribute to the local production of POMC peptides. These data demonstrate functional MC2-R expression in mouse aorta-derived mesenchymal progenitors and implicate both macrophage and mesenchymal cells as relevant sources of local POMC peptides.

Stimulation of glycogen synthesis by heat shock in L6 skeletal-muscle cells: regulatory role of site-specific phosphorylation of glycogen-associated protein phosphatase 1.

Recent evidence suggests that glycogen-associated protein phosphatase 1 (PP-1(G)) is essential for basal and exercise-induced glycogen synthesis, which is mediated in part by dephosphorylation and activation of glycogen synthase (GS). In the present study, we examined the potential role of site-specific phosphorylation of PP-1(G) in heat-shock-induced glycogen synthesis. L6 rat skeletal-muscle cells were stably transfected with wild-type PP-1(G) or with PP-1(G) mutants in which site-1 (S1) Ser(48) and site-2 (S2) Ser(67) residues were substituted with Ala. Cells expressing wild-type and PP-1(G) mutants, S1, S2 and S1/S2, were examined for potential alterations in glycogen synthesis after a 60 min heat shock at 45 degrees C, followed by analysis of [(14)C]glucose incorporation into glycogen at 37 degrees C. PP-1(G) S1 mutation caused a 90% increase in glycogen synthesis on heat-shock treatment, whereas the PP-1(G) S2 mutant was not sensitive to heat stress. The S1/S2 double mutant was comparable with wild-type, which showed a 30% increase over basal. Heat-shock-induced glycogen synthesis was accompanied by increased PP-1 and GS activities. The highest activation was observed in S1 mutant. Heat shock also resulted in a rapid and sustained Akt/ glycogen synthase kinase 3 beta (GSK-3 beta) phosphorylation. Wortmannin blocked heat-shock-induced Akt/GSK-3 beta phosphorylation, prevented 2-deoxyglucose uptake and abolished the heat-shock-induced glycogen synthesis. Muscle glycogen levels regulate GS activity and glycogen synthesis and were found to be markedly depleted in S1 mutant on heat-shock treatment, suggesting that PP-1(G) S1 Ser phosphorylation may inhibit glycogen degradation during thermal stimulation, as S1 mutation resulted in excessive glycogen synthesis on heat-shock treatment. In contrast, PP-1(G) S2 Ser phosphorylation may promote glycogen breakdown under stressful conditions. Heat-shock-induced glycogenesis appears to be mediated via phosphoinositide 3-kinase/Akt-dependent GSK-3 beta inactivation as well as phosphoinositide 3-kinase-independent PP-1 activation.

Bile acid elevation after Roux-en-Y gastric bypass is associated with cardio-protective effect in Zucker Diabetic Fatty rats

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.

Roux-en-Y gastric bypass attenuates the progression of cardiometabolic complications in obese diabetic rats via alteration in gastrointestinal hormones

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.

Systemic and local ACTH produced during inflammatory states promotes osteochondrogenic mesenchymal cell differentiation contributing to the pathologic progression of calcified atherosclerosis

There are many well-known roles for the proopiomelanocortin (POMC) derived peptides and their receptors, the melanocortin receptors (MC-R). The focus here is on the evolving role of the melanocortin system in inflammation. Chronic inflammatory states such as those occurring in diabetes and obesity are associated with both a hyperactive hypothalamic-pituitary-adrenal (HPA) axis as well as increased incidence of atherosclerosis. An inflammation-induced hyperactive HPA axis along with increased leukocyte infiltration can lead to significant exposure to melanocortin peptides, particularly ACTH, in an inflamed vasculature. Mesenchymal progenitor cells are present throughout the vasculature, express receptors for the melanocortin peptides, and respond to ACTH with increased osteochondrogenic differentiation. Coupled to the increased exposure to ACTH during HPA hyperactivity is increased glucocorticoid (GC) exposure. GCs also promote chondrogenic differentiation of mesenchymal progenitors and increase their expression of MC-R as well as their expression of POMC and its cleavage products. It is hypothesized that during inflammatory states systemically produced ACTH and glucocorticoid as well as ACTH produced locally by macrophage and other immune cells, can influence and potentiate mesenchymal progenitor cell differentiation along the osteochondrogenic lineages. In turn the increase in osteochondrogenic matrix contributes to the pathophysiological progression of the calcified atherosclerotic plaque. The roles of the melanocortin system in inflammation and its resolution have just begun to be explored. Investigations into the ACTH-induced matrix changes among mesenchymal cell populations are warranted. ACTH signaling through the MC-R represents a new therapeutic target for the prevention and treatment of calcified atherosclerosis.

Role of Lipocalin-type prostaglandin D2 synthase (L-PGDS) and its metabolite, prostaglandin D2, in preterm birth

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.