Fat Pads Of Rats Research Articles

Rice bran protein concentrate can reduce fat accumulation in rats fed high‐fat diet

Rice is a staple food and major source for energy intake in Korean population. Furthermore, it would be the highest protein source in Korean diet, because the white rice is the most consumed food according to 2005 KNHANES. Rice bran, outer shell of the rice milling process has 10~16% highly nutritional protein itself. Rice bran protein has been reported to have anti‐obesitic effect, However the feasibility of rice protein is not well known. Therefore, we examined the effect of rice bran protein on body far accumulation in rats fed high‐fat diet based on a AIN 93M(CO), and high fat diet with rice bran protein 0% (CA), 10%(RB10) 20%(RB20), and with soy isolated protein(SP). After 50 days, the rats were sacrificed. The complete substitution of casein(CA) with rice bran protein(RB20) reduced significantly final weight and epididymal fat pad of rats fed high‐fat diets. The fecal fat excretion was the highest in the RB20 group among experimental groups. Total lipid and triglyceride concentration in liver of RB10 and RB20 groups were significantly lower than those of CA. The concentration of albumin and total protein in plasma and liver did not show any differences by the replacement of casein with rice bran protein. This study suggests that the reduction of fat accumulation can be achieved in rats by replacing casein protein with rice bran protein without reducing visceral protein content.Grant Funding Source:No

Intradermal injection of GFP-producing adipose stromal cells promotes survival of random-pattern skin flaps in rats

Background Tissue necrosis is a common complication in operations that use skin flaps for reconstructive surgery. Here we demonstrate the beneficial effect of autologous genetically modified adipose-derived stromal cells (ASCs) in the survival of random-pattern skin flaps. Methods ASCs were isolated from the inguinal fat pad of Wistar rats and genetically modified in order to permanently produce green fluorescent protein (GFP) using the Sleeping Beauty transposon technology. Autologous GFP-producing cells were then injected intradermally into random-pattern skin flaps planned on the dorsal area of rats. Results Injection of ASCs resulted in significant improvement of skin flap survival. Histological analysis showed that the connective tissue was almost intact in skin flaps treated with ASCs in contrast to disorganized tissues from mocktreated skin flaps. GFP ASCs were detected in the endothelium of blood vessels co-expressing the endothelial marker von Willebrand factor, thus suggesting that they promote blood vessel regeneration. Conclusions These findings indicate that transplantation of autologous GFP ASCs improve survival of skin flaps. This methodology suggests that the use of genetically modified ASCs producing, e.g., angiogenic factors may facilitate survival and integration of flaps in plastic surgery.

Treatment with heme arginate alleviates adipose tissue inflammation and improves insulin sensitivity and glucose metabolism in a rat model of Human primary aldosteronism

Visceral adiposity and insulin resistance are common pathophysiological denominators in patients with primary aldosteronism. Although we recently reported the antidiabetic effects of heme oxygenase (HO), no study has examined the effects of upregulating HO on visceral adiposity in uninephrectomized (UnX) deoxycorticosterone acetate (DOCA-salt) hypertensive rats, a model of human primary aldosteronism characterized by elevated endothelin (ET-1) and oxidative/inflammatory events. Here, we report the effects of the HO inducer heme arginate and the HO blocker chromium mesoporphyrin (CrMP) on visceral adipose tissue obtained from retroperitoneal fat pads of UnX DOCA-salt rats. UnX DOCA-salt rats were hypertensive but normoglycemic. Heme arginate reduced visceral adiposity and enhanced HO activity and cGMP in the adipose tissue, but suppressed ET-1, nuclear-factor κB (NF-κB), activating-protein (AP-1), c-Jun-NH2-terminal kinase (JNK), macrophage chemoattractant protein-1 (MCP-1), intercellular adhesion molecule-1 (ICAM-1), and 8-isoprostane. These were associated with reduced glycemia, increased insulin, and the insulin-sensitizing protein adiponectin, with corresponding reduction in insulin resistance. In contrast, the HO inhibitor, CrMP, abolished the effects of heme arginate, aggravating insulin resistance, suggesting a role for the HO system in insulin signaling. Importantly, the effects of the HO system on ET-1, NF-κB, AP-1, JNK, MCP-1, and ICAM-1 in visceral or retroperitoneal adiposity in UnX-DOCA-salt rats have not been reported. Because 8-isoprostane stimulates ET-1 to enhance oxidative insults, and increased oxidative events deplete adiponectin and insulin levels, the suppression of oxidative/inflammatory mediators such as 8-isoprostane, NF-κB, AP-1, MCP-1, ICAM-1, and JNK, an inhibitor of insulin biosynthesis, may account for the potentiation of insulin signaling/glucose metabolism by heme arginate. These data indicate that although UnX DOCA-salt rats were normoglycemic, insulin signaling was impaired, suggesting that dysfunctional insulin signaling may be a forerunner to overt diabetes in primary aldosteronism.

Transplantation of Adipose‐Derived Stem Cells is Associated with Neural Differentiation and Functional Improvement in a Rat Model of Intracerebral Hemorrhage

To examine whether transplantation of adipose-derived stem cells (ADSCs) induces neural differentiation and improves neural function in a rat intracerebral hemorrhage (ICH) model. Adipose-derived stem cells cells were isolated from inguinal fat pad of rat. ICH was induced by injection of collagenase type IV into the right basal ganglia of rat. Forty-eight hours after ICH, ADSCs cells (10 μL of 2-4 × 10(7) cells/mL) were injected into the right lateral cerebral ventricle. The differentiation of ADSCs was detected in vitro and in vivo. The neural function was evaluated with Zea Longa 5-grade scale at day 1, 3, 7, 14, or 28. Our data demonstrated that ADSCs differentiated into cells that shared the similarities of neurons or astrocytes in vitro. Transplantation of ADSCs decreased cell apoptosis and the transplanted ADSCs were able to differentiate into neuron-like and astrocyte-like cells around the hematoma, accompanied with upregulation of vascular endothelial growth factor expression and improvement of neural function. Our data suggest that transplantation of ADSCs could be a therapeutic approach for ICH stroke.

YC-1, a potent antithrombotic agent, induces lipolysis through the PKA pathway in rat visceral fat cells

This study investigated the effects of 3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole (YC-1), a soluble guanylyl cyclase (sGC) activator and potential antithrombotic agent, on lipolysis in isolated visceral fat cells of the rat. Visceral fat cells were isolated from epididymal fat pads of rats and treated with YC-1 at different doses and times. Glycerol release, and intracellular cAMP and cGMP levels were analyzed by specific kits. Moreover, several inhibitors or drugs were used to examine the signal transduction pathways of YC-1-induced lipolysis in adipocytes. Herein we report that YC-1 stimulated glycerol release in dose- and time-dependent manners. Intracellular cAMP and cGMP levels of adipocytes both increased in time-dependent manners, but elevation of the cGMP level was faster and higher than that of the cAMP level after YC-1 treatment. An sGC inhibitor (ODQ) inhibited YC-1-induced glycerol release, indicating the involvement of sGC in YC-1-induced lipolysis. Administration of insulin, an activator of type-3B phosphodiesterase (PDE-3B), attenuated YC-1-induced lipolysis, indicating that elevation of the cAMP level is an important step in the lipolytic effect of YC-1. In addition, YC-1-induced lipolysis was inhibited by a protein kinase A (PKA) inhibitor (KT5720) but not by a PKG inhibitor (KT5823), indicating that YC-1-induced lipolysis occurs through a PKA-dependent pathway. A Western blot analysis showed that extracellular signal-regulated kinase was not phosphorylated by YC-1 treatment. In conclusion, our results suggest that YC-1 might stimulate lipolysis via activation of sGC/cGMP and then activation of the cAMP/PKA signaling cascade in isolated rat visceral adipocytes.

In vivo microdialysis sampling of adipokines from rat mammary fat pad. Comparison of dialysates to whole tissue concentrations

ObjectiveMicrodialysis sampling was used to collect adipokines, CCL2 (MCP‐1) and IL‐6, from mammary fat pads of female rats (Figure 1). A critical issue with microdialysis sampling is the calibration and determining actual tissue concentrations. Tissue concentrations of the adipokines were obtained and compared with dialysates.MethodsCMA 20 polyethersulfone, 100 kDa MWCO, probes were implanted in the right abdominal mammary fat pad of female Sprague‐Dawley rats (220–250g). Tissue around the probe was harvested for protein extraction. CCL2 and IL‐6 were quantified using Luminex and ELISA kits.Results and discussionConcentrations of IL‐6 and CCL2 increased after probe implantation (Table 1). Interestingly, IL‐6 levels were not different, but CCL2 levels were between the implanted vs. control tissue. This may suggest that IL‐6 levels are not influenced by the probe insertion trauma.ConclusionsAdipokines can be collected from mammary fat pad using microdialysis sampling. Up to ~50 fold differences exist between tissue and dialysate levels.Funding: COBRE 1P30RR031154‐01 and Arkansas Biosciences Institute.

The effect of flutamide on expression of androgen and estrogen receptors in the gubernaculum and surrounding structures during testicular descent

Background/Purpose Inguinoscrotal testicular descent is controlled by androgens between embryonic days E16-19, but androgen receptor (AR) and estrogen receptor (ER) locations are unknown. We aimed to find AR, ER α, and ER β in the gubernaculum and inguinal fat pad (IFP) in normal rats and after flutamide treatment. Methods Sprague-Dawley timed-mated rats were injected with flutamide (75 mg/kg body weight/5% ethanol + oil) on E16-19 or vehicle alone. Male fetuses or pups (5-10/group) were collected at E16; E19; and postnatal (P) days 0, 2, 4, 8. Sections were prepared for hematoxylin and eosin or immunohistochemistry for AR, ERα, and ERβ. Receptor labeling was quantitated as distinct nuclear labeling/100 μm 2 in gubernaculum and IFP. Results There was minimal gubernacular AR-labeling until E19, dramatically increasing postnatally. By contrast, at E16-E19 there was significant IFP AR immunoreactivity suppressed by flutamide ( P < .05). No ERα expression was observed, but ERβ was expressed in both gubernaculum and IFP, maximally at E16, but unchanged by flutamide. Conclusions During the androgen sensitivity window (E16-19), the gubernaculum contains ERβ but minimal ERα or AR, while the IFP, which is supplied by the genitofemoral nerve, contains abundant AR that are flutamide-sensitive. These results suggest that the IFP could be the site of androgenic action controlling gubernacular development.

Sex differences in brain cholinergic activity in MSG-obese rats submitted to exercise.

Obesity is an epidemic disease most commonly caused by a combination of increased energy intake and lack of physical activity. The cholinergic system has been shown to be involved in the regulation of food intake and energy expenditure. Moreover, physical exercise promotes a reduction of fat pads and body mass by increasing energy expenditure, but also influences the cholinergic system. The aim of this study is to evaluate the interaction between physical exercise (swimming) and central cholinergic activity in rats treated with monosodium glutamate (MSG, a model for obesity) during infancy. Our results show that MSG treatment is able to induce obesity in male and female rats. Specifically, MSG-treated rats presented a reduced body mass and nasoanal length, and increased perigonadal and retroperitoneal fat pads in relation to the body mass. Physical exercise was able to reduce body mass in both male and female rats, but did not change the fat pads in MSG-treated rats. Increased food intake was only seen in MSG-treated females submitted to exercise. Cholinergic activity was increased in the cortex of MSG-treated females and physical exercise was able to reduce this activity. Thalamic cholinergic activity was higher in sedentary MSG-treated females and exercised MSG-treated males. Hypothalamic cholinergic activity was higher in male and female MSG-treated rats, and was not reduced by exercise in the 2 sexes. Taken together, these results show that MSG treatment and physical exercise have different effects in the cholinergic activity of males and females.

Atrial natriuretic peptide enhances microvascular albumin permeability by the caveolae-mediated transcellular pathway

AimsCardiac atrial natriuretic peptide (ANP) participates in the maintenance of arterial blood pressure and intravascular volume homeostasis. The hypovolaemic effects of ANP result from coordinated actions in the kidney and systemic microcirculation. Hence, ANP, via its guanylyl cyclase-A (GC-A) receptor and intracellular cyclic GMP as second messenger, stimulates endothelial albumin permeability. Ultimately, this leads to a shift of plasma fluid into interstitial pools. Here we studied the role of caveolae-mediated transendothelial albumin transport in the hyperpermeability effects of ANP.Methods and resultsIntravital microscopy studies of the mouse cremaster microcirculation showed that ANP stimulates the extravasation of fluorescent albumin from post-capillary venules and causes arteriolar vasodilatation. The hyperpermeability effect was prevented in mice with conditional, endothelial deletion of GC-A (EC GC-A KO) or with deleted caveolin-1 (cav-1), the caveolae scaffold protein. In contrast, the vasodilating effect was preserved. Concomitantly, the acute hypovolaemic action of ANP was abolished in EC GC-A KO and Cav-1−/− mice. In cultured microvascular rat fat pad and mouse lung endothelial cells, ANP stimulated uptake and transendothelial transport of fluorescent albumin without altering endothelial electrical resistance. The stimulatory effect on albumin uptake was prevented in GC-A- or cav-1-deficient pulmonary endothelia. Finally, preparation of caveolin-enriched lipid rafts from mouse lung and western blotting showed that GC-A and cGMP-dependent protein kinase I partly co-localize with Cav-1 in caveolae microdomains.ConclusionANP enhances transendothelial caveolae-mediated albumin transport via its GC-A receptor. This ANP-mediated cross-talk between the heart and the microcirculation is critically involved in the regulation of intravascular volume.

Abstract 4378: Defining the role of mammary stem cells and progenitor cells in the development of estrogen-induced mammary cancer in the rat

Abstract Substantial data implicate both estrogens and progestins in the etiology of breast cancer. However, the mechanisms through which these steroid hormones elicit their effects on breast cancer risk have not been elucidated. The ACI rat serves as a unique model for investigating the mechanisms through which estrogens and progestins contribute to breast cancer development and for defining the genetic bases of inherited risk of breast cancer. When treated with physiologic levels of 17β-estradiol (E2), female ACI rats develop mammary cancer at an incidence that approaches 100%. Interestingly, rapid development of mammary cancer in E2 treated ACI rats is dependent upon progesterone. The mammary cancers that develop in E2 treated ACI rats are estrogen dependent, frequently exhibit non-random patterns of genome instability, and are invasive. Because each of these phenotypes is also commonly exhibited by breast cancers in humans, the ACI rat appears to be highly relevant to the study of breast cancer etiology. Whereas female ACI rats are highly susceptible to E2-induced mammary cancer, female Brown Norway (BN) rats are resistant. Genetic studies performed by our laboratory localized a total of six quantitative trait loci (QTL), designated Emca3 through Emca9, that account for a large fraction of inherited susceptibility in F2 progeny generated in intercrosses between ACI and BN rats. The purpose of this study is to test the hypothesis that ACI and BN rats exhibit differences in the responsiveness of their mammary stem cell (MaSCs) and/or progenitor cell (MaPCs) populations to E2 and/or progesterone. By using a combination of markers CD31, CD45, CD24, CD29 and fluorescence-activated cell sorting, we have identified four distinct populations of mammary cells. To quantify the numbers of MaSC and MaPC in these populations, we are evaluating the ability of the basal (Lin-CD24+CD29high) and luminal (Lin-CD24+CD29low) cell populations to form colonies when cultured in vitro and to generate mamamry gland outgrowths when transplanted into the interscapular fat pad of recipient syngenaic or (ACIxBN)F1 rats. Any observed differeces in MaSC and/or MaPC number or function will be evaluated further using a set of congenic rat strains that harbor BN alleles across an individual Emca locus on the ACI genetic background. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4378. doi:10.1158/1538-7445.AM2011-4378

Low-serum culture system improves the adipogenic ability of visceral adipose tissue-derived stromal cells

In obese adipose tissue, infiltrating macrophages release proinflammatory cytokines that trigger insulin resistance. An adipocyte-based platform from visceral fat would be useful to elucidate the pathology of adipose inflammation and to develop therapeutic drugs for insulin resistance. ADSCs (adipose tissue-derived mesenchymal stromal cells) expanded from subcutaneous fat are intensively studied as sources for regenerative medicine. However, the adipocyte culture system from visceral fat tissue has not been utilized yet. We aimed to establish the bioactive adipocyte platform using ADSCs from visceral fat pad. Stromal vascular fractions were processed from epididymal fat pads of Sprague-Dawley rats and three human omental fat pads, and the ADSCs were expanded using a low-serum culture method. The responses of ADSCs and ADSC-adipocytes (their adipogenic lineages) to pioglitazone, a therapeutic drug for diabesity, were evaluated by gene expression and ELISA. ADSCs (1×108) were expanded from 10 g of rat epididymal fat pads or human omental fat pads over five passages. Cell surface marker expressions revealed that visceral ADSCs were equivalent to mesenchymal stem cells. ADSC-adipocytes expanded in the low-serum culture system significantly showed higher expression of adipogenic markers [PPAR (peroxisome proliferator-activated receptor) γ, LPL (lipoprotein lipase) and FABP4 (fatty acid-binding protein 4)] and adipocytokines [adiponectin, resistin, leptin, PAI-1 (plasminogen-activator inhibitor 1) and IL (interleukin)-10] than those expanded in a high-serum culture system. Pioglitazone accelerated the adipogenic induction and increased adiponectin expression in human ADSCs by 57.9±5.8-fold (mean±S.E.M.) relative to control cells (P<0.001). Both in rat and human ADSC-adipocytes, TNF-α significantly induced proinflammatory cytokines [MCP-1 (monocyte chemoattractant protein-1) and IL-6] and suppressed adiponectin expression, while pioglitazone antagonized these effects. The present findings suggest that visceral ADSC-adipocytes expanded in low-serum culture would be useful for adiposcience and pharmacological evaluations.

Imaging the Endothelial Glycocalyx In Vitro by Rapid Freezing/Freeze Substitution Transmission Electron Microscopy

Recent publications questioned the validity of endothelial cell (EC) culture studies of glycocalyx (GCX) function because of findings that GCX in vitro may be substantially thinner than GCX in vivo. The assessment of thickness differences is complicated by GCX collapse during dehydration for traditional electron microscopy. We measured in vitro GCX thickness using rapid freezing/freeze substitution (RF/FS) transmission electron microscopy (TEM), taking advantage of the high spatial resolution provided by TEM and the capability to stably preserve the GCX in its hydrated configuration by RF/FS. Bovine aortic EC (BAEC) and rat fat pad EC were subjected to conventional or RF/FS-TEM. Conventionally preserved BAEC GCX was ≈0.040 μm in thickness. RF/FS-TEM revealed impressively thick BAEC GCX of ≈11 μm and rat fat pad EC GCX of ≈5 μm. RF/FS-TEM also discerned GCX structure and thickness variations due to heparinase III enzyme treatment and extracellular protein removal, respectively. Immunoconfocal studies confirmed that the in vitro GCX is several micrometers thick and is composed of extensive and well-integrated heparan sulfate, hyaluronic acid, and protein layers. New observations by RF/FS-TEM reveal substantial GCX layers on cultured EC, supporting their continued use for fundamental studies of GCX and its function in the vasculature.

Microarray analysis of the mammalian thromboxane receptor-Trypanosoma cruzi interaction

Trypanosoma cruzi, the etiological agent of Chagas disease, causes vasculopathy and cardiomyopathy in humans and is associated with elevated levels of several vasoactive molecules such as nitric oxide, endothelin-1 and thromboxane A2 (TXA2). Parasite derived TXA2 modulates vasculopathy and other pathophysiological features of Chagasic cardiomyopathy. Previously, we demonstrated that in response to infection with T. cruzi, TXA2 receptor (TP) null mice displayed increased parasitemia; mortality and cardiac pathology compared with wild type (WT) and TXA2 synthase null mice. In order to further study the role of TXA2-TP signaling in the development of Chagas disease, GeneChip microarrays were used to detect transcriptome changes in rat fat pad endothelial cells (RFP-ECs) which is incapable of TXA2 signaling (TP null) to that of control (wild type) and RFP-EC with reconstituted TP expression. Genes that were significantly regulated due to infection were identified using a time course of 2, 18 and 48 hrs post infection. We identified several key genes such as suppressor of cytokine signaling (SOCS-5), several cytokines (CSF-1, CXCF ligands), and MAP kinases (MAPK-1, Janus kinase) that were upregulated in the absence of TP signaling. These data underscore the importance of the interaction of the parasite with mammalian TP and may explain the increased mortality and cardiovascular pathology observed in infected TP null mice.

Soya protein ameliorates the metabolic abnormalities of dysfunctional adipose tissue of dyslipidaemic rats fed a sucrose-rich diet

The present study investigates whether the replacement of dietary casein by soya protein isolate could be able to improve and/or even revert the morphological and metabolic abnormalities underlying the adipose tissue dysfunction of dyslipidaemic rats chronically fed (8 months) a sucrose-rich (62·5%) diet (SRD). For this purpose, Wistar rats were fed a SRD for 4 months. From months 4 to 8, half the animals continued with the SRD and the other half were fed a SRD in which the source of protein, casein, was substituted by soya. The control group received a diet in which the source of carbohydrate was maize starch. Compared with the SRD-fed group, the results showed that: (1) soya protein decreased body-weight gain, limited the accretion of visceral adiposity and decreased adipose tissue cell volume without changes in total cell number; (2) soya protein increased the protein mass expression of PPARγ, which was significantly reduced in the fat pad of the SRD-fed rats; (3) the activity of the enzymes involved in the de novo lipogenesis of adipose tissue was significantly decreased/normalised; (4) soya protein corrected the inhibitory effect of SRD upon the anti-lipolytic action of insulin, reduced basal lipolysis and normalised the protein mass expression of GLUT-4. Dyslipidaemia, glucose homeostasis and plasma leptin levels returned to control values. The present study provides data showing the beneficial effects of soya protein to improve and/or revert the adipose tissue dysfunction of a dyslipidaemic insulin-resistant rat model and suggests that soya could maintain the functionality of the adipose tissue-liver axis improving/reverting lipotoxicity.

Phosphorylation of VE-cadherin controls endothelial phenotypes via p120-catenin coupling and Rac1 activation

To establish the role of vascular endothelial (VE)-cadherin in the regulation of endothelial cell functions, we investigated the effect of phosphorylation of a VE-cadherin site sought to be involved in p120-catenin binding on vascular permeability and endothelial cell migration. To this end, we introduced either wild-type VE-cadherin or Y658 phosphomimetic (Y658E) or dephosphomimetic (Y658F) VE-cadherin mutant constructs into an endothelial cell line (rat fat pad endothelial cells) lacking endogenous VE-cadherin. Remarkably, neither wild-type- nor Y658E VE-cadherin was retained at cell-cell contacts because of p120-catenin preferential binding to N-cadherin, resulting in the targeting of N-cadherin to cell-cell junctions and the exclusion of VE-cadherin. However, Y658F VE-cadherin was able to bind p120-catenin and to localize at adherence junctions displacing N-cadherin. This resulted in an enhanced barrier function and a complete abrogation of Rac1 activation and lamellipodia formation, thereby inhibiting cell migration. These findings demonstrate that VE-cadherin, through the regulation of Y658 phosphorylation, competes for junctional localization with N-cadherin and controls vascular permeability and endothelial cell migration.

Neutral Aminopeptidase and Dipeptidyl Peptidase IV Activities in Plasma of Monosodium Glutamate Obese and Food‐deprived Rats

Biometric parameters, glycemia and activity levels of plasma neutral aminopeptidase (APN) and dipeptidyl peptidase IV (DPPIV) were measured in monosodium glutamate obese and food-deprived rats (MSG-FD), to analyze the involvement of these enzymes in such situations. Plasma APN was distinguished as sensitive (PSA) (K(m) = 7.8 x 10(-5) mol/l) and predominantly insensitive (APM) (K(m) = 21.6 x 10(-5) mol/l) to puromycin, whereas DPPIV was sensitive (DPPIV-DS) (K(m) = 0.24 x 10(-5) mol/l) and predominantly insensitive (DPPIV-DI) (K(m) = 7.04 x 10(-5) mol/l) to diprotin A. Although unchanged in the MSG and food-deprived animals, APM activity levels were closely correlated with body mass, Lee index, and mass of retroperitoneal fat pad in the food deprived, but not in the MSG animals. DPPIV-DI activity levels decreased by 33% and were correlated with body mass, Lee index, and mass of periepididymal fat pad in the food-deprived MSG rats. These data suggest that APM and DPPIV-DI are respectively related to the downregulation of somatostatin in food-deprived rats, and to the recovery of energy balance in MSG obese rats during food deprivation.

Pharmacokinetics and Disposition of a Localized Lymphatic Polymeric Hyaluronan Conjugate of Cisplatin in Rodents

Cisplatin (CDDP) is an effective anticancer agent for many solid tumors but has significant systemic toxicity limiting its use in many patients. We have designed a loco-regional delivery system to increase platinum levels in the lymphatics, where early metastasis is most likely to occur, while reducing systemic toxicities. CDDP was conjugated to a biocompatible polymer hyaluronan (HA), with a conjugation degree of approximately 20% (w/w). Conjugates were delivered via subcutaneous injection into the mammary fat pad of rats. Intravenous hyaluronan-cisplatin (HA-Pt) exhibited an increased plasma area under the curve (AUC) 2.7-fold compared to conventional CDDP but with a reduced peak plasma level (C(max)), and HA-Pt increased the ipsilateral lymph node AUC by 3.8-fold compared to CDDP. Urine creatinine was unchanged over 30 days following dosing of HA-Pt. This study demonstrates that intralymphatic drug delivery with polymer-conjugated platinum may provide greater tissue and systemic plasma concentrations of platinum than intravenous CDDP. In addition, localized particle delivery augmented distribution in the loco-regional tissue basin where tumor burden predominates, while renal toxicity compared to standard intravenous CDDP was significantly reduced.

The Endothelial Glycocalyx In Vitro: Its Structure and The Role of Heparan Sulfate and Glypican‐1 in eNOS Activation by Flow

The endothelial glycocalyx (GCX) mediates flow‐induced nitric oxide release via heparan sulfate (HS), but the GCX structure is unclear and the specific HS core protein(s) involved in this mechanotransduction is unknown. Our study tests the hypotheses that flow regulates GCX thickness and organization and that the HS glypican‐1 core protein mediates flow‐induced activation of endothelial nitric oxide synthase (eNOS). Monolayers of bovine aortic and rat fat pad endothelial cells with intact GCX, enzymatically degraded HS, or RNA‐silenced glypican‐1 were exposed to 15 dyne/cm2 uniform shear stress for 3 hr. Confocal immunocytochemistry and cryo‐transmission electron microscopy revealed a GCX that was 2.5 to 4.0 μm thick. Sheared GCX was well organized and aligned perpendicular to the cell surface, while unsheared GCX was disorganized. Western blot and confocal microscopy demonstrate that when the GCX is intact shear stress increases eNOS activation (ser1177 phosphorylation) and membrane localization, which are blocked by HS degradation. Baseline and flow‐induced levels of activated eNOS are attenuated when glypican‐1 is silenced. This work further elucidates GCX structure and role in endothelial cell mechanotransduction. Supported by NIH: 5T32HL007675, HL73732, HL57093.

Immunohistochemical Detection of Antitumor, Antimetastasis, and Antiangiogenesis Effects of a Vascular Endothelial Growth Factor Receptor 2 Kinase Inhibitor in an Orthotopic Breast Cancer Metastasis Model

Antiangiogenic agents such as vascular endothelial growth factor receptor 2 (VEGFR2) inhibitors may prove most efficacious in the setting of early disease and in the prevention of dissemination and growth of micrometastases. This hypothesis was tested in a metastatic orthotopic rat model of breast cancer with the use of a novel orally bioavailable VEGFR2 kinase inhibitor, MK-0888. Mat B III rat mammary cancer cells were implanted into the mammary fat pads of syngeneic female F344 rats. Primary tumor growth was very aggressive, with micrometastases detected 8 days after cell implantation in ipsilateral axillary and inguinal lymph nodes. Lung metastases were detected 15 days after cell implantation by histological analysis. MK-0888 suppressed primary and metastatic tumor growth and reduced the incidence of metastasis in a dose- and schedule-dependent manner. Inhibitions of primary and metastatic tumor growth, as well as intratumoral antiangiogenesis effects, were detected in situ by immunohistochemical analysis of tumor cells, endothelial cell proliferation, microvascular density, and blood vessel maturity. In the Mat B III rat mammary cancer metastasis model, our results provide further evidence supporting the ongoing clinical development of VEGFR2 kinase inhibitors, as well as clinically applicable in situ detection and verification of the inhibitor effect in tumor and metastasis biopsies. (The J Histotechnol 33(1):15–24, 2010)Submitted November 20, 2009; accepted February 8, 2010.

Mal-Development of the Penis and Loss of Fertility in Male Rats Treated Neonatally with Female Contraceptive 17α-Ethinyl Estradiol: A Dose-Response Study and a Comparative Study with a Known Estrogenic Teratogen Diethylstilbestrol

The objectives of this study were to find a minimal dose of 17alpha-ethinyl estradiol (EE) that is detrimental to the developing penis and fertility and to compare estrogenic effects between EE and diethylstilbestrol (DES). Neonatal rats received EE at 10 ng (1 microg/kg), 100 ng, 1 microg, or 10 microg per pup on alternate days from postnatal days 1 to 11 (dose-response study) or received EE or DES at 100 ng per pup daily from postnatal days 1 to 6 (comparative study). Effects of EE were dose dependent, with > or = 100-ng dose inducing significant (p < 0.05) reductions in penile length, weight, and diameter. Additionally, the penis was malformed, characterized by underdeveloped os penis and accumulation of fat cells. Fertility was 0% in the > or = 1-microg groups, in contrast to 60% in the 100-ng group and 100% in the 10-ng and control groups. Animals treated with > or = 10 ng had significant reductions in the weight of bulbospongious muscle, testis, seminal vesicle, epididymal fat pad, and in epididymal sperm numbers. A comparison of EE and DES effects showed similar reductions in penile weight and length and the weight of bulbospongiosus muscle, testis, seminal vesicle, epididymis, and epididymal fat pad in both adolescent and adult rats. While 5/6 control males sired, only 1/6 in the EE group and 0/6 in the DES group sired. Hence, neonatal exposure to EE at 10 ng (environmentally relevant dose) adversely affects male reproductive organs. A dose ten times higher than this leads to permanently mal-developed penis and infertility. Furthermore, EE and DES exposures show similar level of toxicity to male reproductive organs.