Receptor Protein Abundance Research Articles

Atretic preovulatory follicles could be precursors of ovarian lutein cysts in the pig

Ovarian cysts contribute to reduced reproductive performance in pigs. Unfortunately, the mechanism of lutein cysts formation remains unknown. Here, we compared the endocrine and molecular milieus of intact, healthy preovulatory follicles (PF), gonadotropin (eCG/hCG)-induced healthy and atretic-like PF, as well as gonadotropin-provoked and spontaneous ovarian cysts in gilts. Several endocrine and molecular indicators and microRNA were compared in walls of PF and cysts. Intact and healthy PF, showed high estradiol/androstendione and low progesterone levels associated with CYP17A1, HSD17B1, and CYP19A1 elevation and reduced StAR/HSD3B1 protein expression. In contrast, low estradiol/androstendione and high progesterone concentrations, accompanied by decreased CYP17A1, HSD17B1, CYP19A1 and increased HSD3B1 protein abundance, appeared in atretic-like PF, gonadotropin-induced and spontaneous cysts. High progesterone receptor (PGR) protein abundance was maintained in intact and healthy PF, while it dropped in atretic-like PF, gonadotropins-induced and spontaneous cysts. The atretic PF showed high level of TNFα compared to healthy PF. In conclusion, follicular lutein cysts could be recruited from atretic-like PF with lost estrogenic milieu and inability to ovulate. Ovulatory cascade was presumably disrupted by a low PGR and high TNFα levels associated with earlier luteinization of follicular walls. These results suggest a novel mechanism of lutein ovarian cysts development in pigs and, perhaps, other species.

Tripeptide IRW Improves AMPK/eNOS Signaling Pathway via Activating ACE2 in the Aorta of High-Fat-Diet-Fed C57BL/6 Mice.

This study aims to investigate the effect of tripeptide IRW on the local renin-angiotensin system (RAS), particularly angiotensin-converting enzyme 2 (ACE2), and their association with signaling pathways in the aorta of a high-fat-diet (HFD)-induced insulin-resistant mouse model. C57BL/6 mice were fed HFD (45% of the total calories) for six weeks, and then IRW was added to the diet (45 mg/kg body weight (BW)) for another eight weeks. ACE2 mRNA expression and protein level(s) were increased (p < 0.05), while angiotensin II receptor (AT1R) and angiotensin-converting enzyme (ACE) protein abundance was significantly reduced (p < 0.05) in the aorta of HFD mice treated by IRW. IRW supplementation also improved glucose transporter 4 (GLUT4) abundance (p < 0.05) alongside AMP-activated protein kinase (AMPK) (p < 0.05), Sirtuin 1 (SIRT1) (p < 0.05), and endothelial nitric oxide synthase (eNOS) (p < 0.05) expression. IRW downregulated the levels of endothelin 1 (ET-1) and p38 mitogen-activated protein kinases (p38 MAPK, p < 0.05). Furthermore, the levels of AMPK and eNOS in vascular smooth muscle cells (VSMCs) were significantly reduced in ACE2 knockdown cells treated with or without IRW (p < 0.01). In conclusion, this study provided new evidence of the regulatory role of IRW on the aortic ACE2 against metabolic syndrome (MetS) in an HFD-induced insulin-resistant model.

Abstract 3912: cProSite: A web based interactive platform for on-line proteomics and phosphoproteomics data analysis

Abstract We have developed a web based interactive platform cProSite (The Cancer Proteogenomic Data Analysis Site). It provides on-line proteomics and phosphoproteomics analysis for the data in The National Cancer Institute’s Clinical Proteomic Tumor Analysis Consortium (CPTAC), with a focus on tumor and adjacent normal comparisons and correlations between different protein or phosphorylation levels or between protein abundances and mRNA expressions. The web platform is designed for biologists and clinicians wishing to make rapid searches in a user friendly environment. The platform analysis has several advantages compared with standard analytical methods: 1) Faster analysis on-line; 2) More user-friendly; 3) Less need for bioinformatics expertise to perform analyses. Current cProSite includes 11 tumor types*. The web platform includes the following features: 1) Can compare selected protein abundance between tumors and normal adjacent tissues and visualization of fold-changes between paired tumor and control tissue. 2) Can compare selected levels of individual phosphorylation sites or levels of phosphorylation site per protein abundance and visualization of fold-changes between tumors and normal adjacent tissues. 3) Can correlate protein abundance, phosphorylation site levels, or phosphorylation site level per protein abundance between two selected proteins. 4) Can correlate selected protein abundance with its mRNA expression level if the data are available. The cProSite export feature allows users to download the analyzed data. In addition, search outcomes from cProSite can be used for clinical data validation and novel target identification. For example, cProSite can be used to validate findings from other studies; the upregulation of the Hyaluronan Mediated Motility Receptor (Rhamm/HMMR) protein abundance in non-small cell lung adenocarcinoma detected by tissue microarrays was confirmed by cProSite analysis. cProSite can also be used for hypothesis-generation and lead to hypothesis testing. In one example, cProSite identified several upregulated phosphorylation sites in the ECT2 (G2/M phase protein Epithelia Cell Transforming 2) protein in several tumor types. Consistent with that observation, mass spectrometry results showed increased phosphorylation levels from those phosphorylation sites in synchronized lung cancer cell lines in the G2/M phase. * The 11 tumor types in cProSite are: breast cancer, colon cancer, head and neck cancer, liver cancer, kidney cancer, lung adenocarcinoma, lung squamous carcinoma, ovarian cancer, pancreatic ductal adenocarcinoma, stomach cancer, and uterine cancer. Citation Format: Dunrui Wang, Xiaolan Qian, Yi-Chieh Nancy Du, Beatriz Sanchez-Solana, Kailing Chen, Brian Park, Ben Chen, Lisa Jenkins, Jason Luo, Brajendra K. Tripathi, Marian E. Durkin, Douglas R. Lowy. cProSite: A web based interactive platform for on-line proteomics and phosphoproteomics data analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3912.

Comparison of methods and resources for cell-cell communication inference from single-cell RNA-Seq data

The growing availability of single-cell data, especially transcriptomics, has sparked an increased interest in the inference of cell-cell communication. Many computational tools were developed for this purpose. Each of them consists of a resource of intercellular interactions prior knowledge and a method to predict potential cell-cell communication events. Yet the impact of the choice of resource and method on the resulting predictions is largely unknown. To shed light on this, we systematically compare 16 cell-cell communication inference resources and 7 methods, plus the consensus between the methods’ predictions. Among the resources, we find few unique interactions, a varying degree of overlap, and an uneven coverage of specific pathways and tissue-enriched proteins. We then examine all possible combinations of methods and resources and show that both strongly influence the predicted intercellular interactions. Finally, we assess the agreement of cell-cell communication methods with spatial colocalisation, cytokine activities, and receptor protein abundance and find that predictions are generally coherent with those data modalities. To facilitate the use of the methods and resources described in this work, we provide LIANA, a LIgand-receptor ANalysis frAmework as an open-source interface to all the resources and methods.

Increased uterine androgen receptor protein abundance results in implantation and mitochondrial defects in pregnant rats with hyperandrogenism and insulin resistance

In this study, we show that during normal rat pregnancy, there is a gestational stage-dependent decrease in androgen receptor (AR) abundance in the gravid uterus and that this is correlated with the differential expression of endometrial receptivity and decidualization genes during early and mid-gestation. In contrast, exposure to 5α-dihydrotestosterone (DHT) and insulin (INS) or DHT alone significantly increased AR protein levels in the uterus in association with the aberrant expression of endometrial receptivity and decidualization genes, as well as disrupted implantation. Next, we assessed the functional relevance of the androgen-AR axis in the uterus for reproductive outcomes by treating normal pregnant rats and pregnant rats exposed to DHT and INS with the anti-androgen flutamide. We found that AR blockage using flutamide largely attenuated the DHT and INS-induced maternal endocrine, metabolic, and fertility impairments in pregnant rats in association with suppressed induction of uterine AR protein abundance and androgen-regulated response protein and normalized expression of several endometrial receptivity and decidualization genes. Further, blockade of AR normalized the expression of the mitochondrial biogenesis marker Nrf1 and the mitochondrial functional proteins Complexes I and II, VDAC, and PHB1. However, flutamide treatment did not rescue the compromised mitochondrial structure resulting from co-exposure to DHT and INS. These results demonstrate that functional AR protein is an important factor for gravid uterine function. Impairments in the uterine androgen-AR axis are accompanied by decreased endometrial receptivity, decidualization, and mitochondrial dysfunction, which might contribute to abnormal implantation in pregnant PCOS patients with compromised pregnancy outcomes and subfertility.Key messagesThe proper regulation of uterine androgen receptor (AR) contributes to anormal pregnancy process, whereas the aberrant regulation of uterine AR mightbe linked to polycystic ovary syndrome (PCOS)-induced pregnancy-relatedcomplications.In the current study, we found that during normal rat pregnancy there isa stage-dependent decrease in AR abundance in the gravid uterus and that thisis correlated with the differential expression of the endometrial receptivityand decidualization genes Spp1, Prl, Igfbp1,and Hbegf.Pregnant rats exposed to 5α-dihydrotestosterone (DHT) and insulin (INS)or to DHT alone show elevated uterine AR protein abundance and implantationfailure related to the aberrant expression of genes involved in endometrialreceptivity and decidualization in early to mid-gestation.Treatment with the anti-androgen flutamide, starting frompre-implantation, effectively prevents DHT + INS-induced defects in endometrialreceptivity and decidualization gene expression, restores uterine mitochondrialhomeostasis, and increases the pregnancy rate and the numbers of viablefetuses.This study adds to our understanding of the mechanisms underlying poorpregnancy outcomes in PCOS patients and the possible therapeutic use ofanti-androgens, including flutamide, after spontaneous conception.

Resting in bed - how quickly does the muscle lose its nerve?

Resting in bed - how quickly does the muscle lose its nerve?

Lysophosphatidic acid as a regulator of endometrial connective tissue growth factor and prostaglandin secretion during estrous cycle and endometrosis in the mare

BackgroundEquine endometrosis is a chronic degenerative condition, described as endometrial fibrosis that forms in the stroma, under the basement membrane and around the endometrial glands. The role of lysophosphatidic acid (LPA) in the development of tissue fibrosis varies depending on the organ, and its profibrotic role in mare endometrosis remains unclear. The study aimed to establish the endometrial presence of LPA and its receptors (LPAR1–4), together with its effects on connective tissue growth factor (CTGF) and prostaglandins (PG) secretion from equine endometrium under physiological (estrous cycle), or pathological conditions (endometrosis). Mare endometria in the mid-luteal phase (n = 5 for each category I, IIA, IIB, III of Kenney and Doig) and in the follicular phase (n = 5 for each category I, IIA, III and n = 4 for IIB) were used. In experiment 1, the levels of LPA, LPAR1–4 mRNA level and protein abundance were investigated in endometria at different stages of endometrosis. In experiment 2, the in vitro effect of LPA (10− 9 M) on the secretion of CTGF and PGs from endometrial tissue explants at different stages of endometrosis were determined.ResultsEndometrial LPA concentration was higher in the mid-luteal phase compared to the follicular phase in category I endometrium (P < 0.01). There was an alteration in endometrial concentrations of LPA and LPAR1–4 protein abundance in the follicular phase at different stages of endometrosis (P < 0.05). Additionally, LPA increased the secretion of PGE2 from category I endometrium in both phases of the estrous cycle (P < 0.05). The effect of LPA on the secretion of CTGF and PGF2α from endometrial tissue was altered depending on different stages of endometrosis (P < 0.05).ConclusionOur data indicate that endometrosis disturbs proper endometrial function and is associated with altered endometrial LPA concentration, its receptor expression and protein abundance, PGE2/PGF2α ratio, and CTGF secretion in response to LPA. These changes could influence several physiological events occurring in endometrium in mare during estrous cycle and early pregnancy.

Role for ovarian hormones in purinoceptor-dependent natriuresis

BackgroundPremenopausal women have a lower risk of hypertension compared to age-matched men and postmenopausal women. P2Y2 and P2Y4 purinoceptor can be considered potential contributors to hypertension due to their emerging roles in regulating renal tubular Na+ transport. Activation of these receptors inhibits epithelial Na+ channel activity (ENaC) via a phospholipase C (PLC)-dependent pathway resulting in natriuresis. We recently reported that activation of P2Y2 and P2Y4 receptors in the renal medulla by UTP promotes natriuresis in male and ovariectomized (OVX) rats, but not in ovary-intact females. This led us to hypothesize that ovary-intact females have greater basal renal medullary activity of P2 (P2Y2 and P2Y4) receptors regulating Na+ excretion compared to male and OVX rats.MethodsTo test our hypothesis, we determined (i) the effect of inhibiting medullary P2 receptors by suramin (750 μg/kg/min) on urinary Na+ excretion in anesthetized male, ovary-intact female, and OVX Sprague Dawley rats, (ii) mRNA expression and protein abundance of P2Y2 and P2Y4 receptors, and (iii) mRNA expression of their downstream effectors (PLC-1δ and ENaCα) in renal inner medullary tissues obtained from these three groups. We also subjected cultured mouse inner medullary collecting duct cells (segment 3, mIMCD3) to different concentrations of 17ß-estradiol (E2, 0, 10, 100, and 1000 nM) to test whether E2 increases mRNA expression of P2Y2 and P2Y4 receptors.ResultsAcute P2 inhibition attenuated urinary Na+ excretion in ovary-intact females, but not in male or OVX rats. We found that P2Y2 and P2Y4 mRNA expression was higher in the inner medulla from females compared to males or OVX. Inner medullary lysates showed that ovary-intact females have higher P2Y2 receptor protein abundance, compared to males; however, OVX did not eliminate this sex difference. We also found that E2 dose-dependently upregulated P2Y2 and P2Y4 mRNA expression in mIMCD3.ConclusionThese data suggest that ovary-intact females have enhanced P2Y2 and P2Y4-dependent regulation of Na+ handling in the renal medulla, compared to male and OVX rats. We speculate that the P2 pathway contributes to facilitated renal Na+ handling in premenopausal females.

Two-Component Biosensors: Unveiling the Mechanisms of Predictable Tunability.

Many studies have been devoted to the engineering of cellular biosensors by exploiting intrinsic natural sensors. However, biosensors rely not only on input detection but also on an adequate response range. It is therefore often necessary to tune natural systems to meet the demands of specific applications in a predictable manner. In this study, we explored the customizability of two-component bacterial biosensors by modulating the main biosensor component, i.e., the receptor protein. We developed a mathematical model that describes the functional relationship between receptor abundance and activation threshold, sensitivity, dynamic range, and operating range. The defined mathematical framework allows the design of the genetic architecture of a two-component biosensor that can perform as required with minimal genetic engineering. To experimentally validate the model and its predictions, a library of biosensors was constructed. The good agreement between theoretical designs and experimental results indicates that modulation of receptor protein abundance allows optimization of biosensor designs with minimal genetic engineering.

The impact of progesterone and RU-486 on classic pro-labour proteins & contractility in human myometrial tissues during 24-hour exposure to tension & interleukin-1β

Increased expression of pro-labour genes that encode cyclooxygenase-2 (COX-2), oxytocin receptor (OTR) and connexin-43 (Cx43) at parturition is often attributed to P4 functional withdrawal, based on findings from animal models and human primary myometrial cells. However, the cause of reduced myometrial P4 responsiveness that promotes contractions at labour is not fully determined. Uterine stretch occurs with advancing gestation but most in vitro experimental models do not take this into consideration. We aimed to examine whether tissue-level myometrial stretch influences the ability of P4 to regulate pro-labour protein abundance by using myometrial biopsies from term gestation pregnant women to assess the impact of 24 h exposure to combinations of (i) stretch-mediated tension, (ii) P4 (100 nM) and (iii) an anti-progestin, RU-486 (1 μM). Firstly, we observed baseline COX-2 and Cx43 protein levels increased, whereas P4 content along with calponin-1 and progesterone receptor (PR) protein abundance decreased, in vehicle-treated tissues. P4 supplementation subtly reduced COX-2 levels in un-stretched tissues. Spontaneous and oxytocin-augmented contractility were unchanged by tissue culture exposure to P4 and/or RU-486. Interleukin-1β (IL-1β; 1 ng/ml) enhanced COX-2 protein and PGE2 content in un-stretched tissues. Overall, tissue stretch may, in part, regulate P4-sensitive pro-labour protein levels, but this is likely to be reliant on interaction with other in utero factors that were absent in our tissue cultures. More complex culture conditions should be evaluated in future to aid further development of a physiologically relevant model to improve our understanding of in utero myometrial P4 responsiveness.

Impact of heat stress during the follicular phase on porcine ovarian steroidogenic and phosphatidylinositol-3 signaling.

Environmental conditions that impede heat dissipation and increase body temperature cause heat stress (HS). The study objective was to evaluate impacts of HS on the follicular phase of the estrous cycle. Postpubertal gilts (126.0 ± 21.6 kg) were orally administered altrenogest to synchronize estrus, and subjected to either 5 d of thermal-neutral (TN; 20.3 ± 0.5 °C; n = 6) or cyclical HS (25.4 - 31.9 °C; n = 6) conditions during the follicular phase preceding behavioral estrus. On d 5, blood samples were obtained, gilts were euthanized, and ovaries collected. Fluid from dominant follicles was aspirated and ovarian protein homogenates prepared for protein abundance analysis. HS decreased feed intake (22%; P = 0.03) and while plasma insulin levels did not differ, the insulin:feed intake ratio was increased 3-fold by HS (P = 0.02). Insulin receptor protein abundance was increased (29%; P < 0.01), but insulin receptor substrate 1, total and phosphorylated protein kinase B, superoxide dismutase 1, and acyloxyacyl hydrolase protein abundance were unaffected by HS (P > 0.05). Plasma and follicular fluid 17β-estradiol, progesterone, and lipopolysaccharide-binding protein concentrations as well as abundance of steroid acute regulatory protein, cytochrome P450 19A1, and multidrug resistance-associated protein 1 were not affected by HS (P > 0.05). HS increased estrogen sulfotransferase protein abundance (44%; P = 0.02), toll-like receptor 4 (36%; P = 0.05), and phosphorylated REL-associated protein (31%; P = 0.02). Regardless of treatment, toll-like receptor 4 protein was localized to mural granulosa cells in the porcine ovary. In conclusion, HS altered ovarian signaling in postpubertal gilts during their follicular phase in ways that likely contributes to seasonal infertility.

Downregulated Translation Initiation Signaling Predisposes Low-Birth-Weight Neonatal Pigs to Slower Rates of Muscle Protein Synthesis.

Low-birth-weight (LBWT) neonates experience restricted muscle growth in their perinatal life. Our aim was to investigate the mechanisms that contribute to slower skeletal muscle growth of LBWT neonatal pigs. Twenty-four 1-day old male LBWT (816 ± 55 g) and normal-birth-weight (NBWT; 1,642 ± 55 g) littermates (n = 12) were euthanized to collect blood and longissimus dorsi (LD) muscle subsamples. Plasma glucose, insulin, and insulin-like growth factor-I (IGF-I) were lower in LBWT compared with NBWT pigs. Muscle IGF-I mRNA expression were lower in LBWT than NBWT pigs. However, IGF-I receptor mRNA and protein abundance was greater in LD of LBWT pigs. Abundance of myostatin and its receptors, and abundance and phosphorylation of smad3 were lower in LBWT LD by comparison with NBWT LD. Abundance of eukaryotic initiation factor (eIF) 4E binding protein 1 and mitogen-activated protein kinase-interacting kinases was lower in muscle of LBWT pigs compared with NBWT siblings, while eIF4E abundance and phosphorylation did not differ between the two groups. Furthermore, phosphorylation of ribosomal protein S6 kinase 1 (S6K1) was less in LBWT muscle, possibly due to lower eIF3e abundance. In addition, abundance and phosphorylation of eIF4G was reduced in LBWT pigs by comparison with NBWT littermates, suggesting translation initiation complex formation is compromised in muscle of LBWT pigs. In conclusion, diminished S6K1 activation and translation initiation signaling are likely the major contributors to impaired muscle growth in LBWT neonatal pigs. The upregulated IGF-I R expression and downregulated myostatin signaling seem to be compensatory responses for the reduction in protein synthesis signaling.

MicroRNA-433 Dampens Glucocorticoid Receptor Signaling, Impacting Circadian Rhythm and Osteoblastic Gene Expression

Serum glucocorticoids play a critical role in synchronizing circadian rhythm in peripheral tissues, and multiple mechanisms regulate tissue sensitivity to glucocorticoids. In the skeleton, circadian rhythm helps coordinate bone formation and resorption. Circadian rhythm is regulated through transcriptional and post-transcriptional feedback loops that include microRNAs. How microRNAs regulate circadian rhythm in bone is unexplored. We show that in mouse calvaria, miR-433 displays robust circadian rhythm, peaking just after dark. In C3H/10T1/2 cells synchronized with a pulse of dexamethasone, inhibition of miR-433 using a tough decoy altered the period and amplitude of Per2 gene expression, suggesting that miR-433 regulates rhythm. Although miR-433 does not directly target the Per2 3'-UTR, it does target two rhythmically expressed genes in calvaria, Igf1 and Hif1α. miR-433 can target the glucocorticoid receptor; however, glucocorticoid receptor protein abundance was unaffected in miR-433 decoy cells. Rather, miR-433 inhibition dramatically enhanced glucocorticoid signaling due to increased nuclear receptor translocation, activating glucocorticoid receptor transcriptional targets. Last, in calvaria of transgenic mice expressing a miR-433 decoy in osteoblastic cells (Col3.6 promoter), the amplitude of Per2 and Bmal1 mRNA rhythm was increased, confirming that miR-433 regulates circadian rhythm. miR-433 was previously shown to target Runx2, and mRNA for Runx2 and its downstream target, osteocalcin, were also increased in miR-433 decoy mouse calvaria. We hypothesize that miR-433 helps maintain circadian rhythm in osteoblasts by regulating sensitivity to glucocorticoid receptor signaling.

Prepubertal tamoxifen treatment affects development of heifer reproductive tissues and related signaling pathways

Prepubertal exposure of the developing ovaries and reproductive tract (RT) to estrogen or xenoestrogens can have acute and long-term consequences that compromise the reproductive performance of cattle. This research examined effects of the selective estrogen receptor modulator tamoxifen (TAM) on gene and protein abundance in prepubertal ovaries and RT, with a particular focus on signaling pathways that affect morphology. Tamoxifen was administered to Holstein heifer calves (n=8) daily (0.3mg/kg subcutaneously) from 28 to 120 d of age, when tissues were collected. Control calves (n=7) received an equal volume of excipient. Weight, gross measurements, and samples of reproductive tissues were collected, and protein and mRNA were extracted from snap-frozen samples of vagina, cervix, uterus, oviduct, ovary, and liver. Neither estradiol nor insulin-like growth factor I (IGFI) concentrations in the serum were affected by TAM treatment. Tamoxifen treatment reduced ovarian weight independently from effects on antral follicle populations, as there was no difference in visible antral follicle numbers on the day of collection. Estrogen receptor α (ESR1) and β (ESR2) mRNA, ESR1 protein, IGFI, progesterone receptor, total growth hormone receptor, WNT4, WNT5A, and WNT7A mRNA, in addition to mitogen-activated protein kinase (MAPK) and phosphorylated MAPK proteins were affected differently depending on the tissue examined. However, neither IGFI receptor mRNA nor protein abundance were affected by TAM treatment. Results indicate that reproductive development in prepubertal Holstein heifer calves is TAM-sensitive, and that bovine RT and ovarian development are supported, in part, by estrogen receptor-dependent mechanisms during the period studied here. Potential long-term consequences of such developmental disruption remain to be defined.

Heat Stress‐induced Insulin Sensitivity in Oxidative Skeletal Muscle

Heat‐related complications continue to be a major health concern for humans and animals and lead to potentially life‐threatening conditions ranging from heat exhaustion, heat stroke and even death. Heat stress have been shown to alter metabolic and energetic parameters in mammals and may alter glucose metabolism and insulin sensitivity. Therefore, the purpose of this investigation was to determine the extent to which short‐term HS altered markers of insulin signaling in oxidative skeletal muscle. To address this, crossbred gilts (n=8/group) were assigned to thermoneutral (TN; 24° C), HS (37° C), or pair‐fed to HS and kept under TN conditions (PFTN) groups for 12 hours. Following treatment, animals were euthanized and the semitendinosus red (STR) was recovered. HS‐induced changes were not caused by reduced feed intake as TN and PFTN were similar for nearly all measures. Twelve hours of HS increased insulin receptor protein abundance by 54% (p&lt;0.05) and phosphorylated insulin receptor substrate (pIRS1S307) by 56% (p&lt;0.05) compared to TN. Phosphorylation of IRS1 at this site is a negative regulator of insulin signaling. Relative protein abundance and phosphorylation of phosphoinositide 3‐kinase (PI3K) and 3‐phosphoinositide dependent protein kinase‐1 (PDK1) were similar between groups. Phosphorylation of protein kinase B (pAKTs473) was decreased by 53% (p&lt;0.05) in HS compared to TN, which indicates that insulin signaling was suppressed through pIRS1. Conversely, HS increased phosphorylation of protein kinase C (PKC)λ/ζ and pPKCδ protein abundance by 128% and 205%, respectively, compared to TN which are known to stimulate pIRS1S307. Sarcolemma glucose transporter‐4 (Glut4) was decreased by 47% (p&lt;0.05) in HS group compared to TN suggesting reduced glucose uptake. These data suggest that HS induces insulin sensititvity by disturbing insulin signaling via an AKT‐mediated pathway.Support or Funding InformationThis work is supported by USDA grants 2014‐67015‐21627 and 2011‐67003‐30007

Tau Tubulin Kinase TTBK2 Sensitivity of Glutamate Receptor GluK2

Background/Aims: Inherited, autosomal dominant spinocerebellar ataxia type 11 (SCA11) is caused by loss of function mutations of TTBK2 (tau tubulin kinase 2). Mutations observed in patients with SCA11 include truncated TTBK2(450). The present study explored the possibility that TTBK2 influences the function of the glutamate receptor GluK2. Methods: GluK2 was expressed in Xenopus oocytes without and with additional expression of wild type TTBK2, the truncated mutant TTBK2(450), or the kinase dead mutants TTBK2(KD) and TTBK2(450/KD). GluK2 current was determined by dual electrode voltage clamp and GluK2 protein abundance in the cell membrane utilizing confocal microscopy. Results: Glutamate exposure of GluK2 expressing oocytes generated a current, which was significantly lower in oocytes expressing GluK2 together with TTBK2 wt or TTBK2(KD) than in oocytes expressing GluK2 alone or together with either TTBK2(450) or TTBK2(450/KD). According to confocal microscopy of EGFP-tagged GluK2, TTBK2 wt decreased the GluK2 protein abundance in the cell membrane. Overexpression of an inactive RAB5(N133I) mutant but not RAB5wt could reverse the TTBK2 effect on GluK2 suggesting that RAB5 function is required for the effect. Conclusions: TTBK2 down-regulates GluK2 activity by decreasing the receptor protein abundance in the cell membrane via RAB5-dependent endocytosis, an effect that may protect against neuroexcitotoxicity.

Identification of the (Pro)renin Receptor as a Novel Regulator of Low-Density Lipoprotein Metabolism.

The (pro)renin receptor ([P]RR) interacts with (pro)renin at concentrations that are >1000× higher than observed under (patho)physiological conditions. Recent studies have identified renin-angiotensin system-independent functions for (P)RR related to its association with the vacuolar H(+)-ATPase. To uncover renin-angiotensin system-independent functions of the (P)RR. We used a proteomics-based approach to purify and identify (P)RR-interacting proteins. This resulted in identification of sortilin-1 (SORT1) as a high-confidence (P)RR-interacting protein, a finding which was confirmed by coimmunoprecipitation of endogenous (P)RR and SORT1. Functionally, silencing (P)RR expression in hepatocytes decreased SORT1 and low-density lipoprotein (LDL) receptor protein abundance and, as a consequence, resulted in severely attenuated cellular LDL uptake. In contrast to LDL, endocytosis of epidermal growth factor or transferrin remained unaffected by silencing of the (P)RR. Importantly, reduction of LDL receptor and SORT1 protein abundance occurred in the absence of changes in their corresponding transcript level. Consistent with a post-transcriptional event, degradation of the LDL receptor induced by (P)RR silencing could be reversed by lysosomotropic agents, such as bafilomycin A1. Our study identifies a renin-angiotensin system-independent function for the (P)RR in the regulation of LDL metabolism by controlling the levels of SORT1 and LDL receptor.

Abstract P102: Type I Diabetes Mellitus is More Sensitive to Ang II Due to Increased AT1 Receptor Expression in Afferent Arteriole

The prevalence of hypertension is about twice in diabetic subjects than that in non-diabetics. In the presence of hypertension, the progress of diabetic nephropathy has been shown to be more severe and rapid. But the mechanisms for the development of hypertension in diabetes have not been elucidated. We hypothesized that angiotensin II receptor type 1 (AT1 receptor) expression level in renal afferent arteriole is elevated, which enhances the responses to angiotensin II (Ang II) stimulation and contributes to the development of hypertension in diabetes. First, we measured mean arterial pressure (MAP) with telemetry in diabetic and non-diabetic mice with Ang II infusion for 8 weeks. MAP was 26.7 mmHg higher in diabetic mice (157.4±11.3 mmHg) than in non-diabetic mice (130.7±6.4 mmHg). Next, we induced 2-kidney-1-clip Goldblatt hypertension in diabetic mice and non-diabetic mice and measured MAP with telemetry for 8 weeks. MAP was 17.6 mmHg higher in diabetic mice (151.1±9.5 mmHg) than in non-diabetic mice (133.5±4.2 mmHg), indicating blood pressure of diabetic mice is also more responsive to both exogenous and endogenous Ang II than no-diabetic mice. To investigate the mechanisms of the enhanced sensitivity to Ang II, we dissected afferent arterioles from non-diabetic mice and diabetic mice and measured the expression of AT1 receptor mRNA by real-time PCR. AT1 receptor mRNA level was 6 times higher in diabetic mice that in non-diabetic control. AT1 receptor protein abundance in renal cortex was also 5 times higher in diabetic mice than controls. In conclusion, the blood pressure of the diabetic mice are more sensitive to Ang II than non-diabetic controls. Increased AT1 receptor expression in the afferent arterioles may contribute to the development of hypertension in diabetes.

GW25-e1704 Fructose Diet Elevates Circulating PCSK9 and LDL-cholesterol Levels and Reduces Liver LDL Receptor Protein Abundance in Hamsters through a Novel Mechanism

The liver secreted serum protein PCSK9 binds to hepatic LDL receptor (LDLR) and triggers its intracellular degradation. Thus, circulating PCSK9 concentrations influence plasma LDL-c levels and impact cardiovascular disease risk. High fructose diet (HFD) induces dyslipidemia and insulin resistance in animals and humans; however, whether PCSK9 has a causal role in the development of dyslipidmia by high fructose consumption remains elusive. In this study, we showed that serum levels of LDL-c were elevated in hamsters fed HFD as compared to animals fed a normal diet (ND). The increase in circulating LDL-c was accompanied by a 50% reduction of liver LDLR protein abundance in HFD group without changes in LDLR mRNA levels. Examinations of serum PCSK9 levels and liver PCSK9 expressions revealed that hepatic PCSK9 mRNA and protein were reduced by HFD, but serum PCSK9 levels in HFD group were 2.2-fold higher than ND group. PCSK9 in hamster serum was detected as two forms of ~60 kDa and ~53 kDa, the latter being the dominant one. It was previously reported that proprotein convertases furin and PC5A cleave human PCSK9 at Arg 218 -Gln 219 to produce PCSK9-ΔN218 (53 kDa) that circulates in blood along with the intact form. To assess the activity of the equivalent hamster 53 kDa PCSK9-ΔN224 in degrading hepatic LDLR, we conducted in vitro experiments in Huh7 cells by exogenous overexpression of hamster PCSK9 alone or with furin/PC5A. Western blotting of culture medium from transfected Huh7 cells with anti-hamster PCSK9 antibody demonstrated that coexpression of hamster PCSK9 with either furin or PC5A increased the 53 kDa form to 82% and 70% of total PCSK9 species respectively, compared to 18% of the PCSK9 species in the absence of furin or PC5A coexpression. Importantly, the corresponding LDLR protein abundances were reduced by similar extents in Huh7 cells expressing hamster PCSK9 without or with furin/PC5A coexpression. Comparable results were obtained by treating naive Huh7 cells with culture medium of HEK293 cells expressing hamster PCSK9 alone or with furin/PC5A. Altogether, these new findings suggest that HFD may reduce serum LDL-c uptake by LDLR in hamster liver through a novel mechanism that elevates circulating PCSK9 concentration without increasing its hepatic synthesis.

Insulin-Like Growth Factor-I Regulates LH Release by Modulation of Kisspeptin and NMDA-Mediated Neurotransmission in Young and Middle-Aged Female Rats

This study investigated potential mechanisms by which age and IGF-I receptor (IGF-Ir) signaling in the neuroendocrine hypothalamus affect estradiol-positive feedback effects on GnRH neuronal activation and on kisspeptin and N-methyl-D-aspartate (NMDA)-induced LH release and on the abundance of NMDA receptor subunits Nr1 and Nr2b and Kiss1r transcript and protein in the hypothalamus of young and middle-aged female rats. We infused vehicle, IGF-I, or JB-1, a selective antagonist of IGF-Ir, into the third ventricle of ovariectomized female rats primed with estradiol or vehicle and injected with vehicle, kisspeptin (3 or 30 nmol/kg), or NMDA (15 or 30 mg/kg). Regardless of dose, NMDA and kisspeptin resulted in significantly more LH release, GnRH/c-Fos colabeling, and c-Fos immunoreative cells in young than in middle-aged females. Estradiol priming significantly increased Kiss1r, Nr1, and Nr2b receptor transcript and protein abundance in young but not middle-aged female hypothalamus. JB-1 attenuated kisspeptin and NMDA-induced LH release, numbers of GnRH/c-Fos and c-Fos cells, and Kiss1r, Nr1, and Nr2b transcript and protein abundance in young females to levels observed in middle-aged females. IGF-I significantly enhanced NMDA and kisspeptin-induced LH release in middle-aged females without increasing numbers of GnRH/c-Fos or c-Fos immunoreactive cells. IGF-I infusion in middle-aged females also increased Kiss1r, Nr1, and Nr2b protein and transcript to levels that were equivalent to young estradiol-primed females. These findings indicate that age-related changes in estradiol-regulated responsiveness to excitatory input from glutamate and kisspeptin reflect reduced IGF-Ir signaling.