The Effects of Sex Hormones on the Size of Intestinal Lipoproteins

Larger intestinal lipoproteins are more likely to be retained longer in the intestinal wall, allowing more time for their fat to be hydrolyzed and subsequently taken up by the abdominal viscera. Since men generally accumulate more abdominal visceral fat than women, we sought to determine if males produce larger intestinal lipoproteins compared to females. Using the conscious lymph fistula mouse model, we discovered that the male mice indeed produced larger intestinal lipoproteins than the female mice when they were intraduodenally infused with lipid emulsion. We then employed our differentiated Caco-2 cell model with semipermeable membrane system to determine the effects of sex hormones on the size of intestinal lipoproteins. Lipoprotein size was quantitatively measured by calculating the ratio of triglycerides (TG)/Apolipoprotein B (ApoB) and by analyzing their transmission electron micrographs. Our studies showed that while there was no dose-dependent effect of estrogen and progesterone, testosterone significantly increased the size of lipoproteins. When these hormones were combined to resemble the physiological concentrations observed in males and the different ovarian cycle phases in premenopausal females, both the male and luteal groups had significantly larger lipoproteins than the ovulatory group; and the male group also had significantly larger lipoproteins than the follicular group. The ovulatory group secreted a significantly lower amount of TG than the male and luteal groups. ApoB was comparable among all these groups. These findings support our hypothesis that, through their testosterone effects, males are more likely to produce larger intestinal lipoproteins. Larger lipoproteins tend to remain longer in the intestinal wall and may facilitate fat uptake preferentially by the abdominal viscera. Our studies may partly explain why men are more prone to accumulating abdominal visceral fat, which is an independent predictor of mortality

Interactions among Endothelial Nitric Oxide Synthase, Cardiovascular System, and Nociception during Physiological and Pathophysiological States

Nitric oxide synthase (NOS) plays important roles within the cardiovascular system in physiological states as well as in pathophysiologic and specific cardiovascular (CV) disease states, such as hypertension (HTN), arteriosclerosis, and cerebrovascular accidents. This review discusses the roles of the endothelial NOS (eNOS) and its effect on cardiovascular responses that are induced by nociceptive stimuli. The roles of eNOS enzyme in modulating CV functions while experiencing pain will be discussed. Nociception, otherwise known as the subjective experience of pain through sensory receptors, termed “nociceptors”, can be stimulated by various external or internal stimuli. In turn, events of various cascade pathways implicating eNOS contribute to a plethora of pathophysiological responses to the noxious pain stimuli. Nociception pathways involve various regions of the brain and spinal cord, including the dorsolateral periaqueductal gray matter (PAG), rostral ventrolateral medulla (RVLM), caudal ventrolateral medulla, and intermediolateral column of the spinal cord. These pathways can interrelate in nociceptive responses to pain stimuli. The alterations in CV responses that affect GABAergic and glutamatergic pathways will be discussed in relation to mechanical and thermal (heat and cold) stimuli. Overall, this paper will discuss the aggregate recent and past data regarding pain pathways and the CV system

Chylomicrons Produced by Caco-2 Cells Contained ApoB-48 With Diameter of 80-200 nm

The small intestine generally transports dietary fats to circulation in triglyceride (TG)-rich lipoproteins. The two main intestinal lipoproteins are chylomicron (CM) and very low-density lipoprotein (VLDL). Unfortunately, studies on the CM biogenesis and intestinal transport of dietary fats have been hampered by the lack of an adequate in vitro model. In this study, we investigated the possible factors that might increase the efficiency of CM production by Caco-2 cells. We utilized sequential NaCl gradient ultracentrifugation to isolate the CMs that were secreted by the Caco-2 cells. To confirm the successful isolation of the CMs, we performed Fat Red 7B staining, TG reading, apolipoprotein B (ApoB) measurement, and transmission electron microcopy (TEM) analysis. We then tested the effects of cell differentiation, oleic acid, mono-olein, egg lecithin, incubation time, and collagen matrix on CM secretion. We found that cell differentiation, oleic acid, and lecithin were critical for CM secretion. Using the Transwell system, we further confirmed that the CMs produced by our Caco-2 cells contained significant amount of TGs and ApoB-48 such that they could be detected without the use of isotope labeling. In conclusion, when fully differentiated Caco-2 were challenged with oleic acid, lecithin, and sodium taurocholate, 21% of their total number of lipoproteins were CMs with the diameter of 80-200 nm

Chylomicrons Produced by Caco-2 Cells Contained ApoB-48 with Diameter of 80-200 nm

The small intestine generally transports dietary fats to circulation in triglyceride (TG)-rich lipoproteins. The two main intestinal lipoproteins are chylomicron (CM) and very low-density lipoprotein (VLDL). Unfortunately, studies on the CM biogenesis and intestinal transport of dietary fats have been hampered by the lack of an adequate in vitro model. In this study, we investigated the possible factors that might increase the efficiency of CM production by Caco-2 cells. We utilized sequential NaCl gradient ultracentrifugation to isolate the CMs that were secreted by the Caco-2 cells. To confirm the successful isolation of the CMs, we performed Fat Red 7B staining, TG reading, apolipoprotein B (ApoB) measurement, and transmission electron microcopy (TEM) analysis. We then tested the effects of cell differentiation, oleic acid, mono-olein, egg lecithin, incubation time, and collagen matrix on CM secretion. We found that cell differentiation, oleic acid, and lecithin were critical for CM secretion. Using the Transwell system, we further confirmed that the CMs produced by our Caco-2 cells contained significant amount of TGs and ApoB-48 such that they could be detected without the use of isotope labeling. In conclusion, when fully differentiated Caco-2 were challenged with oleic acid, lecithin, and sodium taurocholate, 21% of their total number of lipoproteins were CMs with the diameter of 80-200 nm

Anaphylactic Reactions to Oligosaccharides in Red Meat: a Syndrome in Evolution

OBJECTIVE: While most allergic responses to food are directed against protein epitopes and occur within 30 minutes of ingesting the allergen, recent studies suggest that delayed reactions may occur, sometimes mediated by IgE antibodies directed against carbohydrate moieties. The objective of this review is to summarize the clinical features and management of delayed hypersensitivity reactions to mammalian meat mediated by IgE antibodies to galactose-alpha 1,3-galactose (alpha-gal), an oligosaccharide. METHODS: A PubMed search was conducted with MeSH terms: galactosyl-(1,3) galactose, oligosaccharides, cetuximab, allergy/hypersensitivity, and anaphylaxis. Reported cases with alpha-gal-mediated reactions were reviewed. This research study was approved by the Institutional Review Board of East Tennessee State University. RESULTS: Thirty-two cases of adults presenting with red-meat induced allergy thought to be related to oligosaccharides have been reported in the literature so far, making this a rare and evolving syndrome. Most of these patients demonstrated delayed reactions to beef, as was seen in the case reported by us in this manuscript. IgE specific to alpha-gal was identified in most patients with variable response to skin testing with beef and pork. Inhibition studies in some cases showed that the IgE antibodies to beef were directed towards alpha-gal in the meat rather than the protein. The patients often reported history of tick bites, the significance of which is unclear at present. Reactions to cetuximab, a monoclonal antibody, are mediated by a similar mechanism, with IgE antibodies directed against an alpha-gal moiety incorporated in the drug structure. CONCLUSION: Alpha-gal is an oligosaccharide recently incriminated in delayed anaphylactic reactions to mammalian meats such as to beef, pork, and lamb. It appears that anaphylactic reactions to the anti-cancer biological agent, cetuximab, may be linked mechanistically to the same process. More studies are required to understand the underlying molecular basis for these delayed reactions in specific, and their broader implications for host defense in general

Anaphylactic Reactions to Oligosaccharides in Red Meat: A Syndrome in Evolution

Objective: While most allergic responses to food are directed against protein epitopes and occur within 30 minutes of ingesting the allergen, recent studies suggest that delayed reactions may occur, sometimes mediated by IgE antibodies directed against carbohydrate moieties. The objective of this review is to summarize the clinical features and management of delayed hypersensitivity reactions to mammalian meat mediated by IgE antibodies to galactose-alpha 1,3-galactose (alpha-gal), an oligosaccharide.Methods: A PubMed search was conducted with MeSH terms: galactosyl-(1,3) galactose, oligosaccharides, cetuximab, allergy/hypersensitivity, and anaphylaxis. Reported cases with alpha-gal-mediated reactions were reviewed. This research study was approved by the Institutional Review Board of East Tennessee State University.Results: Thirty-two cases of adults presenting with red-meat induced allergy thought to be related to oligosaccharides have been reported in the literature so far, making this a rare and evolving syndrome. Most of these patients demonstrated delayed reactions to beef, as was seen in the case reported by us in this manuscript. IgE specific to alpha-gal was identified in most patients with variable response to skin testing with beef and pork. Inhibition studies in some cases showed that the IgE antibodies to beef were directed towards alpha-gal in the meat rather than the protein. The patients often reported history of tick bites, the significance of which is unclear at present. Reactions to cetuximab, a monoclonal antibody, are mediated by a similar mechanism, with IgE antibodies directed against an alpha-gal moiety incorporated in the drug structure.Conclusion: Alpha-gal is an oligosaccharide recently incriminated in delayed anaphylactic reactions to mammalian meats such as to beef, pork, and lamb. It appears that anaphylactic reactions to the anti-cancer biological agent, cetuximab, may be linked mechanistically to the same process. More studies are required to understand the underlying molecular basis for these delayed reactions in specific, and their broader implications for host defense in general

Cilostazol Prevents Endothelin-Induced Smooth Muscle Constriction and Proliferation

Cilostazol is a phosphodiesterase inhibitor that has been shown to inhibit platelet activation. Endothelin is known to be the most potent endogenous growth promoting and vasoactive peptide. In patients and animal models with stroke, the level of circulating endothelin increases and complicates the recovery progress contributed by vascular constriction (an immediate pathology) and vascular proliferation (a long-term pathology). However, the effects of cilostazol on endothelin have not been explored. To demonstrate the dual-antagonizing effects of cilostazol on vasoconstriction and cell proliferation induced by endothelin, we used primary culture of mouse vascular smooth muscle cells in vitro, mouse femoral artery ex vivo, and intracranial basilar artery ex vivo. We show that the dual-inhibition effects of cilostazol are mediated by blocking endothelin-induced extracellular calcium influx. Although cilostazol does not inhibit endothelin-induced intraorganellar calcium release, blockade of extracellular calcium influx is sufficient to blunt endothelin-induced vasoconstriction. We also show that cilostazol inhibits endothelin-induced cellular proliferation by blocking extracellular calcium influx. Inhibition of cAMP-dependent protein kinase (PKA) can block anti-proliferation activity of cilostazol, confirming the downstream role of PKA in cellular proliferation. To further demonstrate the selectivity of the dual-antagonizing effects of cilostazol, we used a different phosphodiesterase inhibitor. Interestingly, sildenafil inhibits endothelin-induced vasoconstriction but not cellular proliferation in smooth muscle cells. For the first time, we show selective dual-antagonizing effects of cilostazol on endothelin. We propose that cilostazol is an excellent candidate to treat endothelin-associated diseases, such as stroke

Cilostazol Prevents Endothelin-Induced Smooth Muscle Constriction and Proliferation

Cilostazol is a phosphodiesterase inhibitor that has been shown to inhibit platelet activation. Endothelin is known to be the most potent endogenous growth promoting and vasoactive peptide. In patients and animal models with stroke, the level of circulating endothelin increases and complicates the recovery progress contributed by vascular constriction (an immediate pathology) and vascular proliferation (a long-term pathology). However, the effects of cilostazol on endothelin have not been explored. To demonstrate the dual-antagonizing effects of cilostazol on vasoconstriction and cell proliferation induced by endothelin, we used primary culture of mouse vascular smooth muscle cells in vitro, mouse femoral artery ex vivo, and intracranial basilar artery ex vivo. We show that the dual-inhibition effects of cilostazol are mediated by blocking endothelin-induced extracellular calcium influx. Although cilostazol does not inhibit endothelin-induced intraorganellar calcium release, blockade of extracellular calcium influx is sufficient to blunt endothelin-induced vasoconstriction. We also show that cilostazol inhibits endothelin-induced cellular proliferation by blocking extracellular calcium influx. Inhibition of cAMP-dependent protein kinase (PKA) can block anti-proliferation activity of cilostazol, confirming the downstream role of PKA in cellular proliferation. To further demonstrate the selectivity of the dual-antagonizing effects of cilostazol, we used a different phosphodiesterase inhibitor. Interestingly, sildenafil inhibits endothelin-induced vasoconstriction but not cellular proliferation in smooth muscle cells. For the first time, we show selective dual-antagonizing effects of cilostazol on endothelin. We propose that cilostazol is an excellent candidate to treat endothelin-associated diseases, such as stroke

Rapamycin Treatment Correlates Changes in Primary Cilia Expression with Cell Cycle Regulation in Epithelial Cells

Primary cilia are sensory organelles that regulate cell cycle and signaling pathways. In addition to its association with cancer, dysfunction of primary cilia is responsible for the pathogenesis of polycystic kidney disease (PKD) and other ciliopathies. Because the association between cilia formation or length and cell cycle or division is poorly understood, we here evaluated their correlation in this study. Using Spectral Karyotyping (SKY) technique, we showed that PKD and the cancer/tumorigenic epithelial cells PC3, DU145, and NL20-TA were associated with abnormal ploidy. We also showed that PKD and the cancer epithelia were highly proliferative. Importantly, the cancer epithelial cells had a reduction in the presence and/or length of primary cilia relative to the normal kidney (NK) cells. We then used rapamycin to restore the expression and length of primary cilia in these cells. Our subsequent analyses indicated that both the presence and length of primary cilia were inversely correlated with cell proliferation. Collectively, our data suggest that restoring the presence and/or length of primary cilia may serve as a novel approach to inhibit cancer cell proliferation

Dopaminergic Signaling Within the Primary Cilia in the Renovascular System

Activation of dopamine receptor type-5 (DR5) has been known to reduce systemic blood pressure, most likely by increasing renal vasodilation and enhancing natriuresis in the kidney. However, the mechanism of DR5 in natriuresis and vasodilation was not clearly known. We have previously shown that DR5 is localized to primary cilia of proximal renal epithelial and vascular endothelial cells. We here show that selective activation of DR5 specifically induces calcium influx only in the primary cilia, whereas non-selective activation of dopamine receptor induces calcium fluxes in both cilioplasm and cytoplasm. Cilia-independent signaling induced by thrombin only shows calcium signaling within cytoplasm. Furthermore, calcium activation in the cilioplasm by DR5 increases length and mechanosensory function of primary cilia, leading to a greater response to fluid-shear stress. We therefore propose a new mechanism by which DR5 induces vasodilation via chemical and mechanical properties that are specific to primary cilia