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.

The use of advanced spectral imaging to reveal nanoparticle identity in biological samples.

Nanoparticles (NPs) have been used in drug delivery therapies, medical diagnostic strategies, and as current Covid-19 vaccine carriers. Many microscope-based imaging systems have been introduced to facilitate detection and visualization of NPs. Unfortunately, none can differentiate the core and the shell of NPs. Spectral imaging has been used to distinguish a drug molecule and its metabolite. We have recently integrated this technology to a resolution of 9 nm by using artificial intelligence-driven analyses. Such a resolution allowed us to collect many robust datapoints for each pixel of an image. Our analyses could recognize 45 spectral points within a pixel to detect unlabeled Ag-NPs and Au-NPs in single live cells and tissues (liver, heart, spleen and kidneys). The improved resolution and software provided a more specific fingerprinting for each single molecule, allowing simultaneous analyses of 990 complex interactions from the 45 points for each molecule within a pixel of an image. This in turn allowed us to detect surface-functionalization of Ag-NPs to distinguish the core from the shell of Ag-NPs for the first time. Our studies were validated using various laborious and time-consuming conventional techniques. We propose that spectral imaging has tremendous potential to study NP localization and identification in biological samples at a high temporal and spatial resolution, based primarily on spectral identity information.

Sexual dimorphism in intestinal absorption and lymphatic transport of dietary lipids.

Although the basic process of intestinal lipid absorption and transport is understood, many critical aspects remain unclear. One question in particular is whether intestinal lipid absorption and transport differ between the sexes. Using a well-established lymph fistula model, we found that intact female mice exhibited lower lymphatic output of triacylglycerol (TAG) than male mice. Further analysis revealed that the female mice segregated into two groups: the high group having similar lymphatic TAG transport to the males, and the low group having significantly less lymphatic output, implying the impact of cyclical variation of ovarian hormonal levels. These led us to examine whether oestradiol (E2) and progesterone (P) affect intestinal absorption and lymphatic transport of dietary lipids. In ovariectomized (OVX) rats, E2 treatment significantly reduced [3 H]-TAG lymphatic output through reducing TAG transport; and P treatment decreased [14 C]cholesterol (Chol) lymphatic output by inhibiting Chol absorption, compared to vehicle treatment. Gene expression data suggested that E2 enhances vascular endothelial growth factor-A (VEGF-A) signalling to reduce the permeability of lacteals, leading to reduced CM transport through the lymphatic system. Interestingly, E2 treatment also increased lymphatic output of apolipoprotein A-I (apoA-I), but not apoB-48 and apoA-IV, in the OVX rats. Collectively, these data suggested that ovarian hormone-induced reductions of intestinal lipid absorption and lymphatic transport, as well as increased lymphatic output of apoA-I, may contribute to a beneficial protection from atherosclerosis in females. KEY POINTS: Significant differences in intestinal lipid absorption and lymphatic transport were found between female and male animals. Oestrogen treatment significantly reduced [3 H]triacylglycerol (TAG) lymphatic output through suppressing TAG transport in ovariectomized (OVX) rats, and this effect is associated with enhanced vegfa gene expression in the intestine. Progesterone treatment significantly decreased the output of [14 C]cholesterol in lymph by inhibiting cholesterol absorption in the OVX rats. Oestrogen treatment also increased lymphatic output of apolipoprotein A-I (apoA-I) in the OVX rats, which may contribute to the reduced risk of atherosclerosis in females.

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.

Analysis of Student Perceptions of Just-In-Time Teaching Pedagogy in PharmD Microbiology and Immunology Courses.

Just-In-Time Teaching (JiTT) active learning pedagogy is utilized by various disciplines, but its value in a professional pharmacy curriculum has not yet been demonstrated. The purpose of our research study is to implement and evaluate JiTT in a Doctor of Pharmacy (PharmD) program. The impetus in implementing JiTT into a PharmD curriculum was to provide students with an out-of-classroom learning opportunity to enhance knowledge-based skills. The current study summarizes the implementation of JiTT in four distinct instances: two iterations of the required courses "Integrated Microbiology and Virology" (Fall 2016 and Fall 2017) and "Integrated Immunology" (Winter 2016-2017 and Winter 2017-2018). JiTT included knowledge-based questions in multiple-choice format, integrated case studies, and student responses prior to the actual lecture session. After the conclusion of each course, students were asked to provide feedback on the utilization of JiTT by way of an anonymous survey. Following the Fall 2016 iteration of the Microbiology & Virology course, students found the integrated case studies to be beneficial (mean = 3.27 out of a maximum of 4, SD = 0.62), and their overall endorsement of JiTT was high (mean = 3.61 out of 4, SD = 0.50). For the other three courses included in this study, the primary dependent variable was the student's average rating of JiTT, rated on a five-point scale. Aggregating the scores from the Fall 2017 iteration of the Integrated Microbiology & Virology course and both instances of the Immunology course, students rated JiTT very favorably (mean = 4.17 out of a maximum of 5, SD = 0.77). Students' performances in JiTT-based courses were compared against non-JiTT-based courses. Analysis of assessment data for student's performance on knowledge-based questions showed JiTT was helpful for student learning and JiTT-based courses had more consistent exam scores compared to non-JiTT-based courses. The current results are a promising initial step in validating the usefulness of JiTT in a pharmacy program and lays the foundation for future studies aimed at a direct comparison between a traditional lecture style and JiTT pedagogy implemented into PharmD curricula.

Why Do Men Accumulate Abdominal Visceral Fat?

Men have a higher tendency to accumulate abdominal visceral fat compared to pre-menopausal women. The accumulation of abdominal visceral fat in men, which is a strong independent predictor of mortality, is mainly due to the higher dietary fat uptake by their abdominal visceral fat. Since dietary fat is absorbed by the enterocytes and transported to the circulation in the forms of chylomicrons and very low density lipoproteins (VLDLs), it is crucial to understand how these lipoproteins are different between men and women. The chylomicrons in men are generally bigger in size and more in quantity than those in women. During the postprandial state, these chylomicrons congest the lamina propria and the low-pressure lymphatics. In this paper, we propose that this congestion predisposes the chylomicron triglycerides to hydrolysis by lipoprotein lipase (LPL). The liberated fatty acids are then stored by the nearby abdominal visceral adipocytes, leading to the accumulation of abdominal visceral fat. These mechanisms perhaps explain why men, through their bigger and higher production of chylomicrons, are more likely to accumulate abdominal visceral fat than pre-menopausal women. This accumulation eventually leads to belly enlargement, which confers men their apple-shaped body.

Using Caco-2 Cells to Study Lipid Transport by the Intestine.

Studies of dietary fat absorption are generally conducted by using an animal model equipped with a lymph cannula. Although this animal model is widely accepted as the in vivo model of dietary fat absorption, the surgical techniques involved are challenging and expensive. Genetic manipulation of the animal model is also costly and time consuming. The alternative in vitro model is arguably more affordable, timesaving, and less challenging. Importantly, the in vitro model allows investigators to examine the enterocytes as an isolated system, reducing the complexity inherent in the whole organism model. This paper describes how human colon carcinoma cells (Caco-2) can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs and vitamins. It explains the proper maintenance of Caco-2 cells and the preparation of their lipid mixture; and it further discusses the valuable option of using the permeable membrane system. Since differentiated Caco-2 cells are polarized, the main advantage of using the permeable membrane system is that it separates the apical from the basolateral compartment. Consequently, the lipid mixture can be added to the apical compartment while the lipoproteins can be collected from the basolateral compartment. In addition, the effectiveness of the lentivirus expression system in upregulating gene expression in Caco-2 cells is discussed. Lastly, this paper describes how to confirm the successful isolation of intestinal lipoproteins by transmission electron microscopy (TEM).

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.

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.

Ciliotherapy: a novel intervention in polycystic kidney disease.

BACKGROUND: Ciliopathies are a group of diseases associated with abnormal structure or function of primary cilia. Ciliopathies include polycystic kidney disease (PKD), a pathology associated with vascular hypertension. We previously showed that cilia length regulates cilia function, and cilia function is required for nitric oxide (NO) biosynthesis in endothelial cells. Because patients with PKD show abnormal sensory cilia function, the aim of our current study was to search for a targeted therapy focused on primary cilia, which we refer to as 'ciliotherapy'. METHODS AND RESULTS: In the present studies, our in vitro analyses refined fenoldopam as an equipotent and more specific dopaminergic agonist to regulate cilia length and function. Our in vivo studies indicated that fenoldopam increased cilia length and serum NO thereby reducing blood pressure in a PKD mouse model. Our crossover, multicenter, double-blind and placebo-controlled clinical study further indicated that cilia-targeting therapy showed an overall reduction in mean arterial pressure in PKD patients. CONCLUSIONS: Overall, our studies provide the first evidence of ciliotherapy as an innovative intervention in patients with abnormal primary cilia.

Intestinal transport as a potential determinant of drug bioavailability.

Orally administered drugs are generally absorbed by the small intestine and transported either to the lymphatic system or to the hepatic portal system. In general, lipid soluble drugs and vitamins are transported by the small intestine to the lymphatics, and water-soluble drugs are transported to the hepatic portal system. By avoiding the early hepatic first pass effect, the lymphatic transport system may increase drug bioavailability. In addition to its transport systems, the small intestine may affect drug bioavailability through drug uptake, intestinal first pass effect, recruitment of drugs by chylomicrons, formation and secretion of chylomicrons, and enterohepatic circulation. All of these factors should be considered when formulating orally administered lipophilic drugs. Our data also suggest that Caco-2 cells may serve as a valuable in vitro model to study the intestinal transport of orally administered drugs.

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.

A Comparative Study of Embedded and Anesthetized Zebrafish in vivo on Myocardiac Calcium Oscillation and Heart Muscle Contraction.

The zebrafish (Danio rerio) has been used as a model for studying vertebrate development in the cardiovascular system. In order to monitor heart contraction and cytosolic calcium oscillations, fish were either embedded in methylcellulose or anesthetized with tricaine. Using high-resolution differential interference contrast and calcium imaging microscopy, we here show that dopamine and verapamil alter calcium signaling and muscle contraction in anesthetized zebrafish, but not in embedded zebrafish. In anesthetized fish, dopamine increases the amplitude of cytosolic calcium oscillation with a subsequent increase in heart contraction, whereas verapamil decreases the frequency of calcium oscillation and heart rate. Interestingly, verapamil also increases myocardial contraction. Our data further indicate that verapamil can increase myocardial calcium sensitivity in anesthetized fish. Taken together, our data reinforce in vivo cardiac responses to dopamine and verapamil. Furthermore, effects of dopamine and verapamil on myocardial calcium and contraction are greater in anesthetized than embedded fish. We suggest that while the zebrafish is an excellent model for a cardiovascular imaging study, the cardio-pharmacological profiles are very different between anesthetized and embedded fish.

Why does the gut choose apolipoprotein B48 but not B100 for chylomicron formation?

Chylomicrons produced by the human gut contain apolipoprotein (apo) B48, whereas very-low-density lipoproteins made by the liver contain apo B100. To study how these molecules function during lipid absorption, we examined the process as it occurs in apobec-1 knockout mice (able to produce only apo B100; KO) and in wild-type mice (of which the normally functioning intestine makes apo B48, WT). Using the lymph fistula model, we studied the process of lipid absorption when animals were intraduodenally infused with a lipid emulsion (4 or 6 micromol/h of triolein). KO mice transported triacylglycerol (TG) as efficiently as WT mice when infused with the lower lipid dose; when infused with 6 micromol/h of triolein, however, KO mice transported significantly less TG to lymph than WT mice, leading to the accumulation of mucosal TG. Interestingly, the size of lipoprotein particles from both KO and WT mice were enlarged to chylomicron-size particles during absorption of the higher dose. These increased-size particles produced by KO mice were not associated with increased apo AIV secretion. However, we found that the gut of the KO mice secreted fewer apo B molecules to lymph (compared with WT), during both fasting and lipid infusion, leading us to conclude that the KO gut produced fewer numbers of TG-rich lipoproteins (including chylomicron) than the wild-type animals. The reduced apo B secretion in KO mice was not related to reduced microsomal triglyceride transfer protein lipid transfer activity. We propose that apo B48 is the preferred protein for the gut to coat chylomicrons to ensure efficient chylomicron formation and lipid absorption.

CD36 is important for chylomicron formation and secretion and may mediate cholesterol uptake in the proximal intestine.

BACKGROUND & AIMS: Studies are aimed to determine the role of CD36 in intestinal lipid absorption. METHODS: Knock-out (KO) and wild-type (WT) lymph fistula mice were used to study fatty acids (FA) and cholesterol uptake, and chylomicron formation and secretion. Uptake of FA and cholesterol was studied by using sucrose polybehenate and fecal dual isotope methods, respectively. RESULTS: The CD36 KO exhibited significant accumulation of dietary cholesterol in the intestinal lumen at the end of 6-hour lipid infusion and significant reduction of dietary cholesterol transport into the lymph. Fecal dual isotope studies, however, did not show any significant difference in cholesterol uptake, suggesting that given sufficient time, the KO intestine could compensate for the reduced cholesterol uptake observed in the acute lymph fistula studies. Recovery of dietary FA in the intestinal lumen was comparable between WT and KO, consistent with the sucrose polybehenate study. However, the KO mice accumulated more, albeit not significantly, dietary triacylglycerols in the intestine, followed by a significant reduction in lymphatic transport. The ratio of intestinal dietary triacylglycerols to FA was not higher in WT than KO, arguing against impaired lipid esterification. It is rather a deficiency in the formation and secretion of chylomicrons, as supported by the significantly less apolipoprotein B-48 and the smaller, albeit not significantly, lipoprotein particles secreted into the lymph of the KO. CONCLUSIONS: CD36 may play an important role in chylomicron formation and secretion and may also facilitate cholesterol uptake in the proximal intestine.

CD36 deficiency impairs intestinal lipid secretion and clearance of chylomicrons from the blood.

CD36 mediates the transfer of fatty acids (FAs) across the plasma membranes of muscle and adipose cells, thus playing an important role in regulating peripheral FA metabolism in vivo. In the proximal intestine, CD36 is localized in abundant quantities on the apical surface of epithelial cells, a pattern similar to that of other proteins implicated in the uptake of dietary FAs. To define the role of CD36 in the intestine, we examined FA utilization and lipoprotein secretion by WT and CD36-null mice in response to acute and chronic fat feeding. CD36-null mice given a fat bolus by gavage or fed a high-fat diet accumulated neutral lipid in the proximal intestine, which indicated abnormal lipid processing. Using a model in which mice were equipped with lymph fistulae, we obtained evidence of defective lipoprotein secretion by directly measuring lipid output. The secretion defect appeared to reflect an impaired ability of CD36-null enterocytes to efficiently synthesize triacylglycerols from dietary FAs in the endoplasmic reticulum. In the plasma of intact mice, the reduced intestinal lipid secretion was masked by slow clearance of intestine-derived lipoproteins. The impaired clearance occurred despite normal lipoprotein lipase activity and likely reflected feedback inhibition of the lipase by FAs due to their defective removal from the plasma. We conclude that CD36 is important for both secretion and clearance of intestinal lipoproteins. CD36 deficiency results in hypertriglyceridemia both in the postprandial and fasting states and in humans may constitute a risk factor for diet-induced type 2 diabetes and cardiovascular disease.

Intestinal alkaline phosphatase release is not associated with chylomicron formation.

Intestinal alkaline phosphatase (IAP) is one of the major sources of alkaline phosphatase in circulation. It is secreted into the intestinal lumen, serum, and lymph. After the ingestion of lipid, lymphatic alkaline phosphatase secretion increases significantly. We have found that the nonabsorbable fat olestra is unable to stimulate lymphatic alkaline phosphatase secretion. We also found that the hydrophobic surfactant Pluronic L-81, which blocks chylomicron formation, fails to inhibit this increase in lymphatic alkaline phosphatase secretion. These results suggest that it is the lipid uptake into the mucosa and/or reesterification to form triacylglycerols, but not the formation of chylomicrons, that is necessary for the stimulation of the secretion of alkaline phosphatase into the lymph.