A TMPRSS2 inhibitor acts as a pan-SARS-CoV-2 prophylactic and therapeutic.

The COVID-19 pandemic caused by the SARS-CoV-2 virus remains a global public health crisis. Although widespread vaccination campaigns are underway, their efficacy is reduced owing to emerging variants of concern1,2. Development of host-directed therapeutics and prophylactics could limit such resistance and offer urgently needed protection against variants of concern3,4. Attractive pharmacological targets to impede viral entry include type-II transmembrane serine proteases (TTSPs) such as TMPRSS2; these proteases cleave the viral spike protein to expose the fusion peptide for cell entry, and thus have an essential role in the virus lifecycle5,6. Here we identify and characterize a small-molecule compound, N-0385, which exhibits low nanomolar potency and a selectivity index of higher than 106 in inhibiting SARS-CoV-2 infection in human lung cells and in donor-derived colonoids7. In Calu-3 cells it inhibits the entry of the SARS-CoV-2 variants of concern B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma) and B.1.617.2 (Delta). Notably, in the K18-human ACE2 transgenic mouse model of severe COVID-19, we found that N-0385 affords a high level of prophylactic and therapeutic benefit after multiple administrations or even after a single administration. Together, our findings show that TTSP-mediated proteolytic maturation of the spike protein is critical for SARS-CoV-2 infection in vivo, and suggest that N-0385 provides an effective early treatment option against COVID-19 and emerging SARS-CoV-2 variants of concern.

Roles of Cholesterol in Early and Late Steps of the Nipah Virus Membrane Fusion Cascade.

Cholesterol has been implicated in various viral life cycle steps for different enveloped viruses, including viral entry into host cells, cell-cell fusion, and viral budding from infected cells. Enveloped viruses acquire their membranes from their host cells. Although cholesterol has been associated with the binding and entry of various enveloped viruses into cells, cholesterol's exact function in the viral-cell membrane fusion process remains largely elusive, particularly for the paramyxoviruses. Furthermore, paramyxoviral fusion occurs at the host cell membrane and is essential for both virus entry (virus-cell fusion) and syncytium formation (cell-cell fusion), central to viral pathogenicity. Nipah virus (NiV) is a deadly member of the Paramyxoviridae family, which also includes Hendra, measles, mumps, human parainfluenza, and various veterinary viruses. The zoonotic NiV causes severe encephalitis, vasculopathy, and respiratory symptoms, leading to a high mortality rate in humans. We used NiV as a model to study the role of membrane cholesterol in paramyxoviral membrane fusion. We used a combination of methyl-beta cyclodextrin (MßCD), lovastatin, and cholesterol to deplete or enrich cell membrane cholesterol outside cytotoxic concentrations. We found that the levels of cellular membrane cholesterol directly correlated with the levels of cell-cell fusion induced. These phenotypes were paralleled using NiV/vesicular stomatitis virus (VSV)-pseudotyped viral infection assays. Remarkably, our mechanistic studies revealed that cholesterol reduces an early F-triggering step but enhances a late fusion pore formation step in the NiV membrane fusion cascade. Thus, our results expand our mechanistic understanding of the paramyxoviral/henipaviral entry and cell-cell fusion processes.IMPORTANCE Cholesterol has been implicated in various steps of the viral life cycle for different enveloped viruses. Nipah virus (NiV) is a highly pathogenic enveloped virus in the Henipavirus genus within the Paramyxoviridae family, capable of causing a high mortality rate in humans and high morbidity in domestic and agriculturally important animals. The role of cholesterol for NiV or the henipaviruses is unknown. Here, we show that the levels of cholesterol influence the levels of NiV-induced cell-cell membrane fusion during syncytium formation and virus-cell membrane fusion during viral entry. Furthermore, the specific role of cholesterol in membrane fusion is not well defined for the paramyxoviruses. We show that the levels of cholesterol affect an early F-triggering step and a late fusion pore formation step during the membrane fusion cascade. Thus, our results expand our mechanistic understanding of the viral entry and cell-cell fusion processes, which may aid the development of antivirals.

Third Helical Domain of the Nipah Virus Fusion Glycoprotein Modulates both Early and Late Steps in the Membrane Fusion Cascade.

Medically important paramyxoviruses, such as measles, mumps, parainfluenza, Nipah, and Hendra viruses, infect host cells by directing fusion of the viral and cellular plasma membranes. Upon infection, paramyxoviruses cause a second type of membrane fusion, cell-cell fusion (syncytium formation), which is linked to pathogenicity. Host cell receptor binding causes conformational changes in the attachment glycoprotein (HN, H, or G) that trigger a conformational cascade in the fusion (F) glycoprotein that mediates membrane fusion. F, a class I fusion protein, contains the archetypal heptad repeat regions 1 (HR1) and 2 (HR2). It is well established that binding of HR1 and HR2 is key to fusing viral and cellular membranes. In this study, we uncovered a novel fusion-modulatory role of a third structurally conserved helical region (HR3) in F. Based on its location within the F structure, and structural differences between its prefusion and postfusion conformations, we hypothesized that the HR3 modulates triggering of the F conformational cascade (still requiring G). We used the deadly Nipah virus (NiV) as an important paramyxoviral model to perform alanine scan mutagenesis and a series of multidisciplinary structural/functional analyses that dissect the various states of the membrane fusion cascade. Remarkably, we found that specific residues within the HR3 modulate not only early F-triggering but also late extensive fusion pore expansion steps in the membrane fusion cascade. Our results characterize these novel fusion-modulatory roles of the F HR3, improving our understanding of the membrane fusion process for NiV and likely for the related Henipavirus genus and possibly Paramyxoviridae family members.IMPORTANCE The Paramyxoviridae family includes important human and animal pathogens, such as measles, mumps, and parainfluenza viruses and the deadly henipaviruses Nipah (NiV) and Hendra (HeV) viruses. Paramyxoviruses infect the respiratory tract and the central nervous system (CNS) and can be highly infectious. Most paramyxoviruses have a limited host range. However, the biosafety level 4 NiV and HeV are highly pathogenic and have a wide mammalian host range. Nipah viral infections result in acute respiratory syndrome and severe encephalitis in humans, leading to 40 to 100% mortality rates. The lack of licensed vaccines or therapeutic approaches against NiV and other important paramyxoviruses underscores the need to understand viral entry mechanisms. In this study, we uncovered a novel role of a third helical region (HR3) of the NiV fusion glycoprotein in the membrane fusion process that leads to viral entry. This discovery sets HR3 as a new candidate target for antiviral strategies for NiV and likely for related viruses.

Solution phase conformation and proteolytic stability of amide-linked neuraminic acid analogues.

Amide-linked homopolymers of sialic acid offer the advantages of stable secondary structure and increased bioavailability making them useful constructs for pharmaceutical design and drug delivery. Defining the structural characteristics that give rise to secondary structure in aqueous solution is challenging in homopolymeric material due to spectral overlap in NMR spectra. Having previously developed computational tools for heteroologomers with resolved spectra, we now report that application of these methods in combination with circular dichroism, NH/ND NMR exchange rates and nOe data has enabled the structural determination of a neutral, δ-amide-linked homopolymer of a sialic acid analogue called Neu2en. The results show that the inherent planarity of the pyranose ring in Neu2en brought about by the α,δ-conjugated amide bond serves as the primary driving force of the overall conformation of the homooligomer. This peptide surrogate has an excellent bioavailability profile, with half-life of ∼12 h in human blood serum, which offers a viable peptide scaffold that is resistant to proteolytic degradation. Furthermore, a proof-of-principle study illustrates that Neu2en oligomers are functionalizable with small molecule ligands using 1,3-dipolar cycloaddition chemistry.

Cell-Cell Fusion Assays to Study Henipavirus Entry and Evaluate Therapeutics.

Henipaviruses include the deadly zoonotic Nipah (NiV) and Hendra (HeV) paramyxoviruses, which have caused recurring outbreaks in human populations. A hallmark of henipavirus infection is the induction of cell-cell fusion (syncytia), caused by the expression of the attachment (G) and fusion (F) glycoproteins on the surface of infected cells. The interactions of G and F with each other and with receptors on cellular plasma membranes drive both viral entry and syncytia formation and are thus of great interest. While F shares structural and functional homologies with class I fusion proteins of other viruses such as influenza and human immunodeficiency viruses, the intricate interactions between the G and F glycoproteins allow for unique approaches to studying the class I membrane fusion process. This allows us to study cell-cell fusion and viral entry kinetics for BSL-4 pathogens such as NiV and HeV under BSL-2 conditions using recombinant DNA techniques. Here, we present approaches to studying henipavirus-induced membrane fusion for currently identified and emerging henipaviruses, including more traditional syncytia counting-based cell-cell fusion assay and a new heterologous fluorescent dye exchange cell-cell fusion assay.

Novel Roles of the Nipah Virus Attachment Glycoprotein and Its Mobility in Early and Late Membrane Fusion Steps.

The Paramyxoviridae family comprises important pathogens that include measles (MeV), mumps, parainfluenza, and the emerging deadly zoonotic Nipah virus (NiV) and Hendra virus (HeV). Paramyxoviral entry into cells requires viral-cell membrane fusion, and formation of paramyxoviral pathognomonic syncytia requires cell-cell membrane fusion. Both events are coordinated by intricate interactions between the tetrameric attachment (G/H/HN) and trimeric fusion (F) glycoproteins. We report that receptor binding induces conformational changes in NiV G that expose its stalk domain, which triggers F through a cascade from prefusion to prehairpin intermediate (PHI) to postfusion conformations, executing membrane fusion. To decipher how the NiV G stalk may trigger F, we introduced cysteines along the G stalk to increase tetrameric strength and restrict stalk mobility. While most point mutants displayed near-wild-type levels of cell surface expression and receptor binding, most yielded increased NiV G oligomeric strength, and showed remarkably strong defects in syncytium formation. Furthermore, most of these mutants displayed stronger F/G interactions and significant defects in their ability to trigger F, indicating that NiV G stalk mobility is key to proper F triggering via moderate G/F interactions. Also remarkably, a mutant capable of triggering F and of fusion pore formation yielded little syncytium formation, implicating G or G/F interactions in a late step occurring post fusion pore formation, such as the extensive fusion pore expansion required for syncytium formation. This study uncovers novel mechanisms by which the G stalk and its oligomerization/mobility affect G/F interactions, the triggering of F, and a late fusion pore expansion step-exciting novel findings for paramyxoviral attachment glycoproteins. IMPORTANCE The important Paramyxoviridae family includes measles, mumps, human parainfluenza, and the emerging deadly zoonotic Nipah virus (NiV) and Hendra virus (HeV). The deadly emerging NiV can cause neurologic and respiratory symptoms in humans with a >60% mortality rate. NiV has two surface proteins, the receptor binding protein (G) and fusion (F) glycoproteins. They mediate the required membrane fusion during viral entry into host cells and during syncytium formation, a hallmark of paramyxoviral and NiV infections. We previously discovered that the G stalk domain is important for triggering F (via largely unknown mechanisms) to induce membrane fusion. Here, we uncovered new roles and mechanisms by which the G stalk and its mobility modulate the triggering of F and also unexpectedly affect a very late step in membrane fusion, namely fusion pore expansion. Importantly, these novel findings may extend to other paramyxoviruses, offering new potential targets for therapeutic interventions.

Multivalent viral particles elicit safe and efficient immunoprotection against Nipah Hendra and Ebola viruses.

Experimental vaccines for the deadly zoonotic Nipah (NiV), Hendra (HeV), and Ebola (EBOV) viruses have focused on targeting individual viruses, although their geographical and bat reservoir host overlaps warrant creation of multivalent vaccines. Here we explored whether replication-incompetent pseudotyped vesicular stomatitis virus (VSV) virions or NiV-based virus-like particles (VLPs) were suitable multivalent vaccine platforms by co-incorporating multiple surface glycoproteins from NiV, HeV, and EBOV onto these virions. We then enhanced the vaccines' thermotolerance using carbohydrates to enhance applicability in global regions that lack cold-chain infrastructure. Excitingly, in a Syrian hamster model of disease, the VSV multivalent vaccine elicited safe, strong, and protective neutralizing antibody responses against challenge with NiV, HeV, or EBOV. Our study provides proof-of-principle evidence that replication-incompetent multivalent viral particle vaccines are sufficient to provide protection against multiple zoonotic deadly viruses with high pandemic potential.

Controlling nonclassical content of clathrate hydrates through the choice of molecular guests and temperature.

Low-temperature, low-pressure studies of clathrate hydrates (CHs) have revealed that small ether and other proton-acceptor guests greatly enhance rates of clathrate hydrate nucleation and growth; rapid formation and transformations are enabled at temperatures as low as 110 K, and cool moist vapors containing small ether molecules convert to mixed-gas CHs on a subsecond time scale. More recently, FTIR spectroscopic studies of the tetrahydrofuran (THF)-HCN double clathrate hydrate revealed a sizable frequency shift accompanied by a four-fold intensification of the C-N stretch-mode absorption of the small cage HCN, behavior that is enhanced by cooling and which correlates precisely with similar significant changes of the ether C-O/C-C stretch modes. These temperature-dependent correlated changes in the infrared spectra have been attributed to equilibrated extensive hydrogen bonding of neighboring large- and small-cage guest molecules with water molecules of the intervening wall. An ether guest functions as a proton acceptor, particularly so when complemented by the action of a proton-donor (HCN)/electron-acceptor (SO(2)) small-cage guest. Because guest molecules of the classic clathrate hydrates do not participate in hydrogen bonds with the host water, this H-bonding of guests has been labeled "nonclassical". The present study has been enriched by comparing observed FTIR spectra with high-level molecular orbital computational results for guests and hydrogen-bonded guest-water dimers. Vibrational frequency shifts, from heterodimerization of ethers and water, correlate well with the corresponding observed classical to nonclassical shifts. The new spectroscopic data reveal that the nonclassical structures can contribute at observable levels to CH infrared spectra for a remarkable range of temperatures and choice of guest molecules. By the choice of guest molecules, it is now possible to select the abundance levels of nonclassical configurations, ranging from ∼0 to 100%, for a given temperature. This ability is expected to hasten understanding of the role of guest-induced nonclassical structures in the acceleration or inhibition of the rates of CH formation and transformation.

Communication: quantitative Fourier-transform infrared data for competitive loading of small cages during all-vapor instantaneous formation of gas-hydrate aerosols.

A simple method has been developed for the measurement of high quality FTIR spectra of aerosols of gas-hydrate nanoparticles. The application of this method enables quantitative observation of gas hydrates that form on subsecond timescales using our all-vapor approach that includes an ether catalyst rather than high pressures to promote hydrate formation. The sampling method is versatile allowing routine studies at temperatures ranging from 120 to 210 K of either a single gas or the competitive uptake of different gas molecules in small cages of the hydrates. The present study emphasizes hydrate aerosols formed by pulsing vapor mixtures into a cold chamber held at 160 or 180 K. We emphasize aerosol spectra from 6 scans recorded an average of 8 s after "instantaneous" hydrate formation as well as of the gas hydrates as they evolve with time. Quantitative aerosol data are reported and analyzed for single small-cage guests and for mixed hydrates of CO(2), CH(4), C(2)H(2), N(2)O, N(2), and air. The approach, combined with the instant formation of gas hydrates from vapors only, offers promise with respect to optimization of methods for the formation and control of gas hydrates.

Rapid Detection of Viral Envelope Lipids Using Lithium Adducts and AP-MALDI High-Resolution Mass Spectrometry.

There is an unmet need to develop analytical strategies that not only characterize the lipid composition of the viral envelope but also do so on a time scale that would allow for high-throughput analysis. With that in mind, we report the use of atmospheric pressure (AP) matrix-assisted laser desorption/ionization (MALDI) high-resolution mass spectrometry (HRMS) combined with lithium adduct consolidation to profile total lipid extracts rapidly and confidently from enveloped viruses. The use of AP-MALDI reduced the dependency of using a dedicated MALDI mass spectrometer and allowed for interfacing the MALDI source to a mass spectrometer with the desired features, which included high mass resolving power (>100000) and tandem mass spectrometry. AP-MALDI combined with an optimized MALDI matrix system, featuring 2',4',6'-trihydroxyacetophenone spiked with lithium salt, resulted in a robust and high-throughput lipid detection platform, specifically geared to sphingolipid detection. Application of the developed workflow included the structural characterization of prominent sphingolipids and detection of over 130 lipid structures from Influenza A virions. Overall, we demonstrate a high-throughput workflow for the detection and structural characterization of total lipid extracts from enveloped viruses using AP-MALDI HRMS and lithium adduct consolidation.

A novel highly potent inhibitor of TMPRSS2-like proteases blocks SARS-CoV-2 variants of concern and is broadly protective against infection and mortality in mice.

The COVID-19 pandemic caused by the SARS-CoV-2 virus remains a global public health crisis. Although widespread vaccination campaigns are underway, their efficacy is reduced against emerging variants of concern (VOCs) 1,2 . Development of host-directed therapeutics and prophylactics could limit such resistance and offer urgently needed protection against VOCs 3,4 . Attractive pharmacological targets to impede viral entry include type-II transmembrane serine proteases (TTSPs), such as TMPRSS2, whose essential role in the virus lifecycle is responsible for the cleavage and priming of the viral spike protein 5-7 . Here, we identify and characterize a small-molecule compound, N-0385, as the most potent inhibitor of TMPRSS2 reported to date. N-0385 exhibited low nanomolar potency and a selectivity index of >10 6 at inhibiting SARS-CoV-2 infection in human lung cells and in donor-derived colonoids 8 . Importantly, N-0385 acted as a broad-spectrum coronavirus inhibitor of two SARS-CoV-2 VOCs, B.1.1.7 and B.1.351. Strikingly, single daily intranasal administration of N-0385 early in infection significantly improved weight loss and clinical outcomes, and yielded 100% survival in the severe K18-human ACE2 transgenic mouse model of SARS-CoV-2 disease. This demonstrates that TTSP-mediated proteolytic maturation of spike is critical for SARS-CoV-2 infection in vivo and suggests that N-0385 provides a novel effective early treatment option against COVID-19 and emerging SARS-CoV-2 VOCs.

Instant conversion of air to a clathrate hydrate: CO(2) hydrates directly from moist air and moist CO(2)(g).

The rapid conversion of vapor mixtures containing the gases CO(2), H(2)S, and HCN to clathrate hydrates was reported recently. The novel method is based on the pulsing of warm vapor mixtures, including a carrier gas, into a cold condensation chamber. With cooling, the vapors, which also include ∼1% water and either tetrahydrofuran or trimethylene oxide as a catalyst, nucleate aqueous solution nanodroplets that, on a millisecond time scale, crystallize as hydrate nanoparticles that consume 100% of the water. Humid air approximates the content of mixtures used successfully in the vapor-to-hydrate conversions. FTIR spectra are examined for gas hydrates formed directly from air and air enriched with CO(2), as well as hydrate particles for which CO(2)(g) serves as both guest and aerosol medium. In each instance all of the water in the condensed phase converts to a clathrate hydrate. The subsecond ether-catalyzed formation of the hydrates near 230 K requires only a few percent of the CO(2) pressure used in conventional processes that yield fractional amounts of gas hydrates on an hour time scale in the same temperature range.

SialoPen peptides are new cationic foldamers with remarkable cell permeability.

The ability to access intracellular targets is of vital importance as the number of identified druggable intracellular targets increases every year. However, intracellular delivery poses a formidable barrier, as many potential therapeutics are impermeable to cell membranes, which hinders their practical application in drug development. Herein we present de novo-designed unnatural cell penetrating peptide foldamers utilizing a 2,3-Didehydro-2-deoxyneuraminic acid (Neu2en) scaffold. Conveniently, this scaffold is amenable to standard Fmoc-based solid-phase peptide synthesis, with the advantages of tunable secondary structures and enhanced biostability. Flow cytometry and live-cell confocal microscopy studies showed that these Neu2en-based peptides, hereinafter termed SialoPen peptides, have significantly superior uptake in HeLa and primary neuronal hippocampal cells, outperforming the classical cell permeable peptides penetratin and HIV-TAT.

Clathrate hydrates with hydrogen-bonding guests.

Clathrate hydrates (CHs) are inclusion compounds in which "tetrahedrally" bonded H(2)O forms a crystalline host lattice composed of a periodic array of cages. The structure is stabilized by guest particles which occupy the cages and interact with cage walls via van der Waals interactions. A host of atoms or small molecules can act as guests; here the focus is on guests that are capable of strong to intermediate H-bonding to water (small ethers, H(2)S, etc.) but nevertheless "choose" this hydrate crystal form in which H-bonding is absent from the equilibrium crystal structure. These CHs can form by exposure of ice to guest molecules at temperatures as low as 100-150 K, at the (low) guest saturation pressure. This is in contrast to the "normal" CHs whose formation typically requires temperatures well above 200 K and at least moderate pressures. The experimental part of this study addresses formation kinetics of CHs with H-bonding guests, as well as transformation kinetics between different CH forms, studied by CH infrared spectroscopy. The accompanying computational study suggests that the unique properties of this family of CHs are due to exceptional richness of the host lattice in point defects, caused by defect stabilization by H-bonding of water to the guests.

Serum carboxy-terminal propeptide of procollagen type I is a marker of myocardial fibrosis in hypertensive heart disease.

BACKGROUND: This study was designed to investigate whether the serum concentration of the carboxy-terminal propeptide of procollagen type I (PIP), a marker of collagen type I synthesis, is related to myocardial fibrosis in hypertensive patients. METHODS AND RESULTS: The study was performed in 26 patients with essential hypertension in which ischemic cardiomyopathy was excluded after a complete medical workup. Right septal endomyocardial biopsies were performed in hypertensive patients to quantify collagen content. Collagen volume fraction (CVF) was determined on picrosirius red-stained sections with an automated image analysis system. The serum concentration of PIP was measured by specific radioimmunoassay. Compared with normotensives, both serum PIP and CVF were increased (P<0.001) in hypertensives. A direct correlation was found between CVF and serum PIP (r=0.471, P<0.02) in all hypertensives. Histological analysis revealed the presence of 2 subgroups of patients: 8 with severe fibrosis and 18 with nonsevere fibrosis. Serum PIP was higher (P<0.05) in patients with severe fibrosis than in patients with nonsevere fibrosis. Using receiver operating characteristic curves, we observed that a cutoff of 127 microg/L for PIP provided 78% specificity and 75% sensitivity for predicting severe fibrosis with a relative risk of 4.80 (95% CI, 1.19 to 19.30). CONCLUSIONS: These results show a strong correlation between myocardial collagen content and the serum concentration of PIP in essential hypertension. Although preliminary, these findings suggest that the determination of PIP may be an easy and reliable method for the screening and diagnosis of severe myocardial fibrosis associated with arterial hypertension.

One-pot SSA-catalyzed ß-elimination: an efficient and inexpensive protocol for easy access to the glycal of sialic acid.

Neu5Ac2en1Me per-OAc, the fully protected glycal of sialic acid, is a key intermediate in the discovery of therapeutics and diagnostics, including anti-influenza drugs and proteolysis resistant peptidomimetic foldamers. The synthesis of this sialic acid derivative, however, still relies on standard sugar chemistry that utilizes multi-step methodologies. Herein we report a facile and highly efficient microwave-assisted preparation of Neu5Ac1Me using silica sulfuric acid (SSA) as solid-supported acid catalyst that is one- to two-orders of magnitude faster than standard procedures. We also describe the microwave-assisted and SSA-catalyzed one-pot, rapid, solvent free reaction that combines both peracetylation and ß-elimination reactions in one step to generate the glycal from Neu5Ac1Me. We coined the term One-pot SSA-catalyzed Technology for ß-Elimination Protocol (OneSTEP) to describe this least laborious, most efficient, and practical preparation to date of Neu5Ac2en1Me per-OAc in terms of yield, time, reagent cost, and waste generation.

Branched dimerization of Tat peptide improves permeability to HeLa and hippocampal neuronal cells.

A dimeric branched peptide TATp-D designed as an analogue of the HIV-Tat protein transduction domain (TATp), a prototypical cell penetrating peptide (CPP), demonstrates significantly enhanced cell uptake at 0.25 to 2.5 µM. Live cell confocal laser scanning microscopy revealed that multivalency dramatically improved the permeation potency of TATp-D to HeLa and primary hippocampal neuronal cells. The observed enhanced ability of TATp-D to translocate through the membrane is highlighted by a non-linear dependence on concentration, exhibiting the greatest uptake at sub-micromolar concentrations as compared to TATp. Multimerization via bis-Fmoc Lysine offered a synthetically straightforward method to investigate the effects of multivalent CPPs while offering orthogonal handles for cargo attachment, increasing the utility of CPPs at significantly lower concentrations.

Hormonal implications of the hypocholesterolemic effect of intake of field beans (Vicia faba L.) by young men with hypercholesterolemia.

This study examined the hypocholesterolemic effect and hormonal changes resulting from 30 d of supplementation with Vicia faba L. (field bean) flour of diets of young men (aged 18-21 y; n = 40) with borderline-high or high serum cholesterol values. All subjects (groups A-D) consumed the same basic diet. Additionally, volunteers in the control group (A) consumed 90 g control flour/d whereas those in the three bean groups received either 90 g cooked field bean flour (groups B and C) or 90 g raw field bean flour (group D) daily. Groups A and B included volunteers with borderline-high cholesterol values [5.2-6.2 mmol total cholesterol/L and 3.4-4.1 mmol low-density-lipoprotein (LDL) cholesterol/L]. Subjects in groups C and D had high serum cholesterol concentrations (total cholesterol > 6.2 mmol/L and LDL cholesterol > 4.1 mmol/L). After 30 d, serum glucose, insulin, triacylglycerol, total, LDL-cholesterol, and very-low-density-lipoprotein (VLDL)-cholesterol values were significantly lower than initial values in all subjects who consumed diets containing field bean flour (P < or = 0.0001, except for LDL-cholesterol concentrations in group C, for which P < or = 0.0007). Legume intake also resulted in a significant increase (P < or = 0.0001) in glucagon and high-density-lipoprotein cholesterol. Neither cortisol nor thyroid hormone values changed significantly. The results suggest that the hypocholesterolemic effect of field bean intake depends at least partly on a concomitant increase in glucagon and decrease in insulin values. The more marked reduction in triacylglycerol and VLDL-cholesterol concentrations in subjects who consumed raw field beans indicates a coparticipation of their thermolabile components.

Is the tissue availability of circulating insulin-like growth factor I involved in organ damage and glucose regulation in hypertension?

BACKGROUND: Besides its capacity to regulate organ and tissue growth, the insulin-like growth factor I exerts biologic actions that resemble those of insulin. Tissue access of the factor depends on the distribution of the circulating bound factor between its binding protein 3 that remains within the intravascular space and its binding protein 1 that is able to cross the endothelium. OBJECTIVE: To investigate whether the distribution of the circulating factor between its binding proteins is altered in patients with essential hypertension and whether this is related to changes in organ damage and glucose regulation in these patients. DESIGN: The study subjects were 30 never-treated patients with essential hypertension and 27 age- and sex-matched normotensive controls. METHODS: Serum insulin-like growth factor I-binding proteins 3 and 1 and plasma insulin-like growth factor I levels were determined by specific radioimmunoassays. RESULTS: Insulin-like growth factor I levels were significantly higher in the hypertensive patients than they were in the normotensive controls. Whereas the serum level of binding protein 1 was significantly higher in hypertensives than it was in controls, we found no differences in the level of binding protein 3 between the two groups. With the upper 100% confidence limit of the normotensive population as the cut-off point, a subgroup of 16 hypertensives had an abnormally high serum level of binding protein 1. Compared with patients with normal binding protein 1 levels, patients with increased binding protein 1 levels were characterized by the following: lower fasting glucose and insulin levels, lower insulin: glucose ratios, lower triglyceride levels, higher left ventricular mass indexes, higher creatinine clearances and higher urinary albumin excretion rates. The serum binding protein 1 level was correlated inversely to the plasma insulin level for the whole group of hypertensives. CONCLUSIONS: These results show that the distribution of circulating insulin-like growth factor I between its binding proteins 1 and 3 is altered in essential hypertension. Thus, there is a subgroup (53%) of hypertensive patients with increased serum levels of insulin-like growth factor I-binding protein 1. Access of the circulating factor to tissues is more easily achieved in these patients. The clinical characteristics of this subgroup of patients suggest that the tissue availability of insulin-like growth factor I is a determinant of organ damage and insulin sensitivity in essential hypertension.

Tissue availability of insulin-like growth factor I is inversely related to insulin resistance in essential hypertension: effects of angiotensin converting enzyme inhibition.

BACKGROUND: The insulin-like growth factor I possesses biologic actions that resemble those of insulin. Tissue access of the factor depends on the distribution of the circulating bound factor between its binding protein 3 that remains within the intravascular space and its binding protein I that is able to cross the endothelium. Preliminary results have shown that tissue availability of insulin-like growth factor I is a determinant of glucose regulation in essential hypertension OBJECTIVE: To investigate whether the tissue availability of circulating insulin-like growth factor I in patients with essential hypertension is related to insulin resistance and whether chronic angiotensin converting enzyme inhibition influences tissue availability of the factor and insulin resistance in these patients. DESIGN AND METHODS: We studied 29 patients with essential hypertension and 20 age-matched and sex-matched normotensive subjects. The measurements were repeated for 25 patients after 12 months of treatment with lisinopril. Tissue availability of circulating insulin-like growth factor I was assessed by analyzing its distribution between its binding proteins 3 and 1. An insulin resistance index was estimated using the homeostasis model analysis of fasting insulin-glucose interactions. Levels of serum insulin-like growth factor I binding proteins 3 and 1, plasma insulin-like growth factor I, and insulin were determined by specific radioimmunoassays. RESULTS: Baseline insulin resistance index was significantly higher in the hypertensive patients than it was in the normotensive controls. With the upper 100% confidence limit of the normotensive population as the cutoff point, a subgroup of 12 hypertensives had an abnormally high insulin resistance index. Compared with patients with normal insulin resistance indexes, patients with greater than normal indexes were characterized by lower binding protein 1 levels, similar binding protein 3 levels, lower binding protein 1 : binding protein 3 ratio and similar insulin-like growth factor I levels. The serum binding protein 1 level and the binding protein 1 : binding protein 3 ratio were inversely correlated to the insulin resistance index for the whole group of hypertensives. After treatment with lisinopril hypertensive patients with higher than normal insulin resistance indexes at baseline exhibited normalization of this parameter and significant increases of binding protein 1 levels and binding protein 1 : binding protein 3 ratio, with no significant changes in insulin-like growth factor I levels. These parameters remained unchanged for the remaining patients. CONCLUSIONS: These results suggest that tissue availability of circulating insulin-like growth factor I is a determinant of insulin sensitivity in patients with essential hypertension. Whereas the patients with normal insulin sensitivity exhibit greater than normal tissue access of circulating insulin-like growth factor I, patients with insulin resistance present normal tissue access of the factor. Our findings suggest that the ability of angiotensin converting enzyme inhibitors to restore insulin sensitivity in essential hypertensives may be related to their ability to facilitate the tissue availability of circulating insulin-like growth factor I.