Interorgan Interactions Research Articles

A microfluidic gradient mixer-flow chamber as a new tool to study biofilm development under defined solute gradients.

Understanding the dynamics of biofilm development in response to chemical cues and signals is required toward the development of controllable biofilm-mediated bioprocesses. In this study, we report a new biofilm growth system that integrates a microfluidic gradient mixer with a biofilm growth chamber. The biofilm growth system allows biofilms to grow under defined solute gradients and enables nondestructive monitoring of the biofilm development dynamics in response to the defined gradients. The solute gradients generated in the system were simulated and then validated experimentally. We then demonstrated the applicability of the biofilm growth system in studying biofilm development under defined solute gradients. Specifically, we examined biofilm development of Shewanella oneidensis and Comamonas testosteroni under a defined calcium and nitrate gradient, respectively. Using two C. testosteroni strains (WDL7 and I2), we further demonstrated the applicability of our biofilm growth system to study the development of coculture biofilms under a defined solute gradient. Our results show that the biofilm growth system we have developed here can be a promising tool to reveal the dynamics of biofilm development in response to chemical cues and signals as well as the interorganism interactions in coculture biofilms.

Modeling organ-specific vasculature with organ-on-a-chip devices

Organ-on-a-chip devices, also known as microphysiological systems, have gained significant attention in recent years. Recent advances in tissue engineering and microfabrication have enabled these devices to provide more precise control over cellular microenvironments to mimic the tissue-level or organ-level function of the human body. These more complex tissue models can provide either an improvement in the functional expression and maturation of cells or an avenue to probe biological events and function that would otherwise be difficult to visualize and mechanistically study. This high-value information, when complimented with the existing gold-standards of cell-based assays and animal models, could potentially lead to more informed decision-making in drug development. A prevalent biological component in many organ-on-a-chip devices is an engineered vascular interface that is present in almost all organs of the human body. The vasculature and the vascular interface are particularly susceptible to biomechanical forces, they function as the conduits for inter-cellular and inter-organ interactions, and regulate drug transport. In this review, we examine the various approaches taken to model the human vasculature with an emphasis on the engineering of organ-specific vasculatures, and discuss various challenges and opportunities ahead as the field advances.

COEXISTENCE OF THREE OSCILLATORY MODES OF INSULIN SECRETION: MATHEMATICAL MODELING AND RELEVANCE TO GLUCOSE REGULATION

Insulin secretion in pancreatic [Formula: see text]-cells exhibits three oscillatory modes with distinct period ranges, called fast, slow, and ultradian modes. To unveil the mechanism underlying such oscillatory behaviors and their roles in blood glucose regulation, we propose a combined model for the glucose–insulin regulation system, incorporating both the cell-level insulin secretion mechanism and inter-organ interactions in the blood glucose regulation. Special emphasis is placed on the identification of the mechanism of the slow oscillation and its role associated with the whole-body glucose regulation. Via extensive numerical simulations, we obtain macroscopic behaviors of the three types of insulin/glucose oscillations in the whole-body as well as microscopic behaviors of the membrane potential and the calcium concentration in the [Formula: see text]-cell. Finally, optimal regulatory strategies for the blood glucose level are discussed on the basis of the quantitative information obtained from the mathematical modeling and numerical simulations.

Hyperpolarized 13C Magnetic Resonance Treatment Response Monitoring: A New Paradigm for Multiorgan Metabolic Assessment of Pharmacological Interventions?

Metformin is one of the most used hyperglycemic control therapeutics in patients with diabetes, although the exact mechanism of action is still not fully understood (1,2). Metformin is the preferred antihyperglycemic drug in patients with type 2 diabetes; its use in patients with type 1 diabetes is limited however (3,4). A potential serious side effect of metformin treatment is lactic acidosis, which has reduced the applicability in renal-impaired patients; however, this has been questioned recently (5,6). These together support further investigations of, first, the exact mechanism of action and, second, the noninvasive methods for monitoring the treatment, in particular the organ-specific modulations imposed by metformin and their complex interorgan interactions, which historically have been especially difficult to assess. This is particularly true in diseases where several organs are simultaneously affected, such as the cardio-renal syndrome, where dysfunction of one organ affects the other and vice versa and where the use of pharmacological interventions in the treatment of one organ can have detrimental effects on the other and vice versa (7,8). Thus the understanding of the organ-specific phenotypic characteristics in diabetes and the therapy-induced alterations is essential …

Thousands of microbial genomes shed light on interconnected biogeochemical processes in an aquifer system.

The subterranean world hosts up to one-fifth of all biomass, including microbial communities that drive transformations central to Earth's biogeochemical cycles. However, little is known about how complex microbial communities in such environments are structured, and how inter-organism interactions shape ecosystem function. Here we apply terabase-scale cultivation-independent metagenomics to aquifer sediments and groundwater, and reconstruct 2,540 draft-quality, near-complete and complete strain-resolved genomes that represent the majority of known bacterial phyla as well as 47 newly discovered phylum-level lineages. Metabolic analyses spanning this vast phylogenetic diversity and representing up to 36% of organisms detected in the system are used to document the distribution of pathways in coexisting organisms. Consistent with prior findings indicating metabolic handoffs in simple consortia, we find that few organisms within the community can conduct multiple sequential redox transformations. As environmental conditions change, different assemblages of organisms are selected for, altering linkages among the major biogeochemical cycles.

New technologies - new insights into the pathogenesis of hepatic encephalopathy.

Hepatic encephalopathy (HE) is a neuropsychiatric syndrome which frequently accompanies acute or chronic liver disease. It is characterized by a variety of symptoms of different severity such as cognitive deficits and impaired motor functions. Currently, HE is seen as a consequence of a low grade cerebral oedema associated with the formation of cerebral oxidative stress and deranged cerebral oscillatory networks. However, the pathogenesis of HE is still incompletely understood as liver dysfunction triggers exceptionally complex metabolic derangements in the body which need to be investigated by appropriate technologies. This review summarizes technological approaches presented at the ISHEN conference 2014 in London which may help to gain new insights into the pathogenesis of HE. Dynamic in vivo 13C nuclear magnetic resonance spectroscopy was performed to analyse effects of chronic liver failure in rats on brain energy metabolism. By using a genomics approach, microRNA expression changes were identified in plasma of animals with acute liver failure which may be involved in interorgan interactions and which may serve as organ-specific biomarkers for tissue damage during acute liver failure. Genomics were also applied to analyse glutaminase gene polymorphisms in patients with liver cirrhosis indicating that haplotype-dependent glutaminase activity is an important pathogenic factor in HE. Metabonomics represents a promising approach to better understand HE, by capturing the systems level metabolic changes associated with disease in individuals, and enabling monitoring of metabolic phenotypes in real time, over a time course and in response to treatment, to better inform clinical decision making. Targeted fluxomics allow the determination of metabolic reaction rates thereby discriminating metabolite level changes in HE in terms of production, consumption and clearance.

Diverse uncultivated ultra-small bacterial cells in groundwater.

Bacteria from phyla lacking cultivated representatives are widespread in natural systems and some have very small genomes. Here we test the hypothesis that these cells are small and thus might be enriched by filtration for coupled genomic and ultrastructural characterization. Metagenomic analysis of groundwater that passed through a ~0.2-μm filter reveals a wide diversity of bacteria from the WWE3, OP11 and OD1 candidate phyla. Cryogenic transmission electron microscopy demonstrates that, despite morphological variation, cells consistently have small cell size (0.009±0.002 μm(3)). Ultrastructural features potentially related to cell and genome size minimization include tightly packed spirals inferred to be DNA, few densely packed ribosomes and a variety of pili-like structures that might enable inter-organism interactions that compensate for biosynthetic capacities inferred to be missing from genomic data. The results suggest that extremely small cell size is associated with these relatively common, yet little known organisms.

Fluid Therapy: Double-Edged Sword during Critical Care?

Fluid therapy is still the mainstay of acute care in patients with shock or cardiovascular compromise. However, our understanding of the critically ill pathophysiology has evolved significantly in recent years. The revelation of the glycocalyx layer and subsequent research has redefined the basics of fluids behavior in the circulation. Using less invasive hemodynamic monitoring tools enables us to assess the cardiovascular function in a dynamic perspective. This allows pinpointing even distinct changes induced by treatment, by postural changes, or by interorgan interactions in real time and enables individualized patient management. Regarding fluids as drugs of any other kind led to the need for precise indication, way of administration, and also assessment of side effects. We possess now the evidence that patient centered outcomes may be altered when incorrect time, dose, or type of fluids are administered. In this review, three major features of fluid therapy are discussed: the prediction of fluid responsiveness, potential harms induced by overzealous fluid administration, and finally the problem of protocol-led treatments and their timing.

Renocardiac syndromes: physiopathology and treatment stratagems.

Purpose of reviewBidirectional inter-organ interactions are essential for normal functioning of the human body; however, they may also promote adverse conditions in remote organs. This review provides a narrative summary of the epidemiology, physiopathological mechanisms and clinical management of patients with combined renal and cardiac disease (recently classified as type 3 and 4 cardiorenal syndrome). Findings are also discussed within the context of basic research in animal models with similar comorbidities.Sources of informationPertinent published articles were identified by literature search of PubMed, MEDLINE and Google Scholar. Additional data from studies in the author’s laboratory were also consulted.FindingsThe prevalence of renocardiac syndrome throughout the world is increasing in part due to an aging population and to other risk factors including hypertension, diabetes and dyslipidemia. Pathogenesis of this disorder involves multiple bidirectional interactions between the kidneys and heart; however, participation of other organs cannot be excluded. Our own work supports the hypothesis that the uremic milieu, caused by kidney dysfunction, produces major alterations in vasoregulatory control particularly at the level of the microvasculature that results in impaired oxygen delivery and blood perfusion.LimitationsRecent clinical literature is replete with articles discussing the necessity to clearly define or characterize what constitutes cardiorenal syndrome in order to improve clinical management of affected patients. Patients are treated after onset of symptoms with limited available information regarding etiology. While understanding of mechanisms involved in pathogenesis of inter-organ crosstalk remains a challenging objective, basic research data remains limited partly because of the lack of animal models.ImplicationsPreservation of microvascular integrity may be the most critical factor to limit progression of multi-organ disorders including renocardiac syndrome. More fundamental studies are needed to help elucidate physiopathological mechanisms and for development of treatments to improve clinical outcomes.

Transcriptomic signatures of peroxisome proliferator-activated receptor α (PPARα) in different mouse liver models identify novel aspects of its biology.

BackgroundThe peroxisome proliferator-activated receptor alpha (PPARα) is a ligand-activated transcription factor that regulates lipid catabolism and inflammation and is hepatocarcinogenic in rodents. It is presumed that the functions of PPARα in liver depend on cross-talk between parenchymal (hepatocytes) and non-parenchymal (Kupffer and endothelial cells) fractions as well as inter-organ interactions. In order to determine how cellular composition and inter-organ interactions influence gene expression upon pharmacological activation of PPARα, we performed a meta-analysis of transcriptomics data obtained from mouse hepatocytes (containing only the parenchymal fraction), mouse liver slices (containing both fractions), and mouse livers exposed to a PPARα agonist. The aim was to obtain a comprehensive view of common and model-specific PPARα-dependent genes and biological processes to understand the impact of cross-talk between parenchymal and non-parenchymal fractions as well as the effect of inter-organ interactions on the hepatic PPARα transcriptome.To this end we analyzed microarray data of experiments performed in mouse primary hepatocytes treated with the PPARα agonist Wy14643 for 6 or 24 h (in vitro), mouse precision cut liver slices treated with Wy14643 for 24 h (ex vivo), and livers of wild type and Ppara knockout mice treated with Wy14643 for 6 h or 5 days (in vivo).ResultsIn all models, activation of PPARα significantly altered processes related to various aspects of lipid metabolism. In ex vivo and in vivo models, PPARα activation significantly regulated processes involved in inflammation; these processes were unaffected in hepatocytes. Only in vivo models showed significant regulation of genes involved in coagulation, carcinogenesis, as well as vesicular trafficking and extracellular matrix.ConclusionsPPARα-dependent regulation of genes/processes involved in lipid metabolism is mostly independent of the presence of non-parenchymal cells or systemic factors, as it was observed in all liver models. PPARα-dependent regulation of inflammatory genes requires the presence of non-parenchymal cells, as it was observed only ex vivo and in vivo. However, the full spectrum of PPARα biology at the level of lipid metabolism, immunity, carcinogenesis, as well as novel aspects of PPARα signaling such as coagulation, vesicular trafficking and the extracellular matrix, seems to require systemic factors, as it was observed exclusively in vivo.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-1106) contains supplementary material, which is available to authorized users.

Precision-cut Intestinal Slices as In Vitro Tool for Studies on Drug Metabolism

The role of the intestine in drug metabolism has long been underestimated as a consequence of the technical difficulty to discern the role of the intestine from that of the liver in in vivo experiments and of the lack of in vitro models that are sufficiently viable and fully representing the physiology and anatomy of the intestine. Recently the precision-cut slice model, which is widely used for liver and kidney, was also adapted for the small and large intestine. In this review the application of precision-cut intestinal slices (PCIS) for research in drug metabolism and transport is discussed. PCIS can be prepared from animal and human tissues from all regions of the intestine allowing investigation of species differences and regional gradients of activities of metabolizing enzymes. They are viable for 8-24 h of incubation and show high activity of drug metabolizing enzymes, representative for the in vivo activity. They have been successfully used to study drug-drug interactions such as induction, inhibition and regulation of drug metabolizing enzymes, transporters and nuclear factors. Moreover they appear to be a suitable model for studies on cold preservation of donor organs for transplantation, and allow exploring inter-organ interactions by co-incubation with precision-cut slices of other organs. Their application as model for drug-induced intestinal toxicity is still in its infancy but appears to be promising. PCIS, prepared from human and animal tissues, represent a powerful translational model for drug metabolism, transport and toxicity studies and as such contributes to the reduction and replacement of animal experiments.

Precision-cut Intestinal Slices as In Vitro Tool for Studies on Drug Metabolism

The role of the intestine in drug metabolism has long been underestimated as a consequence of the technical difficulty to discern the role of the intestine from that of the liver in in vivo experiments and of the lack of in vitro models that are sufficiently viable and fully representing the physiology and anatomy of the intestine. Recently the precision-cut slice model, which is widely used for liver and kidney, was also adapted for the small and large intestine. In this review the application of precision-cut intestinal slices (PCIS) for research in drug metabolism and transport is discussed. PCIS can be prepared from animal and human tissues from all regions of the intestine allowing investigation of species differences and regional gradients of activities of metabolizing enzymes. They are viable for 8-24 h of incubation and show high activity of drug metabolizing enzymes, representative for the in vivo activity. They have been successfully used to study drug-drug interactions such as induction, inhibition and regulation of drug metabolizing enzymes, transporters and nuclear factors. Moreover they appear to be a suitable model for studies on cold preservation of donor organs for transplantation, and allow exploring inter-organ interactions by co-incubation with precision-cut slices of other organs. Their application as model for drug-induced intestinal toxicity is still in its infancy but appears to be promising. PCIS, prepared from human and animal tissues, represent a powerful translational model for drug metabolism, transport and toxicity studies and as such contributes to the reduction and replacement of animal experiments.

Microfluidics Enables Small-Scale Tissue-Based Drug Metabolism Studies with Scarce Human Tissue

Early information on the metabolism and toxicity properties of new drug candidates is crucial for selecting the right candidates for further development. Preclinical trials rely on cell-based in vitro tests and animal studies to characterize the in vivo behavior of drug candidates, although neither are ideal predictors of drug behavior in humans. Improving in vitro systems for preclinical studies both from a technological and biological model standpoint thus remains a major challenge. This article describes how microfluidics can be exploited to come closer to this goal in combination with precision-cut liver slices (PCLS) as an improved organomimetic system. Recently, we developed a novel microfluidic-based system incorporating a microchamber for slice perifusion to perform drug metabolism studies with mammalian PCLS under continuous flow. In the present study, the viability and metabolism of human PCLS were assessed by the measurement of the leakage of liver-specific enzymes and metabolism of four different substrates: lidocaine, 7-hydroxycoumarin, 7-ethoxycoumarin, and testosterone. All experiments were verified with well plates, an excellent benchmark for these experiments. Clearly, however, human tissue is not readily available, and it is worth considering how to perform a maximum number of informative experiments with small amounts of material. In one approach, the microfluidic system was coupled to an HPLC system to allow on-line monitoring and immediate detection of unstable metabolites, something that is generally not possible with conventional well-plate systems. This novel microfluidic system also enables the in vitro measurement of interorgan interactions by connecting microchambers containing different organ slices in series for sequential perfusion. This versatile experimental system has the potential to yield more information about the metabolic profiles of new drug candidates in human and animal tissues in an early stage of development compared with well plates alone.

Microfluidic devices for in vitro studies on liver drug metabolism and toxicity

Microfluidic technologies enable the fabrication of advanced in vitro systems incorporating liver tissue or cells to perform metabolism and toxicity studies for drugs and other xenobiotics. The use of microfluidics provides the possibility to utilize a flow of medium, thereby creating a well-controlled microenvironment. The general goals of most in vitro systems in drug research are to optimally mimic the in vivo situation, and to minimize the number of animals required for preclinical studies. Moreover, they may contribute to a reduced attrition rate of drugs at a late stage of the drug development process; this is especially true if human tissue or cells are used. A number of factors are important in achieving good in vivo predictability in microfluidic systems, of which the biological system itself (cells or tissue) and the incubation conditions are the most important. The last couple of years have seen various microfluidic-based in vitro systems being developed to incorporate many different cells and/or tissues. In this review, microfluidics-based in vitro systems realized to study liver metabolism and toxicity are summarized and discussed with respect to their applications, advantages, and limitations. The biological basis of these systems is evaluated, and incubation conditions considered. Precise control of the cell or tissue microenvironment is a key advantage of using microfluidic technologies, and the benefits of exposing the cells to medium flow are demonstrated. Special attention is also paid to the incorporation of multiple cell types or tissues into a microfluidic device for the investigation of interorgan interactions, which are difficult if not impossible to study in conventional systems.

Biochemical Conservation and Evolution of Germacrene A Oxidase in Asteraceae

Sesquiterpene lactones are characteristic natural products in Asteraceae, which constitutes approximately 8% of all plant species. Despite their physiological and pharmaceutical importance, the biochemistry and evolution of sesquiterpene lactones remain unexplored. Here we show that germacrene A oxidase (GAO), evolutionarily conserved in all major subfamilies of Asteraceae, catalyzes three consecutive oxidations of germacrene A to yield germacrene A acid. Furthermore, it is also capable of oxidizing non-natural substrate amorphadiene. Co-expression of lettuce GAO with germacrene synthase in engineered yeast synthesized aberrant products, costic acids and ilicic acid, in an acidic condition. However, cultivation in a neutral condition allowed the de novo synthesis of a single novel compound that was identified as germacrene A acid by gas and liquid chromatography and NMR analyses. To trace the evolutionary lineage of GAO in Asteraceae, homologous genes were further isolated from the representative species of three major subfamilies of Asteraceae (sunflower, chicory, and costus from Asteroideae, Cichorioideae, and Carduoideae, respectively) and also from the phylogenetically basal species, Barnadesia spinosa, from Barnadesioideae. The recombinant GAOs from these genes clearly showed germacrene A oxidase activities, suggesting that GAO activity is widely conserved in Asteraceae including the basal lineage. All GAOs could catalyze the three-step oxidation of non-natural substrate amorphadiene to artemisinic acid, whereas amorphadiene oxidase diverged from GAO displayed negligible activity for germacrene A oxidation. The observed amorphadiene oxidase activity in GAOs suggests that the catalytic plasticity is embedded in ancestral GAO enzymes that may contribute to the chemical and catalytic diversity in nature.

AMD biofilms: using model communities to study microbial evolution and ecological complexity in nature

Similar to virtually all components of natural environments, microbial systems are inherently complex and dynamic. Advances in cultivation-independent molecular methods have provided a route to study microbial consortia in their natural surroundings and to begin resolving the community structure, dominant metabolic processes and inter-organism interactions. However, the utility of these methods generally scales inversely with community complexity. By applying genomics-enabled methods to the study of natural microbial communities with reduced levels of species richness, a relatively comprehensive understanding of the metabolic networks and evolutionary processes within these communities can be attained. In such well-defined model systems, it is also possible to link emergent ecological patterns to their molecular and evolutionary underpinnings, facilitating construction of predictive ecosystem models. In this study, we review over a decade of research on one such system-acid mine drainage biofilm communities. We discuss the value and limitations of tractable model microbial communities in developing molecular methods for microbial ecology and in uncovering principles that may explain behavior in more complex systems.

A microfluidic approach for in vitro assessment of interorgan interactions in drug metabolism using intestinal and liver slices

Over the past two decades, it has become increasingly clear that the intestine, in addition to the liver, plays an important role in the metabolism of xenobiotics. Previously, we developed a microfluidic-based in vitro system for the perifusion of precision-cut liver slices for metabolism studies. In the present study, the applicability of this system for the perifusion of precision-cut intestinal slices, and for the sequential perifusion of intestinal and liver slices, all from rat, was tested to mimic the in vivo first pass situation. Intestinal and liver slices, exposed to the substrates 7-ethoxycoumarin (7-EC), 7-hydroxycoumarin (7-HC) and lidocaine (Li), exhibited similar metabolic rates in the biochip and in the well plates for periods of at least 3 h. The metabolic rate remained the same when two slices were placed in adjacent microchambers and perifused sequentially. In addition, the system has been adapted to sequentially perifuse intestinal and liver tissue slices in a two-compartment co-culture perfusion system with a continuous flow of medium. It becomes possible to direct metabolites or other excreted compounds formed by an intestinal slice in the first compartment to the second compartment containing a liver slice. The intestine does not influence liver metabolism for these substrates. The interplay between these two organs was demonstrated by exposing the slices to the primary bile acid, chenodeoxycholic acid (CDCA). CDCA induced the expression of fibroblast growth factor 15 (FGF15) in the intestinal slice, which resulted in a stronger down-regulation of the enzyme, cytochrome P450 7A1 (CYP7A1), in the liver slice in the second compartment than when the liver slice was exposed to CDCA in a single-microchamber biochip. We thus demonstrate in this paper that intestinal slices, in addition to liver slices, remain functional in the biochip under flow conditions, and that the two-microchamber biochip has great potential for the study of interorgan effects. This is the first example of the incorporation of both liver and intestinal slices in a microfluidic device. Use of this microfluidic system will improve our insight into interorgan interactions and elucidate as yet unknown mechanisms involved in toxicity, gene regulation and drug-drug interactions.

Trading molecules and tracking targets in symbiotic interactions.

It is probable that nearly every natural product structure results from interactions between organisms. Symbiosis, a subset of inter-organism interactions involving closely associated partners, has recently provided new and interesting experimental systems for the study of these interactions. This review discusses new observations about natural product function and structural evolution that emerge from the study of symbiotic systems. In particular, these advances directly address long-standing 'how' and 'why' questions about natural products, providing fundamental insights about the evolution, origin and purpose of natural products that are inaccessible by other methods.

Interaction networks: coordinating responses to xenobiotic exposure.

In the last decade the increased usage of '-omic' technologies, plus the sequencing of over 800 complete genomes has led to a vast increase in the amount of information available to the researcher for examining cellular responses to xenobiotics. Much effort has been put into the identification and analysis of expression profiles associated with pathobiological conditions and/or xenobiotic exposure. These profiles are commonly used in two applications. Firstly, comparative profile experiments are used to classify pathobiological states and for the screening of novel chemical entities to predict their action(s) on the body. Secondly, mechanistic investigations will gain information on the molecular mechanisms underlying toxic responses/pathobiological states. During the course of such analysis it has become increasingly clear that a series of highly refined interaction networks exist within the body, regulating both the sensitivity and selectivity of the body's response to pathobiological states/xenobiotic exposure. These interaction networks exist at several levels: Firstly, within individual cells, the interaction between factors that transmit xenobiotics signals will determine the overall cellular response. Secondly, intraorgan communication occurs between the different cell types/sub-types which makes up an organ, coordinating the overall organ response. Finally, interorgan interactions provide axes of response through the body.

Dietary and lifestyle interventions in the management of the metabolic syndrome: present status and future perspective.

To review the mechanisms underlying the metabolic syndrome, or syndrome X, in humans, and to delineate dietary and environmental strategies for its prevention. Review of selected papers of the literature. Hyperinsulinemia and insulin resistance play a key role in the development of the metabolic syndrome. Strategies aimed at reducing insulin resistance may be effective in improving the metabolic syndrome. They include low saturated fat intake, consumption of low-glycemic-index foods, physical exercise and prevention of obesity. Future research, in particular the genetic basis of the metabolic syndrome and the interorgan interactions responsible for insulin resistance, is needed to improve therapeutic strategies for the metabolic syndrome.