Functional and genomic comparative study of the bitter taste receptor family TAS2R: Insight into the role of human TAS2R5.

Bitterness is perceived in humans by 25 subtypes of bitter taste receptors (hTAS2R) that range from broadly tuned to more narrowly tuned receptors. hTAS2R5 is one of the most narrowly tuned bitter taste receptors in humans. In this study, we review the literature on this receptor and show there is no consensus about its role. We then compare the possible role of hTAS2R5 with that of the proteins of the TAS2R family in rat, mouse, and pig. A phylogenetic tree of all mammalian TAS2R domain-containing proteins showed that human hTAS2R5 has no ortholog in pig, mouse, or rat genomes. By comparing the agonists that are common to hTAS2R5 and other members of the family, we observed that hTAS2R39 is the receptor that shares most agonists with hTAS2R5. In mouse, some of these agonists activate mTas2r105 and mTas2r144, which are distant paralogs of hTAS2R5. mTas2r144 seems to be the receptor that is most similar to hTAS2R5 because they are both activated by the same agonists and have affinities in the same range of values. Then, we can conclude that hTAS2R5 has a unique functional specificity in humans as it is activated by selective agonists and that its closest functional homolog in mouse is the phylogenetically distant mTas2r144.

GSPE Pre-Treatment Exerts Long-Lasting Preventive Effects against Aging-Induced Changes in the Colonic Enterohormone Profile of Female Rats.

The impact that healthy aging can have on society has raised great interest in understanding aging mechanisms. However, the effects this biological process may have on the gastrointestinal tract (GIT) have not yet been fully described. Results in relation to changes observed in the enteroendocrine system along the GIT are controversial. Grape seed proanthocyanidin extracts (GSPE) have been shown to protect against several pathologies associated with aging. Based on previous results, we hypothesized that a GSPE pre-treatment could prevent the aging processes that affect the enteroendocrine system. To test this hypothesis, we treated 21-month-old female rats with GSPE for 10 days. Eleven weeks after the treatment, we analyzed the effects of GSPE by comparing these aged animals with young animals. Aging induced a greater endocrine response to stimulation in the upper GIT segments (cholecystokinin (CCK) and glucagon-like peptide-1 (GLP-1)), a decrease in the mRNA abundance of GLP-1, peptide YY (PYY) and chromogranin A (ChgA) in the colon, and an increase in colonic butyrate. GSPE-treated rats were protected against a decrease in enterohormone expression in the colon. This effect is not directly related to the abundance of microbiome or short-chain fatty acids (SCFA) at this location. GSPE may therefore be effective in preventing a decrease in the colonic abundance of enterohormone expression induced by aging.

Administration of Alphitobius diaperinus or Tenebrio molitor before meals transiently increases food intake through enterohormone regulation in female rats.

BACKGROUND: It has been previously shown that acutely administered insect Alphitobius diaperinus protein increases food intake in rats and modifies the ex vivo enterohormone secretory profile differently than beef or almond proteins. In this study, we aimed to evaluate whether these effects could be maintained for a longer period and determine the underlying mechanisms. RESULTS: We administered two different insect species to rats for 26 days and measured food intake at different time points. Both insect species increased food intake in the first week, but the effect was later lost. Glucagon-like peptide 1 (GLP-1) and ghrelin were measured in plasma and ex vivo, and no chronic effects on their secretion or desensitization were found. Nevertheless, digested A. diaperinus acutely modified GLP-1 and ghrelin secretion ex vivo. CONCLUSION: Our results suggest that increases in food intake could be explained by a local ghrelin reduction acting in the small intestine. © 2022 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

GSPE pre-treatment protects against long-term cafeteria diet-induced mitochondrial and inflammatory affectations in the hippocampus of rats.

Deregulations like the loss of sensitivity to insulin (insulin resistance) and chronic inflammation are alterations very commonly found in sporadic forms of neurodegenerative pathologies. Thus, finding strategies to protect against them, may lead to a reduction in the incidence and/or affectation of these pathologies. The grape seed-derived proanthocyanidins extract (GSPE) is a mixture of compounds highly enriched in polyphenols and flavonoids that have shown to have a wide range of therapeutic benefits due to their antioxidant and anti-inflammatory properties. OBJECTIVES: This study aimed to assess the protective effects of a short pre-treatment of GSPE in the hippocampus against a prolonged feeding with cafeteria diet. METHODS: GSPE was administered for 10 days followed by 12 weeks of cafeteria diet. We analyzed transcriptional activity of genes and protein expression of key mediators of neurodegeneration in brain samples. RESULTS: Results indicated that GSPE was able to protect against cellular damage through the activation of AKT, as well as promote the maintenance of mitochondrial function by conserving the OXPHOS complexes and upregulating the antioxidant SOD. DISCUSSION: We observed that GSPE decreased inflammatory activation as observed through the downregulation of JNK, IL6 and TNFα, just like the reduction in reactive profile of astrocytes. Overall, the data presented here offers an interesting and hopeful initial step for future long-term studies on the beneficial effects of a supplementation of common diets with polyphenol and flavonoid substances for the amelioration of typical early hallmarks of neurodegeneration.

GLP1 Exerts Paracrine Activity in the Intestinal Lumen of Human Colon.

GLP1 produced in the upper part of the gut is released after food intake and acts by activating insulin secretion, but the role of GLP1 in the colon, where it is predominantly produced, remains unknown. Here we characterized the apical versus basolateral secretion of GLP1 and PYY and the paracrine mechanisms of action of these enterohormones in the human colon. We stimulated human colon tissue in different ex vivo models with meat peptone and we used immunofluorescence to study the presence of canonical and non-canonical receptors of GLP1. We found that PYY and GLP1 are secreted mainly at the gut lumen in unstimulated and stimulated conditions. We detected DPP4 activity and found that GLP1R and GCGR are widely expressed in the human colon epithelium. Unlike GLP1R, GCGR is not expressed in the lamina propria, but it is located in the crypts of Lieberkühn. We detected GLP1R expression in human colon cell culture models. We show that the apical secretion of PYY and GLP1 occurs in humans, and we provide evidence that GLP1 has a potential direct paracrine function through the expression of its receptors in the colon epithelium, opening new therapeutic perspectives in the use of enterohormones analogues in metabolic pathologies.

Application of emerging technologies to obtain legume protein isolates with improved techno-functional properties and health effects.

Current demand of consumers for healthy and sustainable food products has led the industry to search for different sources of plant protein isolates and concentrates. Legumes represent an excellent nonanimal protein source with high-protein content. Legume species are distributed in a wide range of ecological conditions, including regions with drought conditions, making them a sustainable crop in a context of global warming. However, their use as human food is limited by the presence of antinutritional factors, such as protease inhibitors, lectins, phytates, and alkaloids, which have adverse nutritional effects. Antitechnological factors, such as fiber, tannins, and lipids, can affect the purity and protein extraction yield. Although most are removed or reduced during alkaline solubilization and isoelectric precipitation processes, some remain in the resulting protein isolates. Selection of appropriate legume genotypes and different emerging and sustainable facilitating technologies, such as high-power ultrasound, pulsed electric fields, high hydrostatic pressure, microwave, and supercritical fluids, can be applied to increase the removal of unwanted compounds. Some technologies can be used to increase protein yield. The technologies can also modify protein structure to improve digestibility, reduce allergenicity, and tune technological properties. This review summarizes recent findings regarding the use of emerging technologies to obtain high-purity protein isolates and the effects on techno-functional properties and health.

Protective properties of grape-seed proanthocyanidins in human ex vivo acute colonic dysfunction induced by dextran sodium sulfate.

PURPOSE: Anti-inflammatory and barrier-protective properties have been attributed to proanthocyanidins in the context of intestinal dysfunction, however little information is available about the impact of these phytochemicals on intestinal barrier integrity and immune response in the human. Here we assessed the putative protective properties of a grape-seed proanthocyanidin extract (GSPE) against dextran sodium sulfate (DSS)-induced acute dysfunction of the human colon in an Ussing chamber system. METHODS: Human proximal and distal colon tissues from colectomized patients were submitted ex vivo for a 30-min preventive GSPE treatment (50 or 200 µg mL-1) followed by 1-h incubation with DSS (12% w v-1). Transepithelial electrical resistance (TEER), permeation of a fluorescently-labeled dextran (FD4) and proinflammatory cytokine release [tumor necrosis factor (TNF)-α and interleukin (IL)-1ß] of colonic tissues were determined. RESULTS: DSS reduced TEER (45-52%) in both the proximal and distal colon; however, significant increments in FD4 permeation (fourfold) and TNF-α release (61%) were observed only in the proximal colon. The preventive GSPE treatment decreased DSS-induced TEER loss (20-32%), FD4 permeation (66-73%) and TNF-α release (22-33%) of the proximal colon dose-dependently. The distal colon was not responsive to the preventive treatment but showed a reduction in IL-1ß release below basal levels with the highest GSPE concentration. CONCLUSIONS: Our results demonstrate potential preventive effects of GSPE on human colon dysfunction. Further studies are required to test whether administering GSPE could be a complementary therapeutic approach in colonic dysfunction associated with metabolic disorders and inflammatory bowel disease.

Glucagon-like peptide-1 regulation by food proteins and protein hydrolysates.

Glucagon-like peptide-1 (GLP-1) is an enterohormone with a key role in several processes controlling body homeostasis, including glucose homeostasis and food intake regulation. It is secreted by the intestinal cells in response to nutrients, such as glucose, fat and amino acids. In the present review, we analyse the effect of protein on GLP-1 secretion and clearance. We review the literature on the GLP-1 secretory effects of protein and protein hydrolysates, and the mechanisms through which they exert these effects. We also review the studies on protein from different sources that has inhibitory effects on dipeptidyl peptidase-4 (DPP4), the enzyme responsible for GLP-1 inactivation, with particular emphasis on specific sources and treatments, and the gaps there still are in knowledge. There is evidence that the protein source and the hydrolytic processing applied to them can influence the effects on GLP-1 signalling. The gastrointestinal digestion of proteins, for example, significantly changes their effectiveness at modulating this enterohormone secretion in both in vivo and in vitro studies. Nevertheless, little information is available regarding human studies and more research is required to understand their potential as regulators of glucose homeostasis.

Effects of flavanols on the enteroendocrine system: Repercussions on food intake.

Flavanols are plant-derived bioactive compounds for which several beneficial effects have been described. When ingested, they reach the gastrointestinal tract, where they can interact with the enteroendocrine cells. In this paper, we consider the possibility that flavanols modulate enterohormone secretion. Because the regulation of food intake is among the principal functions of the hormones that are secreted in the gastrointestinal tract, we also compile the literature that covers how the effects of flavanols on food intake are measured. Although there are some papers showing the effects of flavanols on the regulation of enterohormones, there are very few papers that have addressed the specific effects at the food intake level. Instead, most of the findings are secondary to the study of the action of flavanols on body weight, which makes it difficult to reach a clear conclusion regarding the effects of flavanols on food intake.

A specific dose of grape seed-derived proanthocyanidins to inhibit body weight gain limits food intake and increases energy expenditure in rats.

PURPOSE: Several studies have suggested that flavanols may have antiobesity effects; however, those effects clearly depend on the experimental conditions. In a previous study, we found that a single acute dose of grape seed proanthocyanidin extract (GSPE) has satiating effects. We therefore hypothesise that satiating doses of GSPE could be used to reduce body weight gain, and our present objective was to define the most effective dose. METHODS: We assayed two GSPE doses in aged male Wistar rats. First we performed a subchronic (8-day) treatment by intragastric administration, which was repeated after a washout period. We measured body weight, energy intake and faeces composition; we performed indirect calorimetry; and we analysed the mRNA expression of genes involved in lipid metabolism to determine the target tissue for the GSPE. RESULTS: We observed that 0.5 g GSPE/kg BW significantly reduced food intake and thus the amount of energy absorbed. This dosage also increased lipid oxidation in subcutaneous adipose tissue, thus causing a higher total energy expenditure. These combined effects caused a decrease in body weight. Conversely, 1 g GSPE/kg BW, which also reduced energy absorption after the first treatment, had a rebound effect on body weight gain which resulted in a lower response to the proanthocyanidin extract. That is, after the second treatment, the GSPE did not reduce the energy absorbed or modify energy expenditure and body weight. CONCLUSION: GSPE at a dose of 0.5 g/kg can reduce body weight by limiting food intake and activating energy expenditure in subcutaneous adipose tissue.

Effects of flavonoids on intestinal inflammation, barrier integrity and changes in gut microbiota during diet-induced obesity.

Diet-induced obesity is associated with low-grade inflammation, which, in most cases, leads to the development of metabolic disorders, primarily insulin resistance and type 2 diabetes. Although prior studies have implicated the adipose tissue as being primarily responsible for obesity-associated inflammation, the latest discoveries have correlated impairments in intestinal immune homeostasis and the mucosal barrier with increased activation of the inflammatory pathways and the development of insulin resistance. Therefore, it is essential to define the mechanisms underlying the obesity-associated gut alterations to develop therapies to prevent and treat obesity and its associated diseases. Flavonoids appear to be promising candidates among the natural preventive treatments that have been identified to date. They have been shown to protect against several diseases, including CVD and various cancers. Furthermore, they have clear anti-inflammatory properties, which have primarily been evaluated in non-intestinal models. At present, a growing body of evidence suggests that flavonoids could exert a protective role against obesity-associated pathologies by modulating inflammatory-related cellular events in the intestine and/or the composition of the microbiota populations. The present paper will review the literature to date that has described the protective effects of flavonoids on intestinal inflammation, barrier integrity and gut microbiota in studies conducted using in vivo and in vitro models.

Antioxidant effects of proanthocyanidin-rich natural extracts from grape seed and cupuassu on gastrointestinal mucosa.

BACKGROUND: The gastrointestinal tract (GI) is constantly exposed to reactive species released by the GI tract itself, and those present in food and beverages. Phenolic compounds may help in protecting the GI tract against damage produced by the reactive species. In this paper we have analyzed the effects of a grape seed proanthocyanidin extract (GSPE) on reactive oxygen species (ROS) production in two different intestinal cell types: the absorptive cell line Caco-2 and the enteroendocrine cell line STC-1. RESULTS: We show that GSPE prevents tert-butylhydroperoxide-induced oxidative stress in both cell lines, and that the effects are dose and time dependent. We have also analyzed whether GSPE has any in vivo effect, and found that 25 mg kg(-1) body weight cannot counteract the increase in intestinal ROS induced by the cafeteria diet. However, an acute (1 h) treatment of 1 g GSPE kg(-1) body weight reduced ROS in fasted animals and also decreased ROS induction by food. These effects were found only after a short-term treatment. Furthermore, we have compared the in vitro GSPE effects with those of another proanthocyanidin-rich extract from cupuassu seeds, though it has compounds with different structures. Cupuassu extract also shows antioxidant effects in both cell types, which suggests different mechanisms from those of GSPE. CONCLUSION: Natural proanthocyanidin-rich extracts have an antioxidant effect in the GI tract, acting on absorptive cells and enterohormone-secreting cells, although the effects depend on the dose and period of treatment. © 2015 Society of Chemical Industry.

Grape-seed procyanidins modulate cellular membrane potential and nutrient-induced GLP-1 secretion in STC-1 cells.

Grape-seed procyanidin extracts (GSPE) modulate glucose homeostasis, and it was suggested that GSPE may achieve this by enhancing the secretion of incretin hormones such as glucagon-like peptide-1 (GLP-1). Therefore, the aim of the present study is to examine in detail the effects of GSPE on intestinal endocrine cells (STC-1). GSPE was found to modulate plasma membrane potential in enteroendocrine cells, inducing depolarization at low concentrations (0.05 mg/l) and hyperpolarization at high concentrations (50 mg/l), and surprisingly this was also accompanied by suppressed GLP-1 secretion. Furthermore, how GSPE affects STC-1 cells under nutrient-stimulated conditions (i.e., glucose, linoleic acid, and l-proline) was also explored, and we found that the higher GSPE concentration was effective in limiting membrane depolarization and reducing GLP-1 secretion. Next, it was also examined whether GSPE affected mitochondrial membrane potential, and it was found that this too is altered by GSPE; however, this does not appear to explain the observed effects on plasma membrane potential and GLP-1 secretion. In conclusion, our results show that grape-seed procyanidins modulate cellular membrane potential and nutrient-induced enteroendocrine hormone secretion in STC-1 cells.

Gallic acid is an active component for the anticarcinogenic action of grape seed procyanidins in pancreatic cancer cells.

The aim of the present work was to evaluate the effects of a grape seed procyanidin extract (GSPE) on proliferation and apoptosis in the pancreatic adenocarcinoma cell line MIA PaCa-2 and identify the components of the extract with higher activity. The effects of the extract were analyzed on the proliferation and apoptosis processes in MIA PaCa-2 cells, as well as in the levels of the apoptosis markers Bcl-2 and Bax, the mitochondrial membrane potential, and reactive oxygen species levels. Finally, the components of the extract with higher effects were elucidated using enriched fractions of the extract and pure compounds. The results showed that GSPE inhibits cell proliferation and increases apoptosis in MIA PaCa-2 cells, which is primarily mediated by the downregulation of the antiapoptotic protein Bcl-2 and the depolarization of the mitochondrial membrane. GSPE also reduced the formation of reactive oxygen species. The component of the extract that possesses the highest antiproliferative and proapoptotic activity was gallic acid. In conclusion, GSPE acts as anticarcinogenic in MIA PaCa-2 cells, with gallic acid as the major single active constituent of the extract.

Intestinal Taste Receptor Expression and Its Implications for Health: An Integrative Analysis in Female Rats after Chronic Insect Supplementation.

Taste receptors are found in the gastrointestinal tract, where they are susceptible to dietary modulation, a key point that is crucial for diet-related responses. Insects are sustainable and good-quality protein sources. This study analyzed the impact of insect consumption on the modulation of taste receptor expression across various segments of the rat intestine under healthy or inflammatory conditions. Female Wistar rats were supplemented with Tenebrio molitor (T) or Alphitobius diaperinus (B), alongside a control group (C), over 21 days under healthy or LPS-induced inflammation. The present study reveals, for the first time, that insect consumption modulates taste receptor gene expression, mainly in the ascending colon. This modulation was not found under inflammation. Integrative analysis revealed colonic Tas1r1 as a key discriminator for insect consumption (C = 1.04 ± 0.32, T = 1.78 ± 0.72, B = 1.99 ± 0.82, p-value <0.05 and 0.01, respectively). Additionally, correlation analysis showed the interplay between intestinal taste receptors and metabolic and inflammatory responses. These findings underscore how insect consumption modulates taste receptors, influencing intestinal function and broader physiological mechanisms.

Assessing the impact of insect protein sources on intestinal health and disease: insights from human ex vivo and rat in vivo models.

The exploration of edible insects, specifically Alphitobius diaperinus and Tenebrio molitor, as sustainable sources of protein for human consumption is an emerging field. However, research into their effects on intestinal health, especially in relation to inflammation and permeability, remains limited. Using ex vivo and in vivo models of intestinal health and disease, in this study we assess the impact of the above insects on intestinal function by focusing on inflammation, barrier dysfunction and morphological changes. Initially, human intestinal explants were exposed to in vitro-digested extracts of these insects, almond and beef. Immune secretome analysis showed that the inflammatory response to insect-treated samples was comparatively lower than it was for samples exposed to almond and beef. Animal studies using yellow mealworm (Tenebrio molitor) and buffalo (Alphitobius diaperinus) flours were then used to evaluate their safety in healthy rats and LPS-induced intestinal dysfunction rats. Chronic administration of these insect-derived flours showed no adverse effects on behavior, metabolism, intestinal morphology or immune response (such as inflammation or allergy markers) in healthy Wistar rats. Notably, in rats subjected to proinflammatory LPS-induced intestinal dysfunction, T. molitor consumption did not exacerbate symptoms, nor did it increase allergic responses. These findings validate the safety of these edible insects under healthy conditions, demonstrate their innocuity in a model of intestinal dysfunction, and underscore their promise as sustainable and nutritionally valuable dietary protein sources.

Inhibition of DPP-IV Activity and Stimulation of GLP-1 Release by Gastrointestinally Digested Black Soldier Fly Prepupae.

The beneficial effects of an insect-based diet on human health and, in particular, the regulatory ability of digested insects' proteins on the glycaemic response in humans are topics that need to be investigated deeper. In this work, we performed an in vitro study on the modulatory activity of gastrointestinal digested black soldier fly (BSF) prepupae on the enterohormone GLP-1 and its natural inhibitor, DPP-IV. We verified whether actions intended to valorise the starting insect biomass, i.e., insect-optimised growth substrates and prior fermentation, can positively impact human health. Our results highlight that the digested BSF proteins from all the prepupae samples had a high stimulatory and inhibitory ability on the GLP-1 secretion and the DPP-IV enzyme in the human GLUTag cell line. Gastrointestinal digestion significantly improved the DPP-IV inhibitory capacity of the whole insect protein. Moreover, it was seen that optimised diets or fermentation processes preceding the digestion, in any case, did not positively affect the efficacy of the answer. BSF was already considered one of the edible insects more suitable for human consumption for its optimal nutritional profile. The BSF bioactivity here shown, after simulated digestion, on glycaemic control systems makes this species even more promising.

Bitter taste receptors along the gastrointestinal tract: comparison between humans and rodents.

For decades bitter taste receptors (TAS2R) were thought to be located only in the mouth and to serve as sensors for nutrients and harmful substances. However, in recent years Tas2r have also been reported in extraoral tissues such as the skin, the lungs, and the intestine, where their function is still uncertain. To better understand the physiological role of these receptors, in this paper we focused on the intestine, an organ in which their activation may be similar to the receptors found in the mouth. We compare the relative presence of these receptors along the gastrointestinal tract in three main species of biomedical research (mice, rats and humans) using sequence homology. Current data from studies of rodents are scarce and while more data are available in humans, they are still deficient. Our results indicate, unexpectedly, that the reported expression profiles do not always coincide between species even if the receptors are orthologs. This may be due not only to evolutionary divergence of the species but also to their adaptation to different dietary patterns. Further studies are needed in order to develop an integrated vision of these receptors and their physiological functionality along the gastrointestinal tract.

Procyanidins improve some disrupted glucose homoeostatic situations: an analysis of doses and treatments according to different animal models.

This review analyses the potential beneficial effects of procyanidins, the main class of flavonoids, in situations in which glucose homeostasis is disrupted. Because the disruption of glucose homeostasis can occur as a result of various causes, we critically review the effects of procyanidins based on the specific origin of each type of disruption. Where little or no insulin is present (Type I diabetic animals), summarized studies of procyanidin treatment suggest that procyanidins have a short-lived insulin-mimetic effect on the internal targets of the organism, an effect not reproduced in normoglycemic, normoinsulinemic healthy animals. Insulin resistance (usually linked to hyperinsulinemia) poses a very different situation. Preventive studies using fructose-fed models indicate that procyanidins may be useful in preventing the induction of damage and thus in limiting hyperglycemia. But the results of other studies using models such as high-fat diet treated rats or genetically obese animals are controversial. Although the effects on glucose parameters are hazy, it is known that procyanidins target key tissues involved in its homeostasis. Interestingly, all available data suggest that procyanidins are more effective when administered in one acute load than when mixed with food.

The effects of a cafeteria diet on insulin production and clearance in rats.

The aim of the present study was to determine the effects of a cafeteria diet on the function and apoptosis of the pancreas, and the activity and expression of the insulin-degrading enzyme (IDE). Female Wistar rats were fed either with a cafeteria diet or a control diet for 17 weeks, and blood and tissues were then collected for analysis. The cafeteria diet-treated rats had higher plasma insulin and C-peptide levels (P<0·05), showing increased insulin secretion by the pancreas. Insulin protein and gene expression levels were higher in the pancreas of obese rats, as was its transcriptional controller, pancreatic duodenal homeobox 1 (P<0·05). Feeding a cafeteria diet down-regulated the gene expression of the anti-apoptotic marker B-cell/lymphoma 2 (BCL2), and up-regulated the protein levels of BCL2-associated X protein, a pro-apoptotic marker (P<0·05). The cafeteria diet caused lipid accumulation in the pancreas and modified the expression of key genes that control lipid metabolism. To assay whether insulin clearance was also modified, we checked the activity of the IDE, one of the enzymes responsible for insulin clearance. We found increased liver IDE activity (P<0·05) in the cafeteria diet-fed animals, which could, in part, be due to an up-regulation of its gene expression. Conversely, IDE gene expression was unmodified in the kidney and adipose tissue; although when the adipose tissue weight was considered, the insulin clearance potential was higher in the cafeteria diet-treated rats. In conclusion, treatment with a cafeteria diet for 17 weeks in rats mimicked a pre-diabetic state, with ectopic lipid accumulation in the pancreas, and increased the IDE-mediated insulin clearance capability.