Allomyrina dichotoma larval extract has protective effects against gut permeability of dextran sulfate sodium-fed Drosophila by E-cadherin and armadillo.

ETHNOPHARMACOLOGICAL RELEVANCE: Larvae of the rhinoceros beetle (Allomyrina dichotoma) (ADL) are used ethnopharmacologically to treat gut-related disorders in Korea and China since 1596 and are also approved as a safe novel food with high nutritional value. AIM OF THE STUDY: We investigated the protective effects of ADL extract against leaky gut disease using a Drosophila model and sought to elucidate the underlying biological mechanisms. MATERIALS AND METHODS: We examined the protective effects of ADL extract (2 mg/mL) against the leaky gut disease using a dextran sulfate sodium (DSS)-induced leaky gut Drosophila melanogaster model. RESULTS: We found that oral administration of ADL extracts significantly increase the survival rate of DSS-fed Drosophila. Under conditions of DSS-induced gut damage, ADL extract reduced gut cell apoptosis and gut permeability, resulting in the maintenance of gut tissue homeostasis. Furthermore, we observed that oral administration of ADL extract can induce high levels of E-cadherin gene expression and also restored the original membrane localization of DSS-disrupted E-cadherin contiguous with the armadillo. CONCLUSION: We concluded that ADL extract plays an important role in maintaining gut homeostasis through the up-regulation of E-cadherin and that it may have a protective effect against leaky gut syndrome.

Toxicological safety evaluation of freeze-dried Protaetia brevitarsis larva powder.

White-spotted flower chafer (Protaetia brevitarsis) is an edible insect and its larva was used as a traditional Asian medicine. It's a promising material as a novel food source because of its nutritional components. In this study, as part of the preclinical toxicity program, we evaluated the toxicity of freeze-dried P. brevitarsis larva powder to develop a novel food material. In a single-dose oral toxicity study in rats, there were no changes in mortality, clinical observations, and body weight in rats administered 5000 mg/kg P. brevitarsis larva powder. In a 13-week oral repeated dose toxicity study in rats, there were no adverse effects or changes in mortality, clinical observations, body weight, food consumption, ophthalmology, clinical pathology, necropsy, organ weight, and histopathology at doses of 300, 1000, and 3000 mg/kg/day. In identification of allergic reactions, P. brevitarsis larva powder induced no increases of serum immunoglobulin E and histamine concentrations over 13 weeks of oral administration in rats. In a genotoxicity assessment, P. brevitarsis larva powder didn't provoke bacterial reverse mutations, chromosomal aberrations, and micronucleated reticulocytes. Therefore, freeze-dried P. brevitarsis larva powder shows no evidence of toxic and mutagenic changes under the experimental conditions of the present in vitro and in vivo studies.

BACE1 Inhibition by Genistein: Biological Evaluation, Kinetic Analysis, and Molecular Docking Simulation.

ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) plays a role in generating amyloid ß (Aß), thus playing a major part early in the pathogenesis of Alzheimer's disease (AD). BACE1 has emerged as a crucial therapeutic target for decreasing the Aß concentration in the AD brain. To explore natural BACE1 inhibitors, the present study concentrated on isoflavones, including genistein, formononetin, glycitein, daidzein, and puerarin. In this study, in vitro anti-AD activities were assessed using BACE1 inhibition assays, as well as enzyme kinetic predictions. Molecular docking analysis was applied to design potential BACE1 inhibitors. Among the major isoflavones, genistein exerted a notable BACE1 inhibition through reversible noncompetitive mechanism, while other compounds were less potent against BACE1. The docking study revealed that genistein had negative binding energy (-8.5 kcal/mol) and was stably positioned in the allosteric domains of BACE1 residues. It interacted with important amino acid residues in BACE1, such as ASN37, GLN73, and TRP76, through hydrogen bonding. The results suggested that genistein may be beneficial for preventing and/or treating AD. Furthermore, it may provide potential guidelines for the design of new BACE1 inhibitors.

Central Administration of 1-Deoxynojirimycin Attenuates Hypothalamic Endoplasmic Reticulum Stress and Regulates Food Intake and Body Weight in Mice with High-Fat Diet-Induced Obesity.

The α-glucosidase inhibitor, 1-deoxynojirimycin (DNJ), is widely used for its antiobesity and antidiabetic effects. Researchers have demonstrated that DNJ regulates body weight by increasing adiponectin levels, which affects energy intake and prevents diet-induced obesity. However, the mechanism by which centrally administered DNJ exerts anorexigenic effects has not been studied until now. We investigated the effect of DNJ in the hypothalamus of mice with high-fat diet-induced obesity. Results showed that intracerebroventricular (ICV) administration of DNJ reduced hypothalamic ER stress, which activated the leptin-induced Janus-activated kinase 2 (JAK2)/signal transducers and activators of transcription 3 (STAT3) signaling pathway to cause appetite suppression. We conclude that DNJ may reduce obesity by moderating feeding behavior and ER stress in the hypothalamic portion of the central nervous system (CNS).

Tenebrio molitor Larvae Inhibit Adipogenesis through AMPK and MAPKs Signaling in 3T3-L1 Adipocytes and Obesity in High-Fat Diet-Induced Obese Mice.

Despite the increasing interest in insect-based bioactive products, the biological activities of these products are rarely studied adequately. Larvae of Tenebrio molitor, the yellow mealworm, have been eaten as a traditional food and provide many health benefits. Therefore, we hypothesized that T. molitor larvae might influence adipogenesis and obesity-related disorders. In the present study, we investigated the anti-adipogenic and antiobesity effects of T. molitor larvae in vitro and in vivo. The lipid accumulation and triglyceride content in mature adipocytes was reduced significantly (up to 90%) upon exposure to an ethanol extract of T. molitor larvae, without a reduction in cell viability. Exposure also resulted in key adipogenic and lipogenic transcription factors. Additionally, in adipogenic differentiation medium the extract induced phosphorylation of adenosine monophosphate (AMP)-activated protein kinase and mitogen-activated protein kinases. Daily oral administration of T. molitor larvae powder to obese mice fed high-fat diet attenuated body weight gain. We also found that the powder efficiently reduced hepatic steatosis as well as aspartate and alanine transaminase enzyme levels in mice fed a high-fat diet. Our results suggest that T. molitor larvae extract has an antiobesity effect when administered as a food supplement and has potential as a therapeutic agent for obesity.

Allomyrina Dichotoma Larvae Regulate Food Intake and Body Weight in High Fat Diet-Induced Obese Mice Through mTOR and Mapk Signaling Pathways.

Recent evidence has suggested that the Korean horn beetle (Allomyrina dichotoma) has anti-hepatofibrotic, anti-neoplastic, and antibiotic effects and is recognized as a traditional medicine. In our previous works, Allomyrina dichotoma larvae (ADL) inhibited differentiation of adipocytes both in vitro and in vivo. However, the anorexigenic and endoplasmic reticulum(ER) stress-reducing effects of ADL in obesity has not been examined. In this study, we investigated the anorexigenic and ER stress-reducing effects of ADL in the hypothalamus of diet-induced obese (DIO) mice. Intracerebroventricular (ICV) administration of ethanol extract of ADL (ADE) suggested that an antagonizing effect on ghrelin-induced feeding behavior through the mTOR and MAPK signaling pathways. Especially, ADE resulted in strong reduction of ER stress both in vitro and in vivo. These findings strongly suggest that ADE and its constituent bioactive compounds are available and valuable to use for treatment of various diseases driven by prolonged ER stress.

Molecular Characterization of Endoplasmic Reticulum Oxidoreductin 1 from Bombyx mori.

We isolated a complementary DNA (cDNA) clone encoding endoplasmic reticulum oxidoreductin 1 (bERO1, a specific oxidant of protein disulfide isomerase (PDI)) from Bombyx mori. This protein has a putative open reading frame (ORF) of 489 amino acids and a predicted size of 57.4 kDa. Although bERO1 protein shares less than 57% amino acid sequence homology with other reported ERO1s, it contains two conserved redox active motifs, a Cys-X-X-X-X-Cys motif of N-terminal and Cys-X-X-Cys-X-X-Cys motif of C-terminal. Both motifs are typically present in ERO1 protein family members. The bEro1 mRNA expression was highest in posterior silk gland on the sixth day of the 5th instar larvae. Expression of bEro1 mRNA also markedly increased during endoplasmic reticulum (ER) stress induced by stimulation with antimycin, calcium ionophore A23187, dithiothreitol, H2O2, monencin, and tunicamycin. In addition, expression levels of bEro1 exactly coincided with that of bPdi. This is the first result suggesting that bERO1 plays an essential role in ER quality control through the combined activities of bERO1 and bPDI as a catalyst of protein folding in the ER and sustaining cellular redox homeostasis.

Immune modulation of glycosaminoglycan derived from P. lewisi in TNF-α stimulated cells.

Poecilocoris lewisi (Korean name: "Kwangdaenolinjae") is a red-striped gold stink bug (insect) which has been used as a crude drug in traditional medicine of East Asia and Korea. In this study, ethanol extract and glycosaminoglycan from P. lewisi (Pl GAG), as an active substance among its components, were investigated for their potential anti-inflammatory actions. They were found to be a potent inducer of nitric oxide (NO) production from calf pulmonary artery endothelial (CPAE) cells and a stimulator of endothelial nitric oxide synthase in a dose-dependent manner. The anti-inflammatory activities were also evaluated by determining the level of adhesion molecules related to atherogenesis and pro-inflammatory cytokines, such as vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), secretory phospholipase A2, and prostaglandin E2, stimulated by tumor necrosis factor (TNF)-α in human umbilical vein endothelial cells (HUVEC). They also showed inhibitory effects on vascular endothelial growth factor (VEGF) production in HUVECs. Matrix metalloproteinases (MMP-2 and 9) were also inhibited by treatment with this extract or glycosaminoglycan. Furthermore, this GAG showed cytotoxicity against CT-26 colon cancer cells whereas having no cytotoxicity in CHO normal cells. Monosaccharide (amino, acidic, neutral monosaccharides) composition of used GAG was characterized by trimethylsilylated GC-MS analysis method.

Anticancer Activity of the Antimicrobial Peptide Scolopendrasin VII Derived from the Centipede, Scolopendra subspinipes mutilans.

Previously, we performed de novo RNA sequencing of Scolopendra subspinipes mutilans using high-throughput sequencing technology and identified several antimicrobial peptide candidates. Among them, a cationic antimicrobial peptide, scolopendrasin VII, was selected based on its physicochemical properties, such as length, charge, and isoelectric point. Here, we assessed the anticancer activities of scolopendrasin VII against U937 and Jurkat leukemia cell lines. The results showed that scolopendrasin VII decreased the viability of the leukemia cells in MTS assays. Furthermore, flow cytometric analysis and acridine orange/ethidium bromide staining revealed that scolopendrasin VII induced necrosis in the leukemia cells. Scolopendrasin VII-induced necrosis was mediated by specific interaction with phosphatidylserine, which is enriched in the membrane of cancer cells. Taken together, these data indicated that scolopendrasin VII induced necrotic cell death in leukemia cells, probably through interaction with phosphatidylserine. The results provide a useful anticancer peptide candidate and an efficient strategy for new anticancer peptide development.

Antimicrobial peptides in the centipede Scolopendra subspinipes mutilans.

The centipede Scolopendra subspinipes mutilans is an environmentally beneficial and medically important arthropod species. Although this species is increasingly applied as a reliable source of new antimicrobial peptides, the transcriptome of this species is a prerequisite for more rational selection of antimicrobial peptides. In this report, we isolated total RNA from the whole body of adult centipedes, S. subspinipes mutilans, that were nonimmunized and immunized against Escherichia coli, and we generated a total of 77,063 pooled contigs and singletons using high-throughput sequencing. To screen putative antimicrobial peptides, in silico analyses of the S. subspinipes mutilans transcriptome were performed based on the physicochemical evidence of length, charge, isoelectric point, and in vitro and in vivo aggregation scores together with the existence of continuous antimicrobial peptide stretches. Moreover, we excluded some transcripts that showed similarity with both previously known antimicrobial peptides and the human proteome, had a proteolytic cleavage site, and had downregulated expression compared with the nonimmunized sample. As a result, we selected 17 transcripts and tested their antimicrobial activity with a radial diffusion assay. Among them, ten synthetic peptides experimentally showed antimicrobial activity against microbes and no toxicity to mouse erythrocytes. Our results provide not only a useful set of antimicrobial peptide candidates and an efficient strategy for novel antimicrobial peptide development but also the transcriptome data of a big centipede as a valuable resource.

Oleic acid and linoleic acid from Tenebrio molitor larvae inhibit BACE1 activity in vitro: molecular docking studies.

In our ongoing research to find therapeutic compounds for Alzheimer's disease (AD) from natural resources, the inhibitory activity of the BACE1 enzyme by Tenebrio molitor larvae and its major compounds were evaluated. The T. molitor larvae extract and its fractions exhibited strong BACE1 suppression. The major components of hexane fraction possessing both high yield and strong BACE1 inhibition were determined by thin layer chromatography, gas chromatography, and nuclear magnetic resonance analysis. A remarkable composition of unsaturated long chain fatty acids, including oleic acid and linoleic acid, were identified. Oleic acid, in particular, noncompetitively attenuated BACE1 activity with a half-maximal inhibitory concentration (IC50) value of 61.31 µM and Ki value of 34.3 µM. Furthermore, the fatty acids were stably interacted with BACE1 at different allosteric sites of the enzyme bound with the OH of CYS319 and the NH3 of TYR320 for oleic acid and with the C=O group of GLN304 for linoleic acid. Here, we first revealed novel pharmacophore features of oleic acids and linoleic acid to BACE1 by in silico docking studies. The present findings would clearly suggest potential guidelines for designing novel BACE1 selective inhibitors.

Antimicrobial activity of the synthetic peptide scolopendrasin ii from the centipede Scolopendra subspinipes mutilans.

The centipede Scolopendra subpinipes mutilans is a medicinally important arthropod species. However, its transcriptome is not currently available and transcriptome analysis would be useful in providing insight into a molecular level approach. Hence, we performed de novo RNA sequencing of S. subpinipes mutilans using next-generation sequencing. We generated a novel peptide (scolopendrasin II) based on a SVM algorithm, and biochemically evaluated the in vitro antimicrobial activity of scolopendrasin II against various microbes. Scolopendrasin II showed antibacterial activities against gram-positive and -negative bacterial strains, including the yeast Candida albicans and antibiotic-resistant gram-negative bacteria, as determined by a radial diffusion assay and colony count assay without hemolytic activity. In addition, we confirmed that scolopendrasin II bound to the surface of bacteria through a specific interaction with lipoteichoic acid and a lipopolysaccharide, which was one of the bacterial cell-wall components. In conclusion, our results suggest that scolopendrasin II may be useful for developing peptide antibiotics.

Blood-pressure-lowering effect of glycosaminoglycan derived from Isaria sinclairii in spontaneously hypertensive rats.

The antihypertensive effects of both extracts and glycosaminoglycan derived from Isaria sinclairii (IS) were investigated in a spontaneously hypertensive rat (SHR) model. Groups of rats were treated orally with 30 mg/kg each of: (1) saline control or extracts of (2) water-IS (3) methanol-IS, (4) butanol-IS, (5) ethyl acetate-IS, or (6) captopril as positive control. The 30-mg/kg dose was administered with a standard diet every day for a period of 2 wk. The antihypertensive effects of the individual extracts were in the following order: methanol > water > ethyl acetate > butanol. Glycosaminoglycan (GAG) obtained from IS as a water-soluble alcohol precipitation fraction produced an antihypertensive effect. One month following administration of GAG derived from IS to SHR animals there was a marked decrease in systolic blood pressure from 183 to 105 mm Hg and reduced diastolic blood pressure from 148 to 80 mm Hg compared to untreated control SHR rats. It was found that GAG produced an antihypertensive effect, which was more effective than the positive control captopril. In the SHR animal model a fall of 19% in body weight was observed in the group that received GAG. Data thus indicate that GAG derived from I. sinclairii may be a potent, naturally occurring antihypertensive agent.

The insect peptide coprisin prevents Clostridium difficile-mediated acute inflammation and mucosal damage through selective antimicrobial activity.

Clostridium difficile-associated diarrhea and pseudomembranous colitis are typically treated with vancomycin or metronidazole, but recent increases in relapse incidence and the emergence of drug-resistant strains of C. difficile indicate the need for new antibiotics. We previously isolated coprisin, an antibacterial peptide from Copris tripartitus, a Korean dung beetle, and identified a nine-amino-acid peptide in the α-helical region of it (LLCIALRKK) that had antimicrobial activity (J.-S. Hwang et al., Int. J. Pept., 2009, doi:10.1155/2009/136284). Here, we examined whether treatment with a coprisin analogue (a disulfide dimer of the nine peptides) prevented inflammation and mucosal damage in a mouse model of acute gut inflammation established by administration of antibiotics followed by C. difficile infection. In this model, coprisin treatment significantly ameliorated body weight decreases, improved the survival rate, and decreased mucosal damage and proinflammatory cytokine production. In contrast, the coprisin analogue had no apparent antibiotic activity against commensal bacteria, including Lactobacillus and Bifidobacterium, which are known to inhibit the colonization of C. difficile. The exposure of C. difficile to the coprisin analogue caused a marked increase in nuclear propidium iodide (PI) staining, indicating membrane damage; the staining levels were similar to those seen with bacteria treated with a positive control for membrane disruption (EDTA). In contrast, coprisin analogue treatment did not trigger increases in the nuclear PI staining of Bifidobacterium thermophilum. This observation suggests that the antibiotic activity of the coprisin analogue may occur through specific membrane disruption of C. difficile. Thus, these results indicate that the coprisin analogue may prove useful as a therapeutic agent for C. difficile infection-associated inflammatory diarrhea and pseudomembranous colitis.

Clinical importance of Bcl-6-positive non-deep-site involvement in non-HIV-related primary central nervous system diffuse large B-cell lymphoma.

In several studies of primary central nervous system lymphoma (PCNSL), deep-site involvement of the brain, as well as age and performance status (PS), were found to be independent prognostic factors. In immunocompetent patients, most primary central nervous system lymphomas (PCNSL) are diffuse large B-cell lymphomas (DLBCL), and recent studies have shown that Bcl-6 would be a favorable prognostic biomarker in PCNS-DLBCL. The objective of this study is to evaluate the clinical importance of the central nervous system (CNS) involvement pattern combined with Bcl-6 expression in PCNS-DLBCL patients. This study included 65 immunocompetent patients with PCNS-DLBCL who underwent treatment with high-dose methotrexate with whole-brain radiotherapy. Immunochemistry was performed for the Bcl-6 and Ki-67 antigens. Forty-four patients were male and 21 patients were female, with median age of 59 years. During the median follow-up period of 26 months, progression-free survival (PFS) was 25% and overall survival (OS) was 31%. Of 65 cases that could be subclassified, 31 patients were Bcl-6 positive and 34 patients were negative. Deep-site involvement of the brain was observed in 31 patients. The Bcl-6-positive group and the group having non-deep-site involvement of the brain were associated with favorable progression-free survival (PFS) (P < 0.001; P < 0.001) and overall survival (OS) (P = 0.001; P < 0.001). Results of univariate analysis showed that age above 60 years, Eastern Cooperative Oncology Group (ECOG) PS above 2, elevated lactate dehydrogenase (LDH) state, complete response (CR), and Bcl-6-positive and deep-site involvement were prognostic factors associated with PFS and OS. Results of multivariate analysis revealed that age above 60 years, ECOG above 2, elevated LDH state, Bcl-6 positivity, and deep-site involvement were independent prognostic factors for prediction of outcome. According to the combined prognostic value of Bcl-6 expression and the deep-site involvement pattern, the subgroup having Bcl-6-positive non-deep-site involvement of the brain showed more favorable PFS and OS than the other subgroups (P < 0.001, P < 0.001), whereas differences of survival among the other three subgroups were not significant (P = 0.054, P = 0.056). Bcl-6 positivity was found to be an independent prognostic factor for survival. Bcl-6 expression was associated with higher PFS and OS in patients having non-deep-site involvement. However, this was counteracted in the group of patients having deep-site involvement of the brain.

Antidiabetic effects and gene expression profiling in obese mice treated with Isaria sinclairii over a 6-month period.

The molecular mechanisms underlying the glucose-lowering effects of Isaria sinclairii (Cicada Dongchunghacho), a fungus cultured on silkworm, are not fully elucidated. Thus the glucose-lowering effects of I. sinclairii as potential an antidiabetic agent were investigated in C57BL/6 obese (ob/ob) mice over a 6-mo period. For a period of 26 wk, ob mice were administered either 5 or 10% (w/w) I. sinclairii powder (IS), 10% dry mulberry leaf powder (ML), or 10% silkworm (SW) powder in the standard diet while a control group received only standard diet. The ML and SW preparations served as positive controls. Isaria sinclairii at 10% in the diet was more effective in reducing body weight compared to 10% ML, 10% SW, or 5% I. sinclairii. The fall in blood glucose levels in the groups treated for 26 wk was greater in both IS groups at 1 mo compared to ML or SW but equal in all groups at 6 mo. Microarray analyses were performed with a mouse 7.4K cDNA clone set array to identify the gene-expression profiles for the IS-, ML-, and SW-exposed ob mouse liver. The 10% IS group, compared to control, showed that 15 genes including glucokinase (Gk-rs1) and LDL receptor relating protein 1 were upregulated and 12 genes including cell translocation gene2 (antiproliferative) and hydroxyprostaglandin dehydrogenase (Hpgd 15) were downregulated. Upregulation of Gk-rs 1 and downregulation of Hpgd 15 were previously shown to occur in drug-induced suppression of diabetes. With ML, Lepr (leptin receptor), Pik3cb (phosphatidylinositol 3-kinase), and Prodh (proline dehydrogenase), related to suppression of diabetes, were upregulated. In the case of SW, the enzymes (G2an, alpha glucosidase 2) and Mmp9 (matrix metalloproteinase 9) involved in elevation of blood glucose levels were both downregulated. Data suggest that I. sinclarii is effective in lowering blood glucose due to the upregulation of glucokinase (Gk-rs1) and downregulation of hydroxyprostaglandin dehydrogenase (Hpgd 15), both associated with suppression of diabetes, indicating that microarray analysis is a useful tool to assess pharmacological potency of therapeutic compounds.

Molecular cloning and characterization of ATX1 cDNA from the mole cricket, Gryllotalpa orientalis.

To search for an insect homologue of antioxidant protein 1 (ATX1), a mole cricket, Gryllotalpa orientalis, cDNA library was screened and a cDNA clone, which encodes a 73 amino acid polypeptide with a predicted molecular mass of 8.0 kDa and pI of 5.68, was isolated. The G. orientalis ATX1 (GoATX1) cDNA features both a MTCXXC copper-binding site in the N-terminus and a KTGK lysine-rich region in the C-terminus. The deduced amino acid sequence of the GoATX1 cDNA showed 63% identity to Drosophila melanogaster ATX1 and 55% to Ixodes pacificus ATX1. Northern blot analysis revealed the presence of GoATX1 transcripts in midgut, fat body, and epidermis. When H2O2 was injected into the body cavity of G. orientalis adult, GoATX1 mRNA expression was up-regulated in the fat body tissue. Fat body expression level of GoATX1 mRNA in the fat body was increased following exposure to low (4 degrees C) and high (37 degrees C) temperatures, suggesting that GoATX1 plays a protective role against oxidative stress caused by temperature shock. This is the first report about a functional role of insect ATX1 in antioxidant defense.

Molecular characterization of a Bombyx mori protein disulfide isomerase (bPDI).

We have isolated a complementary deoxyribonucleic acid clone that encodes the protein disulfide isomerase of Bombyx mori (bPDI). This protein has a putative open reading frame of 494 amino acids and a predicted size of 55.6 kDa. In addition, 2 thioredoxin active sites, each with a CGHC sequence, and an endoplasmic reticulum (ER) retention signal site with a KDEL motif were found at the C-terminal. Both sites are typically found in members of the PDI family of proteins. The expression of bPDI messenger ribonucleic acid (mRNA) was markedly increased during ER stress induced by stimulation with calcium ionophore A23187, tunicamycin, and dithiothreitol, all of which are known to cause an accumulation of unfolded proteins in the ER. We also examined the tissue distribution of bPDI mRNA and found pronounced expression in the fat body of insects. Hormonal regulation studies showed that juvenile hormone, insulin, and a combination of juvenile hormone and transferrin (although not transferrin alone) affected bPDI mRNA expression. A challenge with exogenous bacteria also affected expression, and the effect peaked 16 hours after infection. These results suggest that bPDI is a member of the ER-stress protein group, that it may play an important role in exogenous bacterial infection of the fat body, and that its expression is hormone regulated.