Curcumae Radix Decreases Neurodegenerative Markers through Glycolysis Decrease and TCA Cycle Activation.

Neurodegenerative diseases (ND) are being increasingly studied owing to the increasing proportion of the aging population. Several potential compounds are examined to prevent neurodegenerative diseases, including Curcumae radix, which is known to be beneficial for inflammatory conditions, metabolic syndrome, and various types of pain. However, it is not well studied, and its influence on energy metabolism in ND is unclear. We focused on the relationship between ND and energy metabolism using Curcumae radix extract (CRE) in cells and animal models. We monitored neurodegenerative markers and metabolic indicators using Western blotting and qRT-PCR and then assessed cellular glycolysis and metabolic flux assays. The levels of Alzheimer's disease-related markers in mouse brains were reduced after treatment with the CRE. We confirmed that neurodegenerative markers decreased in the cerebrum and brain tumor cells following low endoplasmic reticulum (ER) stress markers. Furthermore, glycolysis related genes and the extracellular acidification rate decreased after treatment with the CRE. Interestingly, we found that the CRE exposed mouse brain and cells had increased mitochondrial Tricarboxylic acid (TCA) cycle and Oxidative phosphorylation (OXPHOS) related genes in the CRE group. Curcumae radix may act as a metabolic modulator of brain health and help treat and prevent ND involving mitochondrial dysfunction.

The aqueous fraction of Castanea crenata inner shell extract reduces obesity and intramuscular lipid accumulation via induction of mitochondrial respiration and fatty acid oxidation in muscle.

BACKGROUND: Skeletal muscle is responsible for free fatty acid (FFA) disposal via mitochondrial respiration and fatty acid oxidation (FAO). Obesity triggers high levels of circulating FFAs, which can cause intramuscular lipid (IMCL) deposition. Diverse phytochemicals, including crude Castanea crenata inner shell extract (CCE), have been shown to possess an anti-obesity effect. PURPOSE: We aimed to demonstrate whether the aqueous fraction of CCE (ACCE) provides an anti-obesity effect with a decrease in plasma FFAs and reduces IMCL. METHODS: High-fat-fed C57BL/6 mice received ACCE via water intake. A204 cells incubated with fatty acids were treated with ACCE. Lipid accumulation and mitochondrial metabolism were assessed using histological and molecular techniques. RESULTS: ACCE possessed a notably higher gallic acid content than CCE among the constituents. ACCE-administered mice exhibited reduced plasma FFA levels, adiposity, and IMCL. Muscle lipotoxicity was suppressed, including apoptosis, ER stress, and inflammation. The anti-lipid effect of ACCE was observed with the induction of mitochondrial respiration and fatty acid oxidation in muscle. CONCLUSIONS: ACCE increases mitochondrial respiration and FAO in skeletal muscle and protects muscle from IMCL and lipotoxicity, reducing plasma FFA and adiposity.

Letrozole Accelerates Metabolic Remodeling through Activation of Glycolysis in Cardiomyocytes: A Role beyond Hormone Regulation.

Estrogen receptor-positive (ER+) breast cancer patients are recommended hormone therapy as a primary adjuvant treatment after surgery. Aromatase inhibitors (AIs) are widely administered to ER+ breast cancer patients as estrogen blockers; however, their safety remains controversial. The use of letrozole, an AI, has been reported to cause adverse cardiovascular effects. We aimed to elucidate the effects of letrozole on the cardiovascular system. Female rats exposed to letrozole for four weeks showed metabolic changes, i.e., decreased fatty acid oxidation, increased glycolysis, and hypertrophy in the left ventricle. Although lipid oxidation yields more ATP than carbohydrate metabolism, the latter predominates in the heart under pathological conditions. Reduced lipid metabolism is attributed to reduced ß-oxidation due to low circulating estrogen levels. In letrozole-treated rats, glycolysis levels were found to be increased in the heart. Furthermore, the levels of glycolytic enzymes were increased (in a high glucose medium) and the glycolytic rate was increased in vitro (H9c2 cells); the same was not true in the case of estrogen treatment. Reduced lipid metabolism and increased glycolysis can lower energy supply to the heart, resulting in predisposition to heart failure. These data suggest that a letrozole-induced cardiac metabolic remodeling, i.e., a shift from ß-oxidation to glycolysis, may induce cardiac structural remodeling.

Dietary Intake of 17α-Ethinylestradiol Promotes HCC Progression in Humanized Male Mice Expressing Sex Hormone-Binding Globulin.

Hepatocellular carcinoma (HCC) is a male-oriented malignancy; its progression is affected by sex hormones. 17α-ethinylestradiol (EE2) is a synthetic estrogen widely used as an oral contraceptive; however, it is unknown whether EE2 regulates sex hormone action in HCC. We investigated whether EE2 influences HCC risk in male androgenic environments, using mice expressing human sex hormone-binding globulin (SHBG). Two-week-old male mice were injected with diethyl-nitrosamine (DEN, 25 mg/kg) and fed an EE2 diet for 10 weeks from 30 weeks of age. Development and characteristics of liver cancer were evaluated in 40-week-old mice via molecular and histological analyses. Although EE2 did not increase HCC progression in wild-type mice, SHBG mice exhibited remarkably higher HCC risk when fed EE2. The livers of EE2-treated SHBG mice exhibited substantially increased pro-inflammatory necrosis with high plasma levels of ALT and HMGB1, and intrahepatic injury and fibers. Additionally, increased androgen response and androgen-mediated proliferation in the livers of EE2-treated SHBG mice and EE2-exposed hepatocytes under SHBG conditions were observed. As a competitor of SHBG-androgen binding, EE2 could bind with SHBG and increase the bioavailability of androgen. Our results revealed that EE2 is a novel risk factor in androgen-dominant men, predisposing them to HCC risk.

Hepatic LKB1 Reduces the Progression of Non-Alcoholic Fatty Liver Disease via Genomic Androgen Receptor Signaling.

The incidence of non-alcoholic fatty liver disease (NAFLD) increases in males aged >45 years, which indicates that androgens are associated with the development and/or progression of NAFLD, although excess dietary intake is the primary causative factor. However, it is uncertain how androgens are involved in the metabolic process of NAFLD, which is associated with the state of steatosis in hepatocytes. To investigate whether androgen receptor (AR) signaling influences NAFLD development, the state of steatosis was monitored in mouse livers and hepatocytes with or without androgens. As a result, hepatic lipid droplets, expression of AR, and phosphorylation of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) increased in the presence of testosterone. Concurrently, the expression of LKB1, an upstream regulator of AMPK, was increased by testosterone treatment. We observed that the fluctuation of AMPK-ACC signaling, which plays an important role in lipogenesis, depends on the presence of testosterone and AR. Additionally, we demonstrated that testosterone bound AR was recruited to the promoter of the LKB1 gene and induced LKB1 expression. Our study highlights a novel mechanism by which testosterone modulates NAFLD development by inducing the mRNA expression of LKB1.

Loss of PGRMC1 Delays the Progression of Hepatocellular Carcinoma via Suppression of Pro-Inflammatory Immune Responses.

Pgrmc1 is a non-canonical progesterone receptor related to the lethality of various types of cancer. PGRMC1 has been reported to exist in co-precipitated protein complexes with epidermal growth factor receptor (EGFR), which is considered a useful therapeutic target in hepatocellular carcinoma (HCC). Here, we investigated whether Pgrmc1 is involved in HCC progression. In clinical datasets, PGRMC1 transcription level was positively correlated with EGFR levels; importantly, PGRMC1 level was inversely correlated with the survival duration of HCC patients. In a diethylnitrosamine (DEN)-induced murine model of HCC, the global ablation of Pgrmc1 suppressed the development of HCC and prolonged the survival of HCC-bearing mice. We further found that increases in hepatocyte death and suppression of compensatory proliferation in the livers of DEN-injured Pgrmc1-null mice were concomitant with decreases in nuclear factor κB (NF-κB)-dependent production of interleukin-6 (IL-6). Indeed, silencing of Pgrmc1 in murine macrophages led to reductions in NF-κB activity and IL-6 production. We found that the anti-proinflammatory effect of Pgrmc1 loss was mediated by reductions in EGFR level and its effect was not observed after exposure of the EGFR inhibitor erlotinib. This study reveals a novel cooperative role of Pgrmc1 in supporting the EGFR-mediated development of hepatocellular carcinoma, implying that pharmacological suppression of Pgrmc1 may be a useful strategy in HCC treatment.

Progesterone receptor membrane component 1 reduces cardiac steatosis and lipotoxicity via activation of fatty acid oxidation and mitochondrial respiration.

Obesity is implicated in cardiovascular disease and heart failure. When fatty acids are transported to and not adequately oxidized in cardiac cells, they accumulate, causing lipotoxicity in the heart. Since hepatic progesterone receptor membrane component 1 (Pgrmc1) suppressed de novo lipogenesis in a previous study, it was questioned whether cardiac Pgrmc1 protects against lipotoxicity. Hence, we focused on the role of cardiac Pgrmc1 in basal (Resting), glucose-dominant (Refed) and lipid-dominant high-fat diet (HFD) conditions. Pgrmc1 KO mice showed high FFA levels and low glucose levels compared to wild-type (WT) mice. Pgrmc1 KO mice presented low number of mitochondrial DNA copies in heart, and it was concomitantly observed with low expression of TCA cycle genes and oxidative phosphorylation genes. Pgrmc1 absence in heart presented low fatty acid oxidation activity in all conditions, but the production of acetyl-CoA and ATP was in pronounced suppression only in HFD condition. Furthermore, HFD Pgrmc1 KO mice resulted in high cardiac fatty acyl-CoA levels and TG level. Accordingly, HFD Pgrmc1 KO mice were prone to cardiac lipotoxicity, featuring high levels in markers of inflammation, endoplasmic reticulum stress, oxidative stress, fibrosis, and heart failure. In vitro study, it was also confirmed that Pgrmc1 enhances rates of mitochondrial respiration and fatty acid oxidation. This study is clinically important because mitochondrial defects in Pgrmc1 KO mice hearts represent the late phase of cardiac failure.

Absence of progesterone receptor membrane component 1 reduces migration and metastasis of breast cancer.

BACKGROUND: Progesterone receptor membrane component 1 (Pgrmc1) is a non-classical progesterone receptor associated with the development of the mammary gland and xenograft-induced breast cancer. Importantly, Pgrmc1 is associated with the expression of estrogen receptor alpha and can be used for predicting the prognosis of breast cancer. Whether the genetic deletion of Pgrmc1 affects the progression of breast cancer is still unclear. METHODS: We used MMTV-PyMT transgenic mice that spontaneously develop breast tumors. In backcrossed FVB Pgrmc1 knockout (KO) mice, we monitored the development of the primary tumor and lung metastasis. In MCF-7 and MDA-MB-231 tumor cell lines, the migratory activity was evaluated after Pgrmc1 knockdown. RESULTS: There was no significant difference in the development of breast cancer in terms of tumor size at 13 weeks of age between WT and Pgrmc1 KO mice. However, Pgrmc1 KO mice had a significantly longer survival duration compared with WT mice. Furthermore, Pgrmc1 KO mice exhibited a significantly lower degree of lung metastasis. Compared with those of WT mice, the tumors of Pgrmc1 KO mice had a low expression of focal adhesion kinase and epithelial-mesenchymal transition markers. PGRMC1 knockdown resulted in a significantly reduced migration rate in breast cancer cell lines. CONCLUSIONS: Pgrmc1 KO mice with breast cancer had a prolonged survival, which was accompanied by a low degree of lung metastasis. PGRMC1 showed a significant role in the migration of breast cancer cells, and may serve as a potential therapeutic target in breast cancer. Video Abstract.

Progesterone increases blood glucose via hepatic progesterone receptor membrane component 1 under limited or impaired action of insulin.

Hepatic gluconeogenesis is the main pathway for blood glucose maintenance activated during fasting. Retardation of insulin action, such as in diabetes mellitus, activates gluconeogenesis during the fed state. While the role of progesterone (P4) in diabetes is controversial, the P4 receptor, progesterone receptor membrane component 1 (PGRMC1), is known to stimulate pancreatic insulin secretion. We investigated the role of P4, via hepatic PGRMC1, during gluconeogenesis. The PGRMC1 binding chemical, AG-205, induced PGRMC1 monomer (25 kDa) abundance, and increased PEPCK expression and glucose production in parallel with cyclic AMP (cAMP) induction in Hep3B cells. PGRMC1-mediated cyclic AMP was inhibited by an adenylate cyclase inhibitor (MDL-12,330A). PEPCK suppression in Pgrmc1 KO hepatocyte was not observed after treatment of MDL-12,330A. PGRMC1 knockdown or overexpression systems in Hep3B cells confirmed that PGRMC1 mediates PEPCK expression via phosphorylation of cAMP-response element binding protein (CREB). CREB phosphorylation and PEPCK expression in primary hepatocytes were greater than that in PGRMC1 knock-out hepatocytes. Progesterone increased PGRMC1 expression, which induced cAMP and PEPCK induction and glucose production. In vivo, P4 suppressed gluconeogenesis following plasma insulin induction under normal conditions in a mouse model. However, P4 increased blood glucose via gluconeogenesis in parallel with increases in PGRMC1 and PEPCK expression in mice in both insulin-deficient and insulin-resistant conditions. We conclude that P4 increases hepatic glucose production via PGRMC1, which may exacerbate hyperglycaemia in diabetes where insulin action is limited.

On-Line Ion Exchange Liquid Chromatography as a Process Analytical Technology for Monoclonal Antibody Characterization in Continuous Bioprocessing.

Combining process analytical technology (PAT) with continuous production provides a powerful tool to observe and control monoclonal antibody (mAb) fermentation and purification processes. This work demonstrates on-line liquid chromatography (on-line LC) as a PAT tool for monitoring a continuous biologics process and forced degradation studies. Specifically, this work focused on ion exchange chromatography (IEX), which is a critical separation technique to detect charge variants. Product-related impurities, including charge variants, that impact function are classified as critical quality attributes (CQAs). First, we confirmed no significant differences were observed in the charge heterogeneity profile of a mAb through both at-line and on-line sampling and that the on-line method has the ability to rapidly detect changes in protein quality over time. The robustness and versatility of the PAT methods were tested by sampling from two purification locations in a continuous mAb process. The PAT IEX methods used with on-line LC were a weak cation exchange (WCX) separation and a newly developed shorter strong cation exchange (SCX) assay. Both methods provided similar results with the distribution of percent acidic, main, and basic species remaining unchanged over a 2 week period. Second, a forced degradation study showed an increase in acidic species and a decrease in basic species when sampled on-line over 7 days. These applications further strengthen the use of on-line LC to monitor CQAs of a mAb continuously with various PAT IEX analytical methods. Implementation of on-line IEX will enable faster decision making during process development and could potentially be applied to control in biomanufacturing.

Development of a liver-targeted siRNA delivery platform with a broad therapeutic window utilizing biodegradable polypeptide-based polymer conjugates.

The greatest challenge standing in the way of effective in vivo siRNA delivery is creating a delivery vehicle that mediates a high degree of efficacy with a broad therapeutic window. Key structure-activity relationships of a poly(amide) polymer conjugate siRNA delivery platform were explored to discover the optimized polymer parameters that yield the highest activity of mRNA knockdown in the liver. At the same time, the poly(amide) backbone of the polymers allowed for the metabolism and clearance of the polymer from the body very quickly, which was established using radiolabeled polymers to demonstrate the time course of biodistribution and excretion from the body. The fast degradation and clearance of the polymers provided for very low toxicity at efficacious doses, and the therapeutic window of this poly(amide)-based siRNA delivery platform was shown to be much broader than a comparable polymer platform. The results of this work illustrate that the poly(amide) platform has a promising future in the development of a siRNA-based drug approved for human use.