Increased AMPK Phosphorylation Research Articles

Mechanistic studies on the alleviation of lipid accumulation and oxidative stress by Majia pomelo seed oil

In this study, hot-pressed Majia pomelo seed oil (MPSO), which has high oxidative stability and antioxidant capacity, was used to study the effects and potential mechanisms of lipid accumulation and oxidative stress. The results indicated that the low-density lipoprotein cholesterol (LDL-C), total cholesterol (TC), and triglycerides (TG) levels in HepG2 cells were lowest when the MPSO concentration was 0.5 mg/mL. At this concentration, MPSO could alleviate oleic acid (OA)-induced lipid accumulation, reduce intracellular lipid level, and decrease lipid and cholesterol synthesis by increasing AMPK phosphorylation and down-regulating the expression levels of genes and proteins of ACC, SREBP-1c, PPAR-γ, and FAS. Meanwhile, MPSO can reduce reactive oxygen species (ROS) and malondialdehyde (MDA) content and enhance superoxide dismutase (SOD) content in HepG2 cells by down-regulating the expression levels of Nrf2, γ-GCS, and HO-1 genes and proteins, thus acting as an anti-oxidative stress. In addition, molecular docking results indicated that the active ingredients of MPSO could effectively bind AMPK protein and Keap1 protein, which further demonstrated that MPSO could alleviate lipid accumulation and oxidative stress.

Mangiferin prevents glucolipotoxicity-induced pancreatic beta-cell injury through modulation of autophagy via AMPK-mTOR signaling pathway

The aim of this study was to investigate the protective effects of Mangiferin (MG) on glucolipotoxicity-induced pancreatic beta-cell injury. In vivo administration of MG significantly reduced the level of blood glucose in high-fat diet (HFD)-fed mice. MG treatment inhibited beta-cell apoptosis in HFD-treated mice. In vitro, MG protected INS-1 cells against apoptosis and impairment of insulin secretion following High glucose/Palmitic acid (HG/PA) treatment. MG treatment enhanced autophagy flux which was blocked by HG/PA treatment. Inhibition of autophagosome formation by 3-Methyladenine or blockade of autolysosome by Chloroquine reversed the protective effects of MG on INS-1 cells. MG treatment increased AMPK phosphorylation and reduced mTOR activation in INS-1 cells. Administration of the AMPK blocker abrogated MG-induced autophagy, and similar results were observed in INS-1 cells after cotreatment with MG and mTOR activator. In conclusion, MG ameliorated pancreatic beta-cell injury induced by glucolipotoxicity through modulation of autophagy via the AMPK-mTOR pathway.

Strength Training Protects High-Fat-Fed Ovariectomized Mice against Insulin Resistance and Hepatic Steatosis.

Menopause is characterized by a reduction in sex hormones in women and is associated with metabolic changes, including fatty liver and insulin resistance. Lifestyle changes, including a balanced diet and physical exercise, are necessary to prevent these undesirable changes. Strength training (ST) has been widely used because of the muscle and metabolic benefits it provides. Our study aims to evaluate the effects of ST on hepatic steatosis and insulin resistance in ovariectomized mice fed a high-fat diet (HFD) divided into four groups as follows: simulated sedentary surgery (SHAM-SED), trained simulated surgery (SHAM-EXE), sedentary ovariectomy (OVX-SED), and trained ovariectomy (OVX-EXE). They were fed an HFD for 9 weeks. ST was performed thrice a week. ST efficiently reduced body weight and fat percentage and increased lean mass in OVX mice. Furthermore, ST reduced the accumulation of ectopic hepatic lipids, increased AMPK phosphorylation, and inhibited the de novo lipogenesis pathway. OVX-EXE mice also showed a better glycemic profile, associated with greater insulin sensitivity identified by the euglycemic-hyperinsulinemic clamp, and reduced markers of hepatic oxidative stress compared with sedentary animals. Our data support the idea that ST can be indicated as a non-pharmacological treatment approach to mitigate metabolic changes resulting from menopause.

USP10 alleviates Nε-carboxymethyl-lysine-induced vascular calcification and atherogenesis in diabetes mellitus by promoting AMPK activation

Vascular calcification (VC) is a characteristic feature in patients with diabetes mellitus (DM) and is closely associated with the osteogenic differentiation of vascular smooth muscle cells (VSMCs). Ubiquitin-Specific Protease 10 (USP10) has been shown to regulate multiple cellular processes; however, its relationship with diabetic VC remains unclear. This study aims to elucidate the role of USP10 in VC development and the underlying regulatory mechanisms. Nε-carboxymethyl lysine (CML) was significantly increased in calcified ateries from diabetic atherosclerosis ApoE−/− mice fed with high-fat diets. CML downregulated USP10 expression in VSMCs and calcified mice coronary arteries, as assessd by Western blotting, RT-qPCR,immunofluorescence and immunohistochemistry. Loss-and gain-of-function experiments were conducted both in vitro and in vivo to verify the biological functions of USP10. Ectopic expression of USP10 mitigated the severity of VC. With regard to the mechanism, the interaction between USP10 and AMPKα was investigated through double-label immunofluorescence and Co-immunoprecipitation. In vitro ubiquitination assay revealed that USP10 was capable of mediating AMPKα ubiquitination and caused increased AMPKα phosphorylation level at Thr172. Moreover, the anticalcification effect of USP10 was reversed by pharmacological inhibition of AMPK signaling pathway. The current fundings suggest an important role of USP10 in diabetic VC progression, at least in part, via mediating the ubiquitination and activation of AMPKα. USP10 may serve as a novel therapeutic target for the treatment of diabetic VC.

Abstract 2049: Regulation Of MTP-ATGL Interactions In Adipocytes And Their Impact On Hepatic Lipid Metabolism

Introduction: We have shown that microsomal triglyceride transfer protein (MTP) in adipocytes regulates lipolysis by inhibiting adipose triglyceride lipase (ATGL). Adipose MTP knockout mice (A- Mttp -/- ) exhibit smaller adipocytes, more thermogenesis, and less weight gain on an obesogenic diet. Aim: To investigate the MTP-ATGL interaction in adipocytes and its impact on hepatic lipid metabolism. Methods: We studied MTP-ATGL interactions using in silico modeling, ELISA, and co-IP. Lipoprotein profiles were analyzed using FPLC. Adipose-liver cross-talk was assessed through co-culture experiments, adipokine profiling and lipidomics. Hepatic triglyceride (TG) production and fatty acid (FA) oxidation were assessed using poloxamer-407 and 14 C-palmitate. Statistical analyses were done using GraphPad Prism (P <.05 significant). Results: Our study suggests MTP-ATGL interactions are hydrophobic involving C-terminal of ATGL and N-terminal β-barrel of MTP. These interactions vary in different metabolic states like fasting and adipocyte differentiation. A- Mttp -/- mice had higher plasma TG content in VLDL and LDL fractions. Despite increased movement of FAs from adipose to the liver as indicated by lipidomics of eWAT, plasma and liver, A- Mttp -/- mice liver showed less steatosis. Adipokine profiling showed significantly higher amounts of adiponectin in the plasma of A- Mttp -/- mice. Reduced ceramide levels and significantly increased AMPK phosphorylation suggest higher adiponectin activity in A- Mttp -/- livers. A- Mttp -/- livers showed higher expression of FA uptake and utilization related genes, but no change in lipid synthesis. Hepatocytes treated with Mttp -/- adipocytes conditioned media (CM) showed higher FA oxidation than those treated with Mttp f/f adipocytes CM. Mttp -/- adipocytes CM showed significantly higher PPARα reporter activity than Mttp f/f adipocytes CM. Conclusion: Our study reveals dynamic hydrophobic interactions between MTP and ATGL that vary with metabolic states. Enhanced movement of PPARα ligands (FAs) and adiponectin from A- Mttp -/- adipocytes boosts VLDL production and FA oxidation, to protect livers from hepatic steatosis. This highlights a novel role of adipose MTP in the regulation of hepatic lipid homeostasis.

Jiangqi Pingxiao formula regulates dendritic cell apoptosis in an autophagy-dependent manner through the AMPK/mTOR pathway in a murine model of OVA-induced asthma

Ethnopharmacological relevanceAllergic asthma is a recurring respiratory condition that typically manifests during childhood or adolescence. It is characterized by a dominant type II immune response triggered by the identification and capturing of inhaled allergens by dendritic cells (DCs). Jiangqi Pingxiao Formula (JQPXF), a prescription medicine used for the treatment of pediatric asthma, has been clinically proven to be both safe and effective. However, its mechanism of action in the treatment of asthma has not been fully been fully elucidated. Recent research suggests that several natural compounds have the potential to target dendritic cells (DCs) and alleviate ovalbumin (OVA)-induced asthma, which may also be found within JQPXF. Aim of the studyThis study aimed to elucidate the effect of JQPXF on OVA-induced asthma model and its molecular mechanism targeting DCs. Materials and methodsThe main constituents of JQPXF were analyzed by ultra performance liquid chromatography (UPLC). An asthma model was established by OVA. Hematoxylin-eosin staining and measurement of respiratory function was used to evaluate the treatment effect of JQPXF on asthmatic mice. Cytokine (IL-5, IL-13 and IgE) concentrations were determined by enzyme-linked immunosorbent assay (ELISA). Flow cytometry was employed to evaluate inflammatory cell infiltration (T helper 2 cells and DCs) in vivo and DC survival in vivo and vitro. Western blot and immunofluorescence were used to verify the molecular mechanisms. ResultsThe results suggest that JQPXF can ameliorate pathological conditions and improve lung function in asthmatic mice, as well as the Th2 cells. Treatment with JQPXF significantly reduced the number of DCs and increased the number of Propidium iodide+ (PI) DCs. Furthermore, JQPXF upregulated protein levels of the pro-apoptotic factors Cleaved-caspase-3 and Bax, while downregulating the anti-apoptotic factor Bcl-2. Simultaneously, JQPXF increased autophagy levels by facilitating p62 degradation and promoting translation from LC3B I to LC3B II of DCs in vitro, as well as reducing the integrated optical density (IOD) of p62 within the CD11c-positive area in the lung. 3-Methyladenine (3-MA) was used to block autophagic flux and the apoptotic effect of JQPXF on DCs was abolished in vitro, with the number of DCs decreased by JQPXF being reversed in vivo. We further investigated the upstream key regulator of autophagy, the AMPK/mTOR pathway, and found that JQPXF increased AMPK phosphorylation while decreasing mTOR phosphorylation levels. Additionally, we employed Compound C (CC) as an AMPK inhibitor to inhibit this signaling pathway, and our findings revealed that both autophagic flux and apoptotic levels in DCs were abolished in vitro. ConclusionsIn summary, we have demonstrated that JQPXF could alleviate type II inflammation in an asthmatic model by promoting the apoptosis of DCs through an autophagy-dependent mechanism, achieved by regulating the AMPK/mTOR signaling pathway.

Xanthohumol relieves arthritis pain in mice by suppressing mitochondrial-mediated inflammation.

Chronic pain is the most common symptom for people who suffer from rheumatoid arthritis and it affects approximately 1% of the global population. Neuroinflammation in the spinal cord induces chronic arthritis pain. In this study, a collagen-induced arthritis (CIA) mice model was established through intradermally injection of type II collagen in complete Freund's adjuvant solution. Following CIA inducement, the paws and ankles of mice were found to swell, mechanical pain and spontaneous pain were induced, and their motor coordination was impaired. The spinal inflammatory reaction was triggered, which presented as severe infiltration of inflammatory cells, and the expression levels of GFAP, IL-1β, NLRP3, and cleaved caspase-1 increased. Oxidative stress in the spinal cord of CIA mice was manifested as reduced Nrf2 and NDUFB11 expression and SOD activity, and increased levels of DHODH and Cyto-C. At the same time, spinal AMPK activity was decreased. In order to explore the potential therapeutic options for arthritic pain, Xanthohumol (Xn) was intraperitoneally injected into mice for three consecutive days. Xn treatment was found to reduce the number of spontaneous flinches, in addition to elevating mechanical pain thresholds and increasing latency time. At the same time, Xn treatment in the spinal cord reduced NLRP3 inflammasome-mediated inflammation, increased the Nrf2-mediated antioxidant response, and decreased mitochondrial ROS level. In addition, Xn was found to bind with AMPK via two electrovalent bonds and increased AMPK phosphorylation at Thr174. In summary, the findings indicate that Xn treatment activates AMPK, increases Nrf2-mediated antioxidant response, reduces Drp1-mediated mitochondrial dysfunction, suppresses neuroinflammation, and can serve to relieve arthritis pain.

Effect of electroacupuncture on colonic autophagy and AMPK/mTOR signaling pathway in rats with acute ulcerative colitis

To observe the effect of electroacupuncture （EA） at "Zhongwan" （CV12）, "Tianshu" （ST25） and "Shangjuxu" （ST37） （an acupoint prescription "Changbingfang" for treatment of intestinal disorders） on autophagy and expression of AMPK/mTOR signaling pathway in rats with ulcerative colitis （UC）, so as to explore its mechanism underlying improvement of UC. Thirty-two male SD rats were randomly divided into control, model, medication and EA groups, with 8 rats in each group. The UC model was established by free drinking of 5% dextran sulfate sodium salt solution for 7 days. EA stimulation （10 Hz/50 Hz） was delivered to CV12, ST25 and ST37 for 20 min, once a day for 3 consecutive days. Rats of the medication group received gavage of mesalazine suspension （200 mg/kg） once a day, 3 times in total. The rats' general conditions were recorded for calculating the disease activity index （DAI） score （0-4 points）. Histomorphological changes of colon were observed via HE staining. The levels of serum interleukin 6 （IL-6）, tumor necrosis factor-α （TNF-α） and IL-10 were measured by ELISA. The mRNA expressions of LC3B and p62 were tested by fluorescence quantitative PCR. Western blot was used to detect the expression levels of LC3B, p62 and AMPK/mTOR pathway related proteins in colon tissues. Compared with the control group, the DAI score, contents of serum IL-6 and TNF-α, the expression levels of p62 protein and mRNA, ratio of p-mTOR/mTOR were significantly increased （P<0.01）； while the content of serum IL-10, the expression levels of LC3B mRNA, ratio of LC3BⅡ/LC3BⅠ and p-AMPK/AMPK were decreased （P<0.01, P<0.05） in the model group. Relevant to the model group, modeling-induced increases of DAI score, serum IL-6, TNF-α and IL-10 contents, expressions of p62 protein and mRNA, LC3B mRNA, ratio of p-mTOR/mTOR, LC3BⅡ/LC3BⅠ and p-AMPK/AMPK were reversed in both medication and EA groups （P<0.01, P<0.05）. The effect of EA was apparently superior to that of mesalazine in up-regulating ratio of LC3BⅡ/LC3BⅠ and p-AMPK/AMPK, p62 mRNA expression （P<0.01, P<0.05）, and in down-regulating ratio of p-mTOR/mTOR （P<0.05）. H.E. staining showed severe damage of the colonic mucosal barrier with infiltration of a large number of inflammatory cells in the model group, which was milder in medication and EA groups. EA of acupoint recipe "Changbingfang" can improve the symptoms in UC rats, which may be related to its functions in promoting colonic autophagy, increasing AMPK phosphorylation level, and decreasing mTOR phosphorylation level.

Characterization and anti-aging effects of polysaccharide from Gomphus clavatus Gray

In this study, a highly branched polysaccharide (GPF, 112.0 kDa) was isolated and purified from Gomphus clavatus Gray fruiting bodies. GPF was primarily composed of mannose, galactose, arabinose, xylose, and glucose at a molar ratio of 3.2:1.9:1.6:1.2:1.0. GPF was a highly branched heteropolysaccharide composed of 13 glucosidic bonds, with a degree of branching (DB) of 48.85 %. GPF exhibited anti-aging activity in vivo, significantly increased antioxidant enzymes activities (SOD, CAT and GSH-Px), improved total antioxidant capability (T-AOC) and decreased MDA level in the serum and brain of d-Gal induced aging mice. Behavioral experiments showed that GPF effectively improved learning and memory deficits in d-Gal induced aging mice. Mechanistic studies indicated that GPF could activate AMPK by increasing AMPK phosphorylation and upregulating SIRT1 and PGC-1α expression. These findings suggest that GPF has significant potential as a natural candidate to slow down aging and prevent aging-related diseases.

Could Metformin and Resveratrol Support Glioblastoma Treatment? A Mechanistic View at the Cellular Level.

Glioblastoma, a malignant brain tumor, is a common primary brain tumor in adults, with diabetes mellitus being a crucial risk factor. This review examines how the antidiabetic drug metformin and dietary supplement resveratrol can benefit the treatment of glioblastoma. Metformin and resveratrol have demonstrated action against relevant pathways in cancer cells. Metformin and resveratrol inhibit cell proliferation by downregulating the PI3K/Akt pathway, activating mTOR, and increasing AMPK phosphorylation, resulting in lower proliferation and higher apoptosis levels. Metformin and resveratrol both upregulate and inhibit different cascades in the MAPK pathway. In vivo, the drugs reduced tumor growth and volume. These actions show how metformin and resveratrol can combat cancer with both glucose-dependent and glucose-independent effects. The pre-clinical results, alongside the lack of clinical studies and the rise in novel delivery mechanisms, warrant further clinical investigations into the applications of metformin and resveratrol as both separate and as a combination complement to current glioblastoma therapies.

Tanshinone IIA inhibited intermittent hypoxia induced neuronal injury through promoting autophagy via AMPK-mTOR signaling pathway

Ethnopharmacological relevanceChronic intermittent hypoxia (CIH) is the primary pathophysiological process of obstructive sleep apnea (OSA) and is closely linked to neurocognitive dysfunction. Tanshinone IIA (Tan IIA) is extracted from Salvia miltiorrhiza Bunge and used in Traditional Chinese Medicine (TCM) to improve cognitive impairment. Studies have shown that Tan IIA has anti-inflammatory, anti-oxidant, and anti-apoptotic properties and provides protection in intermittent hypoxia (IH) conditions. However, the specific mechanism is still unclear. Aim of the studyTo assess the protective effect and mechanism of Tan IIA treatment on neuronal injury in HT22 cells exposed to IH. Materials and methodsThe study established an HT22 cell model exposed to IH (0.1% O2 3 min/21% O2 7 min for six cycles/h). Cell viability was determined using the Cell Counting Kit-8, and cell injury was determined using the LDH release assay. Mitochondrial damage and cell apoptosis were observed using the Mitochondrial Membrane Potential and Apoptosis Detection Kit. Oxidative stress was assessed using DCFH-DA staining and flow cytometry. The level of autophagy was assessed using the Cell Autophagy Staining Test Kit and transmission electron microscopy (TEM). Western blot was used to detect the expressions of the AMPK-mTOR pathway, LC3, P62, Beclin-1, Nrf2, HO-1, SOD2, NOX2, Bcl-2/Bax, and caspase-3. ResultsThe study showed that Tan IIA significantly improved HT22 cell viability under IH conditions. Tan IIA treatment improved mitochondrial membrane potential, decreased cell apoptosis, inhibited oxidative stress, and increased autophagy levels in HT22 cells under IH conditions. Furthermore, Tan IIA increased AMPK phosphorylation and LC3II/I, Beclin-1, Nrf2, HO-1, SOD2, and Bcl-2/Bax expressions, while decreasing mTOR phosphorylation and NOX2 and cleaved caspase-3/caspase-3 expressions. ConclusionThe study suggested that Tan IIA significantly ameliorated neuronal injury in HT22 cells exposed to IH. The neuroprotective mechanism of Tan IIA may mainly be related to inhibiting oxidative stress and neuronal apoptosis by activating the AMPK/mTOR autophagy pathway under IH conditions.

Chaya (Cnidoscolus aconitifolius (Mill.) I.M. Johnst) leaf extracts regulate mitochondrial bioenergetics and fatty acid oxidation in C2C12 myotubes and primary hepatocytes

Ethnopharmacological relevanceChaya (Cnidoscolus aconitifolius (Mill.) I.M. Johnst) is an important component of the regular diet and traditional medicine of indigenous communities in Mexico. Customarily, Chaya is consumed as a beverage made of macerated leaf, cooked, or prepared in teas or infusions to empirically treat obesity, diabetes, gastrointestinal disorders, and kidney stones. The beneficial effects of Chaya can be attributed to the presence of protein, dietary fiber, vitamins, and especially polyphenols, which regulate mitochondrial function. Therefore, polyphenols present in Chaya extracts could be used to develop novel strategies to prevent and treat metabolic alterations related to mitochondrial dysfunction in the muscle and liver of subjects with obesity, type 2 diabetes, and cardiovascular diseases. However, limited information is available concerning the effect of Chaya extracts on mitochondrial activity in those tissues. Aim of the studyThe aim of this study was to evaluate the antioxidant capacity of an aqueous extract (AE) or mixed (methanol/acetone/water) extract (ME) of Chaya leaf and their effect on C2C12 myotubes and primary hepatocyte mitochondrial bioenergetics and fatty acid oxidation (FAO). Materials and methodsTotal polyphenol content and antioxidant activity were determined using the Folin–Ciocalteu method and the oxygen radical absorbance capacity assay, respectively. The effect of AE and ME from Chaya leaf on mitochondrial activity and FAO of C2C12 myotubes and primary hepatocytes was evaluated using an extracellular flux analyzer. ResultsThe AE and ME from Chaya leaf exhibited antioxidant activity and a polyphenol content similar to nopal, another plant used in Mexican traditional medicine. AE significantly (p < 0.05) decreased the maximal respiration and spare respiratory capacity (SRC) of C2C12 cells, whereas ME had little effect on C2C12 mitochondrial function. Conversely, ME significantly (p < 0.05) decreased SRC in primary hepatocytes, whereas AE increased maximal respiration and SRC at low doses (5 and 10 μM). Moreover, low doses of Chaya AE significantly (p < 0.05) increased AMPK phosphorylation, acyl-coenzyme A oxidase protein abundance, and palmitate oxidation in primary hepatocytes. ConclusionThe AE of Chaya leaf increases mitochondrial function and FAO of primary hepatocytes, indicating its potential to treat hepatic mitochondrial dysfunction underlying metabolic diseases.

Antimicrobial protein REG3A regulates glucose homeostasis and insulin resistance in obese diabetic mice

Innate immune mediators of pathogen clearance, including the secreted C-type lectins REG3 of the antimicrobial peptide (AMP) family, are known to be involved in the regulation of tissue repair and homeostasis. Their role in metabolic homeostasis remains unknown. Here we show that an increase in human REG3A improves glucose and lipid homeostasis in nutritional and genetic mouse models of obesity and type 2 diabetes. Mice overexpressing REG3A in the liver show improved glucose homeostasis, which is reflected in better insulin sensitivity in normal weight and obese states. Delivery of recombinant REG3A protein to leptin-deficient ob/ob mice or wild-type mice on a high-fat diet also improves glucose homeostasis. This is accompanied by reduced oxidative protein damage, increased AMPK phosphorylation and insulin-stimulated glucose uptake in skeletal muscle tissue. Oxidative damage in differentiated C2C12 myotubes is greatly attenuated by REG3A, as is the increase in gp130-mediated AMPK activation. In contrast, Akt-mediated insulin action, which is impaired by oxidative stress, is not restored by REG3A. These data highlight the importance of REG3A in controlling oxidative protein damage involved in energy and metabolic pathways during obesity and diabetes, and provide additional insight into the dual function of host-immune defense and metabolic regulation for AMP.

Exposure to dipentyl phthalate in utero disrupts the adrenal cortex function of adult male rats by inhibiting SIRT1/PGC-1α and inducing AMPK phosphorylation.

Di-n-pentyl phthalate (DPeP) is an endocrine-disrupting phthalate plasticizer. The objective of this study was to investigate the effect of DPeP on adrenocortical function in adult male rats following in utero exposure. DPeP (0, 10, 50, 100, and 500 mg/kg/day) was administered by gavage to pregnant Sprague-Dawley rats from gestational day 14 to 21. The morphology and function of the adrenal cortex in 56-day-old male offspring were studied. DPeP at 100 and 500 mg/kg/day significantly reduced serum aldosterone levels and at 500 mg/kg/day markedly reduced corticosterone and adrenocorticotropic hormone levels. DPeP at 10-500 mg/kg markedly reduced the thickness of zona glomerulosa without affecting the thickness of zona fasciculata. DPeP significantly downregulated the expression of Agtr1a, Mc2r, Scarb1, Cyp11a1, Hsd3b1, Cyp21, Cyp11b1, Cyp11b2, Nr5a1, Nr4a2, and Bcl2 genes as well as their proteins. DPeP at 500 mg/kg/day significantly increased phosphorylated AMPK, while DPeP at 100 mg/kg/day and higher doses reduced phosphorylated AKT1 and total SIRT1 level. DPeP at 100 and 500 μM markedly induced reactive oxygen species and apoptosis in H295R cells after 24 h of culture. In conclusion, in utero exposure to DPeP disrupts adrenocortical function of the adult male offspring by (1) increasing AMPK phosphorylation and decreasing AKT1 phosphorylation and SIRT1 levels, (2) reducing adrenocorticotropic hormone levels, and (3) possibly inducing oxidative stress and apoptosis.

Inhibition of USP1 activates ER stress through Ubi-protein aggregation to induce autophagy and apoptosis in HCC

The deubiquitinating enzyme USP1 (ubiquitin-specific protease 1) plays a role in the progression of various tumors, emerging as a potential therapeutic target. This study aimed to determine the role of USP1 as a therapeutic target in hepatocellular carcinoma (HCC). We detected USP1 expression in the tumor and adjacent tissues of patients with HCC using immunohistochemical staining. We evaluated the effect of the USP1 inhibitor ML-323 on HCC cell proliferation and cell cycle using a CCK-8 cell-counting kit and plate cloning assays, and propidium iodide, respectively. Apoptosis was detected by annexin V-FITC/Propidium Iodide (PI) staining and caspase 3 (casp3) activity. Transmission electron microscopy and LC3B immunofluorescence were used to detect autophagy. Western blotting was used to detect the accumulation of ubiquitinated proteins, the expression of endoplasmic reticulum (ER) stress-related proteins, and the AMPK-ULK1/ATG13 signaling pathway. We demonstrated that ML-323 inhibits the growth of HCC cells and induces G1 phase cell cycle arrest by regulating cyclin expression. ML-323 treatment resulted in the accumulation of ubiquitinated proteins, induced ER stress, and triggered Noxa-dependent apoptosis, which was regulated by the Activating Transcription Factor 4(ATF4). Moreover, active ER stress induces protective autophagy by increasing AMPK phosphorylation; therefore, we inhibited ER stress using 4-Phenylbutyric acid (4-PBA), which resulted in ER stress reduction, apoptosis, and autophagy in ML-323-treated HCC cells. In addition, blocking autophagy using the AMPK inhibitor compound C (CC), chloroquine (CQ), or bafilomycin A1 (BafA1) enhanced the cytotoxic effect of ML-323. Our findings revealed that targeting USP1 may be a potential strategy for the treatment of HCC.

Larotrectinib induces autophagic cell death through AMPK/mTOR signalling in colon cancer.

Larotrectinib (Lar) is a highly selective and potent small-molecule inhibitor used in patients with tropomyosin receptor kinase (TRK) fusion-positive cancers, including colon cancer. However, the underlying molecular mechanisms specifically in patients with colon cancer have not yet been explored. Our data showed that Lar significantly suppressed proliferation and migration of colon cancer cells. In addition, Lar suppressed the epithelial-mesenchymal transition (EMT) process, as evidenced by elevation in E-cadherin (E-cad), and downregulation of vimentin and matrix metalloproteinase (MMP) 2/9 expression. Furthermore, Lar was found to activate autophagic flux, in which Lar increased the ratio between LC3II/LC3I and decreased the expression of p62 in colon cancer cells. More importantly, Lar also increased AMPK phosphorylation and suppressed mTOR phosphorylation in colon cancer cells. However, when we silenced AMPK in colon cancer cells, Lar-induced accumulation of autolysomes as well as Lar-induced suppression of the EMT process were significantly diminished. An in vivo assay also confirmed that tumour volume and weight decreased in Lar-treated mice than in control mice. Taken together, this study suggests that Lar significantly suppresses colon cancer proliferation and migration by activating AMPK/mTOR-mediated autophagic cell death.

Schisandrin C isolated from Schisandra chinensis fruits inhibits lipid accumulation by regulating adipogenesis and lipolysis through AMPK signaling in 3T3-L1 adipocytes.

In this study, lignans of Schisandra chinensis fruits (SCF) were profiled using HPLC-MS/MS, and the inhibitory effects of nine of these lignans were evaluated on triglyceride (TG) accumulation. We then examined the effects and molecular mechanisms on adipogenesis and lipolysis of schisandrin C (SC), which most inhibited TG levels during adipogenesis of 3T3-L1 cells. Treatment of 3T3-L1 cells with SC markedly decreased adipocyte differentiation but did not influence cell proliferation. During adipogenesis, SC significantly reduced total lipid and TG contents and down-regulated the mRNA expressions of C/EBPα, PPARγ, SREBP1c, aP2, and FAS. In addition, SC significantly increased p-AMPK, and this activation regulated the protein levels of major adipogenic transcription factors (PPARγ and C/EBPα). Furthermore, SC lowered the mRNA expressions of HSL and perilipin and inhibited pancreatic lipase levels, which are both related to lipolysis. PRACTICAL APPLICATIONS: Our results indicate that SC regulates lipogenesis and lipolysis by increasing AMPK phosphorylation and suggest that it may be beneficial for preventing obesity and related metabolic diseases. Thus, this study proposes a mechanical basis for developing SC-containing foods as a beneficial dietary strategy.

DUSP1 inhibition in the treatment of high grade serous ovarian carcinoma (141.5)

<b>Objectives:</b> Dual Specificity Phosphatase (DUSP) proteins regulate signaling cascades involved in tumor progression, but there are limited studies of DUSPs in epithelial ovarian carcinoma, especially in high-grade serous ovarian carcinoma (HGSOC). The study objective was to elucidate <i>DUSP1</i>'s contribution to HGSOC progression and examine a <i>DUSP1/6</i> inhibitor (DUSPi) in therapy-resistant HGSOC. <b>Methods:</b> Bioinformatic analyses were conducted using The Cancer Genome Atlas (TCGA) Ovarian Serous Cystadenocarcinoma Firehose Database and PanCancer Atlas. qPCR was completed to determine the mRNA expression of <i>DUSP1</i>. Fifty percent inhibitory concentration of DUSPi was determined using colony formation assays. Reverse-phase protein array (RPPA) examined the effect of DUSPi on cellular signaling. The antitumoral effect of DUSPi was examined using a patient-derived xenograft (PDX) model. Quantitative data were expressed as mean +/- standard error of the mean. RPPA relative protein levels were determined for each and designated as log2 intensities and subsequently median-centered. Analysis of variance with Tukey correction was used for multiple comparisons. The level of significance was set at 0.05. <b>Results:</b> Of 24 DUSP proteins examined in HGSOC, <i>DUSP1</i> mRNA was significantly upregulated (p<0.001, one-way ANOVA). Progression-free survival (PFS) and overall survival (OS) were significantly higher in patients with low <i>DUSP1</i> expression (PFS: 15.41 vs 22.22 months, Log-rank test, p=0.0010, HR: 1.65, 95% CI: 1.213-2.259; OS: 41.52 vs 52.40 months, Log-rank test, p=0.0183, HR: 1.462, 95% CI: 1.064-2.00). Elevated <i>DUSP1</i> protein expression was noted in our tissue microarray of 137 serous ovarian carcinoma primary and recurrent tumors. Olaparib-resistant HGSOC cells had significantly increased <i>DUSP1</i> expression compared to olaparib-sensitive HGSOC cells (unpaired t-test, p<0.0001). The RPPA analysis revealed 34 proteins with significantly (p<0.05) altered expression after treatment with DUSPi. DUSPi led to the significant downregulation of <i>WEE1</i> and <i>ATR</i>. Phosphor-AMPK (AMPK-a2_pS345), part of the mTORC pathway that can serve both tumor-suppressive and oncogenic roles, was elevated and dual phosphorylated ERK (MAPK_pT202_Y204). RPPA findings were confirmed in TCGA data and by immunoblotting. A therapy-resistant PDX model was used to examine DUSPi <i>in vivo</i> activity. DUSPi led to a decrease in total flux (photons/sec), indicating tumor regression, in the mice treated with DUSPi compared to control. At the end of the study, there was no evidence of ascites or tumor burden in mice treated with DUSPi. <b>Conclusions:</b> <i>DUSP1</i> expression is associated with poor progression-free and overall survival. After treatment with a novel <i>DUSP1/6</i> inhibitor (DUSPi), cell viability was significantly reduced. We defined the differential activation of signaling following DUSPi via RPPA. DUSP inhibition significantly resulted in altering the expression of multiple pathway proteins, including increasing AMPK phosphorylation. Finally, we identified a robust <i>in vivo</i> antitumor response with DUSPi in PDX models.

Abstract 1410: Metabolic modulation of anaplastic thyroid cancer by Berberine: A targeted intervention

Abstract Anaplastic thyroid cancer (ATC) is an undifferentiated thyroid cancer. A hallmark of ATC is increased inflammatory cytokine expression and inflammatory cell infiltration in the tumor microenvironment that is responsible for its aggressive nature. In contrast, papillary thyroid cancer (PTC) is well-differentiated and subsequently significantly less aggressive and more responsive to available therapies. The metabolic differences between ATC and PTC that correlate with differentiation status, and hence metastatic propensity, are poorly understood. Owing to its lack of differentiation, ATC is radioiodine therapy resistant, whereas differentiated thyroid cancers are susceptible. AMPK in the thyroid gland is responsible for glucose and iodine uptake. The AMPK pathway is a potential target for modulation due to the higher glucose uptake and metabolism associated with treatment resistance seen in ATC. Berberine, a naturally occurring plant alkaloid, regulates glycometabolism and lipid metabolism, improves energy expenditure, and reduces body weight. Exploration of a possible connection between the metabolic regulation displayed by BBR and AMPKα expression was warranted. Increased AMPKα signaling, observed through increased AMPKα phosphorylation, has an anti-tumor effect. When comparing PTC (TPC1) and ATC (T238) cell lines there was a 4.75- and 2.34-fold change, respectively, in phosphorylation of AMPKα upon BBR treatment. This demonstrates that BBR increases metabolic regulation in thyroid cancer cell lines, importantly, even when poorly differentiated. Further analysis of the correlation between differentiation status and metabolic characteristics between ATC and PTC was investigated using RNASeq. Differential gene expression was conducted to compare ATC and PTC using human thyroid cancer cell lines T238 and K1, respectively. These thyroid cancer cell lines were treated with 100 μM BBR or the same concentration of DMSO as a vehicle control for 24 hours. RNA was isolated from T238 and K1 cells and RNASeq was conducted by GeneWiz. This differential gene expression will help identify specific genes that are regulating metabolic patterns and is currently ongoing. Berberine (BBR) is a natural compound used widely in Traditional Chinese Medicine for its many pharmacological properties including anti-inflammatory, metabolic, antioxidant, and anti-cancer. BBR may be able to mitigate the metabolic pattern of ATC to potentially affect differentiation status as an intervening product. Intervention of the differentiation status of ATC will impact its progression, specifically metastasis. Therefore, identifying gene patterns that correlate with differentiation status will identify novel intervention targets for BBR. Citation Format: Kaci Kopec, Tara Jarboe, Mordechai Sternman, Nicole R. DeSouza, Sarnath Singh, Augustine Moscatello, Jan Geliebter, Raj K. Tiwari, Xiu-Min Li. Metabolic modulation of anaplastic thyroid cancer by Berberine: A targeted intervention [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1410.

In utero bisphenol AF exposure causes fetal Leydig cell dysfunction and induces multinucleated gonocytes by generating oxidative stress and reducing the SIRT1/PGC1α signals

Bisphenol AF (BPAF) is one of the primary alternatives of bisphenol A. It has been ubiquitously detected in the environment and is an emerging endocrine disrupting compound. However, the effects of BPAF exposure on fetal Leydig cells and germ cells and the underlying mechanisms remain largely unknown. To this end, pregnant Sprague-Dawley rats were exposed to 10, 50, and 200 mg/kg/d BPAF by gavage from gestational days 14 to 21. The neonatal rats were sacrificed on day 1 at birth. The results showed that serum testosterone levels were significantly decreased at 50 and 200 mg/kg/d, the expression of Scarb1, Star, Cyp17a1, Hsd17b3, and Dhh and their proteins were markedly down-regulated at 50 and 100 mg/kg/d. BPAF exposure also significantly increased the incidence of multinucleated gonocytes at 200 mg/kg/d. We further detected significant increase of testicular malondialdehyde levels and reduction of antioxidants, including SOD1, SOD2, and CAT at 50 and/or 200 mg/kg/d. Furthermore, BPAF markedly reduced the levels of SIRT1 and PGC1α at 200 mg/kg/d while significantly increased AMPK phosphorylation in the testes at 50 and 200 mg/kg/d. In conclusion, our results provide novel in vivo data that BPAF can induce fetal Leydig cell dysfunction by interfering with steroidogenic networks and induce the formation of multinucleated gonocytes after suppressing the antioxidant defense system and reducing SIRT1 and PGC1α signals and increasing the phosphorylation of AMPK, which highlights the potential health risk of environmental exposure to BPAF in inducing male reproductive tract malformation.