Unveiling the Role of Oxidative Stress in Cochlear Hair Cell Death: Prospective Phytochemical Therapeutics against Sensorineural Hearing Loss.

Hearing loss represents a multifaceted and pervasive challenge that deeply impacts various aspects of an individual's life, spanning psychological, emotional, social, and economic realms. Understanding the molecular underpinnings that orchestrate hearing loss remains paramount in the quest for effective therapeutic strategies. This review aims to expound upon the physiological, biochemical, and molecular aspects of hearing loss, with a specific focus on its correlation with diabetes. Within this context, phytochemicals have surfaced as prospective contenders in the pursuit of potential adjuvant therapies. These compounds exhibit noteworthy antioxidant and anti-inflammatory properties, which hold the potential to counteract the detrimental effects induced by oxidative stress and inflammation-prominent contributors to hearing impairment. Furthermore, this review offers an up-to-date exploration of the diverse molecular pathways modulated by these compounds. However, the dynamic landscape of their efficacy warrants recognition as an ongoing investigative topic, inherently contingent upon specific experimental models. Ultimately, to ascertain the genuine potential of phytochemicals as agents in hearing loss treatment, a comprehensive grasp of the molecular mechanisms at play, coupled with rigorous clinical investigations, stands as an imperative quest.

Hypoxia promotes proliferation and inhibits myogenesis in broiler satellite cells.

Breast muscle myopathies in broilers compromise meat quality and continue to plague the poultry industry. Broiler breast muscle myopathies are characterized by impaired satellite cell (SC)-mediated repair, and localized tissue hypoxia and dysregulation of oxygen homeostasis have been implicated as contributing factors. The present study was designed to test the hypothesis that hypoxia disrupts the ability of SC to differentiate and form myotubes, both of which are key components of myofiber repair, and to determine the extent to which effects are reversed by restoration of oxygen tension. Primary SC were isolated from pectoralis major of young (5 d) Cobb 700 chicks and maintained in growth conditions or induced to differentiate under normoxic (20% O2) or hypoxic (1% O2) conditions for up to 48 h. Hypoxia enhanced SC proliferation while inhibiting myogenic potential, with decreased fusion index and suppressed myotube formation. Reoxygenation after hypoxia partially reversed effects on both proliferation and myogenesis. Western blotting showed that hypoxia diminished myogenin expression, activated AMPK, upregulated proliferation markers, and increased molecular signaling of cellular stress. Hypoxia also promoted accumulation of lipid droplets in myotubes. Targeted RNAseq identified numerous differentially expressed genes across differentiation under hypoxia, including several genes that have been associated with myopathies in vivo. Altogether, these data demonstrate localized hypoxia may influence SC behavior in ways that disrupt muscle repair and promote the formation of myopathies in broilers.

Ginsenoside Rc from Panax Ginseng Ameliorates Palmitate-Induced UB/OC-2 Cochlear Cell Injury.

By 2050, at least 700 million people will require hearing therapy while 2.5 billion are projected to suffer from hearing loss. Sensorineural hearing loss (SNHL) arises from the inability of the inner ear to convert fluid waves into neural electric signals because of injury to cochlear hair cells that has resulted in their death. In addition, systemic chronic inflammation implicated in other pathologies may exacerbate cell death leading to SNHL. Phytochemicals have emerged as a possible solution because of the growing evidence of their anti-inflammatory, antioxidant, and anti-apoptotic properties. Ginseng and its bioactive molecules, ginsenosides, exhibit effects that suppress pro-inflammatory signaling and protect against apoptosis. In the current study, we investigated the effects of ginsenoside Rc (G-Rc) on UB/OC-2 primary murine sensory hair cell survival in response to palmitate-induced injury. G-Rc promoted UB/OC-2 cell survival and cell cycle progression. Additionally, G-Rc enhanced the differentiation of UB/OC-2 cells into functional sensory hair cells and alleviated palmitate-induced inflammation, endoplasmic reticulum stress, and apoptosis. The current study offers novel insights into the effects of G-Rc as a potential adjuvant for SNHL and warrants further studies elucidating the molecular mechanisms.

The emerging role of PKM in keratinocyte homeostasis and pathophysiology.

Increased aerobic glycolysis in keratinocytes has been reported as a hallmark of skin diseases while its pharmacological inhibition restores keratinocyte homeostasis. Pyruvate kinase muscle (PKM) isoforms are key enzymes in the glycolytic pathway and, therefore, an attractive therapeutic target. Simon Nold and colleagues used CRISPR/Cas9-mediated gene editing to investigate the outcomes of PKM splicing perturbations and specific PKM1 or PKM2 deficiency in human HaCaT keratinocytes. Collectively, the study demonstrated different effects of PKM1 or PKM2 depletion on the reciprocal PKM isoform and on keratinocyte gene expression, metabolism and proliferation. Findings from this study provide novel insights into the role of PKM in keratinocyte homeostasis, warranting additional investigations into the underlying molecular mechanisms and potential therapeutic applications.

Podocyte specific deletion of PKM2 ameliorates LPS-induced podocyte injury through beta-catenin.

BACKGROUND: Acute kidney injury (AKI) is associated with a severe decline in kidney function caused by abnormalities within the podocytes' glomerular matrix. Recently, AKI has been linked to alterations in glycolysis and the activity of glycolytic enzymes, including pyruvate kinase M2 (PKM2). However, the contribution of this enzyme to AKI remains largely unexplored. METHODS: Cre-loxP technology was used to examine the effects of PKM2 specific deletion in podocytes on the activation status of key signaling pathways involved in the pathophysiology of AKI by lipopolysaccharides (LPS). In addition, we used lentiviral shRNA to generate murine podocytes deficient in PKM2 and investigated the molecular mechanisms mediating PKM2 actions in vitro. RESULTS: Specific PKM2 deletion in podocytes ameliorated LPS-induced protein excretion and alleviated LPS-induced alterations in blood urea nitrogen and serum albumin levels. In addition, PKM2 deletion in podocytes alleviated LPS-induced structural and morphological alterations to the tubules and to the brush borders. At the molecular level, PKM2 deficiency in podocytes suppressed LPS-induced inflammation and apoptosis. In vitro, PKM2 knockdown in murine podocytes diminished LPS-induced apoptosis. These effects were concomitant with a reduction in LPS-induced activation of ß-catenin and the loss of Wilms' Tumor 1 (WT1) and nephrin. Notably, the overexpression of a constitutively active mutant of ß-catenin abolished the protective effect of PKM2 knockdown. Conversely, PKM2 knockdown cells reconstituted with the phosphotyrosine binding-deficient PKM2 mutant (K433E) recapitulated the effect of PKM2 depletion on LPS-induced apoptosis, ß-catenin activation, and reduction in WT1 expression. CONCLUSIONS: Taken together, our data demonstrates that PKM2 plays a key role in podocyte injury and suggests that targetting PKM2 in podocytes could serve as a promising therapeutic strategy for AKI. TRIAL REGISTRATION: Not applicable. Video abstract.

Pyruvate kinase M1 regulates butyrate metabolism in cancerous colonocytes.

Colorectal cancer (CRC) cells shift metabolism toward aerobic glycolysis and away from using oxidative substrates such as butyrate. Pyruvate kinase M1/2 (PKM) is an enzyme that catalyzes the last step in glycolysis, which converts phosphoenolpyruvate to pyruvate. M1 and M2 are alternatively spliced isoforms of the Pkm gene. The PKM1 isoform promotes oxidative metabolism, whereas PKM2 enhances aerobic glycolysis. We hypothesize that the PKM isoforms are involved in the shift away from butyrate oxidation towards glycolysis in CRC cells. Here, we find that PKM2 is increased and PKM1 is decreased in human colorectal carcinomas as compared to non-cancerous tissue. To test whether PKM1/2 alter colonocyte metabolism, we created a knockdown of PKM2 and PKM1 in CRC cells to analyze how butyrate oxidation and glycolysis would be impacted. We report that butyrate oxidation in CRC cells is regulated by PKM1 levels, not PKM2. Decreased butyrate oxidation observed through knockdown of PKM1 and PKM2 is rescued through re-addition of PKM1. Diminished PKM1 lowered mitochondrial basal respiration and decreased mitochondrial spare capacity. We demonstrate that PKM1 suppresses glycolysis and inhibits hypoxia-inducible factor-1 alpha. These data suggest that reduced PKM1 is, in part, responsible for increased glycolysis and diminished butyrate oxidation in CRC cells.

Differential Effects of 17,18-EEQ and 19,20-EDP Combined with Soluble Epoxide Hydrolase Inhibitor t-TUCB on Diet-Induced Obesity in Mice.

17,18-Epoxyeicosatetraenoic acid (17,18-EEQ) and 19,20-epoxydocosapentaenoic acid (19,20-EDP) are bioactive epoxides produced from n-3 polyunsaturated fatty acid eicosapentaenoic acid and docosahexaenoic acid, respectively. However, these epoxides are quickly metabolized into less active diols by soluble epoxide hydrolase (sEH). We have previously demonstrated that an sEH inhibitor, t-TUCB, decreased serum triglycerides (TG) and increased lipid metabolic protein expression in the brown adipose tissue (BAT) of diet-induced obese mice. This study investigates the preventive effects of t-TUCB (T) alone or combined with 19,20-EDP (T + EDP) or 17,18-EEQ (T + EEQ) on BAT activation in the development of diet-induced obesity and metabolic disorders via osmotic minipump delivery in mice. Both T + EDP and T + EEQ groups showed significant improvement in fasting glucose, serum triglycerides, and higher core body temperature, whereas heat production was only significantly increased in the T + EEQ group. Moreover, both the T + EDP and T + EEQ groups showed less lipid accumulation in the BAT. Although UCP1 expression was not changed, PGC1α expression was increased in all three treated groups. In contrast, the expression of CPT1A and CPT1B, which are responsible for the rate-limiting step for fatty acid oxidation, was only increased in the T + EDP and T + EEQ groups. Interestingly, as a fatty acid transporter, CD36 expression was only increased in the T + EEQ group. Furthermore, both the T + EDP and T + EEQ groups showed decreased inflammatory NFκB signaling in the BAT. Our results suggest that 17,18-EEQ or 19,20-EDP combined with t-TUCB may prevent high-fat diet-induced metabolic disorders, in part through increased thermogenesis, upregulating lipid metabolic protein expression, and decreasing inflammation in the BAT.

A role for zinc transporter gene SLC39A12 in the nervous system and beyond.

The SLC39A12 gene encodes the zinc transporter protein ZIP12, which is expressed across many tissues and is highly abundant in the vertebrate nervous system. As a zinc transporter, ZIP12 functions to transport zinc across cellular membranes, including cellular zinc influx across the plasma membrane. Genome-wide association and exome sequencing studies have shown that brain susceptibility-weighted magnetic resonance imaging (MRI) intensity is associated with ZIP12 polymorphisms and rare mutations. ZIP12 is required for neural tube closure and embryonic development in Xenopus tropicalis. Frog embryos depleted of ZIP12 by antisense morpholinos develop an anterior neural tube defect and lack viability. ZIP12 is also necessary for neurite outgrowth and mitochondrial function in mouse neural cells. ZIP12 mRNA is increased in brain regions of schizophrenic patients. Outside of the nervous system, hypoxia induces ZIP12 expression in multiple mammalian species, including humans, which leads to endothelial and smooth muscle thickening in the lung and contributes towards pulmonary hypertension. Other studies have associated ZIP12 with other diseases such as cancer. Given that ZIP12 is highly expressed in the brain and that susceptibility-weighted MRI is associated with brain metal content, ZIP12 may affect neurological diseases and psychiatric illnesses such as Parkinson's disease, Alzheimer's disease, and schizophrenia. Furthermore, the induction of ZIP12 and resultant zinc uptake under pathophysiological conditions may be a critical component of disease pathology, such as in pulmonary hypertension. Drug compounds that bind metals like zinc may be able to treat diseases associated with impaired zinc homeostasis and altered ZIP12 function.

Genetic deficiency or pharmacological inhibition of soluble epoxide hydrolase ameliorates high fat diet-induced pancreatic ß-cell dysfunction and loss.

Pancreatic ß-cells are crucial regulators of systemic glucose homeostasis, and their dysfunction and loss are central features in type 2 diabetes. Interventions that rectify ß-cell dysfunction and loss are essential to combat this deadly malady. In the current study, we sought to delineate the role of soluble epoxide hydrolase (sEH) in ß-cells under diet-induced metabolic stress. The expression of sEH was upregulated in murine and macaque diabetes models and islets of diabetic human patients. We postulated that hyperglycemia-induced elevation in sEH leads to a reduction in its substrates, epoxyeicosatrienoic acids (EETs), and attenuates the function of ß-cells. Genetic deficiency of sEH potentiated glucose-stimulated insulin secretion in mice, likely in a cell-autonomous manner, contributing to better systemic glucose control. Consistent with this observation, genetic and pharmacological inactivation of sEH and the treatment with EETs exhibited insulinotropic effects in isolated murine islets ex vivo. Additionally, sEH deficiency enhanced glucose sensing and metabolism with elevated ATP and cAMP concentrations. This phenotype was associated with attenuated oxidative stress and diminished ß-cell death in sEH deficient islets. Moreover, pharmacological inhibition of sEH in vivo mitigated, albeit partly, high fat diet-induced ß-cell loss and dedifferentiation. The current observations provide new insights into the role of sEH in ß-cells and information that may be leveraged for the development of a mechanism-based intervention to rectify ß-cell dysfunction and loss.

The Role of PKM2 in Metabolic Reprogramming: Insights into the Regulatory Roles of Non-Coding RNAs.

Pyruvate kinase is a key regulator in glycolysis through the conversion of phosphoenolpyruvate (PEP) into pyruvate. Pyruvate kinase exists in various isoforms that can exhibit diverse biological functions and outcomes. The pyruvate kinase isoenzyme type M2 (PKM2) controls cell progression and survival through the regulation of key signaling pathways. In cancer cells, the dimer form of PKM2 predominates and plays an integral role in cancer metabolism. This predominance of the inactive dimeric form promotes the accumulation of phosphometabolites, allowing cancer cells to engage in high levels of synthetic processing to enhance their proliferative capacity. PKM2 has been recognized for its role in regulating gene expression and transcription factors critical for health and disease. This role enables PKM2 to exert profound regulatory effects that promote cancer cell metabolism, proliferation, and migration. In addition to its role in cancer, PKM2 regulates aspects essential to cellular homeostasis in non-cancer tissues and, in some cases, promotes tissue-specific pathways in health and diseases. In pursuit of understanding the diverse tissue-specific roles of PKM2, investigations targeting tissues such as the kidney, liver, adipose, and pancreas have been conducted. Findings from these studies enhance our understanding of PKM2 functions in various diseases beyond cancer. Therefore, there is substantial interest in PKM2 modulation as a potential therapeutic target for the treatment of multiple conditions. Indeed, a vast plethora of research has focused on identifying therapeutic strategies for targeting PKM2. Recently, targeting PKM2 through its regulatory microRNAs, long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs) has gathered increasing interest. Thus, the goal of this review is to highlight recent advancements in PKM2 research, with a focus on PKM2 regulatory microRNAs and lncRNAs and their subsequent physiological significance.

Soluble Epoxide Hydrolase Inhibition by t-TUCB Promotes Brown Adipogenesis and Reduces Serum Triglycerides in Diet-Induced Obesity.

Brown adipose tissue (BAT) is an important target for obesity treatment and prevention. Soluble epoxide hydrolase (sEH) converts bioactive epoxy fatty acids (EpFAs) into less active diols. sEH inhibitors (sEHI) are beneficial in many chronic diseases by stabilizing EpFAs. However, roles of sEH and sEHI in brown adipogenesis and BAT activity in treating diet-induced obesity (DIO) have not been reported. sEH expression was studied in in vitro models of brown adipogenesis and the fat tissues of DIO mice. The effects of the sEHI, trans-4-{4-[3-(4-trifluoromethoxy-phenyl)-ureido]-cyclohexyloxy-benzoic acid (t-TUCB), were studied in vitro and in the obese mice via mini osmotic pump delivery. sEH expression was increased in brown adipogenesis and the BAT of the DIO mice. t-TUCB promoted brown adipogenesis in vitro. Although t-TCUB did not change body weight, fat pad weight, or glucose and insulin tolerance in the obese mice, it decreased serum triglycerides and increased protein expression of genes important for lipid metabolism in the BAT. Our results suggest that sEH may play a critical role in brown adipogenesis, and sEHI may be beneficial in improving BAT protein expression involved in lipid metabolism. Further studies using the sEHI combined with EpFA generating diets for obesity treatment and prevention are warranted.

Zyflamend induces apoptosis in pancreatic cancer cells via modulation of the JNK pathway.

BACKGROUND: Current pharmacological therapies and treatments targeting pancreatic neuroendocrine tumors (PNETs) have proven ineffective, far too often. Therefore, there is an urgent need for alternative therapeutic approaches. Zyflamend, a combination of anti-inflammatory herbal extracts, that has proven to be effective in various in vitro and in vivo cancer platforms, shows promise. However, its effects on pancreatic cancer, in particular, remain largely unexplored. METHODS: In the current study, we investigated the effects of Zyflamend on the survival of beta-TC-6 pancreatic insulinoma cells (ß-TC6) and conducted a detailed analysis of the underlying molecular mechanisms. RESULTS: Herein, we demonstrate that Zyflamend treatment decreased cell proliferation in a dose-dependent manner, concomitant with increased apoptotic cell death and cell cycle arrest at the G2/M phase. At the molecular level, treatment with Zyflamend led to the induction of ER stress, autophagy, and the activation of c-Jun N-terminal kinase (JNK) pathway. Notably, pharmacological inhibition of JNK abrogated the pro-apoptotic effects of Zyflamend. Furthermore, Zyflamend exacerbated the effects of streptozotocin and adriamycin-induced ER stress, autophagy, and apoptosis. CONCLUSION: The current study identifies Zyflamend as a potential novel adjuvant in the treatment of pancreatic cancer via modulation of the JNK pathway. Video abstract.

Zyflamend, a unique herbal blend, induces cell death and inhibits adipogenesis through the coordinated regulation of PKA and JNK.

The prevalence of obesity and its comorbidities has sparked a worldwide concern to address rates of adipose tissue accrual. Recent studies have demonstrated a novel role of Zyflamend, a blend of natural herbal extracts, in regulating lipid metabolism in several cancer cell lines through the activation of the AMPK signalling pathway. Yet, the role of Zyflamend in adipogenic differentiation and lipid metabolism remains largely unexplored. The objective of this study is to investigate the effects of Zyflamend on white 3T3-MBX pre-adipocyte differentiation and elucidate the molecular mechanisms. We demonstrate that Zyflamend treatment altered cell cycle progression, attenuated proliferation, and increased cell death of 3T3-MBX pre-adipocytes. In addition, treatment with Zyflamend inhibited lipid accumulation during the differentiation of 3T3-MBX cells, consistent with decreased expression of lipogenic genes and increased lipolysis. Mechanistically, Zyflamend-induced alterations in adipogenesis were mediated, at least in part, through the activation of AMPK, PKA, and JNK. Inhibition of AMPK partially reversed Zyflamend-induced inhibition of differentiation, whereas the inhibition of either JNK or PKA fully restored adipocyte differentiation and decreased lipolysis. Taken together, the present study demonstrates that Zyflamend, as a novel anti-adipogenic bioactive mix, inhibits adipocyte differentiation through the activation of the PKA and JNK pathways. ABBREVIATION: 7-AAD: 7-amino-actinomycin D; ACC: acetyl-CoA carboxylase; AKT: protein kinase B; AMPK: AMP-activated protein kinase; ATGL: adipose triglyceride lipase; C/EBPα: CCAAT-enhancer binding protein alpha; DMEM: Dulbecco's Modified Eagle Medium; DMSO: dimethyl sulphoxide; DTT: dithiothreitol; EGTA: ethylene glycol-bis-(2-aminoethyl)-N,N,N',N'-tetraacetic acid; ERK: extracellular signal-regulated kinases; FASN: fatty acid synthase; FBS: foetal bovine serum; GLUT: glucose transporter; HSL: hormone-sensitive lipase; IR: insulin receptor; IRS: insulin receptor substrate; JNK: c-JUN N-terminal kinase; MGL: monoacylglycerol lipase; NaF: sodium fluoride; NF-κB: nuclear factor kappa-light-chain-enhancer of activated B cells; PBS: phosphate buffered- saline; PCB: pyruvate carboxylase; PDE: phosphodiesterase; PKA: protein kinase cAMP-dependent; PMSF: phenylmethylsulfonyl fluoride; PPARγ: perilipin peroxisome proliferator-activated receptor gamma; PREF-1: pre-adipocyte factor 1; PVDF: polyvinylidene fluoride; RIPA: radio-immunoprecipitation assay; SDS-PAGE: sodium dodecyl sulphate polyacrylamide gel electrophoresis; SEM: standard error of the mean; SOX9: suppressor of cytokine signalling 9; TGs: triacylglycerols.

Role of zinc transporter ZIP12 in susceptibility-weighted brain magnetic resonance imaging (MRI) phenotypes and mitochondrial function.

Brain zinc dysregulation is linked to many neurological disorders. However, the mechanisms regulating brain zinc homeostasis are poorly understood. We performed secondary analyses of brain MRI GWAS and exome sequencing data from adults in the UK Biobank. Coding ZIP12 polymorphisms in zinc transporter ZIP12 (SLC39A12) were associated with altered brain susceptibility weighted MRI (swMRI). Conditional and joint association analyses revealed independent GWAS signals in linkage disequilibrium with 2 missense ZIP12 polymorphisms, rs10764176 and rs72778328, with reduced zinc transport activity. ZIP12 rare coding variants predicted to be deleterious were associated with similar impacts on brain swMRI. In Neuro-2a cells, ZIP12 deficiency by short hairpin RNA (shRNA) depletion or CRISPR/Cas9 genome editing resulted in impaired mitochondrial function, increased superoxide presence, and detectable protein carbonylation. Inhibition of Complexes I and IV of the electron transport chain reduced neurite outgrowth in ZIP12 deficient cells. Transcriptional coactivator PGC-1α, mitochondrial superoxide dismutase (SOD2), and chemical antioxidants α-tocopherol, MitoTEMPO, and MitoQ restored neurite extension impaired by ZIP12 deficiency. Mutant forms of α-synuclein and tau linked to familial Parkinson's disease and frontotemporal dementia, respectively, reduced neurite outgrowth in cells deficient in ZIP12. Zinc and ZIP12 may confer resilience against neurological diseases or premature aging of the brain.

Pyruvate kinase M2: A simple molecule with complex functions.

Pyruvate kinase M2 is a critical enzyme that regulates cell metabolism and growth under different physiological conditions. In its metabolic role, pyruvate kinase M2 catalyzes the last glycolytic step which converts phosphoenolpyruvate to pyruvate with the generation of ATP. Beyond this metabolic role in glycolysis, PKM2 regulates gene expression in the nucleus, phosphorylates several essential proteins that regulate major cell signaling pathways, and contribute to the redox homeostasis of cancer cells. The expression of PKM2 has been demonstrated to be significantly elevated in several types of cancer, and the overall inflammatory response. The unusual pattern of PKM2 expression inspired scientists to investigate the unrevealed functions of PKM2 and the therapeutic potential of targeting PKM2 in cancer and other disorders. Therefore, the purpose of this review is to discuss the mechanistic and therapeutic potential of targeting PKM2 with the focus on cancer metabolism, redox homeostasis, inflammation, and metabolic disorders. This review highlights and provides insight into the metabolic and non-metabolic functions of PKM2 and its relevant association with health and disease.

Idebenone is a cytoprotective insulin sensitizer whose mechanism is Shc inhibition.

When insulin binds insulin receptor, IRS1 signaling is stimulated to trigger the maximal insulin response. p52Shc protein competes directly with IRS1, thus damping and diverting maximal insulin response. Genetic reduction of p52Shc minimizes competition with IRS1, and improves insulin signaling and glucose control in mice, and improves pathophysiological consequences of hyperglycemia. Given the multiple benefits of Shc reduction in vivo, we investigated whether any of 1680 drugs used in humans may function as Shc inhibitors, and thus potentially serve as novel anti-diabetics. Of the 1680, 30 insulin sensitizers were identified by screening in vitro, and of these 30 we demonstrated that 7 bound Shc protein. Of the 7 drugs, idebenone dose-dependently bound Shc protein in the 50-100 nM range, and induced insulin sensitivity and cytoprotection in this same 100 nM range that clinically dosed idebenone reaches in human plasma. By contrast we observe mitochondrial effects of idebenone in the 5,000 nM range that are not reached in human dosing. Multiple assays of target engagement demonstrate that idebenone physically interacts with Shc protein. Idebenone sensitizes mice to insulin in two different mouse models of prediabetes. Genetic depletion of idebenone's target eliminates idebenone's ability to insulin-sensitize in vivo. Thus, idebenone is the first-in-class member of a novel category of insulin-sensitizing and cytoprotective agents, the Shc inhibitors. Idebenone is an approved drug and could be considered for other indications such as type 2 diabetes and fatty liver disease, in which insulin resistance occurs.

Concurrent regulation of LKB1 and CaMKK2 in the activation of AMPK in castrate-resistant prostate cancer by a well-defined polyherbal mixture with anticancer properties.

BACKGROUND: Zyflamend, a blend of herbal extracts, effectively inhibits tumor growth using preclinical models of castrate-resistant prostate cancer mediated in part by 5'-adenosine monophosphate-activated protein kinase (AMPK), a master energy sensor of the cell. Clinically, treatment with Zyflamend and/or metformin (activators of AMPK) had benefits in castrate-resistant prostate cancer patients who no longer responded to treatment. Two predominant upstream kinases are known to activate AMPK: liver kinase B1 (LKB1), a tumor suppressor, and calcium-calmodulin kinase kinase-2 (CaMKK2), a tumor promotor over-expressed in many cancers. The objective was to interrogate how Zyflamend activates AMPK by determining the roles of LKB1 and CaMKK2. METHODS: AMPK activation was determined in CWR22Rv1 cells treated with a variety of inhibitors of LKB1 and CaMKK2 in the presence and absence of Zyflamend, and in LKB1-null HeLa cells that constitutively express CaMKK2, following transfection with wild type LKB1 or catalytically-dead mutants. Upstream regulation by Zyflamend of LKB1 and CaMKK2 was investigated targeting protein kinase C-zeta (PKCζ) and death-associated protein kinase (DAPK), respectively. RESULTS: Zyflamend's activation of AMPK appears to be LKB1 dependent, while simultaneously inhibiting CaMKK2 activity. Zyflamend failed to rescue the activation of AMPK in the presence of pharmacological and molecular inhibitors of LKB1, an effect not observed in the presence of inhibitors of CaMKK2. Using LKB1-null and catalytically-dead LKB1-transfected HeLa cells that constitutively express CaMKK2, ionomycin (activator of CaMKK2) increased phosphorylation of AMPK, but Zyflamend only had an effect in cells transfected with wild type LKB1. Zyflamend appears to inhibit CaMKK2 by DAPK-mediated phosphorylation of CaMKK2 at Ser511, an effect prevented by a DAPK inhibitor. Alternatively, Zyflamend mediates LKB1 activation via increased phosphorylation of PKCζ, where it induced translocation of PKCζ and LKB1 to their respective active compartments in HeLa cells following treatment. Altering the catalytic activity of LKB1 did not alter this translocation. DISCUSSION: Zyflamend's activation of AMPK is mediated by LKB1, possibly via PKCζ, but independent of CaMKK2 by a mechanism that appears to involve DAPK. CONCLUSIONS: Therefore, this is the first evidence that natural products simultaneously and antithetically regulate upstream kinases, known to be involved in cancer, via the activation of AMPK.