AMP-activated protein kinase: An energy sensor and survival mechanism in the reinstatement of metabolic homeostasis.

Cells are programmed to favorably respond towards the nutrient availability by adapting their metabolism to meet energy demands. AMP-activated protein kinase (AMPK) is a highly conserved serine/threonine energy-sensing kinase. It gets activated upon a decrease in the cellular energy status as reflected by an increased AMP/ATP ratio, ADP, and also during the conditions of glucose starvation without change in the adenine nucelotide ratio. AMPK functions as a centralized regulator of metabolism, acting at cellular and physiological levels to circumvent the metabolic stress by restoring energy balance. This review intricately highlights the integrated signaling pathways by which AMPK gets activated allosterically or by multiple non-canonical upstream kinases. AMPK activates the ATP generating processes (e.g., fatty acid oxidation) and inhibits the ATP consuming processes that are non-critical for survival (e.g., cell proliferation, protein and triglyceride synthesis). An integrated signaling network with AMPK as the central effector regulates all the aspects of enhanced stress resistance, qualified cellular housekeeping, and energy metabolic homeostasis. Importantly, the AMPK mediated amelioration of cellular stress and inflammatory responses are mediated by stimulation of transcription factors such as Nrf2, SIRT1, FoxO and inhibition of NF-κB serving as main downstream effectors. Moreover, many lines of evidence have demonstrated that AMPK controls autophagy through mTOR and ULK1 signaling to fine-tune the metabolic pathways in response to different cellular signals. This review also highlights the critical involvement of AMPK in promoting mitochondrial health, and homeostasis, including mitophagy. Loss of AMPK or ULK1 activity leads to aberrant accumulation of autophagy-related proteins and defective mitophagy thus, connecting cellular energy sensing to autophagy and mitophagy.

PHLPP1/Nrf2-Mdm2 axis induces renal apoptosis via influencing nucleo-cytoplasmic shuttling of FoxO1 during diabetic nephropathy.

Impaired PI3K/Akt signaling (insulin resistance) and poor glycemic control (hyperglycemia) are the major risk factors involved in the progression of diabetic nephropathy (DN). This study was designed to identify factors influencing cell survival during DN. We found that high glucose exposure in renal proximal tubular cells (NRK52E) upregulated PHLPP1, an Akt phosphatase (Ser473), causing suppression in Akt and IGF1ß phosphorylation leading to inhibition in insulin signaling pathway. Results demonstrate that sustained activation of PHLPP1 promoted nuclear retention of FoxO1 by preventing its ubiquitination via Mdm2, an Akt/ Nrf2-dependent E3 ligase. Thus, enhanced FoxO1 nuclear stability caused aberration in renal gluconeogenesis and activated apoptotic cascade. Conversely, gene silencing of PHLPP1-enhanced Nrf2 expression and attenuated FoxO1 regulated apoptosis compared to hyperglycemic cells. Mechanistic aspects of PHLPP1-Nrf2/FoxO1 signaling were further validated in STZ-nicotinamide-induced type 2 diabetic Wistar rats. Importantly, we observed via immunoblotting and dual immunocytochemical studies that treatment of Morin (2',3,4',5,7-Pentahydroxyflavone) during diabetes significantly augmented FoxO1 nuclear exclusion, resulting in its ubiquitination via Akt-Nrf2/Mdm2 pathway. Furthermore, lowering of PHLPP1 expression by Morin also prevented FoxO1/Mst1-mediated apoptotic signaling in vitro and in vivo. Morin treatment under the experimental conditions, effectively decreased blood glucose levels, ameliorated insulin resistance, alleviated oxidative stress and attenuated renal apoptosis in diabetic rats comparable to metformin thereby exhibiting tremendous potential against renal complications of diabetes. These novel results further acclaim that inhibition of PHLPP1/FoxO1-Mdm2 axis is critical in the pathogenesis of diabetic nephropathy.

Critical role of mitochondrial dysfunction and impaired mitophagy in diabetic nephropathy.

Mitochondrial dynamics play a critical role in deciding the fate of a cell under normal and diseased condition. Recent surge of studies indicate their regulatory role in meeting energy demands in renal cells making them critical entities in the progression of diabetic nephropathy. Diabetes is remarkably associated with abnormal fuel metabolism, a basis for free radical generation, which if left unchecked may devastate the mitochondria structurally and functionally. Impaired mitochondrial function and their aberrant accumulation have been known to be involved in the manifestation of diabetic nephropathy, indicating perturbed balance of mitochondrial dynamics, and mitochondrial turnover. Mitochondrial dynamics emphasize the critical role of mitochondrial fission proteins such as mitochondrial fission 1, dynamin-related protein 1 and mitochondrial fission factor and fusion proteins including mitofusin-1, mitofusin-2 and optic atrophy 1. Clearance of dysfunctional mitochondria is aided by translocation of autophagy machinery to the impaired mitochondria and subsequent activation of mitophagy regulating proteins PTEN-induced putative kinase 1 and Parkin, for which mitochondrial fission is a prior event. In this review, we discuss recent progression in our understanding of the molecular mechanisms targeting reactive oxygen species mediated alterations in mitochondrial energetics, mitophagy related disorders, impaired glucose transport, tubular atrophy, and renal cell death. The molecular cross talks linking autophagy and renoprotection through an intervention of 5'-AMP-activated protein kinase, mammalian target of rapamycin, and SIRT1 factors are also highlighted here, as in-depth exploration of these pathways may help in deriving therapeutic strategies for managing diabetes provoked end-stage renal disease.

Berberine induced modulation of PHLPP2-Akt-MST1 kinase signaling is coupled with mitochondrial impairment and hepatoma cell death.

Pleckstrin homology domain leucine-rich repeat protein phosphatase 2 (PHLPP2) has been known to exert tumor suppressive activity for long without much knowledge about its regulation and implications. Protein kinase B (Akt), Protein kinase C (PKC) and Ribosomal protein S6 Kinase (S6K) are known downtargets of PHLPP2, regulating a plethora of life processes viz. cell growth, survival and evasion from apoptosis. Present study decoded the crucial role of PHLPP2 in inducing apoptosis by its interaction with the newly found binding partner Mammalian sterile 20-like kinase 1 (Mst1) in berberine (BBR)-treated human hepatoma cells. HepG2 cells were exposed to (50 µM, 100 µM) berberine for different time intervals (18 h, 24 h). The results showed enhanced expression of PHLPP2 at transcriptional (2.13 fold, P < 0.01) and translational level (4 fold, P < 0.001), but not of PHLPP1, in berberine-treated HepG2 cells. Elevated expression of PHLPP2 was reported to inactivate Akt by dephosphorylating it on Ser473 (P < 0.001). As Akt is known to inhibit apoptotic effect of Mst1, we found that PHLPP2 mediated inactivation of Akt releases its repression from Mst1 leading to heightened phosphorylation of Mst1 on its activating site Thr183 (1.5 fold, P < 0.001). Consequently, coordination between PHLPP2, Akt and Mst1 stimulated downstream targets c-jun N-terminal kinase (JNK), Bim and Bak which are direct activators of pro-apoptotic proteins leading to cell death. Further, PHLPP2/Mst1 knock-down efficiently curtailed anti-proliferative effect of berberine by restoring the basal level of downstream anti-apoptotic proteins. In addition, pre-treatment of NAC (5 mM) showed that ROS generation was a primitive event to initiate activation of stress kinases. Thus, our findings suggest that PHLPP2, Akt and Mst1 constitute an autoinhibitory triangle which may be partly responsible for antiproliferative effect of berberine.

Concurrent acetylation of FoxO1/3a and p53 due to sirtuins inhibition elicit Bim/PUMA mediated mitochondrial dysfunction and apoptosis in berberine-treated HepG2 cells.

Post-translational modifications i.e. phosphorylation and acetylation are pivotal requirements for proper functioning of eukaryotic proteins. The current study aimed to decode the impact of acetylation/deacetylation of non-histone targets i.e. FoxO1/3a and p53 of sirtuins (NAD(+) dependent enzymes with lysine deacetylase activity) in berberine treated human hepatoma cells. Berberine (100 µM) inhibited sirtuins significantly (P<0.05) at transcriptional level as well as at translational level. Combination of nicotinamide (sirtuin inhibitor) with berberine potentiated sirtuins inhibition and increased the expression of FoxO1/3a and phosphorylation of p53 tumor suppressor protein. As sirtuins deacetylate non-histone targets including FoxO1/3a and p53, berberine increased the acetylation load of FoxO1/3a and p53 proteins. Acetylated FoxO and p53 proteins transcriptionally activate BH3-only proteins Bim and PUMA (3.89 and 3.87 fold respectively, P<0.001), which are known as direct activator of pro-apoptotic Bcl-2 family protein Bax that culminated into mitochondria mediated activation of apoptotic cascade. Bim/PUMA knock-down showed no changes in sirtuins' expression while cytotoxicity induced by berberine and nicotinamide was curtailed up to 28.3% (P<0.001) and it restored pro/anti apoptotic protein ratio in HepG2 cells. Sirtuins inhibition was accompanied by decline in NAD(+)/NADH ratio, ATP generation, enhanced ROS production and decreased mitochondrial membrane potential. TEM analysis confirmed mitochondrial deterioration and cell damage. SRT-1720 (1-10 µM), a SIRT-1 activator, when pre-treated with berberine (25 µM), reversed sirtuins expression comparable to control and significantly restored the cell viability (P<0.05). Thus, our findings suggest that berberine mediated sirtuins inhibition resulting into FoxO1/3a and p53 acetylation followed by BH3-only protein Bim/PUMA activation may in part be responsible for mitochondria-mediated apoptosis.

Morin mitigates acetaminophen-induced liver injury by potentiating Nrf2 regulated survival mechanism through molecular intervention in PHLPP2-Akt-Gsk3ß axis.

Acetaminophen (APAP) is frequently taken to relieve pain. Staggered APAP overdoses have been reported to cause acetaminophen-induced liver injury (AILI). Identification of efficacious therapeutic modalities to address complications imposed by accidental/intentional long-term APAP ingestion is needed. Morin, a plant-derived phytochemical, possesses a multitude of pharmacological properties including hepatoprotective action; however, the underlying mechanisms have been inadequately explored. Our present report demonstrates significant attenuation of APAP-mediated liver injury by morin supplementation in vivo as indicated by reduction in histological and serum markers of hepatotoxicity. Morin not only limited necroinflammation as revealed by reduced HMGB1 release, NALP3 and caspase-1 maturation, but also suppressed oxidative stress and mitochondrial dysfunction. This suggests that morin may have exerted its cytoprotective role by way of early intervention in the pathway leading to perpetuation of AILI. Morin reinforced cellular defenses by suppressing Nrf2 ubiquitination and promoting nuclear Nrf2 retention as well as ARE-Nrf2 binding affinity. The effects were observed to be a result of molecular intervention in the activity of PHLPP2, a phosphatase previously reported by us to subdue cellular Nrf2 responses via Fyn kinase activation. Morin was observed to inhibit APAP-induced increase in PHLPP2 activity ex vivo as well as its association with cellular target Akt1. As a result, morin prevented oxidative stress induced deactivation of Akt (Ser473) leading to suppression in GSK3ß and Fyn kinase activation. The study supports the inhibitory action of morin against PHLPP2-regulated Nrf2-suppression and hence indentifies Nrf2-potentiating property of morin that may be exploited in developing novel therapeutic strategy to address AILI.

Chitosan/alginate nanogel potentiate berberine uptake and enhance oxidative stress mediated apoptotic cell death in HepG2 cells.

Biopolymer-based nanoscale drug delivery systems have become a promising approach to overcome the limitations associated with conventional chemotherapeutics used for cancer treatment. Herein, we reported to develop a hydrophilic nanogel (NG) composed of Chitosan (Chi) and sodium alginate (Alg) using the ion gelation method for delivering Berberine hydrochloride (BBR), an alkaloid obtained from Berberis aristata roots. The use of different nanocarriers for BBR delivery has been reported previously, but the bioavailability of these carriers was limited due to phagocytic uptake and poor systemic delivery. The developed NG showed enhanced stability and efficient entrapment of BBR ∼92 %, resulting in a significant increase in bioavailability. The pH-dependent release behavior demonstrated sustained and effective release of ∼86 %, ∼74 % and, ∼53 % BBR at pH 5.5, 6.6, and 7.4 respectively after 72h, indicating its potential as a drug carrier. Additionally, the cellular uptake of BBR was significantly higher ∼19 % in the BBR-NG (25 µM) than in bulk BBR (100 µM), leading to enhanced ROS generation, mitochondrial depolarisation, and inhibition of cell proliferation and colony formation in HepG2 cells. In summary, the results suggest that the Chi/Alg biopolymer-based nano-formulation could be an effective approach for delivering BBR and enhancing its cellular uptake, efficacy, and cytotoxicity.

Naringenin prevents high glucose-induced mitochondria-mediated apoptosis involving AIF, Endo-G and caspases.

Oxidative stress is implicated in hyperglycemia-induced alterations in cell signaling pathways. We examined the toxicity of high glucose in primary rat hepatocytes and its amelioration by naringenin. Incubation of hepatocytes with 40 mM glucose for 1.5 h exhibited significant decrease in cell viability confirmed by MTT reduction and Alamar blue assay. At the same time primary rat hepatocytes exhibited significant decrease in mitochondrial membrane potential indicating organelle dysfunction. Enhanced translocation of Cyt-c from mitochondria to cytosol and AIF/Endo-G from mitochondria to nucleus, activation of caspase-9/3, DNA damage, and chromatin condensation were observed in glucose-stressed hepatocytes, indicating the involvement of mitochondrial pathway in high glucose-induced apoptosis. Transcript levels of antioxidant enzymes were significantly altered along with corresponding changes in their enzymatic activities. The level of intracellular antioxidant glutathione as well as superoxide dismutase, catalase, and glutathione peroxidase activities were observed to be significantly decreased in hepatocytes treated with high concentration of glucose. Naringenin, a flavanone, was effective in preventing loss of cell viability, reactive oxygen species generation, and decline in antioxidant defense. Translocation of AIF, Endo-G, and Cyt-c from mitochondria was also inhibited by naringenin in glucose-stressed cells. Messenger RNA expression of anti-apoptotic and apoptotic genes, externalization of phosphatidyl serine, DNA damage, chromatin condensation, and sub-diploid cell population were effectively altered by naringenin indicating its anti-apoptotic potential in vitro. Our data suggests that naringenin can prevent apoptosis induced by high glucose through scavenging of reactive oxygen species and modulation of mitochondria-mediated apoptotic pathway.

Genetic diversity of pinus roxburghii sarg. Collected from different himalayan regions of India assessed by random amplified polymorphic DNA analysis.

Present study was aimed at molecular genetic fingerprint profile of 15 genotypes of three populations of Pinus roxburghii Sarg. from Himalayan regions of India using random amplified polymorphic DNA (RAPD) based markers. Needles of Pinus roxburghii Sarg. were collected from Dharamshala, Himachal Pradesh (HP), Nainital, Uttarakhand (UK) and Darjeeling, West Bengal (WB) regions of India. The samples were subjected to DNA extraction and RAPD analysis using oligonucleotide purification cartridge (OPC) primers. Out of 15 primers tested, nine primers gave scorable bands. Altogether 48 bands were obtained, out of which 43 were found to be polymorphic. Number of amplified fragments with RAPD primers ranged from four to eight with the size of amplicon ranging from 500 to 7,000bp. Investigation of natural diversity at intraspecies level was performed with 15 genotypes. Forty-eight amplification products were scored by RAPD and showed 89.58% polymorphism with a mean intrapopulation genetic diversity (Hpop) of 0.2754. A significant inter- and intrapopulation diversity was observed, with the percentage of polymorphic loci (Pp) ranging from 50.09 to 70.83%, Shannon's information index (I) from 0.3262 to 0.4689 and Nei's gene diversity (h) from 0.2032 to 0.3335 with mean Nei's gene diversity 0.377 and the overall estimate of gene flow being (Nm) 1.3555. Unweighted pair-group method with arithmetic average (UPGMA) analysis based Dendrogram showed single cluster. The variation amongst the samples of the three ecological regions can be attributed to varied climatic conditions and may help in conservation/future cultivation of these species.

Endoplasmic reticulum stress induces degradation of glucose transporter proteins during hyperglycemic hepatotoxicity: Role of PERK-eIF2α-ATF4 axis.

Hyperglycemia induced reactive oxygen species oxidize macromolecules including cellular proteins leading to their accumulation in Endoplasmic Reticulum (ER) lumen which in turn activates unfolded protein response (UPR) sensors including, PERK (Protein Kinase RNA-Like ER Kinase). Activated PERK induces ER associated degradation of misfolded proteins to lower the ER stress. In the present study, we hypothesized that ER stress leads to the degradation of glucose transporter proteins resulting in complex glucose metabolism. In vivo studies were carried out in the experimental model of hyperglycemia using streptozotocin/nicotinamide induced diabetic male Wistar rats. High glucose (30 mM) treated HepG2 cells were used to perform the mechanistic study at different time points. PERK gene knockdown (siRNA transfection) and inhibition by ISRIB (Integrated Stress Response Inhibitor, a potent inhibitor of PERK signaling) confirmed the involvement of PERK axis in regulating the expression and translocation of hepatic glucose transporters. Co-immunoprecipitation and dual immunostaining studies further demonstrated increased degradation of GLUT proteins under high glucose conditions. Moreover, Morin (3,5,7,2',4' pentahydroxyflavone) treatment prevented PERK-eIF2α-ATF4 mediated degradation of glucose transporters and enhanced glucose uptake in both, HepG2 cells and diabetic rats. Targeting aberrant regulation of the expression and translocation of facilitative glucose transporter proteins (GLUT proteins) may provide novel therapeutic strategies for the better management of diabetes.

Endoplasmic reticulum stress-dependent activation of TRB3-FoxO1 signaling pathway exacerbates hyperglycemic nephrotoxicity: Protection accorded by Naringenin.

Endoplasmic reticulum (ER) dysfunction contributes greatly to the pathophysiology of hyperglycemic nephrotoxicity. This study unravels the critical role of Tribbles 3 (TRB3)-Forkhead box O1 (FoxO1) signaling pathway during hyperglycemic renal toxicity. It also uncovers the novel role of Naringenin, a flavanone, in regulating ER stress in proximal tubular cells, NRK 52E, and kidneys of streptozotocin/nicotinamide induced experimental diabetic Wistar rats. Results demonstrate that expression of ER stress marker proteins including phosphorylated protein kinase ER like kinase (p-PERK), phosphorylated eukaryotic Initiation Factor 2α (p-eIF2α), X Box Binding Protein 1 spliced (XBP1s), Activating Transcription Factor 4 (ATF4) and C/EBP Homologous Protein (CHOP) were upregulated in diabetic kidneys indicating the activation of ER stress response due to nephrotoxicity. Treatment with Naringenin reduced the expression of TRB3, an ER stress-inducible pseudokinase, both in vitro and in vivo. Gene silencing of TRB3 enhanced Akt and FoxO1 phosphorylation and alleviated FoxO1 mediated apoptosis during hyperglycemic nephrotoxicity. Notably, TRB3 gene silencing effects were comparable to the response with Naringenin treatment. Prevention of nuclear colocalization of ATF4 and CHOP in Naringenin treated cells was evident. Naringenin also reduced insulin resistance, apoptosis and glycogen accumulation along with enhancement of glucose tolerance in diabetic rats. Prevention of ultrastructural aberrations in the ER of hyperglycemic renal cells by Naringenin confirmed its anti-ER stress effects. These findings affirm that activation of TRB3-FoxO1 signaling is critical in the pathogenesis of hyperglycemia-induced renal toxicity and protective effect of Naringenin via modulation of ER stress may be exploited as a novel approach for its management.

Naringenin alleviates hyperglycemia-induced renal toxicity by regulating activating transcription factor 4-C/EBP homologous protein mediated apoptosis.

Endoplasmic reticulum (ER) dysfunction plays a prominent role in the pathophysiology of diabetic nephropathy (DN). This study aimed to investigate the novel role of Naringenin (a flavanone mainly found in citrus fruits) in modulating ER stress in hyperglycemic NRK 52E cells and STZ/nicotinamide induced diabetes in Wistar rats. The results demonstrated that Naringenin supplementation downregulated the expression of ER stress marker proteins, including p-PERK, p-eIF2α, XBP1s, ATF4 and CHOP during hyperglycemic renal toxicity in vitro and in vivo. Naringenin abrogated hyperglycemia-induced ultrastructural changes in ER, evidencing its anti-ER stress effects. Interestingly, treatment of Naringenin prevented nuclear translocation of ATF4 and CHOP in hyperglycemic renal cells and diabetic kidneys. Naringenin prevented apoptosis in hyperglycemic renal cells and diabetic kidney tissues by downregulating expression of apoptotic marker proteins. Further, photomicrographs of TEM confirmed anti-apoptotic potential of Naringenin as it prevented membrane blebbing and formation of apoptotic bodies in hyperglycemic renal cells. Naringenin improved glucose tolerance, restored serum insulin level and reduced serum glucose level in diabetic rats evidencing its anti-hyperglycemic effects. Histopathological examination of kidney tissues also confirmed prevention of damage after 28 days of Naringenin treatment in diabetic rats. Additionally, Naringenin diminished oxidative stress and improved antioxidant defense response during hyperglycemic renal toxicity. Taken together, our study revealed a novel role of Naringenin in ameliorating ER stress during hyperglycemic renal toxicity along with prevention of apoptosis, cellular and tissue damage. The findings suggest that prevention of ER stress can be exploited as a novel approach for the management of hyperglycemic nephrotoxicity.

Alteration in mitochondrial thiol enhances calcium ion dependent membrane permeability transition and dysfunction in vitro: a cross-talk between mtThiol, Ca(2+), and ROS.

Mitochondrial permeability transition (MPT) and dysfunctions play a pivotal role in many patho-physiological and toxicological conditions. The interplay of mitochondrial thiol (mtThiol), MPT, Ca(2+) homeostasis, and resulting dysfunctions still remains controversial despite studies by several research groups. Present study was undertaken to ascertain the correlation between Ca(2+) homeostasis, mtThiol alteration and reactive oxygen species (ROS) in causing MPT leading to mitochondrial dysfunction. mtThiol depletion significantly enhanced Ca(2+) dependent MPT (swelling) and depolarization of mitochondria resulting in release of pro-apoptotic proteins like Cyt c, AIF, and EndoG. mtThiol alteration and Ca(2+) overload caused reduced mitochondrial electron flow, oxidation of pyridine nucleotides (NAD(P)H) and significantly enhanced ROS generation (DHE and DCFH-DA fluorescence). Studies with MPT inhibitor (Cyclosporin A), Ca(2+) uniport blocker (ruthenium red) and Ca(2+) chelator (BAPTA) indicated that mitochondrial dysfunction was more pronounced under dual stress of altered mtThiol and Ca(2+) overload in comparison with single stress of excessive Ca(2+). Transmission electron microscopy confirmed the changes in mitochondrial integrity under stress. Our findings suggest that the Ca(2+) overload itself is not solely responsible for structural and functional impairment of mitochondria. A multi-factorial cross-talk between mtThiol, Ca(2+) and ROS is responsible for mitochondrial dysfunction. Furthermore, minor depletion of mtThiol was found to be an important factor along with Ca(2+) overload in triggering MPT in isolated mitochondria, tilting the balance towards disturbed functionality.

Berbamine induced activation of the SIRT1/LKB1/AMPK signaling axis attenuates the development of hepatic steatosis in high-fat diet-induced NAFLD rats.

Non-alcoholic fatty liver disease (NAFLD), a chronic metabolic disorder is concomitant with oxidative stress and inflammation. This study aimed to assess the effects of berbamine (BBM), a natural bisbenzylisoquinoline alkaloid with manifold biological activities and pharmacological effects on lipid, cholesterol and glucose metabolism in a rat model of NAFLD, and to explicate the potential mechanisms underlying its activity. BBM administration alleviated the increase in the body weight and liver index of HFD rats. The aberrations in liver function, serum parameters, and microscopic changes in the liver structure of HFD fed rats were significantly improved upon BBM administration. BBM also significantly attenuated oxidative damage and inhibited triglyceride and cholesterol synthesis. The SIRT1 deacetylase activity was also enhanced by BBM through liver kinase B1 and activated AMP-activated protein kinase. Activation of the SIRT1/LKB1/AMPK pathway prevented the downstream target ACC (acetyl-CoA carboxylase) and elevation in the expression of FAS (fatty acid synthase) and SCD1 (steroyl CoA desaturase). BBM also modulated the expression of PPARs maintaining the fatty acid homeostasis regulation. The assessment of berbamine induced ultrastructural changes by TEM analysis and the expression of autophagic markers LC3a/b, Beclin 1 and p62 revealed the induction of autophagy to alleviate fatty liver conditions. These results show novel findings that BBM induced protection against hepatic lipid metabolic disorders is achieved by regulating the SIRT1/LKB1/AMPK pathway, and thus it emerges as an effective phyoconstituent for the management of NAFLD.

Phytochemical profiling and cytotoxic evaluation of Premna serratifolia L. against human liver cancer cell line.

Premna serratifolia L. (Lamiaceae) is a medicinal plant, widely distributed in the tropical and subtropical regions and commonly used in traditional medicine. The current study was focused to evaluate the cytotoxic potential of aqueous extract of root of P. serratifolia (AEPS) against human hepatoblastoma cancer cell line (Hep G2).The yield of the dried extract was 5.8% and used for further studies.Cytotoxic potential of AEPS was analyzed by MTT assay, which exhibits IC50 value 1000 µg/mL after 48 h incubation. Hoechst and AO/EtBr staining, ROS measurement, mitochondrial membrane potential, clonogenic and wound healing assays also confirmed the cytotoxic efficacy of AEPS in dose and time-dependent manner. UPLC-Q-TOF-MS/MS analysis of AEPS confirmed the presence of 12polyphenolic compounds, namely 4-hydroxy-3-methoxycinnamic acid, linarin, peonidin-3,5-O-di-beta-glucopyranoside, diosmin, trans-cinnamic acid, daidzein, saponarin, homoorietin, acacetin, sarsasapogenin, phytol and sissotrin. The cytotoxic potential of AEPS might due to presence of biologically active polyphenolic compounds.

Activation of autophagic flux via LKB1/AMPK/mTOR axis against xenoestrogen Bisphenol-A exposure in primary rat hepatocytes.

Bisphenol-A, an endocrine disruptive chemical widely used to manufacture polycarbonate plastics and epoxy resins, acts via multiple mechanisms that perturb cellular and molecular functions. BPA has the potential to induce hepatotoxicity via generation of ROS and oxidative stress. However, the mechanism of BPA induced oxidative stress and autophagy is still ambiguous at molecular and cellular levels. This study aims to elucidate the impact of BPA exposure (50 and 100 µM) in primary rat hepatocytes. AMP kinase, an intracellular energy sensor and key regulator in cellular signaling were found to be activated during BPA exposure. The increased AMP/ATP ratio and subsequent phosphorylation by its upstream mediator Liver Kinase B1 (LKB1) activates AMPK. BPA down-regulated AMPK downstream molecule i.e. mammalian target of rapamycin (mTOR) by inhibiting its phosphorylation, eventually enhances expression of autophagic markers LC3B, Beclin-1 while lowers p62. Results also revealed that BPA induces mitophagy by promoting accumulation of PINK1 and translocation of Parkin to damaged mitochondria culminating in decreased mitochondrial mass. Ultra-structural changes also confirmed mitochondrial disintegration, enhanced autophagic induction as evident from autophagosome formation. Findings confirm that BPA caused oxidative stress which eventually triggered LKB1/AMPK mediated autophagy and maintains cellular energy balance by mitophagic removal of unhealthy mitochondria in primary rat hepatocytes.

Entrenching role of cell cycle checkpoints and autophagy for maintenance of genomic integrity.

Genomic integrity of the cell is crucial for the successful transmission of genetic information to the offspring and its survival. Persistent DNA damage induced by endogenous and exogenous agents leads to various metabolic manifestations. To combat this, eukaryotes have developed complex DNA damage response (DDR) pathway which senses the DNA damage and activates an arsenal of enzymes for the repair of damaged DNA. The active pathways for DNA repair are nucleotide excision repair (NER), base excision repair (BER) and mismatch repair (MMR) for single-strand break repair whereas homologous recombination (HR) and non-homologous end-joining (NHEJ) for double-strand break repair. OGG1 is a DNA glycosylase which initiates BER while Mre11-Rad50-Nbs1 (MRN) protein complex is the primary responder to DSBs which gets localized to damage sites. DNA damage response is meticulously executed by three related kinases: ATM, ATR, and DNA-PK. ATM- and ATR-dependent phosphorylation of p53, Chk1, and Chk2 regulate the G1/S, intra-S, or G2/M checkpoints of the cell cycle, respectively. Autophagy is an evolutionarily conserved process that plays a pivotal role in the regulation of DNA repair and maintains the cellular homeostasis. Genotoxic stress-induced altered autophagy occurs in a P53 dependent manner which is also the master regulator of genotoxic stress. A plethora of proteins involved in autophagy is regulated by p53 which involve DRAM, DAPK, and AMPK. As evident, the mtDNA is more prone to damage than nuclear DNA because of its close proximity to the site of ROS generation. Depending on the extent of damage either the repair mechanism or mitophagy gets triggered. SIRT1 is the master regulator which directs the stress response to mitophagy. Nix, a LC3 adapter also participates in Parkin mediated mitophagy. This review highlights the intricate crosstalks between DNA damage and cell cycle checkpoints activation. The DNA damage mediated regulation of autophagy and mitophagy is also reviewed in detail.

Berbamine induced AMPK activation regulates mTOR/SREBP-1c axis and Nrf2/ARE pathway to allay lipid accumulation and oxidative stress in steatotic HepG2 cells.

Non-alcoholic fatty liver disease is emanating as a global cataclysm. This study was designed to investigate the antioxidative, anti-inflammatory and fat metabolism-regulating potential of berbamine (BBM), a natural bis-benzylisoquinoline alkaloid. BBM attenuated intracellular lipid accumulation in oleic-acid exposed HepG2 cells (0.5 mM) by inhibiting fatty acid uptake, lipogenesis, and promoting fatty acid ß-oxidation by activating AMP-activated kinase (AMPK) and peroxisome proliferator-activated receptor (PPAR)-α. Berbamine (5 µM) induced AMPK activation (P < 0.001) via LKB1 (Ser-428) and elevated AMP:ATP ratio (P < 0.001). AMPK activation negatively regulated mTOR and also constrained the nuclear translocation of SREBP-1c and inhibited the lipogenic proteins, stearoyl-CoA desaturase-1 (SCD-1) and fatty acid synthase (FAS) (P < 0.001). BBM stimulated nuclear translocation of redox-sensitive nuclear factor erythroid-2-related factor-2 (Nrf2) and increased hepatic expression of Nrf2 responsive enzymes, HO-1 and Nqo-1. BBM treatment reduced the oxidative burst and pro-inflammatory responses by significantly enhancing hepatic antioxidant defenses [SOD (P < 0.001), catalase (P < 0.001) and cellular glutathione (P < 0.01)] and diminishing NF-κB regulated pro-inflammatory cytokines (TNF-α, and IL-6) levels respectively. TEM analysis confirmed the disruption of mitochondrial structure and reduction in mitochondrial size (50.97%, P < 0.001) in steatotic HepG2 cells which was significantly prevented by 5 µM BBM treatment (71.84% as compared to control, P < 0.01). Pre-treatment of Compound C (AMPK inhibitor, 25 µM) greatly repressed the anti-steatotic properties exhibited by BBM confirming the involvement of AMPK signaling pathway. In summary, the results manifest that BBM reduces intracellular lipid accumulation via AMPK/mTOR/SREBP-1c axis mediated regulation of lipid metabolism and upsurged nuclear stability of Nrf2 by promoting AMPK/Nrf2 association to ameliorate oxidative stress/proinflammatory response.

Bacopa monnieri modulates antioxidant responses in brain and kidney of diabetic rats.

Role of oxidative stress has been reported in various diabetic complications including neuropathy, nephropathy and cardiopathy. This study was undertaken to evaluate the protective effect of Bacopa monnieri, a medicinal plant, on tissue antioxidant defense system and lipid peroxidative status in streptozotocin-induced diabetic rats. Extract of B. monnieri was administered orally, once a day for 15 days (at doses 50, 125 and 250mg/(kgbw)) to diabetic rats. Activity of antioxidant enzymes (SOD, Catalase, and GPx), levels of GSH and lipid peroxidation were estimated in kidney, cerebrum, cerebellum and midbrain of diabetic rats and compared to reference drug, Glibenclamide. Administration of plant extract to diabetic rats showed significant reversal of disturbed antioxidant status and peroxidative damage. Significant increase in SOD, CAT, GPx activity and levels of GSH was observed in extract treated diabetic rats. The present study indicates that extract of B. monnieri modulates antioxidant activity, and enhances the defense against ROS generated damage in diabetic rats.

Emerging role of Unfolded Protein Response (UPR) mediated proteotoxic apoptosis in diabetes.

Endoplasmic reticulum (ER) is a crucial single membrane organelle that acts as a quality control system for cellular proteins as it is intricately involved in their synthesis, folding and trafficking to the respective targets. Type 2 diabetes is characterized by enhanced blood glucose level that promotes insulin resistance and hampers cellular glucose metabolism. Hyperglycemia provokes mitochondrial ROS production and glycation of proteins which exert a tremendous load on ER for conventional refolding of misfolded/unfolded and nascent proteins that perturb ER homeostasis resulting in apoptotic cell death. Impairment in ER functions is suspected to be through specific ER membrane-bound proteins known as Unfolded Protein Response (UPR) sensor proteins. Conformational changes in these proteins induce oligomerization and cross-autophosphorylation which facilitate processes required for the restoration of ER homeostatic imbalance. Multiple studies have reported the involvement of UPR mediated autophagy and apoptotic pathways in the progression of metabolic disorders including diabetes, cardiac ischemia/reperfusion injury and hypoxia-mediated cell death. In this review, the involvement of UPR pathways in the progression of diabetes associated complications have been addressed, which underscores molecular crosstalks during neuropathy, nephropathy, hepatic injury and retinopathy. A better understanding of these molecular interventions may reveal advanced therapeutic approaches for preventing diabetic comorbidities. The article also highlights the importance of phytochemicals that are emerging as novel ER stress inhibitors and are being explored for targeted interaction in preventing cell death responses during diabetes.