The role of fenugreek seed extract in alleviating pancreatic toxic effects and altering glucose homeostasis induced by acetamiprid via modulation of oxidative stress, apoptosis, and autophagy.

Acetamiprid (ACMP) is a second-generation neonicotinoid that has been extensively used in the last few years. The present study examined the toxic effects of ACMP on the pancreas and glucose homeostasis through the evaluation of histological and biochemical changes and the possible ameliorative role of fenugreek seed extract (FG). Fifty adult albino rats were divided into 5 groups: negative control, positive control, FG-treated, ACMP-treated, and ACMP + FG-treated groups by oral gavage for 12 weeks. The ACMP-treated group highlighted significant elevations in plasma glucose, glycosylated haemoglobin levels (HbA1c), serum amylase, and serum lipase, along with a decrease in plasma insulin levels. In addition, significant increases in tumour necrosis factor- alpha (TNF-α) and malondialdehyde (MDA) were associated with reductions in the levels of interleukin 10 (IL-10), glutathione peroxidase, and catalase. Moreover, glucose-6-phosphatase and glycogen phosphorylase were significantly increased, with a significant reduction in hexokinase and liver glycogen stores. These biochemical changes were associated with histological changes in pancreatic sections stained by haematoxylin and eosin, Masson stain, and Orcein stain. ACMP-treated cells showed a marked reduction in ß- cell immune reactivity to insulin, with pronounced p53, and beclin 1 immune expression. The use of FG with ACMP induced partial protection except for hexokinase and glycogen phosphorylase.

Juglone promotes antitumor activity against prostate cancer via suppressing glycolysis and oxidative phosphorylation.

The treatments currently used for prostate cancer (PC) do not meet clinical needs, and thus, new therapies with greater effectiveness are urgently required. Metabolic reprogramming of tumor cells is emerging as an exciting field for cancer therapy. Although the Warburg effect is a common feature of glucose metabolism in many cancers, PC cells have a unique metabolic phenotype. Non-neoplastic prostate cells show reduced oxidative phosphorylation (OXPHOS) because large, accumulated zinc inhibits citrate oxidation. During transformation, there are low levels of zinc in PC cells, and the tricarboxylic acid (TCA) cycle is reactivated. However, metastatic PC exhibits the Warburg effect. Due to metabolic differences in prostate tissue, targeting metabolic alterations in PC cells is an attractive therapeutic strategy. In this study, we investigated the effect of juglone on energy metabolism in PC cells. We found that juglone inhibited cell proliferation and induced apoptosis. Mechanistically, we demonstrated that juglone suppressed OXPHOS and glycolysis due to its inhibition of hexokinase (HK), phosphofructokinase (PFK), and pyruvate kinase (PK) activity. Furthermore, downregulation of PFK and PK, but not HK contributed to the inhibition of these enzyme activities. The current study indicates that further development of juglone for PC treatment would be beneficial.

Role of Glycolysis and Fatty Acid Synthesis in the Activation and T Cell-Modulating Potential of Dendritic Cells Stimulated with a TLR5-Ligand Allergen Fusion Protein.

Trained immune responses, based on metabolic and epigenetic changes in innate immune cells, are de facto innate immune memory and, therefore, are of great interest in vaccine development. In previous studies, the recombinant fusion protein rFlaA:Betv1, combining the adjuvant and toll-like receptor (TLR)5-ligand flagellin (FlaA) and the major birch pollen allergen Bet v 1 into a single molecule, significantly suppressed allergic sensitization in vivo while also changing the metabolism of myeloid dendritic cells (mDCs). Within this study, the immune-metabolic effects of rFlaA:Betv1 during mDC activation were elucidated. In line with results for other well-characterized TLR-ligands, rFlaA:Betv1 increased glycolysis while suppressing oxidative phosphorylation to different extents, making rFlaA:Betv1 a suitable model to study the immune-metabolic effects of TLR-adjuvanted vaccines. In vitro pretreatment of mDCs with cerulenin (inhibitor of fatty acid biosynthesis) led to a decrease in both rFlaA:Betv1-induced anti-inflammatory cytokine Interleukin (IL) 10 and T helper cell type (TH) 1-related cytokine IL-12p70, while the pro-inflammatory cytokine IL 1ß was unaffected. Interestingly, pretreatment with the glutaminase inhibitor BPTES resulted in an increase in IL-1ß, but decreased IL-12p70 secretion while leaving IL-10 unchanged. Inhibition of the glycolytic enzyme hexokinase-2 by 2-deoxyglucose led to a decrease in all investigated cytokines (IL-10, IL-12p70, and IL-1ß). Inhibitors of mitochondrial respiration had no effect on rFlaA:Betv1-induced IL-10 level, but either enhanced the secretion of IL-1ß (oligomycin) or decreased IL-12p70 (antimycin A). In extracellular flux measurements, mDCs showed a strongly enhanced glycolysis after rFlaA:Betv1 stimulation, which was slightly increased after respiratory shutdown using antimycin A. rFlaA:Betv1-stimulated mDCs secreted directly antimicrobial substances in a mTOR- and fatty acid metabolism-dependent manner. In co-cultures of rFlaA:Betv1-stimulated mDCs with CD4+ T cells, the suppression of Bet v 1-specific TH2 responses was shown to depend on fatty acid synthesis. The effector function of rFlaA:Betv1-activated mDCs mainly relies on glycolysis, with fatty acid synthesis also significantly contributing to rFlaA:Betv1-mediated cytokine secretion, the production of antimicrobial molecules, and the modulation of T cell responses.

Kaempferol impairs aerobic glycolysis against melanoma metastasis via inhibiting the mitochondrial binding of HK2 and VDAC1.

Metastasis is the leading cause of death in melanoma patients. Aerobic glycolysis is a common metabolic feature in tumor and is closely related to cell growth and metastasis. Kaempferol (KAM) is one of the active ingredients in the total flavonoids of Chinese traditional medicine Sparganii Rhizoma. Studies have shown that it interferes with the cell cycle, apoptosis, angiogenesis and metastasis of tumor cells, but whether it can affect the aerobic glycolysis of melanoma is still unclear. Here, we explored the effects and mechanisms of KAM on melanoma metastasis and aerobic glycolysis. KAM inhibited the migration and invasion of A375 and B16F10 cells, and reduced the lung metastasis of melanoma cells. Extracellular acidification rates (ECAR) and glucose consumption were obviously suppressed by KAM, as well as the production of ATP, pyruvate and lactate. Mechanistically, the activity of hexokinase (HK), the first key kinase of aerobic glycolysis, was significantly inhibited by KAM. Although the total protein expression of HK2 was not significantly changed, the binding of HK2 and voltage-dependent anion channel 1 (VDAC1) on mitochondria was inhibited by KAM through AKT/GSK-3ß signal pathway. In conclusion, KAM inhibits melanoma metastasis via blocking aerobic glycolysis of melanoma cells, in which the binding of HK2 and VDAC1 on mitochondria was broken.

Hypothermia Prevents Cardiac Dysfunction during Acute Ischemia Reperfusion by Maintaining Mitochondrial Bioenergetics and by Promoting Hexokinase II Binding to Mitochondria.

Background: Hypothermia (H), cardioplegia (CP), and both combined (HCP) are known to be protective against myocardial ischemia reperfusion (IR) injury. Mitochondria have molecular signaling mechanisms that are associated with both cell survival and cell death. In this study, we investigated the dynamic changes in proapoptotic and prosurvival signaling pathways mediating H, CP, or HCP-induced protection of mitochondrial function after acute myocardial IR injury. Methods: Rats were divided into five groups. Each group consists of 3 subgroups based on a specific reperfusion time (5, 20, or 60 min) after a 25-min global ischemia. The time control (TC) groups were not subjected to IR but were perfused with 37 °C Krebs-Ringer's (KR) buffer, containing 4.5 mM K+, in a specific perfusion protocol that corresponded with the duration of each IR protocol. The IR group (control) was perfused for 20 min with KR, followed by 25-min global ischemia, and then KR reperfusion for 5, 20, or 60 min. The treatment groups were exposed to 17 °C H, 37 °C CP (16 mM K+), or HCP (17 °C + CP) for 5 min before ischemia and for 2 min on reperfusion before switching to 37 °C KR perfusion for the remainder of each of the reperfusion times. Cardiac function and mitochondrial redox state (NADH/FAD) were monitored online in the ex vivo hearts before, during, and after ischemia. Mitochondria were isolated at the end of each specified reperfusion time, and changes in O2 consumption, membrane potential (ΔΨ m), and Ca2+ retention capacity (CRC) were assessed using complex I and complex II substrates. In another set of hearts, mitochondrial and cytosolic fractions were isolated after a specified reperfusion time to conduct western blot assays to determine hexokinase II (HKII) and Bax binding/translocation to mitochondria, cytosolic pAkt levels, and cytochrome c (Cyto-c) release into the cytosol. Results: H and HCP were more protective of mitochondrial integrity and, concomitantly, cardiac function than CP alone; H and HCP improved post-ischemic cardiac function by (1) maintaining mitochondrial bioenergetics, (2) maintaining HKII binding to mitochondria with an increase in pAkt levels, (3) increasing CRC, and (4) decreasing Cyto-c release during reperfusion. Bax translocation/binding to mitochondria was unaffected by any treatment, regardless of cardiac functional recovery. Conclusions: Hypothermia preserved mitochondrial function and cardiac function, in part, by maintaining mitochondrial bioenergetics, by retaining HKII binding to mitochondria via upstream pAkt, and by reducing Cyto-c release independently of Bax binding to mitochondria.

The HDAC Inhibitor Butyrate Impairs ß Cell Function and Activates the Disallowed Gene Hexokinase I.

Histone deacetylase (HDAC) inhibitors such as butyrate have been reported to reduce diabetes risk and protect insulin-secreting pancreatic ß cells in animal models. However, studies on insulin-secreting cells in vitro have found that butyrate treatment resulted in impaired or inappropriate insulin secretion. Our study explores the effects of butyrate on insulin secretion by BRIN BD-11 rat pancreatic ß cells and examined effects on the expression of genes implicated in ß cell function. Robust HDAC inhibition with 5 mM butyrate or trichostatin A for 24 h in ß cells decreased basal insulin secretion and content, as well as insulin secretion in response to acute stimulation. Treatment with butyrate also increased expression of the disallowed gene hexokinase I, possibly explaining the impairment to insulin secretion, and of TXNIP, which may increase oxidative stress and ß cell apoptosis. In contrast to robust HDAC inhibition (>70% after 24 h), low-dose and acute high-dose treatment with butyrate enhanced nutrient-stimulated insulin secretion. In conclusion, although protective effects of HDAC inhibition have been observed in vivo, potent HDAC inhibition impairs ß cell function in vitro. The chronic low dose and acute high dose butyrate treatments may be more reflective of in vivo effects.

Molecular docking and simulation studies of phytocompounds derived from Centella asiatica and Andrographis paniculata against hexokinase II as mitocan agents.

Hexokinase II (HK2), a glycolytic enzyme is commonly overexpressed in most cancer types. The overexpression of HK2 is reported to promote the survival of cancer cells by facilitating the constant ATP generation and protecting the cancer cell against apoptotic cell death. Hence, HK2 is considered as potential target of many mitochondria targeting anticancerous agents (referred to as mitocans). Most of the existing mitocans are synthetic and hence such compounds are observed to exhibit adverse effects, witnessed through many experimental outcomes. These limitations necessitates hunting for an alternative source of mitocans with minimum/no side effects. The need for an alternative therapy points towards the ethnomedicinal herbs, known for their minimal side effects and effectiveness. Henceforth recent studies have put forth the effort to utilize anticancer herbs in formulating naturally derived mitocans as an add-on to improve cancer therapeutics. So, our study aims to explore the HK2 targeting potential of phytocompounds from the selected anticancerous herbs Andrographis paniculata (AP) and Centella asiatica (CA). 60 phytocompounds collectively from CA and AP were docked against HK2 and drug-likeness prediction of the selected phytocompounds was performed to screen the best possible ligand for HK2. Furthermore, the docked complexes were subjected to molecular dynamics simulations (MDS) to analyse the molecular mechanism of protein-ligand interactions. The results of the study suggest that the natural compounds asiatic acid and bayogenin (from CA) and andrographolide (from AP) can bepotential natural mitocans by targeting HK2. Further experimental studies (in-vitro and in-vivo) are required to validate the results.

Antitumor Effect of Morusin via G1 Arrest and Antiglycolysis by AMPK Activation in Hepatocellular Cancer.

Though Morusin isolated from the root of Morus alba was known to have antioxidant, anti-inflammatory, antiangiogenic, antimigratory, and apoptotic effects, the underlying antitumor effect of Morusin is not fully understood on the glycolysis of liver cancers. Hence, in the current study, the antitumor mechanism of Morusin was explored in Hep3B and Huh7 hepatocellular carcninomas (HCC) in association with glycolysis and G1 arrest. Herein, Morusin significantly reduced the viability and the number of colonies in Hep3B and Huh7 cells. Moreover, Morusin significantly increased G1 arrest, attenuated the expression of cyclin D1, cyclin D3, cyclin E, cyclin-dependent kinase 2 (CDK2), cyclin-dependent kinase 4 (CDK4), and cyclin-dependent kinase 6 (CDK6) and upregulated p21 and p27 in Hep3B and Huh7 cells. Interestingly, Morusin significantly activated phosphorylation of the adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK)/acetyl-CoA carboxylase (ACC) but attenuated the expression of the p-mammalian target of protein kinase B (AKT), rapamycin (mTOR), c-Myc, hexokinase 2(HK2), pyruvate kinases type M2 (PKM2), and lactate dehydrogenase (LDH) in Hep3B and Huh7 cells. Consistently, Morusin suppressed lactate, glucose, and adenosine triphosphate (ATP) in Hep3B and Huh7 cells. Conversely, the AMPK inhibitor compound C reduced the ability of Morusin to activate AMPK and attenuate the expression of p-mTOR, HK2, PKM2, and LDH-A and suppressed G1 arrest induced by Morusin in Hep3B cells. Overall, these findings suggest that Morusin exerts an antitumor effect in HCCs via AMPK mediated G1 arrest and antiglycolysis as a potent dietary anticancer candidate.

Role of zonulin and GLP-1/DPP-IV in alleviation of diabetes mellitus by peptide/polypeptide fraction of Aloe vera in streptozotocin- induced diabetic wistar rats.

ETHNO-PHARMACOLOGICAL RELEVANCE: The genus Aloe has a long history of usage in medicine. Aloe barbadensis Miller, commonly known as Aloe vera, is said to possess anti-diabetic, anti-inflammatory, anti-cancer, anti-microbial, immunomodulation, wound healing properties. AIM OF THE STUDY: In diabetes mellitus, loss in intestinal permeability is observed with high levels of zonulin and low levels of glucagon-like peptide-1 (GLP-1) leading to hyperglycemia. The aim of the study was to understand the role of peptide/polypeptide fraction (PPF) of Aloe vera in the alleviation of diabetes through maintaining the intestinal permeability by regulating the zonulin and GLP-1 levels. MATERIALS AND METHODS: The PPF of Aloe vera was obtained through trichloroacetic acid precipitation. The anti-diabetic potential of the PPF was tested through DPP-IV inhibition, glucose diffusion assay, and by using Rin-m5F cells. The anti-diabetic potential of the PPF was tested at a dose of 0.450 mg/kg bw in vivo using streptozotocin-induced diabetic Wistar rats. The effect of PPF on fasting plasma glucose, insulin, glucagon, Zonulin, GLP-1, DPP-IV, levels were studied in diabetic rats. The histopathological studies of the pancreas, small intestine, and liver were carried out for organ-specific effects. RESULTS: PPF has the ability to reduce fasting plasma glucose levels with concomitant increase in insulin levels in streptozotocin-induced diabetic rats. It was also observed that increase in GLP-1 levels with a decrease in DPP-IV and zonulin levels thereby mitigating the loss of intestinal permeability. These findings correlate with the small intestine's histopathological observation where the excessive proliferation of epithelium in the small intestine of diabetic rats was reduced after PPF treatment. CONCLUSION: These results suggest that the PPF of Aloe vera alleviates diabetes through islet cell rejuvenation via GLP-1/DPP-IV pathway and thereby suggesting the usage of PPF as an alternate medicine for diabetes mellitus with the possibility to reduce the intestinal permeability and zonulin levels.

Bromide impairs the circadian clock and glycolytic homeostasis via disruption of autophagy in rat H9C2 cardiomyocytes.

BACKGROUND: Trace elements function as essential cofactors that are involved in various biochemical processes in mammals. Autophagy is vital for nutrient supplement, which is an important Zeitegber for the circadian homeostasis in heart. Here, we considered the possibility that autophagy, as well as the cardiomyocyte clock and glycolysis are interlinked. Detrimental effects were observed when cardiac system is exposed to bromine containing drugs. This study investigated the effects and mechanisms of bromide on the circadian clock and glycolytic metabolism of H9C2 cardiomyocytes. RESULTS: In the present study, bromide does not affect cell viability and apoptosis of H9C2 cardiomyocytes. Bromide dampens the clock and glycolytic (Hk2 and Pkm2) gene expression rhythmicity in a dose-dependent manner. Additionally, bromide inhibits autophagic process in H9C2 cardiomyocytes. In contrast, rapamycin (an autophagy inducer) dramatically restores the inhibitory effect of NaBr on the mRNA expression levels of clock genes (Bmal1, Cry1 and Rorα) and glycolytic genes (Hk2 and Pkm2). CONCLUSIONS: Our results reveal that bromide represses the clock and glycolytic gene expression patterns, partially through inhibition of autophagy.

Aristolochic acid mutational signature defines the low-risk subtype in upper tract urothelial carcinoma.

Rationale: Dietary exposure to aristolochic acids and similar compounds (collectively, AA) is a significant risk factor for nephropathy and subsequent upper tract urothelial carcinoma (UTUC). East Asian populations, who have a high prevalence of UTUC, have an unusual genome-wide AA-induced mutational pattern (COSMIC signature 22). Integrating mutational signature analysis with clinicopathological information may demonstrate great potential for risk ranking this UTUC subtype. Methods: We performed whole-genome sequencing (WGS) on 90 UTUC Chinese patients to extract mutational signatures. Genome sequencing data for urinary cell-free DNA from 26 UTUC patients were utilized to noninvasively identify the mutational signatures. Genome sequencing for primary tumors on 8 out of 26 patients was also performed. Metastasis-free survival (MFS) and cancer-specific survival (CSS) were measured using Kaplan-Meier methods. Results: Data analysis showed that a substantial proportion of patients harbored the AA mutational signature and were associated with AA-containing herbal drug intake, female gender, poor renal function, and multifocality. Field cancerization was found to partially contribute to multifocality. Nevertheless, AA Sig subtype UTUC patients exhibited favorable outcomes of CSS and MFS compared to the No-AA Sig subtype. Additionally, AA Sig subtype patients showed a higher tumor mutation burden, higher numbers of predicted neoantigens, and infiltrating lymphocytes, suggesting the potential for immunotherapy. We also confirmed the AA signature in AA-treated human renal tubular HK-2 cells. Notably, the AA subtype could be ascertained using a clinically applicable sequencing strategy (low coverage) in both primary tumors and urinary cell-free DNA as a basis for therapy selection. Conclusion: The AA mutational signature as a screening tool defines low-risk UTUC with therapeutic relevance. The AA mutational signature, as a molecular prognostic marker using either ureteroscopy and/or urinary cell-free DNA, is especially useful for diagnostic uncertainty when kidney-sparing treatment and/or immune checkpoint inhibitor therapy were considered.

Therapeutic targeting of YY1/MZF1 axis by MZF1-uPEP inhibits aerobic glycolysis and neuroblastoma progression.

As a hallmark of metabolic reprogramming, aerobic glycolysis contributes to tumorigenesis and aggressiveness. However, the mechanisms and therapeutic strategies regulating aerobic glycolysis in neuroblastoma (NB), one of leading causes of cancer-related death in childhood, still remain elusive. Methods: Transcriptional regulators and their downstream glycolytic genes were identified by a comprehensive screening of publicly available datasets. Dual-luciferase, chromatin immunoprecipitation, real-time quantitative RT-PCR, western blot, gene over-expression or silencing, co-immunoprecipitation, mass spectrometry, peptide pull-down assay, sucrose gradient sedimentation, seahorse extracellular flux, MTT colorimetric, soft agar, matrigel invasion, and nude mice assays were undertaken to explore the biological effects and underlying mechanisms of transcriptional regulators in NB cells. Survival analysis was performed by using log-rank test and Cox regression assay. Results: Transcription factor myeloid zinc finger 1 (MZF1) was identified as an independent prognostic factor (hazard ratio=2.330, 95% confidence interval=1.021 to 3.317), and facilitated glycolysis process through increasing expression of hexokinase 2 (HK2) and phosphoglycerate kinase 1 (PGK1). Meanwhile, a 21-amino acid peptide encoded by upstream open reading frame of MZF1, termed as MZF1-uPEP, bound to zinc finger domain of Yin Yang 1 (YY1), resulting in repressed transactivation of YY1 and decreased transcription of MZF1 and downstream genes HK2 and PGK1. Administration of a cell-penetrating MZF1-uPEP or lentivirus over-expressing MZF1-uPEP inhibited the aerobic glycolysis, tumorigenesis and aggressiveness of NB cells. In clinical NB cases, low expression of MZF1-uPEP or high expression of MZF1, YY1, HK2, or PGK1 was associated with poor survival of patients. Conclusions: These results indicate that therapeutic targeting of YY1/MZF1 axis by MZF1-uPEP inhibits aerobic glycolysis and NB progression.

WD-3 inhibits the proliferation of breast cancer cells by regulating the glycolytic pathway.

Number 3 Prescription (WD-3) is an herbal remedy used in traditional Chinese medicine that has been shown to improve the outcomes of patients with advanced colon and gastric cancers. This study aimed to investigate the effect of WD-3 on proliferation, glycolysis, and hexokinase 2 expression in breast cancer cells. Four breast cancer cell lines (MDA-MB-231, BT-549, MCF-7, and MCF-7/ADR-RES) were treated with different concentrations of WD-3 compared with blank control (phosphate-buffered saline). Each of the breast cancer cell lines was also divided into WD-3, paclitaxel, and blank control group. Cell proliferation and morphology were assessed by MTT assay, nuclear Hoechst 33258 staining, or immunofluorescence. Apoptosis was analyzed by flow cytometry. High performance liquid chromatography was used for measurement of ATP, ADP, and AMP. Hexokinase 2 expression was analyzed by Western blot and quantitative reverse transcription PCR. WD-3 inhibited proliferation and increased apoptosis in all four breast cancer cell lines, in a dose-dependent manner. ATP and EC (energy charge) were significantly decreased in WD-3-treated BT-549 and MDA-MB-231 cells. WD-3 significantly downregulated the protein and mRNA expression of hexokinase II in BT-549 cells, however, not in the other three breast cancer cell lines. Our findings indicate that WD-3 targets the glycolytic pathway in breast cancer cells to exert its antitumor activity.

Glycogen storage in a zebrafish Pompe disease model is reduced by 3-BrPA treatment.

Pompe disease (PD) is an autosomal recessive muscular disorder caused by deficiency of the glycogen hydrolytic enzyme acid α-glucosidase (GAA). The enzyme replacement therapy, currently the only available therapy for PD patients, is efficacious in improving cardiomyopathy in the infantile form, but not equally effective in the late onset cases with involvement of skeletal muscle. Correction of the skeletal muscle phenotype has indeed been challenging, probably due to concomitant dysfunctional autophagy. The increasing attention to the pathogenic mechanisms of PD and the search of new therapeutic strategies prompted us to generate and characterize a novel transient PD model, using zebrafish. Our model presented increased glycogen content, markedly altered motor behavior and increased lysosome content, in addition to altered expression of the autophagy-related transcripts and proteins Beclin1, p62 and Lc3b. Furthermore, the model was used to assess the beneficial effects of 3-bromopyruvic acid (3-BrPA). Treatment with 3-BrPA induced amelioration of the model phenotypes regarding glycogen storage, motility behavior and autophagy-related transcripts and proteins. Our zebrafish PD model recapitulates most of the defects observed in human patients, proving to be a powerful translational model. Moreover, 3-BrPA unveiled to be a promising compound for treatment of conditions with glycogen accumulation.

Deficiency of rice hexokinase HXK5 impairs synthesis and utilization of starch in pollen grains and causes male sterility.

There is little known about the function of rice hexokinases (HXKs) in planta. We characterized hxk5-1, a Tos17 mutant of OsHXK5 that is up-regulated in maturing pollen, a stage when starch accumulates. Progeny analysis of self-pollinated heterozygotes of hxk5-1 and reciprocal crosses between the wild-type and heterozygotes revealed that loss of HXK5 causes male sterility. Homozygous hxk5-1, produced via anther culture, and additional homozygous hxk5-2, hxk5-3 and hxk5-4 lines created by CRISPR/Cas9 confirmed the male-sterile phenotype. In vitro pollen germination ability and in vivo pollen tube growth rate were significantly reduced in the hxk5 mutant pollen. Biochemical analysis of anthers with the mutant pollen revealed significantly reduced hexokinase activity and starch content, although they were sufficient to produce some viable seed. However, the mutant pollen was unable to compete successfully against wild-type pollen. Expression of the catalytically inactive OsHXK5-G113D did not rescue the hxk5 male-sterile phenotype, indicating that its catalytic function was responsible for pollen fertility, rather than its role in sugar sensing and signaling. Our results demonstrate that OsHXK5 contributes to a large portion of the hexokinase activity necessary for the starch utilization pathway during pollen germination and tube growth, as well as for starch biosynthesis during pollen maturation.

Aqueous extract of Digitaria exilis grains ameliorate diabetes in streptozotocin-induced diabetic male Wistar rats.

ETHNOPHARMACOLOGICAL RELEVANCE: The absence of scientific data on the age long folkloric use of Digitaria exilis grains by sufferers of diabetes prompted the present investigation. This study was aimed at evaluating the antidiabetic activity of aqueous extract of Digitaria exilis grains in streptozotocin (STZ)-induced diabetic rats. MATERIAL AND METHODS: Forty two male rats (166.43 ±â€¯3.32 g) were completely randomized into six groups (A-F) of 7 animals each. Animals in group A (control) were administered 0.5 ml of distilled water while those in groups B, C, D, E and F which were induced with diabetes mellitus (by intraperitoneal administration of 60 mg/kg body weight of STZ) were also administered distilled water, 50 mg/kg body weight of metformin (a reference antidiabetic drug), 50, 100 and 200 mg/kg body weight of aqueous extract of D. exilis grains respectively, twice daily for 14 days. Blood glucose levels and some relevant biomolecules were determined 14 days post-administration. RESULTS: Alkaloids, flavonoids, saponins, tannins, anthraquinones, terpenoids, cardiac glycosides, phlobatannins, phenolics and cardenolides were detected in the extract with alkaloids (30.20 mg/ml) occurring the most and phlobatannins (0.22 mg/ml) the least. Streptozotocin significantly (p < 0.05) increased the levels of blood glucose, serum albumin, urea, creatinine and cholesterol, activities of glucose-6-phosphatase and fructose-1,6-bisphosphatase in the liver and intake of feed and water. Body weight, weight of pancreas, pancreatic insulin, liver glycogen content, red blood cell and white blood cell and their related indices, liver hexokinase and phosphofructokinase activities were significantly reduced by STZ. In contrast, the extract significantly reversed all those STZ-treatment induced changes with the 200 mg/kg body weight of the extract producing profound values that compared favourably with the distilled water treated non-diabetic animals and metformin treated diabetic animals. CONCLUSION: Overall, this study revealed that Digitaria exilis grains possess antidiabetic activity via increased insulin secretion, as plasma concentrations of insulin were not determined, enhanced activities of hexokinase and phosphofuctokinase and repletion of hepatic glycogen content.

The Black Truffle, Tuber melanosporum (Ascomycetes), Ameliorates Hyperglycemia and Regulates Insulin Signaling Pathway in STZ-Induced Diabetic Rats.

Black truffle mushroom, Tuber melanosporum, is effective in treating various symptoms associated with diabetes mellitus such as hyperglycemia, oxidative stress, and hyperlipidemia and is used as traditional medicine. The aim of our study is to elucidate the antidiabetic potential of T. melanosporum. Male albino Wistar rats were administered a single dose of STZ (40 mg/kg b.w.) to induce mild diabetes mellitus (DM). After the confirmation of hyperglycemia, rats were treated with three different doses of truffle extract (TE) (200, 400, and 600 mg/kg b.w.) for the duration of 45 days. The various tissues were collected at the end of the study. The levels of glucose, oral glucose tolerance test, insulin, hexokinase, glucose 6 phosphatase, and fructose-1,6-bisphosphatase, and regulation of insulin signaling genes were quantified. The results showed that STZ- induced rats have a higher blood glucose level and a lower insulin level compared with the control groups and TE treated groups. Results also reveal that STZ suppressed the expressions of insulin signaling genes in diabetic rats and TE treatment resulted in a positive regulation of the insulin signaling pathway. The results of TE are similar to the results attained in glibenclamide (GB) group rats. Overall, the study provides scientific evidence for the medicinal properties of black truffle; future clinical studies can warrant a potential antidiabetic drug in the form of diet.

Antihyperglycaemia and related gene expressions of aqueous extract of Gongronema latifolium leaf in alloxan-induced diabetic rats.

Context: Gongronema latifolium Benth (Asclepiadaceae) has been highly utilized in controlling diabetes mellitus traditionally in the eastern part of Nigeria. Objectives: Antihyperglycaemic and related gene expressions of aqueous extract of Gongronema latifolium leaf in alloxan-induced diabetic rats. Materials and methods: Forty-eight female Wistar rats were induced intraperitoneally using alloxan (150 mg/kg body weight). The rats were separated into six groups (n = 8) as follows: non-diabetic control, diabetic control, diabetic rats administered 5 mg/kg body weight of metformin, and diabetic rats administered 6.36, 12.72 and 25.44 mg/kg body weight (ethnobotanical doses) of G. latifolium orally daily. On the 14th day, the animals were sacrificed and different antihyperglycaemic parameters were evaluated as well as its related gene expressions. Results: Diabetic rats administered three doses of aqueous extract of G. latifolium significantly (p < 0.05) lowered the fasting blood glucose, glycated haemoglobin, serum lipid profiles, lipid peroxidation (5.62-1.2 µ/mg protein) levels, as well as gene expression of glucose-6-phosphatase in alloxan-induced diabetic rats. There was a significant (p < 0.05) increase in the liver glycogen content (16.23-112.5 mg glucose/2 g), antioxidant enzymes activities, glucose transporter (GLUT-2 and GLUT-4) levels and relative gene expression of hexokinase in diabetic rats administered different doses of aqueous extract of G. latifolium. Discussion and conclusions: It can be deduced that the aqueous extract of G. latifolium leaf at these doses may be useful in managing diabetes mellitus and its associated complications. Therefore, this extract may be a potent antidiabetic agent in clinical therapy in the future.

Antidiabetic property of miracle fruit plant (Synsepalum dulcificum Shumach. & Thonn. Daniell) leaf extracts in fructose-fed streptozotocin-injected rats via anti-inflammatory activity and inhibition of carbohydrate metabolizing enzymes.

ETHNOPHARMACOLOGICAL RELEVANCE: Miracle fruit plant (Synsepalum dulcificum) has many applications in folk medicine. The leaves are used traditionally to treat diabetes and other diseases. The antidiabetic potential of Synsepalum dulcificum leaves in rats has been studied but the mechanisms involved are yet to be elucidated. AIM: The present study aimed to provide insight into the antidiabetic mechanisms of methanol and flavonoid-rich leaf extracts of Synsepalum dulcificum (MSD and FSD, respectively). METHODS: In vivo effects of administering 30 mg/kg or 60 mg/kg MSD and FSD for 21 consecutive days to rats after type II diabetes was induced through 14 days of fructose feeding and injection of one dose of streptozotocin, were assessed. Glibenclamide (5 mg/kg) served as the reference drug. In addition, in vitro inhibitory activity of MSD and FSD on the carbohydrate metabolizing enzymes, α-amylase and glucokinase, were evaluated, with acarbose as the reference drug. Moreover, in silico analyses to elucidate the contribution of key polyphenolics to the antidiabetic activity of the extracts through docking with glucokinase were performed. RESULTS: MSD and FSD significantly reduced HbA1c and serum levels of interleukin-6 and TNF-α (p < 0.05) in diabetic animals. Conversely, serum level of insulin and hepatic hexokinase activity were increased (p < 0.05) in extract treated groups. Both extracts showed α-amylase and α-glucosidase inhibitory activities. Quercetin, caffeic acid and chlorogenic acid in extracts showed strong binding affinities with glucokinase in the molecular docking analyses. CONCLUSION: Results from this study indicate that increased insulin synthesis, reduction of inflammation and inhibition of carbohydrate metabolizing enzymes are likely mechanisms by which MSD and FSD exert antidiabetic action in type II diabetic rats.

BACH1 Stabilization by Antioxidants Stimulates Lung Cancer Metastasis.

For tumors to progress efficiently, cancer cells must overcome barriers of oxidative stress. Although dietary antioxidant supplementation or activation of endogenous antioxidants by NRF2 reduces oxidative stress and promotes early lung tumor progression, little is known about its effect on lung cancer metastasis. Here, we show that long-term supplementation with the antioxidants N-acetylcysteine and vitamin E promotes KRAS-driven lung cancer metastasis. The antioxidants stimulate metastasis by reducing levels of free heme and stabilizing the transcription factor BACH1. BACH1 activates transcription of Hexokinase 2 and Gapdh and increases glucose uptake, glycolysis rates, and lactate secretion, thereby stimulating glycolysis-dependent metastasis of mouse and human lung cancer cells. Targeting BACH1 normalized glycolysis and prevented antioxidant-induced metastasis, while increasing endogenous BACH1 expression stimulated glycolysis and promoted metastasis, also in the absence of antioxidants. We conclude that BACH1 stimulates glycolysis-dependent lung cancer metastasis and that BACH1 is activated under conditions of reduced oxidative stress.