Carbazole alkaloids with potential cytotoxic activities targeted on PCK2 protein from Murraya microphylla.

From the 95% aqueous ethanol extract of Murraya microphylla, five pairs of new carbazole alkaloid enantiomers, (+/-)-microphylines N-R (1a/1b-5a/5b), were isolated, together with 20 known carbazole alkaloids. The structures of the new compounds were determined by the HRMS and NMR spectroscopic data, along with the calculated electronic circular dichroism (ECD) and Mo2(AcO)4-induced CD data. The known compound (+)-mahanine (21) showed significant cytotoxicities against Du145, HepG2, HeLa, and HCT-116 cell lines, and its possible mechanism was deduced to target on phosphoenolpyruvate carboxykinase 2 (PCK2) protein via surface plasmon resonance (SPR) and molecular docking.

Pharmacology and preclinical validation of a novel anticancer compound targeting PEPCK-M.

BACKGROUND: Phosphoenolpyruvate carboxykinase (PEPCK) catalyzes the decarboxylation of oxaloacetate to phosphoenolpyruvate. The mitochondrial isozyme, PEPCK-M is highly expressed in cancer cells, where it plays a role in nutrient stress response. To date, pharmacological strategies to target this pathway have not been pursued. METHODS: A compound embodying a 3-alkyl-1,8-dibenzylxanthine nucleus (iPEPCK-2), was synthesized and successfully probed in silico on a PEPCK-M structural model. Potency and target engagement in vitro and in vivo were evaluated by kinetic and cellular thermal shift assays (CETSA). The compound and its target were validated in tumor growth models in vitro and in murine xenografts. RESULTS: Cross-inhibitory capacity and increased potency as compared to 3-MPA were confirmed in vitro and in vivo. Treatment with iPEPCK-2 inhibited cell growth and survival, especially in poor-nutrient environment, consistent with an impact on colony formation in soft agar. Finally, daily administration of the PEPCK-M inhibitor successfully inhibited tumor growth in two murine xenograft models as compared to vehicle, without weight loss, or any sign of apparent toxicity. CONCLUSION: We conclude that iPEPCK-2 is a compelling anticancer drug targeting PEPCK-M, a hallmark gene product involved in metabolic adaptations of the tumor.

Current anti-diabetic agents and their molecular targets: A review.

Diabetes mellitus is a medical condition characterized by the body's loss of control over blood sugar. The frequency of diagnosed cases and consequential increases in medical costs makes it a rapidly growing chronic disease that threatens human health worldwide. In addition, its unnerving statistical projections are perilous to both the economy of the nation and man's life expectancy. Type-I and type-II diabetes are the two clinical forms of diabetes mellitus. Type-II diabetes mellitus (T2DM) is illustrated by the abnormality of glucose homeostasis in the body, resulting in hyperglycemia. Although significant research attention has been devoted to the development of diabetes regimens, which demonstrates success in lowering blood glucose levels, their efficacies are unsustainable due to undesirable side effects such as weight gain and hypoglycemia. Over the years, heterocyclic scaffolds have been the basis of anti-diabetic chemotherapies; hence, in this review we consolidate the use of bioactive scaffolds, which have been evaluated for their biological response as inhibitors against their respective anti-diabetic molecular targets over the past five years (2012-2017). Our investigation reveals a diverse target set which includes; protein tyrosine phosphatase 1 B (PTP1B), dipeptidly peptidase-4 (DPP-4), free fatty acid receptors 1 (FFAR1), G protein-coupled receptors (GPCR), peroxisome proliferator activated receptor-γ (PPARγ), sodium glucose co-transporter-2 (SGLT2), α-glucosidase, aldose reductase, glycogen phosphorylase (GP), fructose-1,6-bisphosphatase (FBPase), glucagon receptor (GCGr) and phosphoenolpyruvate carboxykinase (PEPCK). This review offers a medium on which future drug design and development toward diabetes management may be modelled (i.e. optimization via structural derivatization), as many of the drug candidates highlighted show promise as an effective anti-diabetic chemotherapy.

Anti-diabetic activities of cis- and trans-2,3,5,4'-tetrahydroxystilbene 2-O-ß-glucopyranoside from Polygonum multiflorum.

SCOPE: Functional foods can be used alone or in combination with existing therapies in preventing and treating type 2 diabetes (T2D). Trans-2,3,5,4'-tetrahydroxystilbene 2-O-ß-glucopyranoside (trans-THSG), a dominant bioactive compound from Polygonum multiflorum (PM)-a popular medicinal food in Asia, has attracted increasing research interests due to its strong antioxidant activity. The content of naturally occurring cis-THSG (cis-2,3,5,4'-tetrahydroxystilbene 2-O-ß-glucopyranoside) was very low in PM root, but was prepared in this study by mimicking the traditional process of PM. The anti-diabetic effects of trans- and cis-THSG were evaluated in T2D to search for more efficacious food ingredient(s). METHODS AND RESULTS: Trans-THSG was chromatographically purified from PM roots and cis-THSG was prepared with our innovative process via exposure of trans-THSG to UV-light. The anti-diabetic effects of both THSGs were tested with HFD-induced male CF-1 diabetic mice. Cis-THSG was found more effective than trans-THSG in hypoglycemic effect and in ameliorating glucose intolerance and insulin resistance. In HepG2 cells, cis-THSG also demonstrated more potent activity than trans-THSG in suppressing transcription of phosphoenopyruvate carboxykinase (PEPCK). CONCLUSION: Cis-THSG can be an enriched bioactive ingredient in PM roots from post-processing and is significantly more effective against hyperglycemia than trans-THSG. One of the effective pathways was through inhibition of PEPCK.

Neuronal Progenitor Maintenance Requires Lactate Metabolism and PEPCK-M-Directed Cataplerosis.

This study investigated the metabolic requirements for neuronal progenitor maintenance in vitro and in vivo by examining the metabolic adaptations that support neuronal progenitors and neural stem cells (NSCs) in their undifferentiated state. We demonstrate that neuronal progenitors are strictly dependent on lactate metabolism, while glucose induces their neuronal differentiation. Lactate signaling is not by itself capable of maintaining the progenitor phenotype. The consequences of lactate metabolism include increased mitochondrial and oxidative metabolism, with a strict reliance on cataplerosis through the mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M) pathway to support anabolic functions, such as the production of extracellular matrix. In vivo, lactate maintains/induces populations of postnatal neuronal progenitors/NSCs in a PEPCK-M-dependent manner. Taken together, our data demonstrate that, lactate alone or together with other physical/biochemical cues maintain NSCs/progenitors with a metabolic signature that is classically found in tissues with high anabolic capacity.

The Metabolic Regulator Histone Deacetylase 9 Contributes to Glucose Homeostasis Abnormality Induced by Hepatitis C Virus Infection.

Class IIa histone deacetylases (HDACs), such as HDAC4, HDAC5, and HDAC7, provide critical mechanisms for regulating glucose homeostasis. Here we report that HDAC9, another class IIa HDAC, regulates hepatic gluconeogenesis via deacetylation of a Forkhead box O (FoxO) family transcription factor, FoxO1, together with HDAC3. Specifically, HDAC9 expression can be strongly induced upon hepatitis C virus (HCV) infection. HCV-induced HDAC9 upregulation enhances gluconeogenesis by promoting the expression of gluconeogenic genes, including phosphoenolpyruvate carboxykinase and glucose-6-phosphatase, indicating a major role for HDAC9 in the development of HCV-associated exaggerated gluconeogenic responses. Moreover, HDAC9 expression levels and gluconeogenic activities were elevated in livers from HCV-infected patients and persistent HCV-infected mice, emphasizing the clinical relevance of these results. Our results suggest HDAC9 is involved in glucose metabolism, HCV-induced abnormal glucose homeostasis, and type 2 diabetes.

Structural Basis for Small Molecule NDB (N-Benzyl-N-(3-(tert-butyl)-4-hydroxyphenyl)-2,6-dichloro-4-(dimethylamino) Benzamide) as a Selective Antagonist of Farnesoid X Receptor α (FXRα) in Stabilizing the Homodimerization of the Receptor.

Farnesoid X receptor α (FXRα) as a bile acid sensor plays potent roles in multiple metabolic processes, and its antagonist has recently revealed special interests in the treatment of metabolic disorders, although the underlying mechanisms still remain unclear. Here, we identified that the small molecule N-benzyl-N-(3-(tert-butyl)-4-hydroxyphenyl)-2,6-dichloro-4-(dimethylamino) benzamide (NDB) functioned as a selective antagonist of human FXRα (hFXRα), and the crystal structure of hFXRα ligand binding domain (hFXRα-LBD) in complex with NDB was analyzed. It was unexpectedly discovered that NDB induced rearrangements of helix 11 (H11) and helix 12 (H12, AF-2) by forming a homodimer of hFXRα-LBD, totally different from the active conformation in monomer state, and the binding details were further supported by the mutation analysis. Moreover, functional studies demonstrated that NDB effectively antagonized the GW4064-stimulated FXR/RXR interaction and FXRα target gene expression in primary mouse hepatocytes, including the small heterodimer partner (SHP) and bile-salt export pump (BSEP); meanwhile, administration of NDB to db/db mice efficiently decreased the gene expressions of phosphoenolpyruvate carboxykinase (PEPCK), glucose 6-phosphatase (G6-pase), small heterodimer partner, and BSEP. It is expected that our first analyzed crystal structure of hFXRα-LBD·NDB will help expound the antagonistic mechanism of the receptor, and NDB may find its potential as a lead compound in anti-diabetes research.

Chromium-containing traditional Chinese medicine, Tianmai Xiaoke Tablet improves blood glucose through activating insulin-signaling pathway and inhibiting PTP1B and PCK2 in diabetic rats.

OBJECTIVE: Chromium is an essential mineral that is thought to be necessary for normal glucose homeostasis. Numerous studies give evidence that chromium picolinate can modulate blood glucose and insulin resistance. The main ingredient of Tianmai Xiaoke (TMXK) Tablet is chromium picolinate. In China, TMXK Tablet is used to treat type 2 diabetes. This study investigated the effect of TMXK on glucose metabolism in diabetic rats to explore possible underlying molecular mechanisms for its action. METHODS: Diabetes was induced in rats by feeding a high-fat diet and subcutaneously injection with a single dose of streptozotocin (50 mg/kg, tail vein). One week after streptozotocin-injection, model rats were divided into diabetic group, low dose of TMXK group and high dose of TMXK group. Eight normal rats were used as normal control. After 8 weeks of treatment, skeletal muscle was obtained and was analyzed using Roche NimbleGen mRNA array and quantitative polymerase chain reaction (qPCR). Fasting blood glucose, oral glucose tolerance test and homeostasis model assessment of insulin resistance (HOMA-IR) index were also measured. RESULTS: The authors found that the administration of TMXK Tablet can reduce the fasting blood glucose and fasting insulin level and HOMA-IR index. The authors also found that 2 223 genes from skeletal muscle of the high-dose TMXK group had significant changes in expression (1 752 increased, 471 decreased). Based on Kyoto encyclopedia of genes and genomes pathway analysis, the most three significant pathways were "insulin signaling pathway", "glycolysis/gluconeogenesis" and "citrate cycle (TCA)". qPCR showed that relative levels of forkhead box O3 (FoxO3), phosphoenolpyruvate carboxykinase 2 (Pck2), and protein tyrosine phosphatase 1B (Ptp1b) were significantly decreased in the high-dose TMXK group, while v-akt murine thymoma viral oncogene homolog 1 (Akt1) and insulin receptor substrate 2 (Irs2) were increased. CONCLUSION: Our data show that TMXK Tablet reduces fasting glucose level and improves insulin resistance in diabetic rats. The mechanism may be linked to the inactivation of PTP1B and PCK enzymes, or through intracellular pathways, such as the insulin signaling pathway.

Sodium arsenite induces orphan nuclear receptor SHP gene expression via AMP-activated protein kinase to inhibit gluconeogenic enzyme gene expression.

Sodium arsenite has been demonstrated to alter the expression of genes associated with glucose homeostasis in tissues involved in the pathogenesis of type 2 diabetes; however, the underlying molecular mechanism has not been fully elucidated yet. In this study, we report that the sodium arsenite-induced gene expression of the small heterodimer partner (SHP; NR0B2), an atypical orphan nuclear receptor, regulates the expression of hepatic gluconeogenic genes. Sodium arsenite augments hepatic SHP mRNA levels in an AMP-activated protein kinase (AMPK)-dependent manner. Sodium arsenite activated AMPK and was shown to perturb cellular ATP levels. The arsenite-induced SHP mRNA level was blocked by adenoviral overexpression of dominant negative AMPK (Ad-dnAMPKalpha) or by the AMPK inhibitor compound C in hepatic cell lines. We demonstrated the dose-dependent induction of SHP mRNA levels by sodium arsenite and repressed the forskolin/dexamethasone-induced gene expression of the key hepatic gluconeogenic genes phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase). Ad-dnAMPKalpha blocked the repressive effects of arsenite-induced SHP on PEPCK and G6Pase. Sodium arsenite inhibited the promoter activity of PEPCK and G6Pase, and this repression was abolished by small interfering (si)RNA SHP treatments. The knockdown of SHP expression by oligonucleotide siRNA SHP or adenoviral siRNA SHP released the sodium arsenite-mediated repression of forskolin/dexamethasone-stimulated PEPCK and G6Pase gene expression in a variety of hepatic cell lines. Results from our study suggest that sodium arsenite induces SHP via AMPK to inhibit the expression of hepatic gluconeogenic genes and also provide us with a novel molecular mechanism of arsenite-mediated regulation of hepatic glucose homeostasis.

Kupffer cell cytokines interleukin-1beta and interleukin-10 combine to inhibit phosphoenolpyruvate carboxykinase and gluconeogenesis in cultured hepatocytes.

BACKGROUND AND AIMS: Recent evidence suggests that inflammatory cytokines may mediate reduced hepatic glucose production and reduced blood glucose concentrations in sepsis. Therefore the aim of this study is to provide direct evidence of a cytokine-mediated interaction between Kupffer cells and hepatocytes by characterising the effects of lipopolysaccharide-stimulated Kupffer cells on hepatocyte gluconeogenesis, and the activity of key regulatory enzymes of this pathway. METHODS AND RESULTS: Primary isolates of hepatocytes co-cultured with lipopolysaccharide-stimulated Kupffer cells in Transwell inserts showed a 48% inhibition of gluconeogenesis (P < 0.001). RNase protection assay and ELISA of Kupffer cells and the culture media following exposure to lipopolysaccharide showed increased levels of interleukin-1 alpha and beta, tumour necrosis factor alpha and IL-10. The addition of IL-1beta and IL-10 to hepatocyte cultures inhibited gluconeogenesis by 52% (P < 0.001), whereas each cytokine alone was ineffective. To determine whether altered production or activity of phosphoenolpyruvate carboxykinase or pyruvate kinase was responsible for the reduced glucose synthesis, their mRNA, protein levels and enzyme activities were measured. Primary hepatocytes co-cultured with lipopolysaccharide-stimulated Kupffer cells or cultured with a combination of IL-1beta and IL-10 displayed reduced levels of phosphoenolpyruvate carboxykinase mRNA, protein and enzyme activity. In contrast the mRNA, protein levels and enzyme activity of pyruvate kinase were not altered; suggesting that gluconeogenesis was suppressed by downregulation of phosphoenolpyruvate carboxykinase. CONCLUSIONS: Therefore, hypoglycaemia, which is often observed in sepsis, may be mediated by Kupffer cell-derived IL-1beta and IL-10. In addition this study suggests these cytokines inhibit phosphoenolpyruvate carboxykinase production and thereby hepatic gluconeogenesis.

CCAAT/enhancer-binding protein beta is an accessory factor for the glucocorticoid response from the cAMP response element in the rat phosphoenolpyruvate carboxykinase gene promoter.

The cyclic AMP response element (CRE) of the rat phosphoenolpyruvate carboxykinase (PEPCK) gene promoter is required for a complete glucocorticoid response. Proteins known to bind the PEPCK CRE include the CRE-binding protein (CREB) and members of the CCAAT/enhancer-binding protein (C/EBP) family. We took two different approaches to determine which of these proteins provides the accessory factor activity for the glucocorticoid response from the PEPCK CRE. The first strategy involved replacing the CRE of the PEPCK promoter/chloramphenicol acetyltransferase reporter plasmid (pPL32) with a consensus C/EBP-binding sequence. This construct, termed pDeltaCREC/EBP, binds C/EBPalpha and beta but not CREB, yet it confers a nearly complete glucocorticoid response when transiently transfected into H4IIE rat hepatoma cells. These results suggest that one of the C/EBP family members may be the accessory factor. The second strategy involved co-transfecting H4IIE cells with a pPL32 mutant, in which the CRE was replaced with a GAL4-binding sequence (pDeltaCREGAL4), and various GAL4 DNA-binding domain (DBD) fusion protein expression vectors. Although chimeric proteins consisting of the GAL4 DBD fused to either CREB or C/EBPalpha are able to confer an increase in basal transcription, they do not facilitate the glucocorticoid response. In contrast, a fusion protein consisting of the GAL4 DBD and amino acids 1-118 of C/EBPbeta provides a significant glucocorticoid response. Additional GAL4 fusion studies were done to map the minimal domain of C/EBPbeta needed for accessory factor activity to the glucocorticoid response. Chimeric proteins containing amino acid regions 1-84, 52-118, or 85-118 of C/EBPbeta fused to the GAL4 DBD do not mediate a glucocorticoid response. We conclude that the amino terminus of C/EBPbeta contains a multicomponent domain necessary to confer accessory factor activity to the glucocorticoid response from the CRE of the PEPCK gene promoter.