Structure-Activity Relationship Study of Biselyngbyolide B Reveals Mitochondrial Fission-Induced Cytotoxicity in Cancer.

A systematic structure-activity relationship study of the potent anticancer marine macrolide biselyngbyolide B has been accomplished. A total of 11 structural variants of the parent natural product, of which 2 are natural analogues, have been studied against a human colorectal carcinoma cell line. The requisite functional units of the parent molecule responsible for the cytotoxic activities have been disclosed. Biselyngbyolide C, one of the natural analogues of biselyngbyolide B, has been studied in depth to explore its molecular mechanism. Interestingly, the in vitro data demonstrated an induction of dynamin-related protein 1-mediated mitochondrial fission and reactive oxygen species production which led to activation of ASK1/P38/JNK-mediated apoptosis in colon cancer cells as an important pathway for biselyngbyolide B-mediated cytotoxicity. Notably, this study revealed that a macrolide participated in mitochondrial fission to promote apoptosis of cancer cells, providing new insight.

Accelerated export of Dicer1 from lipid-challenged hepatocytes buffers cellular miRNA-122 levels and prevents cell death.

Hepatocytes on exposure to high levels of lipids reorganize the metabolic program while fighting against the toxicity associated with elevated cellular lipids. The mechanism of this metabolic reorientation and stress management in lipid-challenged hepatocytes has not been well explored. We have noted the lowering of miR-122, a liver-specific miRNA, in the liver of mice fed with either a high-fat diet or a methionine-choline-deficient diet that is associated with increased fat accumulation in mice liver. Interestingly, low miR-122 levels are attributed to the enhanced extracellular export of miRNA processor enzyme Dicer1 from hepatocytes in the presence of high lipids. Export of Dicer1 can also account for the increased cellular levels of pre-miR-122-the substrate of Dicer1. Interestingly, restoration of Dicer1 levels in the mouse liver resulted in a strong inflammatory response and cell death in the presence of high lipids. Increasing death of hepatocytes was found to be caused by increased miR-122 levels in hepatocytes restored for Dicer1. Thus, the Dicer1 export by hepatocytes seems to be a key mechanism to combat lipotoxic stress by shunting out miR-122 from stressed hepatocytes. Finally, as part of this stress management, we determined that the Ago2-interacting pool of Dicer1, responsible for mature microribonucleoprotein formation in mammalian cells, gets depleted. miRNA-binder and exporter protein HuR is found to accelerate Ago2-Dicer1 uncoupling to ensure export of Dicer1 via extracellular vesicles in lipid-loaded hepatocytes.

PIMT Controls Insulin Synthesis and Secretion through PDX1.

Pancreatic beta cell function is an important component of glucose homeostasis. Here, we investigated the function of PIMT (PRIP-interacting protein with methyl transferase domain), a transcriptional co-activator binding protein, in the pancreatic beta cells. We observed that the protein levels of PIMT, along with key beta cell markers such as PDX1 (pancreatic and duodenal homeobox 1) and MafA (MAF bZIP transcription factor A), were reduced in the beta cells exposed to hyperglycemic and hyperlipidemic conditions. Consistently, PIMT levels were reduced in the pancreatic islets isolated from high fat diet (HFD)-fed mice. The RNA sequencing analysis of PIMT knockdown beta cells identified that the expression of key genes involved in insulin secretory pathway, Ins1 (insulin 1), Ins2 (insulin 2), Kcnj11 (potassium inwardly-rectifying channel, subfamily J, member 11), Kcnn1 (potassium calcium-activated channel subfamily N member 1), Rab3a (member RAS oncogene family), Gnas (GNAS complex locus), Syt13 (synaptotagmin 13), Pax6 (paired box 6), Klf11 (Kruppel-Like Factor 11), and Nr4a1 (nuclear receptor subfamily 4, group A, member 1) was attenuated due to PIMT depletion. PIMT ablation in the pancreatic beta cells and in the rat pancreatic islets led to decreased protein levels of PDX1 and MafA, resulting in the reduction in glucose-stimulated insulin secretion (GSIS). The results from the immunoprecipitation and ChIP experiments revealed the interaction of PIMT with PDX1 and MafA, and its recruitment to the insulin promoter, respectively. Importantly, PIMT ablation in beta cells resulted in the nuclear translocation of insulin. Surprisingly, forced expression of PIMT in beta cells abrogated GSIS, while Ins1 and Ins2 transcript levels were subtly enhanced. On the other hand, the expression of genes, PRIP/Asc2/Ncoa6 (nuclear receptor coactivator 6), Pax6, Kcnj11, Syt13, Stxbp1 (syntaxin binding protein 1), and Snap25 (synaptosome associated protein 25) associated with insulin secretion, was significantly reduced, providing an explanation for the decreased GSIS upon PIMT overexpression. Our findings highlight the importance of PIMT in the regulation of insulin synthesis and secretion in beta cells.

Butyrate limits inflammatory macrophage niche in NASH.

Immune cell infiltrations with lobular inflammation in the background of steatosis and deregulated gut-liver axis are the cardinal features of non-alcoholic steatohepatitis (NASH). An array of gut microbiota-derived metabolites including short-chain fatty acids (SCFA) multifariously modulates NASH pathogenesis. However, the molecular basis for the favorable impact of sodium butyrate (NaBu), a gut microbiota-derived SCFA, on the immunometabolic homeostasis in NASH remains elusive. We show that NaBu imparts a robust anti-inflammatory effect in lipopolysaccharide (LPS) stimulated or classically activated M1 polarized macrophages and in the diet-induced murine NASH model. Moreover, it impedes monocyte-derived inflammatory macrophage recruitment in liver parenchyma and induces apoptosis of proinflammatory liver macrophages (LM) in NASH livers. Mechanistically, by histone deactylase (HDAC) inhibition NaBu enhanced acetylation of canonical NF-κB subunit p65 along with its differential recruitment to the proinflammatory gene promoters independent of its nuclear translocation. NaBu-treated macrophages thus exhibit transcriptomic signatures that corroborate with a M2-like prohealing phenotype. NaBu quelled LPS-mediated catabolism and phagocytosis of macrophages, exhibited a differential secretome which consequently resulted in skewing toward prohealing phenotype and induced death of proinflammatory macrophages to abrogate metaflammation in vitro and in vivo. Thus NaBu could be a potential therapeutic as well as preventive agent in mitigating NASH.

Ex Vivo Dual-Hit Method for Inflammasome Activation in Liver.

Activation of the inflammasome in hepatocytes and the liver-resident macrophages is associated with drug-induced hepatotoxicity and a plethora of metabolic diseases including nonalcoholic steatohepatitis (NASH). Initiation of this innate immune response requires two concomitant signals resulting in the formation of a molecular assembly that post-transcriptionally maturates a specific set of cytokines. While signal 1 results from the engagement and activation of pattern recognition receptors, signal 2 can be induced by diverse stimuli including adenosine triphosphate (ATP). Among various modules, NOD-like receptor 3 (NLRP3) inflammasome activation followed by caspase-1-dependent proIL-1ß maturation has been observed in both preclinical models and NASH patients suggesting the crucial importance of inflammasome activation in NAFLD progression. The protocol reported here depicts an ex vivo method for investigating the role of inflammasome activation in macrophages and its impact on hepatocytes. We first described a rapid protocol for the isolation of primary Kupffer cells (KC) and hepatocytes from the murine liver. Next, to investigate the crosstalks between KCs and hepatocytes in the context of inflammasome activation, isolated KCs were activated with lipopolysaccharide (LPS), alone or in tandem with ATP, which resulted in inflammasome activation in KCs evident by abundant IL-1ß secretion. Isolated primary hepatocytes were treated with conditioned medium (CM) from activated KCs to investigate the effect of inflammasome activation by various readouts. Moreover, this model also enabled us to investigate the role of specific cytokines by neutralizing them in the CM of inflammasome-activated KC. This precise ex vivo method provides a comprehensive protocol for investigating hepatocellular inflammasome activation.

A nexus of miR-1271, PAX4 and ALK/RYK influences the cytoskeletal architectures in Alzheimer's Disease and Type 2 Diabetes.

Alzheimer's Disease (AD) and Type 2 Diabetes (T2D) share a common hallmark of insulin resistance. Reportedly, two non-canonical Receptor Tyrosine Kinases (RTKs), ALK and RYK, both targets of the same micro RNA miR-1271, exhibit significant and consistent functional down-regulation in post-mortem AD and T2D tissues. Incidentally, both have Grb2 as a common downstream adapter and NOX4 as a common ROS producing factor. Here we show that Grb2 and NOX4 play critical roles in reducing the severity of both the diseases. The study demonstrates that the abundance of Grb2 in degenerative conditions, in conjunction with NOX4, reverse cytoskeletal degradation by counterbalancing the network of small GTPases. PAX4, a transcription factor for both Grb2 and NOX4, emerges as the key link between the common pathways of AD and T2D. Down-regulation of both ALK and RYK through miR-1271, elevates the PAX4 level by reducing its suppressor ARX via Wnt/ß-Catenin signaling. For the first time, this study brings together RTKs beyond Insulin Receptor (IR) family, transcription factor PAX4 and both AD and T2D pathologies on a common regulatory platform.

Interaction of a Triantennary Quinoline Glycoconjugate with the Asialoglycoprotein Receptor.

Targeted intracellular delivery is an efficient strategy for developing therapeutics against cancer and other intracellular infections. Nonspecific drug delivery shows limited clinical applications owing to high dosage, cytotoxicity, nonspecific action, high cost, etc. Therefore, targeted delivery of less cytotoxic drug candidates to hepatocytes through ASGPR-mediated endocytosis could be an efficient strategy to surmount the prevailing shortcomings. In the present work, the gene encoding ASGPR-H1-CRD was amplified from Huh7 cells, cloned into pET 11a vector, and the ASGPR-H1-CRD protein was expressed and purified from E. coli. A novel triantennary galactose-conjugated quinoline derivative 4 was synthesized that demonstrates 17-fold higher binding affinity to isolated ASGPR-H1-CRD protein receptor (Kd ∼54 µM) in comparison to D-galactose (Kd ∼900 µM). Moreover, micro-calorimetric studies for the interaction of glycoconjugate 4 with ASGPR protein on live hepatocytes showed notable thermal response in case of ASGPR-containing Huh7 cells, in comparison to non-ASGPR Chang cells. These results might serve as an approach towards targeted delivery of small glycoconjugates to hepatocytes.

Subcutaneous amyloidoma models for screening potential anti-fibrillating agents in vivo.

Here, we describe a robust protocol using mouse models to screen potential insulin-stabilizers and insulin moieties. We have generated a mouse model of amyloidoma, found in diabetic patients undergoing insulin therapy. This model can be used to screen potential insulin stabilizers and insulin moieties to prevent amyloidoma formation. This protocol can further be used for the preclinical validation of therapeutically relevant insulin stabilizers and formulations. The protocol highlights all the critical steps for generating amyloidoma in a preclinical model. For complete details on the use and execution of this profile, please refer to Mukherjee et al. (2021).

Incretins in fibrocalculous pancreatic diabetes: A unique subtype of pancreatogenic diabetes.

BACKGROUND: Studies evaluating endocrine and exocrine functions in fibrocalculous pancreatic diabetes (FCPD) are scarce. METHODS: Insulin, C-peptide, glucagon, incretin hormones (glucagon-like peptide 1 [GLP-1] and gastric inhibitory peptide [GIP]), and dipeptidyl peptidase IV (DPP-IV) were estimated in patients with FCPD (n = 20), type 2 diabetes mellitus (T2DM) (n = 20), and controls (n = 20) in fasting and 60 minutes after 75 g glucose. RESULTS: Fasting and post-glucose C-peptide and insulin in FCPD were lower than that of T2DM and controls. Plasma glucagon decreased after glucose load in controls (3.72, 2.29), but increased in T2DM (4.01, 5.73), and remained unchanged in FCPD (3.44, 3.44). Active GLP-1 (pmol/L) after glucose load increased in FCPD (6.14 to 9.72, P = <.001), in T2DM (2.87 to 4.62, P < .001), and in controls (3.91 to 6.13, P < .001). Median active GLP-1 in FCPD, both in fasting and post-glucose state (6.14, 9.72), was twice that of T2DM (2.87, 4.62) and 1.5 times that of controls (3.91, 6.13) (P < .001 for all). Post-glucose GIP (pmol/L) increased in all: FCPD (15.83 to 94.14), T2DM (21.85 to 88.29), and control (13.00 to 74.65) (P < .001 for all). GIP was not different between groups. DPP-IV concentration (ng/mL) increased in controls (1578.54, 3012.00) and FCPD (1609.95, 1995.42), but not in T2DM (1204.50, 1939.50) (P = .131). DPP-IV between the three groups was not different. Fecal elastase was low in FCPD compared with T2DM controls. CONCLUSIONS: In FCPD, basal C-peptide and glucagon are low, and glucagon does not increase after glucose load. GLP-1, but not GIP, in FCPD increases 1.5 to 2 times as compared with T2DM and controls (fasting and post glucose) without differences in DPP-IV.

Suppression of poised oncogenes by ZMYND8 promotes chemo-sensitization.

The major challenge in chemotherapy lies in the gain of therapeutic resistance properties of cancer cells. The relatively small fraction of chemo-resistant cancer cells outgrows and are responsible for tumor relapse, with acquired invasiveness and stemness. We demonstrate that zinc-finger MYND type-8 (ZMYND8), a putative chromatin reader, suppresses stemness, drug resistance, and tumor-promoting genes, which are hallmarks of cancer. Reinstating ZMYND8 suppresses chemotherapeutic drug doxorubicin-induced tumorigenic potential (at a sublethal dose) and drug resistance, thereby resetting the transcriptional program of cells to the epithelial state. The ability of ZMYND8 to chemo-sensitize doxorubicin-treated metastatic breast cancer cells by downregulating tumor-associated genes was further confirmed by transcriptome analysis. Interestingly, we observed that ZMYND8 overexpression in doxorubicin-treated cells stimulated those involved in a good prognosis in breast cancer. Consistently, sensitizing the cancer cells with ZMYND8 followed by doxorubicin treatment led to tumor regression in vivo and revert back the phenotypes associated with drug resistance and stemness. Intriguingly, ZMYND8 modulates the bivalent or poised oncogenes through its association with KDM5C and EZH2, thereby chemo-sensitizing the cells to chemotherapy for better disease-free survival. Collectively, our findings indicate that poised chromatin is instrumental for the acquisition of chemo-resistance by cancer cells and propose ZMYND8 as a suitable epigenetic tool that can re-sensitize the chemo-refractory breast carcinoma.

Biselyngbyolides A & C: their total synthesis and anticancer activities.

Convergent strategies for the first total synthesis of biselyngbyolide C and an alternative route for the total synthesis of biselyngbyolide A have been developed. The key strategic feature in this study is Heck macrocyclization. The use of intramolecular Heck coupling for biselyngbyolide B was demonstrated by us earlier; however such a strategy has not been explored further for the other members of this family of natural products, in particular, where sensitive skipped olefins are involved. The other highlights of this synthetic study include iterative Crimmins acetate aldol and Wittig olefination processes, followed by the less explored cobalt-hydride-based reduction of an activated olefin and Shiina esterification. Our synthetic study enabled us to amend the reported NMR data of biselyngbyolides A and C. An evaluation of the anticancer activities of both biselyngbyolides A and C revealed that the apoptosis generated in cancer cells followed an intrinsic pathway.

Resveratrol as a nontoxic excipient stabilizes insulin in a bioactive hexameric form.

Insulin aggregation is the leading cause of considerable reduction in the amount of active drug molecules in liquid formulations manufactured for diabetes management. Phenolic compounds, such as phenol and m-cresol, are routinely used to stabilize insulin in a hexameric form during its commercial preparation. However, long term usage of commercial insulin results in various adverse secondary responses, for which toxicity of the phenolic excipients is primarily responsible. In this study we aimed to find out a nontoxic insulin stabilizer. To that end, we have selected resveratrol, a natural polyphenol, as a prospective nontoxic insulin stabilizer because of its structural similarity with commercially used phenolic compounds. Atomic force microscopy visualization of resveratrol-treated human insulin revealed that resveratrol has a unique ability to arrest hINS in a soluble oligomeric form having discrete spherical morphology. Most importantly, resveratrol-treated insulin is nontoxic for HepG2 cells and it effectively maintains low blood glucose in a mouse model. Cryo-electron microscopy revealed 3D morphology of resveratrol-stabilized insulin that strikingly resembles crystal structures of insulin hexamer formulated with m-cresol. Significantly, we found that, in a condition inductive to amyloid fibrillation at physiological pH, resveratrol is capable of stabilizing insulin more efficiently than m-cresol. Thus, this study describes resveratrol as an effective nontoxic natural molecule that can be used for stabilizing insulin in a bioactive oligomeric form during its commercial formulation.

A novel role of tumor suppressor ZMYND8 in inducing differentiation of breast cancer cells through its dual-histone binding function.

Accumulating evidences indicate the involvement of epigenetic deregulations in cancer. While some epigenetic regulators with aberrant functions in cancer are targeted for improving therapeutic outcome in patients, reinstating the functions of tumor-suppressor-like epigenetic regulators might further potentiate anti-cancer therapies. Epigenetic reader zinc-finger MYND-type-containing 8 (ZMYND8) has been found to be endowed with multiple anti-cancer functions like inhibition of tumor cell migration and proliferation. Here, we report another novel tumor suppressor role of ZMYND8 as an inducer of differentiation in breast cancer cells, by upregulating differentiation genes. Interestingly, we also demonstrated that ZMYND8 mediates all its antitumor roles through a common dual-histone mark binding to H4K16Ac and H3K36Me2. We validated these findings by both biochemical and biophysical analyses. Furthermore, we also confirmed the differentiationinducing potential of ZMYND8 in vivo, using 4T1 murine breast cancer model in Balb/c mice. Differentiation therapy holds great promise in cancer therapy, since it is non-toxic and makes the cancer cells therapysensitive. In this scenario, we propose epigenetic reader ZMYND8 as a potential therapeutic candidate for differentiation therapy in breast cancer.

Metabolic impairment in response to early induction of C/EBPß leads to compromised cardiac function during pathological hypertrophy.

Chronic pressure overload-induced left ventricular hypertrophy in heart is preceded by a metabolic perturbation that prefers glucose over lipid as substrate for energy requirement. Here, we establish C/EBPß (CCAAT/enhancer-binding protein ß) as an early marker of the metabolic derangement that triggers the imbalance in fatty acid (FA) oxidation and glucose uptake with increased lipid accumulation in cardiomyocytes during pathological hypertrophy, leading to contractile dysfunction and endoplasmic reticulum (ER) stress. This is the first study that shows that myocardium-targeted C/EBPß knockdown prevents the impaired cardiac function during cardiac hypertrophy led by maladaptive metabolic response with persistent hypertrophic stimuli, whereas its targeted overexpression in control increases lipid accumulation significantly compared to control hearts. A new observation from this study was the dual and opposite transcriptional regulation of the alpha and gamma isoforms of Peroxisomal proliferator activated receptors (PPARα and PPARγ) by C/EBPß in hypertrophied cardiomyocytes. Before the functional and structural remodeling sets in the diseased myocardium, C/EBPß aggravates lipid accumulation with the aid of the increased FA uptake involving induced PPARγ expression and decreased fatty acid oxidation (FAO) by suppressing PPARα expression. Glucose uptake into cardiomyocytes was greatly increased by C/EBPß via PPARα suppression. The activation of mammalian target of rapamycin complex-1 (mTORC1) during increased workload in presence of glucose as the only substrate was prevented by C/EBPß knockdown, thereby abating contractile dysfunction in cardiomyocytes. Our study thus suggests that C/EBPß may be considered as a novel cellular marker for deranged metabolic milieu before the heart pathologically remodels itself during hypertrophy.

Increased Plasma Dipeptidyl Peptidase-4 (DPP4) Activity Is an Obesity-Independent Parameter for Glycemic Deregulation in Type 2 Diabetes Patients.

Background: Increase in circulating dipeptidyl peptidase-4 (DPP4) activity and levels has been reported to associate both with hyperglycemia and obesity. Here we aim to decipher the role of enhanced plasma DPP4 activity in obese type 2 diabetes (T2DM) patients. Materials and methods: Plasma DPP4 levels and activity were measured in obese and non-obese newly diagnosed T2DM patients (n = 123). Visceral and subcutaneous adipose tissue DPP4 expression and activity were determined in 43 obese subjects (T2DM = 21 and non-T2DM = 22). 20 subjects undergoing Mini-Gastric Bypass (MGB) surgery were followed up over 4-6 weeks for plasma DPP4. Results: Plasma DPP4 levels and activity both were increased in T2DM patients compared to control group. However, DPP4 levels and not DPP4 activity were increased in obese T2DM patients compared to non-obese T2DM (62.49 ± 26.27 µg/ml vs. 48.4 ± 30.98 µg/ml, respectively, p = 0.028). DPP4 activity in visceral adipose tissue (VAT) from obese T2DM and obese non-T2DM groups were similar (5.05 ± 3.96 nmol/min/ml vs. 5.83 ± 4.13 nmol/min/ml respectively, p = 0.548) in spite of having increased DPP4 expression in the obese T2DM group. Moreover, in obese patients, plasma DPP4 levels and activity did not show any significant change after weight reduction and glycemic control following MGB surgery. Conclusion: Enhanced plasma DPP4 activity in T2DM occurs independently of obesity. Thus, adipose derived DPP4 may not be playing any significant role in glycemic deregulation in obese T2DM patients.

PEDF promotes nuclear degradation of ATGL through COP1.

Adipose triglyceride lipase (ATGL) plays a compelling role in hepatic lipid turnover and in the pathophysiology of non-alcoholic fatty liver disease. Hepatic ATGL is post-transcriptionally regulated by E3 ubiquitin ligase constitutive photomorphogenic1 (COP1) through polyubiquitylation and proteasomal degradation. However the physiological cue for COP1-mediated hepatocellular degradation of ATGL remained unknown. Here we checked for the role of pigment epithelium-derived factor (PEDF), a moonlighting hepatokine and the so-called ligand of ATGL for its stability in hepatocytes. We show that PEDF diminishes ATGL protein stability by promoting its proteasomal degradation in COP1-dependent manner. Despite being a secretory glycoprotein, PEDF is also sequestered in the nuclear compartment so as COP1. Interestingly, PEDF enhances nuclear import of predominantly cytosolic ATGL protein for its subsequent proteasomal degradation in the nucleus. PEDF also controls cell autonomous hepatocyte lipid accumulation and mobilization through COP1-ATGL axis, thereby unraveling a novel pathway for hepatic lipid metabolism.

A peptide-modified solid lipid nanoparticle formulation of paclitaxel modulates immunity and outperforms dacarbazine in a murine melanoma model.

Melanoma is a highly aggressive skin cancer. A paclitaxel formulation of solid lipid nanoparticles modified with Tyr-3-octreotide (PSM) is employed to treat melanoma that highly expresses somatostatin receptors (SSTRs). PSM exerts more apoptotic and anti-invasive effects in B16F10 mice melanoma cells as compared to dacarbazine (DTIC), an approved chemotherapeutic drug for treating aggressive melanoma. Besides, PSM induces one of the biomarkers of immunogenic cell death in vitro and in vivo as confirmed by calreticulin exposure on the B16F10 cell surface. We observed a significant number of CD8 positive T cells in the tumor bed of the PSM treated group. As a result, PSM effectively reduces tumor volume in vivo as compared to DTIC. PSM also induces a favorable systemic immune response as determined in the spleen and sera of the treated animals. Importantly, PSM can reduce the number of nodule formations in the experimental lung metastasis model. Our experimentations indicate that the metronomic PSM exhibits remarkable anti-melanoma activities without any observable toxicity. This immune modulation behavior of PSM can be exploited for the therapy of melanoma and probably for other malignancies.

Quinoline-Glycomimetic Conjugates Reducing Lipogenesis and Lipid Accumulation in Hepatocytes.

Nonalcoholic fatty liver disease (NAFLD), which is characterized by excess accumulation of triglyceride in hepatocytes, is the major cause of chronic liver disease worldwide and no approved drug is available. The mechanistic target of rapamycin (mTOR) complexes has been implicated in promoting lipogenesis and fat accumulation in the liver, and thus, serve as attractive drug targets. The generation of non- or low cytotoxic mTOR inhibitors is required because existing cytotoxic mTOR inhibitors are not useful for NAFLD therapy. New compounds based on the privileged adenosine triphosphate (ATP) site binder quinoline scaffold conjugated to glucose and galactosamine derivatives, which have significantly low cytotoxicity, but strong mTORC1 inhibitory activity at low micromolar concentrations, have been synthesized. These compounds also effectively inhibit the rate of lipogenesis and lipid accumulation in cultured hepatocytes. This is the first report of glycomimetic-quinoline derivatives that reduce lipid load in hepatocytes.

Inhibition of mTOR complexes protects cancer cells from glutamine starvation induced cell death by restoring Akt stability.

Glutamine, a well-established oncometabolite, anaplerotically fuels mitochondrial energy metabolism and modulates activity of mammalian/mechanistic target of rapamycin complexes (mTOR). Currently, mTOR inhibitors are in clinical use for certain types of cancer but with limited success. Since glutamine is essential for growth of many cancers, we reasoned that glutamine deprivation under conditions of mTOR inhibition should be more detrimental to cancer cell survival. However, our results show that when cells are deprived of glutamine concomitant with mTOR inhibition, hepatocarcinoma cells elicit an adaptive response which aids in their survival due to enhanced autophagic flux. Moreover, inhibition of mTOR promotes Akt ubiquitination and its proteasomal degradation however we show that Akt degradation is abrogated by increased autophagy following glutamine withdrawal. Under conditions of glutamine deficiency and mTOR inhibition, the enhanced stability of Akt protein may provide survival cues to cancer cells. Thus, our data uncovers a novel molecular link between glutamine metabolism, autophagy and stability of Akt with cancer cell survival.

Significance of circulatory DPP4 activity in metabolic diseases.

Dipeptidyl peptidase 4 (DPP4), also known as CD26 is a type II transmembrane protein that is released from the cell membrane in a nonclassical secretory mechanism. This exopeptidase selectively degrades varieties of substrates including incretin hormones, growth factors, and cytokines. A significant detectable amount of DPP4 activity can be measured in plasma as well as in different tissues such as intestinal epithelium, vascular endothelium, lymphocytes, monocytes, kidney, liver, adipose, lung, thymus, spleen, prostate, etc. Enzymatically active circulatory DPP4 is shed from the plasma membrane via proteolytic cleavage, a process responsible for the enhanced plasma DPP4 levels and activity. Elevated circulatory DPP4 activity as well as levels has been found in wide spectrum of metabolic diseases including diabetes, obesity, cardiovascular diseases, and nonalcoholic fatty liver diseases. Moreover, recent preclinical studies have further expanded the repertoire for the usage of DPP4 inhibitors in the treatment of other metabolic diseases and in their consequent complications. In the present review we highlight the reason behind the elevated circulatory DPP4 levels in metabolic diseases with a focus on the tissue of origin. We also underscore the discrepancy of protein levels with enzyme activity of circulatory DPP4 in metabolic diseases. © 2018 IUBMB Life, 70(2):112-119, 2018.