Postprandial Plasma Lipidomics Reveal Specific Alteration of Hepatic-derived Diacylglycerols in Nonalcoholic Fatty Liver Disease.

BACKGROUND & AIMS: Hepatic energy metabolism is a dynamic process modulated by multiple stimuli. In nonalcoholic fatty liver disease (NAFLD), human studies typically focus on the static fasting state. We hypothesized that unique postprandial alterations in hepatic lipid metabolism are present in NAFLD. METHODS: In a prospective clinical study, 37 patients with NAFLD and 10 healthy control subjects ingested a standardized liquid meal with pre- and postprandial blood sampling. Postprandial plasma lipid kinetics were characterized at the molecular lipid species level by untargeted lipidomics, cluster analysis, and lipid particle isolation, then confirmed in a mouse model. RESULTS: There was a specific increase of multiple plasma diacylglycerol (DAG) species at 4 hours postprandially in patients with NAFLD but not in controls. This was replicated in a nonalcoholic steatohepatitis mouse model, where postprandial DAGs increased in plasma and concomitantly decreased in the liver. The increase in plasma DAGs appears early in the disease course, is dissociated from NAFLD severity and obesity, and correlates with postprandial insulin levels. Immunocapture isolation of very low density lipoprotein in human samples and stable isotope tracer studies in mice revealed that elevated postprandial plasma DAGs reflect hepatic secretion of endogenous, rather than meal-derived lipids. CONCLUSIONS: We identified a selective insulin-related increase in hepatic secretion of endogenously derived DAGs after a mixed meal as a unique feature of NAFLD. DAGs are known to be lipotoxic and associated with atherosclerosis. Although it is still unknown whether the increased exposure to hepatic DAGs contributes to extrahepatic manifestations and cardiovascular risk in NAFLD, our study highlights the importance of extending NAFLD research beyond the fasting state.

Metabolomic profiling of metoprolol hypertension treatment reveals altered gut microbiota-derived urinary metabolites.

INTRODUCTION: Metoprolol succinate is a long-acting beta-blocker prescribed for the management of hypertension (HTN) and other cardiovascular diseases. Metabolomics, the study of end-stage metabolites of upstream biologic processes, yield insight into mechanisms of drug effectiveness and safety. Our aim was to determine metabolomic profiles associated with metoprolol effectiveness for the treatment of hypertension. METHODS: We performed a prospective pragmatic trial (NCT02293096) that enrolled patients between 30 and 80 years with uncontrolled HTN. Patients were started on metoprolol succinate at a dose based upon systolic blood pressure (SBP). Urine and blood pressure measurements were collected weekly. Individuals with a 10% decline in SBP or heart rate (HR) were considered responsive. Genotype for the CYP2D6 enzyme, the primary metabolic pathway for metoprolol, was evaluated for each subject. Unbiased metabolomic analyses were performed on urine samples using UPLC-QTOF mass spectrometry. RESULTS: Urinary metoprolol metabolite ratios are indicative of patient CYP2D6 genotypes. Patients taking metoprolol had significantly higher urinary levels of many gut microbiota-dependent metabolites including hydroxyhippuric acid, hippuric acid, and methyluric acid. Urinary metoprolol metabolite profiles of normal metabolizer (NM) patients more closely correlate to ultra-rapid metabolizer (UM) patients than NM patients. Metabolites did not predict either 10% SBP or HR decline. CONCLUSION: In summary, urinary metabolites predict CYP2D6 genotype in hypertensive patients taking metoprolol. Metoprolol succinate therapy affects the microbiome-derived metabolites.

Moderate Renal Impairment and Toxic Metabolites Produced by the Intestinal Microbiome: Dietary Implications.

OBJECTIVE: Toxic metabolites produced by the intestinal microbiome from animal proteins, carnitine (mainly from red meat), or phosphatidylcholine (mainly from egg yolk), have important adverse effects on cardiovascular disease. These are renally eliminated and may be termed gut-derived uremic toxins (GDUT). We hypothesized that even moderate renal impairment and intake of nutrient precursors would raise plasma levels of GDUT. DESIGN: A cohort study. SETTING: Academic medical center. SUBJECTS: Patients attending stroke prevention clinics at a university medical center were recruited. MAIN OUTCOME MEASURE: Nutrient intake was assessed by the 131-item Harvard Food Frequency Questionnaire; estimated glomerular filtration rate (eGFR) was caculated using the Chronic Kidney Disease-Epidemiology (EPI) equations. Plasma levels of trimethylamine n-oxide, p-cresyl sulfate, hippuric acid, p-cresyl glucuronide, pheny acetyl glutamine, and phenyl sulfate were measured by ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry. RESULTS: Among 316 patients recruited, the mean (standard deviation [SD]) age was 66.74 (10.42) years; 59.7% were men. Mean eGFR was 76.03 ± 20.01; 57 (18%) had eGFR<60 mL/min/1.73 m2. Plasma levels of all GDUT were significantly higher even with moderate reduction of eGFR. Nutrient intake affected plasma levels of some GDUT; the effects differed by eGFR above and below 60 mL/min/1.73 m2. Plasma levels were obtained fasting, so we probably underestimated the effect of nutrient intake. CONCLUSIONS: Even moderate impairment of renal function was associated with higher plasma levels of GDUT. This has dietary implications for patients at risk of atherosclerosis, particularly in those with impaired renal function (including the elderly): they should limit intake of animal protein, red meat, and egg yolk. It also points the way to novel approaches to vascular prevention, including more intensive dialysis, renal transplantation, and modification of the intestinal microbiome with probiotics or fecal transplantation.

Extrahepatic PPARα modulates fatty acid oxidation and attenuates fasting-induced hepatosteatosis in mice.

PPARα (PPARA), expressed in most oxidative tissues, is a major regulator of lipid homeostasis; hepatic PPARA plays a critical role during the adaptive fasting response by promoting FA oxidation (FAO). To clarify whether extrahepatic PPARA activity can protect against lipid overload when hepatic PPARA is impaired, lipid accumulation was compared in WT (Ppara+/+), total body Ppara-null (Ppara-/-), and hepatocyte-specific Ppara-null (PparaΔHep) mice that were fasted for 24 h. Histologic staining indicated reduced lipid accumulation in PparaΔHep versus Ppara-/- mice, and biochemical analyses revealed diminished medium- and long-chain FA accumulation in PparaΔHep mouse livers. Hepatic PPARA target genes were suppressed in both mouse models. Serum FFAs increased in all genotypes after fasting but were highest in Ppara-/- mice. In PparaΔHep mice, FAO genes were increased in brown adipose tissue, heart, and muscle, and total lipase activity was elevated in the muscle and heart, suggesting increased lipid utilization. Thus, extrahepatic PPARA activity reduces systemic lipid load when hepatic lipid metabolism is impaired by elevating FAO and lipase activity in other tissues and, as a result, protects against fasting-induced hepatosteatosis. This has important clinical implications in disease states with impaired hepatic PPARA function, such as nonalcoholic steatohepatitis and nonalcoholic fatty liver disease.

Effect of CKD and dialysis modality on exposure to drugs cleared by nonrenal mechanisms.

BACKGROUND: Patients with kidney disease frequently experience adverse effects from medication exposure, even when drugs are cleared by nonrenal pathways. Although many studies suggest that nonrenal drug clearance is decreased in chronic kidney disease (CKD), there remains a paucity of in vivo studies in patients with varying degrees of decreased kidney function and those comparing the impact of dialysis modality (eg, hemodialysis [HD] and peritoneal dialysis [PD]). STUDY DESIGN: We performed in vivo clinical pharmacokinetic studies of midazolam, a nonrenally cleared specific probe for CYP3A4, and fexofenadine, a nonspecific probe for hepatic and intestinal transporters. SETTING & PARTICIPANTS: Healthy controls (n=8), patients with non-dialysis-dependent (NDD)-CKD (n=8), and patients receiving HD (n=10) or PD (n=8). OUTCOMES: Exposure to midazolam and fexofenadine were quantified using area under the curve (AUC). Comprehensive pharmacokinetic parameters also were calculated for both probes. RESULTS: Midazolam AUC was significantly higher in the HD group (382.8 h·ng/mL) than in the healthy-control (63.0 h·ng/mL; P<0.001), NDD-CKD (84.5 h·ng/mL; P=0.002), and PD (47.4 h·ng/mL; P<0.001) groups. Fexofenadine AUC was significantly higher in each of the NDD-CKD (2,950 h·ng/mL; P=0.003), HD (2,327 h·ng/mL; P=0.01), and PD (2,095 h·ng/mL; P=0.04) groups compared with healthy controls (1,008 h·ng/mL). LIMITATIONS: Small study groups had different proportions of diabetic patients, early stages of CKD not available. CONCLUSIONS: Our data suggest that selection of dialysis modality is a major determinant of exposure to the CYP3A4 probe midazolam. Exposure to the intestinal and hepatic transporter probe fexofenadine is altered in patients with NDD-CKD and PD and HD patients. Thus, drug development and licensing of nonrenally cleared drugs should include evaluation in these 3 patient groups, with these results included in approved product information labeling. This reinforces the critical need for more in vivo studies of humans that evaluate the exposure to drugs cleared by these pathways.

Decreased nuclear receptor activity and epigenetic modulation associates with down-regulation of hepatic drug-metabolizing enzymes in chronic kidney disease.

Patients with chronic kidney disease (CKD) require many medications. CYP2C and CYP3A drug-metabolizing enzymes play a critical role in determining the pharmacokinetics of the majority of prescribed medications. These enzymes are transcriptionally regulated by the nuclear receptors pregnane X receptor (PXR) and hepatic nuclear factor 4α (HNF-4α). Expression of CYP2C and CYP3A is decreased in CKD; however, the mechanisms by which this occurs is unknown. We induced CKD in rats by 5/6 nephrectomy and used chromatin immunoprecipitation (ChIP) to determine nuclear receptor- and epigenetic alteration-mediated differences in the promoter region of the CYP2C and CYP3A genes. RNA polymerase II and HNF-4α binding was decreased 76 and 57% in the CYP2C11 promotor and 71 and 77% in the CYP3A2 promoter, respectively (P<0.05). ChIP also revealed a 57% decrease in PXR binding to the CYP3A2 promoter in CKD rats (P<0.05). The decrease in PXR and HNF-4α binding was accompanied by diminished histone 4 acetylation in the CYP3A2 promoter (48%) and histone 3 acetylation in the CYP2C11 (77%) and CYP3A2 (77%) promoter loci for nuclear receptor activation (P<0.05). This study suggests that decreased nuclear receptor binding and histone acetylation may contribute to the mechanism of drug-metabolizing enzyme down-regulation and altered pharmacokinetics in CKD.

Enzymatic and non-enzymatic mechanisms of dimesna metabolism.

The chemical reduction of the disulfide homodimer dimesna to its constituent mesna moieties is essential for its mitigation of nephrotoxicity associated with cisplatin and ifosfamide anticancer therapies and enhancement of dialytic clearance of the cardiovascular risk factor homocysteine. The objective of this study was to investigate potential enzymatic and non-enzymatic mechanisms of intracellular dimesna reduction. Similar to endogenous intracellular disulfides, dimesna undergoes thiol-disulfide exchange with thiolate anion-forming sulfhydryl groups via the two-step SN2 reaction. Determination of equilibrium constants of dimesna reduction when mixed with cysteine or glutathione provided a mechanistic explanation for dramatic cysteine and homocysteine depletion, but sparing of the endogenous antioxidant glutathione, previously observed during mesna therapy. Dimesna was reduced by recombinant enzymes of the thioredoxin system; however, oxidation of NADPH by the glutaredoxin system was only observed in the presence of combined dimesna and reduced glutathione, suggesting formation of oxidized glutathione following an initial non-enzymatic reduction of dimesna. Production of mesna by enzymatic and non-enzymatic mechanisms in HeLa cell lysate following dimesna incubation was demonstrated by a loss in mesna production following protein denaturation and prediction of residual non-enzymatic mesna production by mathematical modeling of thiol-disulfide exchange reactions. Reaction modeling also revealed that mixed disulfides make up a significant proportion of intracellular thiols, supporting their role in providing additional nephroprotection, independent of direct platinum conjugation.

Protein restoration in low-birth-weight rat offspring derived from maternal low-protein diet leads to elevated hepatic CYP3A and CYP2C11 activity in adulthood.

The World Health Organization has identified hypercholesterolemia to be one of the major symptoms encompassing the metabolic syndrome. Moreover, epidemiologic evidence indicates that low-birth-weight offspring are at greater risk of developing the metabolic syndrome. Previous work in our laboratory demonstrated that maternal protein restriction (MPR) results in impaired fetal growth and hypercholesterolemia in adulthood. This was attributed to repression of hepatic CYP7A1, a rate-limiting enzyme that catabolizes cholesterol to bile acids. Another important function of hepatic cytochrome P450 enzymes is the phase I oxidative metabolism of drugs (i.e., statins for hypercholesterolemia), which can significantly impact pharmacokinetics. We hypothesized that MPR offspring may have altered ability to metabolize drugs in adulthood. To address this hypothesis, we maintained Wistar rats on a 20% protein diet (control) or a low 8% protein diet throughout prenatal and postnatal life (LP1) or exclusively during prenatal life and weaning (LP2). Intriguingly CYP3A and CYP2C11 intrinsic clearance (Vmax/Km) was significantly increased exclusively in LP2 offspring at postnatal day 130 compared with control or LP1 offspring, as evaluated by testosterone enzyme kinetics in liver microsomes. The increase in activity was secondary to an increase in CYP3A23 and CYP2C11 mRNA. Collectively, these findings suggest that a low-birth-weight offspring with postnatal catch-up growth may have a diminished response to xenobiotics metabolized by CYP3A and CYP2C11 enzymes.

N-Acetylcysteine prevents congenital heart defects induced by pregestational diabetes.

BACKGROUND: Pregestational diabetes is a major risk factor of congenital heart defects (CHDs). Glutathione is depleted and reactive oxygen species (ROS) production is elevated in diabetes. In the present study, we aimed to examine whether treatment with N-acetylcysteine (NAC), which increases glutathione synthesis and inhibits ROS production, prevents CHDs induced by pregestational diabetes. METHODS: Female mice were treated with streptozotocin (STZ) to induce pregestational diabetes prior to breeding with normal males to produce offspring. Some diabetic mice were treated with N-acetylcysteine (NAC) in drinking water from E0.5 to the end of gestation or harvesting of the embryos. CHDs were identified by histology. ROS levels, cell proliferation and gene expression in the fetal heart were analyzed. RESULTS: Our data show that pregestational diabetes resulted in CHDs in 58% of the offspring, including ventricular septal defect (VSD), atrial septal defect (ASD), atrioventricular septal defects (AVSD), transposition of great arteries (TGA), double outlet right ventricle (DORV) and tetralogy of Fallot (TOF). Treatment with NAC in drinking water in pregestational diabetic mice completely eliminated the incidence of AVSD, TGA, TOF and significantly diminished the incidence of ASD and VSD. Furthermore, pregestational diabetes increased ROS, impaired cell proliferation, and altered Gata4, Gata5 and Vegf-a expression in the fetal heart of diabetic offspring, which were all prevented by NAC treatment. CONCLUSIONS: Treatment with NAC increases GSH levels, decreases ROS levels in the fetal heart and prevents the development of CHDs in the offspring of pregestational diabetes. Our study suggests that NAC may have therapeutic potential in the prevention of CHDs induced by pregestational diabetes.

CD73-TNAP crosstalk regulates the hypertrophic response and cardiomyocyte calcification due to α1 adrenoceptor activation.

Cluster of differentiation 73 (CD73) is an ecto-5' nucleotidase which catalyzes the conversion of AMP to adenosine. One of the many functions of adenosine is to suppress the activity of tissue nonspecific alkaline phosphatase (TNAP), an enzyme important in regulating intracellular calcification. Since myocardial calcification is associated with various cardiac disease states, we studied the individual roles and crosstalk between CD73 and TNAP in regulating myocyte responses to the α1 adrenoceptor agonist phenylephrine in terms of calcification and hypertrophy. Cultured neonatal rat cardiomyocytes were treated with 10 µM phenylephrine for 24 h in the absence or presence of the stable adenosine analog 2-chloro-adenosine, the TNAP inhibitor tetramisole or the CD73 inhibitor α,ß-methylene ADP. Phenylephrine produced marked hypertrophy as evidenced by significant increases in myocyte surface area and ANP gene expression, as well as calcification determined by Alizarin Red S staining. These responses were associated with reduced CD73 gene and protein expression and CD73 activity. Conversely, TNAP expression and activity were significantly increased although both were suppressed by 2-chloro-adenosine. CD73 inhibition alone significantly reduced myocyte-derived adenosine levels by >50 %, and directly induced hypertrophy and calcification in the absence of phenylephrine. These responses and those to phenylephrine were abrogated by TNAP inhibition. We conclude that TNAP contributes to the hypertrophic effect of phenylephrine, as well as its ability to produce cardiomyocyte calcification. These responses are minimized by CD73-dependent endogenously produced adenosine.

Hepatocyte-specific CCAAT/enhancer binding protein α restricts liver fibrosis progression.

Metabolic dysfunction-associated steatohepatitis (MASH) - previously described as nonalcoholic steatohepatitis (NASH) - is a major driver of liver fibrosis in humans, while liver fibrosis is a key determinant of all-cause mortality in liver disease independent of MASH occurrence. CCAAT/enhancer binding protein α (CEBPA), as a versatile ligand-independent transcriptional factor, has an important function in myeloid cells, and is under clinical evaluation for cancer therapy. CEBPA is also expressed in hepatocytes and regulates glucolipid homeostasis; however, the role of hepatocyte-specific CEBPA in modulating liver fibrosis progression is largely unknown. Here, hepatic CEBPA expression was found to be decreased during MASH progression both in humans and mice, and hepatic CEBPA mRNA was negatively correlated with MASH fibrosis in the human liver. CebpaΔHep mice had markedly enhanced liver fibrosis induced by a high-fat, high-cholesterol, high-fructose diet or carbon tetrachloride. Temporal and spatial hepatocyte-specific CEBPA loss at the progressive stage of MASH in CebpaΔHep,ERT2 mice functionally promoted liver fibrosis. Mechanistically, hepatocyte CEBPA directly repressed Spp1 transactivation to reduce the secretion of osteopontin, a fibrogenesis inducer of hepatic stellate cells. Forced hepatocyte-specific CEBPA expression reduced MASH-associated liver fibrosis. These results demonstrate an important role for hepatocyte-specific CEBPA in liver fibrosis progression, and may help guide the therapeutic discoveries targeting hepatocyte CEBPA for the treatment of liver fibrosis.

Silencing IDO in dendritic cells: a novel approach to enhance cancer immunotherapy in a murine breast cancer model.

Cancer immunotherapeutic agents (vaccines) in the form of antigen-loaded dendritic cells (DCs) reached an important milestone with the recent approval of Provenge, the first DC vaccine for treatment of prostate cancer. Although this heralds a new era of tumor immunotherapy, it also highlights the compelling need to optimize such DC-based therapies as they are increasingly tested and used to treat human patients. In this study we sought to augment and enhance the antitumor activity of a DC-based vaccine using siRNA to silence expression of immunosuppressive enzyme indoleamine 2,3-dioxygenase (IDO) in DCs. We report here that DCs loaded with tumor antigens, but with siRNA-silenced IDO expression, were introduced into 4T1 breast tumor-bearing mice, the treatment: (i) lengthened the time required for tumor onset, (ii) decreased tumor size compared to tumors grown for equal lengths of time in mice treated with antigen-loaded DCs without IDO silencing and (iii) reduced CD4(+) and CD8(+) T cell apoptosis. Furthermore, immunization with IDO-silenced DCs enhanced tumor antigen-specific T cell proliferation and CTL activity, and decreased numbers of CD4(+) CD25(+) Foxp3(+) T(reg). This study provides evidence to support silencing of immunosuppressive genes (IDO) as an effective strategy to enhance the efficacy of DC-based cancer immunotherapeutic.

Metabolomics for the identification of early biomarkers of nephrotoxicity in a mouse model of cisplatin-induced acute kidney injury.

BACKGROUND AND PURPOSE: Cisplatin-induced nephrotoxicity manifests as acute kidney injury (AKI) in approximately one third of patients receiving cisplatin therapy. Current measures of AKI are inadequate in detecting AKI prior to significant renal injury, and better biomarkers are needed for early diagnosis of cisplatin-induced AKI. EXPERIMENTAL APPROACH: C57BL/6 and FVB/N mice were treated with a single intraperitoneal injection of cisplatin (15 mg kg-1) or saline. Plasma, urine, and kidney samples were collected prior to cisplatin injection and 24-, 48-, 72-, and 96-hours following cisplatin injection. Untargeted metabolomics was employed using liquid chromatography-mass spectrometry to identify early diagnostic biomarkers for cisplatin nephrotoxicity. PRINCIPAL RESULTS: There was clear metabolic discrimination between saline and cisplatin-treated mice at all timepoints (day 1 to day 4). In total, 26 plasma, urine, and kidney metabolites were identified as exhibiting early alterations following cisplatin treatment. Several of the metabolites showing early alterations were associated with mitochondrial function and energetics, including intermediates of the tricarboxylic acid cycle, regulators of mitochondrial function and indicators of fatty acid ß-oxidation dysfunction. Furthermore, several metabolites were derived from the gut microbiome. MAJOR CONCLUSIONS: Our results highlight the detrimental effects of cisplatin on mitochondrial function and demonstrate potential involvement of the gut microbiome in the pathophysiology of cisplatin-induced AKI. We provide a panel of metabolites to guide future clinical studies of cisplatin-induced AKI and provide insight into potential mechanisms behind cisplatin nephrotoxicity.

Investigation of N,N,N-Trimethyl-L-alanyl-L-proline Betaine (TMAP) as a Biomarker of Kidney Function.

Glomerular filtration rate (GFR) is the most widely used tool for the measurement of kidney function, but endogenous biomarkers such as cystatin C and creatinine have limitations. A previous metabolomic study revealed N,N,N-trimethyl-L-alanyl-L-proline betaine (TMAP) to be reflective of kidney function. In this study, we developed a quantitative LCMS assay for the measurement of TMAP and evaluated TMAP as a biomarker of GFR. An assay to measure TMAP was developed using liquid chromatography-mass spectrometry. After validation of the method, we applied it to plasma samples from three distinct kidney disease patient cohorts: nondialysis chronic kidney disease (CKD) patients, patients receiving peritoneal and hemodialysis, and living kidney donors. We investigated whether TMAP was conserved in other mammalian and nonmammalian species, by analyzing plasma samples from Wistar rats with diet-induced CKD and searching for putative matches to the m/z for TMAP and its known fragments in the raw sample data repository "Metabolomics Workbench". The assay can measure plasma TMAP at a lower limit of quantitation (100 ng/mL) with an interday precision and accuracy of 12.8 and 12.1%, respectively. In all three patient cohorts, TMAP concentrations are significantly higher in patients with CKD than in controls with a normal GFR. Further, TMAP concentrations are also elevated in rats with CKD and TMAP is present in the sap produced from Acer saccharum trees. TMAP concentration is inversely related to GFR suggesting that it is a marker of kidney function. TMAP is present in nonmammalian species suggesting that it is part of a biologically conserved process.

Down-regulation of hepatic CYP3A and CYP2C mediated metabolism in rats with moderate chronic kidney disease.

Expression and activity of drug-metabolizing enzymes are decreased in severe kidney disease; however, only a small percentage of patients with chronic kidney disease (CKD) are at the final stage of the disease. This study aimed to determine the changes in drug-metabolizing enzyme function and expression in rats with varying degrees of kidney disease. Sprague-Dawley rats were subjected to surgical procedures that allowed the generation of three distinct models of kidney function: normal kidney function, moderate kidney function, and severe kidney disease. Forty-two days after surgery, rats were sacrificed and hepatic CYP3A and CYP2C expression was determined. In addition, enzymatic activity was determined in liver microsomes by evaluating midazolam (CYP3A), testosterone (CYP3A and CYP2C), and tolbutamide (CYP2C) enzyme kinetics. Both moderate and severe kidney disease were associated with a reduction in CYP3A2 and CYP2C11 expression (p < 0.05). Likewise, moderate kidney disease resulted in more than a 60% decrease in enzyme activity (V(max)) for CYP2C11 and CYP3A, compared with controls (p < 0.05). When the degree of kidney disease was correlated with metabolic activity, an exponential decline in CYP2C- and CYP3A-mediated metabolism was observed. Our results demonstrate that CYP3A and CYP2C expression and activity are decreased in both moderate and severe CKD. Our data suggest that drug metabolism is significantly decreased in the earlier stages of CKD and imply that patients with moderate CKD may be subject to unpredictable pharmacokinetics.

Pharmacometabolomics reveals urinary diacetylspermine as a biomarker of doxorubicin effectiveness in triple negative breast cancer.

Triple-negative breast cancer (TNBC) patients receive chemotherapy treatment, including doxorubicin, due to the lack of targeted therapies. Drug resistance is a major cause of treatment failure in TNBC and therefore, there is a need to identify biomarkers that determine effective drug response. A pharmacometabolomics study was performed using doxorubicin sensitive and resistant TNBC patient-derived xenograft (PDX) models to detect urinary metabolic biomarkers of treatment effectiveness. Evaluation of metabolite production was assessed by directly studying tumor levels in TNBC-PDX mice and human subjects. Metabolic flux leading to biomarker production was determined using stable isotope-labeled tracers in TNBC-PDX ex vivo tissue slices. Findings were validated in 12-h urine samples from control (n = 200), ER+/PR+ (n = 200), ER+/PR+/HER2+ (n = 36), HER2+ (n = 81) and TNBC (n = 200) subjects. Diacetylspermine was identified as a urine metabolite that robustly changed in response to effective doxorubicin treatment, which persisted after the final dose. Urine diacetylspermine was produced by the tumor and correlated with tumor volume. Ex vivo tumor slices revealed that doxorubicin directly increases diacetylspermine production by increasing tumor spermidine/spermine N1-acetyltransferase 1 expression and activity, which was corroborated by elevated polyamine flux. In breast cancer patients, tumor diacetylspermine was elevated compared to matched non-cancerous tissue and increased in HER2+ and TNBC compared to ER+ subtypes. Urine diacetylspermine was associated with breast cancer tumor volume and poor tumor grade. This study describes a pharmacometabolomics strategy for identifying cancer metabolic biomarkers that indicate drug response. Our findings characterize urine diacetylspermine as a non-invasive biomarker of doxorubicin effectiveness in TNBC.

Clenbuterol exerts antidiabetic activity through metabolic reprogramming of skeletal muscle cells.

Activation of the sympathetic nervous system causes pronounced metabolic changes that are mediated by multiple adrenergic receptor subtypes. Systemic treatment with ß2-adrenergic receptor agonists results in multiple beneficial metabolic effects, including improved glucose homeostasis. To elucidate the underlying cellular and molecular mechanisms, we chronically treated wild-type mice and several newly developed mutant mouse strains with clenbuterol, a selective ß2-adrenergic receptor agonist. Clenbuterol administration caused pronounced improvements in glucose homeostasis and prevented the metabolic deficits in mouse models of ß-cell dysfunction and insulin resistance. Studies with skeletal muscle-specific mutant mice demonstrated that these metabolic improvements required activation of skeletal muscle ß2-adrenergic receptors and the stimulatory G protein, Gs. Unbiased transcriptomic and metabolomic analyses showed that chronic ß2-adrenergic receptor stimulation caused metabolic reprogramming of skeletal muscle characterized by enhanced glucose utilization. These findings strongly suggest that agents targeting skeletal muscle metabolism by modulating ß2-adrenergic receptor-dependent signaling pathways may prove beneficial as antidiabetic drugs.

Long non-coding RNA Gm15441 attenuates hepatic inflammasome activation in response to PPARA agonism and fasting.

Exploring the molecular mechanisms that prevent inflammation during caloric restriction may yield promising therapeutic targets. During fasting, activation of the nuclear receptor peroxisome proliferator-activated receptor α (PPARα) promotes the utilization of lipids as an energy source. Herein, we show that ligand activation of PPARα directly upregulates the long non-coding RNA gene Gm15441 through PPARα binding sites within its promoter. Gm15441 expression suppresses its antisense transcript, encoding thioredoxin interacting protein (TXNIP). This, in turn, decreases TXNIP-stimulated NLR family pyrin domain containing 3 (NLRP3) inflammasome activation, caspase-1 (CASP1) cleavage, and proinflammatory interleukin 1ß (IL1B) maturation. Gm15441-null mice were developed and shown to be more susceptible to NLRP3 inflammasome activation and to exhibit elevated CASP1 and IL1B cleavage in response to PPARα agonism and fasting. These findings provide evidence for a mechanism by which PPARα attenuates hepatic inflammasome activation in response to metabolic stress through induction of lncRNA Gm15441.

The effect of chronic kidney disease on CYP2B expression and activity in male Wistar rats.

Chronic kidney disease (CKD) is characterized by progressive reduction in kidney function over time. CKD affects greater than 10% of the population and its incidence is on the rise due to the growing prevalence of its risk factors. Previous studies demonstrated CKD alters nonrenal clearance of drugs in addition to reducing renal clearance. We assessed the function and expression of hepatic CYP2B enzymes using a rat model of CKD. CKD was induced in Wistar rats by supplementing their chow with adenine and confirmed through the detection of elevated uremic toxins in plasma. Liver enzymes AST and ALT were unchanged by the adenine diet. Bupropion was used as a probe substrate for hepatic CYP2B function using rat liver microsomes. The resulting metabolite, hydroxy-bupropion, and bupropion were quantified by ultra-performance liquid chromatography coupled to time-of-flight mass spectrometry. Level of mRNA and protein were determined by RT-PCR and Western blot, respectively. The results of our study demonstrate that CYP2B1 is downregulated in a rat model of CKD. CYP2B1 mRNA level was significantly decreased (88%, P < 0.001) in CKD relative to control. Similarly, maximal enzymatic velocity (Vmax) for CYP2B was decreased by 46% in CKD relative to control (P < 0.0001). Previous studies involving patients with CKD demonstrated altered bupropion pharmacokinetics compared to control. Hence, our results suggest that these alterations may be mediated by attenuated CYP2B hepatic metabolism. This finding may partially explain the alterations in pharmacokinetics and nonrenal drug clearance frequently observed in patients with CKD.

Untargeted metabolomics reveals N, N, N-trimethyl-L-alanyl-L-proline betaine (TMAP) as a novel biomarker of kidney function.

The diagnosis and prognosis of chronic kidney disease (CKD) currently relies on very few circulating small molecules, which can vary by factors unrelated to kidney function. In end-stage renal disease (ESRD), these same small molecules are used to determine dialysis dose and dialytic clearance. Therefore, we aimed to identify novel plasma biomarkers to estimate kidney function in CKD and dialytic clearance in ESRD. Untargeted metabolomics was performed on plasma samples from patients with a single kidney, non-dialysis CKD, ESRD and healthy controls. For ESRD patients, pre- and post-dialysis plasma samples were obtained from several dialysis modalities. Metabolomics analysis revealed over 400 significantly different features in non-dialysis CKD and ESRD plasma compared to controls while less than 35 features were significantly altered in patients with a single kidney. N,N,N-trimethyl-L-alanyl-L-proline betaine (TMAP, AUROC = 0.815) and pyrocatechol sulfate (AUROC = 0.888) outperformed creatinine (AUROC = 0.745) in accurately identifying patients with a single kidney. Several metabolites accurately predicted ESRD; however, when comparing pre-and post-hemodialysis, TMAP was the most robust biomarker of dialytic clearance for all modalities (AUROC = 0.993). This study describes TMAP as a novel potential biomarker of kidney function and dialytic clearance across several hemodialysis modalities.