It has been increasingly recognized that tinnitus is likely to be generated by complex network changes. Acoustic trauma that causes tinnitus induces significant changes in multiple metabolic pathways in the brain. However, it is not clear whether those metabolic changes in the brain could also be reflected in blood samples and whether metabolic changes could discriminate acoustic trauma, hyperacusis and tinnitus. We analyzed brain and serum metabolic changes in rats following acoustic trauma or a sham procedure using metabolomics. Hearing levels were recorded before and after acoustic trauma and behavioral measures to quantify tinnitus and hyperacusis were conducted at 4 weeks following acoustic trauma. Tissues from 11 different brain regions and serum samples were collected at about 3 months following acoustic trauma. Among the acoustic trauma animals, eight exhibited hyperacusis-like behavior and three exhibited tinnitus-like behavior. Using Gas chromatography-mass spectrometry and multivariate statistical analysis, significant metabolic changes were found in acoustic trauma animals in both the brain and serum samples with a number of metabolic pathways significantly perturbated. Furthermore, metabolic changes in the serum were able to differentiate sham from acoustic trauma animals, as well as sham from hyperacusis animals, with high accuracy. Our results suggest that serum metabolic profiling in combination with machine learning analysis may be a promising approach for identifying biomarkers for acoustic trauma, hyperacusis and potentially, tinnitus.

High-calorie diet, circadian rhythms and metabolic features are intimately linked. However, the mediator(s) between nutritional status, circadian rhythms and metabolism remain largely unknown. This article aims to clarify the key metabolic pathways bridging nutritional status and circadian rhythms based on a combination of metabolomics and molecular biological techniques. A mouse model of high-fat diet-induced obesity was established and serum samples were collected in obese and normal mice at different zeitgeber times. Gas chromatography/mass spectrometry, multivariate/univariate data analyses and metabolic pathway analysis were used to reveal changes in metabolism. Metabolites involved in the metabolism of purines, carbohydrates, fatty acids and amino acids were markedly perturbed in accordance with circadian related variations, among which purine catabolism showed a typical oscillation. What's more, the rhythmicity of purine catabolism dampened in the high-fat diet group. The expressions of clock genes and metabolic enzymes in the liver were measured. The mRNA expression of Xanthine oxidase (Xor) was highly correlated with the rhythmicity of Clock, Rev-erbα and Bmal1, as well as the metabolites involved in purine catabolism. These data showed that a high-fat diet altered the circadian rhythm of metabolic pathways, especially purine catabolism. It had an obvious circadian oscillation and a high-fat diet dampened its circadian rhythmicity. It was suggested that circadian rhythmicity of purine catabolism is related to circadian oscillations of expression of Xor, Uox and corresponding clock genes.

Epalrestat is an inhibitor of aldose reductase in the polyol pathway and is used for the management of diabetic neuropathy clinically. Our pilot experiments and accumulated evidences showed that epalrestat inhibited polyol pathway and reduced sorbitol production, and suggested the potential renal protection effects of epalrestat on diabetic nephropathy (DN). To evaluate the protective effect of epalrestat, the db/db mice were used and exposed to epalrestat for 8 weeks, both the physiopathological condition and function of kidney were examined. For the first time, we showed that epalrestat markedly reduced albuminuria and alleviated the podocyte foot process fusion and interstitial fibrosis of db/db mice. Metabolomics was employed, and metabolites in the plasma, renal cortex, and urine were profiled using a gas chromatography-mass spectrometry (GC/MS)-based metabolomic platform. We observed an elevation of sorbitol and fructose, and a decrease of myo-inositol in the renal cortex of db/db mice. Epalrestat reversed the renal accumulation of the polyol pathway metabolites of sorbitol and fructose, and increased myo-inositol level. Moreover, the upregulation of aldose reductase, fibronectin, collagen III, and TGF-β1 in renal cortex of db/db mice was downregulated by epalrestat. The data suggested that epalrestat has protective effects on DN, and the inhibition of aldose reductase and the modulation of polyol pathway in nephritic cells be a potentially therapeutic strategy for DN.

Berberrubine (BRB) has a strong lipid-lowering effect and can be extensively metabolized into berberrubine-9-O-β-d-glucuronide (BRBG) in vivo. Recently, pharmacokinetics studies showed that the production of BRBG was significantly decreased in the urine of mice fed with a high-fat diet (HFD), indicating a decreased glucuronidation capacity. Based on the UDP-glucuronosyltransferase (UGT) isoform identification, hepatic and renal microsomal incubation, glucuronidation was examined to suggest the metabolism of BRB in liver and kidneys. The results showed that the renal UGT activity for metabolizing BRB markedly decreased, which may be highly related to the decreased expression and activity of renal Ugt1a7c. Surprisingly, in vitro studies revealed neither BRB nor BRBG inhibited the renal UGT activity. By employing an integrated strategy of metabolomics and pharmacokinetics, we identified and confirmed for the first time the inhibitory effect of some potential endogenous molecules on the renal glucuronidation of C57BL/6J mice, such as glutaric acid (GA) and linoleic acid (LA). By employing recombinant human UGTs, we found that GA and LA efficiently affect the activity of recombinant human UGT1A7, 1A9, and 1A8 at their normal or abnormal physiologic levels in vivo. GA (2 mM) markedly inhibited the activity of UGT1A7 by 89.4% and UGT1A9 by 32.8%. The inhibition rates reached 99.3% for UGT1A9, 48.3% for UGT1A7, and 46.8% for UGT1A8 with LA at 200 μM. It has been suggested that the endogenous molecules have the potential to affect the efficiency of glucuronidation, which might be a key factor contributing to individual differences in drug metabolism.

The early diagnosis of diabetic nephropathy (DN) is rather challenging. Our previous study suggested that citric acid is a potential marker for the early diagnosis of diabetic nephropathy in db/db mice. For the first time, in this study, a surrogate analyte of 13C6-citric acid was employed to generate calibration curves for the quantitative measurement of the endogenous citric acid in the sera of db/db mice and diabetic nephropathy patients by GC/MS after the analytes were extracted, methoximated and trimethylsilylated. The constant response factor of 13C6-citric acid versus citric acid over the linear range indicated the identical ionization efficiency of these two compounds. The full validation assessments suggested that the method is sensitive, specific, reliable, reproducible and has acceptable parameters. Statistical analysis revealed cut-off citric acid concentrations of 29.24 μg/mL with a 95% confidence interval between 32.75 and 39.16 μg/mL in the diabetic nephropathy patients and 16.74 and 22.57 μg/mL in the normal controls. The areas under the receiver operating characteristic curves indicated accuracies of over 90% for the diagnoses of early diabetic nephropathy in both humans and db/db mice, which suggests that the serum citric acid level is potentially a biomarker that could assist in the diagnosis of diabetic nephropathy.

Objective To develop a sensitive and reproducible liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to evaluate the pharmacokinetic behavior of berberrubine (BRB) and its glucuronide (BRBG) in rats. Methods BRB, BRBG and tetrahydroberberine (THB, internal standard) were isolated by liquid-liquid extraction in rat biological samples. Chromatographic separation was achieved on an Agilent Zorbax Eclipse Plus C-18 (2.1 mm x 50 mm, 3.5-Micron) with a gradient mobile phases primarily containing acetonitrile, water with 0.1% formic acid and 5 mm ammonium acetate. The analytes were monitored by MS/MS in positive electrospray ionization mode. Herein, the feasibility of new developed method was validated with respect to specificity, linearity, precision, accuracy, stability, extraction efficiency and matrix effect. The appropriate method was used for the pharmacokinetic study in rats. Results The new developed method could be applied to the pharmacokinetic study of BRB in rats. BRB and BRBG showed good linearity over the ranges of 2-1000 ng/mL and 5-2000 ng/mL, respectively, and precision was no more than 15%. The accuracy, specificity and stability could be acceptable. Conclusion The new method is sensitive and reproducible. In pharmacokinetic study, BRB showed nonlinear elimination property. Meanwhile, BRB was rapidly absorbed and widely distributed in various tissues with the highest exposure of BRB in kidney and liver. The absolute bioavailability of BRB was determined to be 8.2% and at the dose of 40 mg/kg, a total of 44% BRB was excreted in urine and feces.

Epalrestat is clinically applied for the management of diabetic peripheral neuropathy, yet its pharmacokinetic properties are not well understood. In this study, a rapid and sensitive LC–MS/MS method was established for assaying epalrestat in bio-samples of mice. The method was validated and it showed a good linearity over the range of 2–5000 ng/mL, a precision of less than 12.3%, and recovery and matrix effects of 112.5–123.6% and 87.9–89.5%, respectively. After administration of a single dose of epalrestat administered, the exposure level of AUC0–∞ was positively dose-dependent and the mean Cmax, AUC0–12 h, T1/2, and MRT were 36.23 ± 7.39 μg/mL, 29,086.5 μg/L h, 1.2 h and 1.8 h, respectively. Epalrestat was highly exposed in stomach, intestine, liver and kidney, and only a small amount was detected in brain, urine and feces. Multi-dose of epalrestat significantly increased MRT and apparent volume of distribution (Vd) relative to those of a single-dose.

Berberrubine (BRB) is the primary metabolite of berberine (BBR) that has shown a stronger glucose-lowering effect than BBR in vivo . On the other hand, BRB is quickly and extensively metabolized into berberrubine-9- O -β- D -glucuronide (BRBG) in rats after oral administration. In this study we compared the pharmacokinetic properties of BRB and BRBG in rats, and explored the mechanisms underlying their glucose-lowering activities. C57BL/6 mice with HFD-induced hyperglycemia were administered BRB (50 mg·kg −1 ·d −1 , ig) for 6 weeks, which caused greater reduction in the plasma glucose levels than those caused by BBR (120 mg·kg −1 ·d −1 ) or BRB (25 mg·kg −1 ·d −1 ). In addition, BRB dose-dependently decreased the activity of α-glucosidase in gut of the mice. After oral administration of BRB in rats, the exposures of BRBG in plasma at 3 different dosages (10, 40, 80 mg/kg) and in urine at different time intervals (0–4, 4–10, 10–24 h) were dramatically greater than those of BRB. In order to determine the effectiveness of BRBG in reducing glucose levels, we prepared BRBG from the urine pool of rats, and identified and confirmed it through LC-MS-IT-TOF and NMR spectra. In human normal liver cell line L-O2 in vitro , treatment with BRB or BRBG (5, 20, 50 μmol/L) increased glucose consumption, enhanced glycogenesis, stimulated the uptake of the glucose analog 2-NBDG, and modulated the mRNA levels of glucose-6-phosphatase and hexokinase. However, both BBR and BRB improved 2-NBDG uptake in insulin-resistant L-O2 cells, while BRBG has no effect. In conclusion, BRB exerts a stronger glucose-lowering effect than BBR in HFD-induced hyperglycemia mice. Although BRB significantly stimulated the insulin sensitivity and glycolysis in vitro , BRBG may have a greater contribution to the glucose-lowering effect because it has much greater system exposure than BRB after oral administration of BRB. The results suggest that BRBG is a potential agent for reducing glucose levels.

Acoustic trauma is the most common cause of hearing loss and tinnitus in humans. However, the impact of acoustic trauma on system biology is not fully understood. It has been increasingly recognized that tinnitus caused by acoustic trauma is unlikely to be generated by a single pathological source, but rather a complex network of changes involving not only the auditory system but also systems related to memory, emotion and stress. One obvious and significant gap in tinnitus research is a lack of biomarkers that reflect the consequences of this interactive “tinnitus-causing” network. In this study, we made the first attempt to analyse brain metabolic changes in rats following acoustic trauma using metabolomics, as a pilot study prior to directly linking metabolic changes to tinnitus. Metabolites in 12 different brain regions collected from either sham or acoustic trauma animals were profiled using a gas chromatography mass spectrometry (GC/MS)-based metabolomics platform. After deconvolution of mass spectra and identification of the molecules, the metabolomic data were processed using multivariate statistical analysis. Principal component analysis showed that metabolic patterns varied among different brain regions; however, brain regions with similar functions had a similar metabolite composition. Acoustic trauma did not change the metabolite clusters in these regions. When analyzed within each brain region using the orthogonal projection to latent structures discriminant analysis sub-model, 17 molecules showed distinct separation between control and acoustic trauma groups in the auditory cortex, inferior colliculus, superior colliculus, vestibular nucleus complex (VNC), and cerebellum. Further metabolic pathway impact analysis and the enrichment overview with network analysis suggested the primary involvement of amino acid metabolism, including the alanine, aspartate and glutamate metabolic pathways, the arginine and proline metabolic pathways and the purine metabolic pathway. Our results provide the first metabolomics evidence that acoustic trauma can induce changes in multiple metabolic pathways. This pilot study also suggests that the metabolomic approach has the potential to identify acoustic trauma-specific metabolic shifts in future studies where metabolic changes are correlated with the animal's tinnitus status.

Berberrubine (BRB), the active metabolite of berberine (BBR), possesses various pharmacological activities. In this study, we found BRB showed not only a stronger lipid-lowering effect than berberine but also a specific nephrotoxicity in mice fed with high fat diet (HFD). To explore the underlying mechanism, the pharmacokinetics of BRB were evaluated. There was a greater in vivo exposure of BRB in C57BL/6J mice fed with HFD than with routine chows, in terms of Cmax, AUC0-t, levels of BRB in kidney and urinary excretion. Moreover, in vitro assessment clearly showed BRB had a toxic effect on renal cell lines, while the primary metabolite, berberrubine-9-O-β-d-glucuronide (BRBG), did not show any obvious toxicity. These results suggested HFD aggravated BRB-induced nephrotoxicity by promoting the in vivo exposure of BRB especially in urine and kidney. Although our previous study indicated BRB could be metabolized into BRBG, BRBG did not show any obvious toxicity in vitro.

Previous studies suggest that the lipid-lowering effect of berberine (BBR) involves actions on the low-density lipoprotein receptor and the AMP-activated protein kinase signaling pathways. However, the implication of these mechanisms is unclear because of the low bioavailability of BBR. Because the main action site of BBR is the gut and intestinal farnesoid X receptor (FXR) plays a pivotal role in the regulation of lipid metabolism, we hypothesized that the effects of BBR on intestinal FXR signaling pathway might account for its pharmacological effectiveness. Using wild type (WT) and intestine-specific FXR knockout (FXRint-/-) mice, we found that BBR prevented the development of high-fat-diet-induced obesity and ameliorated triglyceride accumulation in livers of WT, but not FXRint-/- mice. BBR increased conjugated bile acids in serum and their excretion in feces. Furthermore, BBR inhibited bile salt hydrolase (BSH) activity in gut microbiota, and significantly increased the levels of tauro-conjugated bile acids, especially tauro-cholic acid(TCA), in the intestine. Both BBR and TCA treatment activated the intestinal FXR pathway and reduced the expression of fatty-acid translocase Cd36 in the liver. These results indicate that BBR may exert its lipid-lowering effect primarily in the gut by modulating the turnover of bile acids and subsequently the ileal FXR signaling pathway. In summary, we provide the first evidence to suggest a new mechanism of BBR action in the intestine that involves, sequentially, inhibiting BSH, elevating TCA, and activating FXR, which lead to the suppression of hepatic expression of Cd36 that results in reduced uptake of long-chain fatty acids in the liver.