Antiviral therapy reduces hepatocellular carcinoma through suppressing hepatitis B virus replication may improve ER stress, mitochondrial and metabolic dysfunctions and decrease p62 in hybridized mice with single HBV transgene and miR-122.

Hepatitis B virus (HBV) hijacks autophagy for its replication. Nucleos(t)ide analogs (NUCs) treatment suppressed HBV replication and reduced hepatocellular carcinoma (HCC) incidence. However, the use of NUCs in chronic hepatitis B (CHB) patients with normal or minimally elevated serum alanine aminotransferase (ALT) levels is still debated. Animal models are crucial for studying the unanswered issue and evaluating new therapies. MicroRNA-122 (miR-122), which regulates fatty acid and cholesterol metabolism, is downregulated during hepatitis and HCC progression. The reciprocal inhibition of miR-122 with HBV highlights its role in HCC development as a tumor suppressor. By crossbreeding HBV-transgenic mice with miR-122 knockout mice, we generated a hybrid mouse model with a high incidence of HCC up to 89% and normal ALT levels before HCC. The model exhibited early-onset hepatic steatosis, progressive liver fibrosis, and impaired late-phase autophagy. Metabolomics and microarray analysis identified metabolic signatures, including dysregulation of lipid metabolism, inflammation, genomic instability, the Warburg effect, reduced TCA cycle flux, energy deficiency, and impaired free radical scavenging. Antiviral treatment reduced HCC incidence in hybrid mice by approximately 30-35% compared to untreated mice. This effect was linked to the activation of ER stress-responsive transcription factor ATF4, clearance of autophagosome cargo p62, and suppression of the CHOP-mediated apoptosis pathway. In summary, this study suggests that despite minimal ALT elevation, HBV replication can lead to liver injury. Endoplasmic reticulum stress, reduced miR-122 levels, mitochondrial and metabolic dysfunctions, blocking protective autophagy resulting in p62 accumulation, apoptosis, fibrosis, and HCC. Antiviral may improve the above-mentioned pathogenesis through HBV suppression.

Vegetable Signatures Derived from Human Urinary Metabolomic Data in Controlled Feeding Studies.

Examination of changes in urinary metabolomic profiles after vegetable ingestion may lead to new methods of assessing plant food intake. To this regard, we developed a proof-of-principle methodology to identify urinary metabolomic signatures for spinach, celery, and onion. Three feeding studies were conducted. In the first study, healthy individuals were fed with spinach, celery, onion, and no vegetables in four separate experiments with pooled urinary samples for metabolite discovery. The same protocol was used to validate the finding at the individual level in the second study and when feeding all three vegetables simultaneously in the third study. An LC-MS-based metabolomics approach was adopted to search for indicative metabolites from urine samples collected during multiple time periods before and after the meal. Consequently, a total of 1, 9, and 3 nonoverlapping urinary metabolites were associated with the intake of spinach, celery, and onion, respectively. The PCA signature of these metabolites followed a similar "time cycle" pattern, which maximized at approximately 2-4 h after intake. In addition, the metabolite profiles for the same vegetable were consistent across samples, regardless of whether it was consumed individually or in combination. The developed methodology along with the identified urinary metabolomic signatures were potential tools for assessing plant food intake.

Metabolomics study in severe extracranial carotid artery stenosis.

BACKGROUND: Significant genetic association has been found in patients with severe carotid artery stenosis (CAS). The present study wished to investigate if metabolites may also act as biomarkers for CAS. METHODS: Consecutive patients with at least one carotid artery stenosis > = 60% on cerebral angiography were prospectively recruited from May 2007 to January 2016. Normal controls were recruited from outpatient clinic who had no stroke and coronary artery disease (CAD) history, and the brain magnetic resonance or computed tomographic angiography showed bilateral CAS < 30%. Risk factor profile, clinical characteristics, age, and clinical features were recorded. All subjects were male, and none had diabetes. 1H-NMR spectroscopy-based metabolomics analysis was carried out for plasma samples. RESULTS: Totally, 130 male subjects were recruited. Age had no significant difference between the controls and CAS group (60.2 ± 5.9 vs. 63.3 ± 6.0, p = 0.050). The CAS group had significantly higher frequency of CAD, hypertension, smoking and alcohol but lower body mass index than the controls (p < 0.05). The laboratory tests showed CAS group had significantly higher level of homocysteine but lower levels of cholesterol, high-density lipoprotein and hemoglobin than the controls (p < 0.05). The 1H-NMR based plasma metabolomics analysis indicated that choline was significantly lower in CAS patients. The VIP values of lipids were greater than 1.0, which were considered significantly different. CONCLUSIONS: Our results suggest homocysteine, choline and lipids in association with traditional risk factors may be involved in the pathogenesis of CAS. Diet adjustment to control homocysteine, choline and lipids may be helpful for the prevention of CAS.

Flux balance analysis predicts Warburg-like effects of mouse hepatocyte deficient in miR-122a.

The liver is a vital organ involving in various major metabolic functions in human body. MicroRNA-122 (miR-122) plays an important role in the regulation of liver metabolism, but its intrinsic physiological functions require further clarification. This study integrated the genome-scale metabolic model of hepatocytes and mouse experimental data with germline deletion of Mir122a (Mir122a-/-) to infer Warburg-like effects. Elevated expression of MiR-122a target genes in Mir122a-/-mice, especially those encoding for metabolic enzymes, was applied to analyze the flux distributions of the genome-scale metabolic model in normal and deficient states. By definition of the similarity ratio, we compared the flux fold change of the genome-scale metabolic model computational results and metabolomic profiling data measured through a liquid-chromatography with mass spectrometer, respectively, for hepatocytes of 2-month-old mice in normal and deficient states. The Ddc gene demonstrated the highest similarity ratio of 95% to the biological hypothesis of the Warburg effect, and similarity of 75% to the experimental observation. We also used 2, 6, and 11 months of mir-122 knockout mice liver cell to examined the expression pattern of DDC in the knockout mice livers to show upregulated profiles of DDC from the data. Furthermore, through a bioinformatics (LINCS program) prediction, BTK inhibitors and withaferin A could downregulate DDC expression, suggesting that such drugs could potentially alter the early events of metabolomics of liver cancer cells.

Dietary Leucine Supplement Ameliorates Hepatic Steatosis and Diabetic Nephropathy in db/db Mice.

Dietary leucine supplementation has been explored for the therapeutic intervention of obesity and obesity-induced metabolic dysfunctions. In this study, we aim to examine the effects of dietary leucine supplementation in db/db mice. Mice were treated with or without leucine (1.5% w/v) in drinking water for 12 weeks. The leucine supplement was found to reduce insulin resistance and hepatic steatosis in db/db mice. Using Nuclear Magnetic Resonance (NMR)-based lipidomics, we found that the reduction of hepatic triglyceride synthesis was correlated with attenuated development of fatty liver. In addition, diabetic nephropathy (DN) was also ameliorated by leucine. Using liquid chromatography⁻time-of-flight mass spectrometry (LC-TOF MS)-based urine metabolomics analysis, we found that the disturbance of the tricarboxylic acid (TCA) cycle was reversed by leucine. The beneficial effects of leucine were probably due to AMP-activated protein kinase (AMPK) activation in the liver and kidneys of db/db mice. Thus, dietary leucine supplementation may potentially be a nutritional intervention to attenuate hepatic steatosis and early DN in type II diabetes.

Recovery of pan-genotypic and genotype-specific amino acid alterations in chronic hepatitis C after viral clearance: transition at the crossroad of metabolism and immunity.

Recovery of amino acid (AA) metabolism and the associated clinical implications in chronic hepatitis C (CHC) patients with sustained virological response (SVR) following anti-hepatitis C virus (HCV) therapy remains elusive. A prospective cohort study was conducted on 222 CHC patients with SVR. Eighty-two age-matched male genotype 1 (G1) and G2 patients underwent paired serum metabolomics analyses with liquid chromatography-tandem mass spectrometry to examine AAs before and 24 weeks after anti-HCV therapy. Before anti-HCV therapy, G1 patients had a higher HCV RNA level than G2 patients. Twenty-four weeks post-therapy versus pre-therapy, repeated-measures ANOVA showed that the levels of alanine aminotransferase and most AAs decreased while those of lipids, glutamine and putrescine increased in CHC patients. The methionine sulfoxide/methionine ratio decreased, while the asymmetric dimethylarginine/arginine, glutamine/glutamate, citrulline/arginine, ornithine/arginine, kynurenine/tryptophan, tyrosine/phenylalanine and Fisher's ratios increased. Genotype-specific subgroup analyses showed that valine and serotonin/tyrosine increased in G1 and that kynurenine and tyrosine/phenylalanine increased and sarcosine decreased in G2 patients. Viral clearance in CHC patients pan-genotypically restored fuel utilization by decelerating the tricarboxylic acid cycle. Following improvement in liver function, the urea, nitric oxide, methionine, and polyamine cycles were accelerated. The cardiometabolic risk attenuated, but the augmented kynurenine pathway activity could increase the oncogenesis risk. The trends in neurotransmitter formation differed between G1 and G2 patients after SVR. Moreover, the HCV-suppressing effect of valine was evident in G1 patients; with the exception of prostate cancer, the oncogenesis risk increased, particularly in G2 patients, at least within 24 weeks post-anti-HCV therapy.

Increased p-cresyl sulfate level is independently associated with poor outcomes in patients with heart failure.

Amino acid-derived metabolites, including protein-bound uremic toxins, may have prognostic value for patients with heart failure (HF). The aim of this study was to investigate whether p-cresyl sulfate (PCS), indoxyl sulfate (IS), and arginine metabolites provided prognostic values in addition to the traditional biomarker, B-type natriuretic peptide (BNP), in patients with HF. Chromatography mass spectrometry was performed to measure tyrosine, tryptophan, arginine, PCS, IS, and asymmetric (ADMA) and symmetric dimethylarginine (SDMA) in the plasma from 51 normal controls and 136 HF patients. Compared to the normal controls, PCS levels significantly increased in HF patients (p = 0.003). During the follow-up (2.3 ± 1.1 years), 35 (25.7 %) patients experienced a composite event of death or HF-related re-hospitalization. In univariable analysis, PCS, estimated glomerular filtration rate (eGFR), BNP, DMA/arginine ratio, and ADMA/arginine ratio were associated with a higher rate of composite events. In the multivariable analysis, PCS was the only independent predictor of composite events [hazard ratio (HR) 1.06 (per 10 µM), 95 % confidence interval (CI) 1.01-1.11, p = 0.02]. Kaplan-Meier curves showed that a PCS level of ≥50 µM was significantly associated with a higher composite event rate than those with a PCS level of <50 µM (Log rank = 5.11, p = 0.024; HR 2.13, 95 % CI 1.09-4.16, p = 0.02). In conclusion, among protein-bound uremic toxins, eGFR, and DMA metabolites, increased PCS is the only independent predictor of HF-related events in patients with HF. A combination of PCS and BNP should better risk-stratify patients with HF.

Metabolite identification for mass spectrometry-based metabolomics using multiple types of correlated ion information.

Metabolite identification remains a bottleneck in mass spectrometry (MS)-based metabolomics. Currently, this process relies heavily on tandem mass spectrometry (MS/MS) spectra generated separately for peaks of interest identified from previous MS runs. Such a delayed and labor-intensive procedure creates a barrier to automation. Further, information embedded in MS data has not been used to its full extent for metabolite identification. Multimers, adducts, multiply charged ions, and fragments of given metabolites occupy a substantial proportion (40-80%) of the peaks of a quantitation result. However, extensive information on these derivatives, especially fragments, may facilitate metabolite identification. We propose a procedure with automation capability to group and annotate peaks associated with the same metabolite in the quantitation results of opposite modes and to integrate this information for metabolite identification. In addition to the conventional mass and isotope ratio matches, we would match annotated fragments with low-energy MS/MS spectra in public databases. For identification of metabolites without accessible MS/MS spectra, we have developed characteristic fragment and common substructure matches. The accuracy and effectiveness of the procedure were evaluated using one public and two in-house liquid chromatography-mass spectrometry (LC-MS) data sets. The procedure accurately identified 89% of 28 standard metabolites with derivative ions in the data sets. With respect to effectiveness, the procedure confidently identified the correct chemical formula of at least 42% of metabolites with derivative ions via MS/MS spectrum, characteristic fragment, and common substructure matches. The confidence level was determined according to the fulfilled identification criteria of various matches and relative retention time.

ADI1, a methionine salvage pathway enzyme, is required for Drosophila fecundity.

BACKGROUND: Methionine, an essential amino acid, is required for protein synthesis and normal cell metabolism. The transmethylation pathway and methionine salvage pathway (MTA cycle) are two major pathways regulating methionine metabolism. Recently, methionine has been reported to play a key role in Drosophila fecundity. RESULTS: Here, we revealed that the MTA cycle plays a crucial role in Drosophila fecundity using the mutant of aci-reductone dioxygenase 1 (DADI1), an enzyme in the MTA cycle. In dietary restriction condition, the egg production of adi1 mutant flies was reduced compared to that of control flies. This fecundity defect in mutant flies was rescued by reintroduction of Dadi1 gene. Moreover, a functional homolog of human ADI1 also recovered the reproduction defect, in which the enzymatic activity of human ADI1 is required for normal fecundity. Importantly, methionine supply rescued the fecundity defect in Dadi1 mutant flies. The detailed analysis of Dadi1 mutant ovaries revealed a dramatic change in the levels of methionine metabolism. In addition, we found that three compounds namely, methionine, SAM and Methionine sulfoxide, respectively, may be required for normal fecundity. CONCLUSIONS: In summary, these results suggest that ADI1, an MTA cycle enzyme, affects fly fecundity through the regulation of methionine metabolism.

Pharmacometabolomic study of drug response to antihypertensive medications for hypertension marker identification in Han Chinese individuals in Taiwan.

Various groups of antihypertensive drugs targeting different pathways have been developed; however, the pharmacometabolic responses to these drugs have rarely been compared to elucidate the common pathway of blood pressure regulation. Here, we performed a comparative multi-dimensional pharmacometabolic study on the four major lines of antihypertensive drugs, namely angiotensin-converting enzyme inhibitors (ACEis), angiotensin receptor blockers (ARBs), calcium channel blockers (CCBs), and diuretics (DIURs), through ultra-performance liquid chromatography coupled to quantum time-of-flight mass spectrometry. Two hundred fifty patients with young-onset hypertension, who were equally divided among five study groups: non-medicated, ACEi, ARB, CCB, and DIUR groups, were recruited. In a metabolome-wide association study conducted through analysis of covariance, 37 molecular features significantly associated with pharmacometabolic responses to antihypertensive drugs were identified. One-third of these features were shared by multiple medications. ACEis, ARBs, and DIURs shared more features than CCB, partially reflecting that ACEis, ARBs, and DIURs affect the renin-angiotensin-aldosterone system. Thirteen molecular features were consistently identified by all four models of the analysis of covariance. A tandem mass spectrometry (or MS/MS) experiment was performed to decipher the chemical structure of these 13 molecular features, including ARB-associated lysophosphatidylcholine (P4135), CCB-associated diacylglycerol(15:0/18:2) (P1175), and DIUR-associated oleamide (P1516). In addition, diacylglycerol(15:0/14:2) (P408) was significantly associated with the pharmacometabolic response to all four antihypertensive drugs. The identified metabolites provide insights into the mechanisms of blood pressure regulation and potential predictive markers of pharmacometabolic responses to antihypertensive drugs.

A novel testosterone catabolic pathway in bacteria.

Forty years ago, Coulter and Talalay (A. W. Coulter and P. Talalay, J. Biol. Chem. 243:3238-3247, 1968) established the oxygenase-dependent pathway for the degradation of testosterone by aerobes. The oxic testosterone catabolic pathway involves several oxygen-dependent reactions and is not available for anaerobes. Since then, a variety of anaerobic bacteria have been described for the ability to degrade testosterone in the absence of oxygen. Here, a novel, oxygenase-independent testosterone catabolic pathway in such organisms is described. Steroidobacter denitrificans DSMZ18526 was shown to be capable of degrading testosterone in the absence of oxygen and was selected as the model organism in this study. In a previous investigation, we identified the initial intermediates involved in an anoxic testosterone catabolic pathway, most of which are identical to those of the oxic pathway demonstrated in Comamonas testosteroni. In this study, five additional intermediates of the anoxic pathway were identified. We demonstrated that subsequent steps of the anoxic pathway greatly differ from those of the established oxic pathway, which suggests that a novel pathway for testosterone catabolism is present. In the proposed anoxic pathway, a reduction reaction occurs at C-4 and C-5 of androsta-1,4-diene-3,17-dione, the last common intermediate of both the oxic and anoxic pathways. After that, a novel hydration reaction occurs and a hydroxyl group is thus introduced to the C-1α position of C(19)steroid substrates. To our knowledge, an enzymatic hydration reaction occurring at the A ring of steroid compounds has not been reported before.

Resveratrol ameliorates metabolic disorders and muscle wasting in streptozotocin-induced diabetic rats.

Diabetes mellitus (DM) is characterized by dysregulated energy metabolism. Resveratrol (RSV) has been shown to ameliorate hyperglycemia and hyperlipidemia in diabetic animals. However, its overall in vivo effects on energy metabolism and the underlying mechanism require further investigation. In the present study, electrospray ionization-tandem mass spectrometry was employed to characterize the urine and plasma metabolomes of control, streptozotocin-induced DM and RSV-treated DM rats. Using principal component analysis (PCA) and heat map analysis, we discovered significant differences among control and experimental groups. RSV treatment significantly reduced the metabolic abnormalities in DM rats. Compared with the age-matched control rats, the level of carnitine was lower, and the levels of acetylcarnitine and butyrylcarnitine were higher in the urine and plasma of DM rats. RSV treatment ameliorated the deranged carnitine metabolism in DM rats. In addition, RSV treatment attenuated the diabetic ketoacidosis and muscle protein degradation, as evidenced from the attenuation of elevated urinary methyl-histidine and plasma branched-chain amino acids levels in DM rats. The beneficial effects of RSV in DM rats were correlated with activation of hepatic AMP-activated protein kinase and SIRT1 expression, increase of hepatic and muscular mitochondrial biogenesis and inhibition of muscle NF-κB activities. We concluded that RSV possesses multiple beneficial metabolic effects in insulin-deficient DM rats, particularly in improving energy metabolism and reducing protein wasting.

Aß40(L17A/F19A) mutant diminishes the aggregation and neurotoxicity of Aß40.

Aggregated ß-amyloid peptides (Aß) are neurotoxic and responsible for neuronal death both in vitro and in vivo. From the structural point of view, Aß self-aggregation involves a conformational change in the peptide. Here, we investigated the relationship between conformational changes and amino acid residues of Aß(40). Urea unfolding in combination with NMR spectroscopy was applied to probe the stabilization of Aß(40) conformation. L17 and F19 residues were found more sensitive to environmental changes than the other residues. Replacement of these two residues with alanine could stabilize the conformation of Aß(40). Further analysis indicated that the Aß(40)(L17A/F19A) mutant could diminish the aggregation and reduce the neurotoxicity. These results suggest that L17 and F19 are the critical residues responsible for conformational changes which may trigger neurotoxic cascade of Aß(40).

Salvianolic acid B inhibits low-density lipoprotein oxidation and neointimal hyperplasia in endothelium-denuded hypercholesterolaemic rabbits.

BACKGROUND: Atherosclerosis and restenosis are inflammatory responses involving free radicals and lipid peroxidation and may be prevented/cured by antioxidant-mediated lipid peroxidation inhibition. Salvianolic acid (Sal B), a water-soluble antioxidant obtained from a Chinese medicinal herb, is believed to have multiple preventive and therapeutic effects against human vascular diseases. In this study the in vitro and in vivo inhibitory effects of Sal B on oxidative stress were determined. RESULTS: In human aortic endothelial cells (HAECs), Sal B reduced oxidative stress, inhibited low-density lipoprotein (LDL) oxidation and reduced oxidised LDL-induced cytotoxicity. Sal B inhibited Cu(2+) -induced LDL oxidation in vitro (with a potency 16.3 times that of probucol) and attenuated HAEC-mediated LDL oxidation as well as reactive oxygen species (ROS) production. In cholesterol-fed New Zealand White rabbits (with probucol as positive control), Sal B intake reduced Cu(2+) -induced LDL oxidation, lipid deposition in the thoracic aorta, intimal thickness of the aortic arch and thoracic aorta and neointimal formation in the abdominal aorta. CONCLUSION: The data obtained in this study suggest that Sal B protects HAECs from oxidative injury-mediated cell death via inhibition of ROS production. The antioxidant activity of Sal B may help explain its efficacy in the treatment of vascular diseases.

Retrospective Analysis of the Effects of Non-Compliance with Denosumab on Changes in Bone Mineral Density During the COVID-19 Pandemic.

PURPOSE: Although denosumab is a safe and effective treatment for osteoporosis in various clinical trials, few studies have investigated its efficacy in specific clinical situations. The effect of non-compliance with the standard six-month dosing regimen for denosumab on bone mineral density (BMD) was assessed in a retrospective study of patients prescribed denosumab during the COVID-19 pandemic. PATIENTS AND METHODS: Between February 2019 and September 2020, 638 patient records were reviewed, with 236 patients meeting the eligibility criteria. Patients were divided into three groups: those who received denosumab injections between five and seven months after their initial subcutaneous injection, those who received denosumab injections between seven and nine months after their initial subcutaneous injection, and those who received denosumab injections more than nine months after their initial subcutaneous injection. A multivariate regression study was conducted to compare the BMD shift (at least one year apart) before and after two denosumab injections between the three pre-specified groups in both the lumbar spine (LS) and the femoral neck (FN). RESULTS: The difference between LS BMD indicates that there is a statistical difference between subjects who received denosumab injections between 5 and 7 months (near-standard dosing interval) and more than 9 months (P=0.03), but not in FN BMD, and no clinically significant association was identified. CONCLUSION: The results of this study show that in special clinical situations, such as the COVID-19 pandemic, clinicians may have some flexibility to prescribe denosumab, but the interval between injections should not exceed 9 months.

Retrospective Study of the Effects of Zoledronic Acid on Muscle Mass in Osteoporosis Patients.

PURPOSE: Several osteoporosis drugs can continuously improve bone mass, but the impact on muscle mass is still unknown. This study aims to investigate how zoledronic acid monotherapy affected muscle mass in osteoporosis patients. PATIENTS AND METHODS: Patients from an osteoporosis database were divided into two groups in this retrospective cohort, case-control study: zoledronic acid-treated patients (n = 113) and a control group without osteoporosis treatment (n = 118). At four years, appendicular skeletal muscle mass (ASM) and appendicular skeletal muscle mass index (ASMI) were calculated using dual-energy X-ray absorptiometry. The differences in muscle mass between the groups were compared. RESULTS: At baseline, there was no difference in sex, ASM, ASMI, and bone mineral density between the zoledronic acid treatment group and the control group. The treatment group's skeletal muscle mass increased by 841 g in ASM and 0.35 kg/m2 in ASMI after three years, while decreased in the control group. CONCLUSION: This study for the first time demonstrated that that zoledronic acid is beneficial not only to the bone but also to muscle.

Profiling of Serum Metabolites of Acute Intermittent Porphyria and Asymptomatic HMBS Mutation Carriers.

This study aims to present the serum metabolite profiles of patients with acute intermittent porphyria (AIP) and identify specific metabolites that could potentially discriminate between AIP, asymptomatic HMBS mutation carriers, and healthy individuals. The study cohort included 46 female participants: 21 AIP patients, 5 asymptomatic carriers, and 20 'normal' participants (without HMBS gene mutation). Serum samples were analyzed for 157 selected metabolites or clinical variables using an assay combining liquid chromatography MS/MS and direct flow injection. AUC analysis was used to distinguish unique variables between the three groups. A total of 15 variables differed significantly between the AIP and normal control group (VIP score > 1.0 and p < 0.05 with FDR correction). In AIP patients, the levels tyrosine, valine, and eGFR were significantly lower, and the levels of sphingomyelin C16:0, C24:0, C24:1, phosphatidylcholine diacyl C32:1, C36:1, C36:3, ornithine, sarcosine, citrulline, blood urea nitrogen AST, and ALT were significantly higher. The AUC of these 15 variables in discriminating between normal and AIP patients ranged between 0.73 and 0.94 (p < 0.05). In conclusion, serum metabolic profiles differ between normal individuals and patients carrying the HMBS mutation. The unique metabolites associated with AIP identified in this study may be useful for monitoring the development of AIP symptoms.

A metabolomics approach to investigate the proceedings of mitochondrial dysfunction in rats from prediabetes to diabetes.

Diabetes mellitus (DM) is a leading cause of preventable cardiovascular disease, but the metabolic changes from prediabetes to diabetes have not been fully clarified. This study implemented a metabolomics profiling platform to investigate the variations of metabolites and to elucidate their global profiling from metabolic syndrome to DM. METHODS: Male Sprague-Dawley rats (n = 44) were divided into four groups. Three groups were separately fed with a normal diet, a high-fructose diet (HF), or a high-fat (HL) diet while one group was treated with streptozotocin. The HF and HL diet were meant to induce insulin resistance, obesity, and dyslipidemia, which known to induce DM. RESULTS: The most significant metabolic variations in the DM group's urine samples were the reduced release of citric acid cycle intermediates, the increase in acylcarnitines, and the decrease in urea excretion, all of which indicated energy metabolism abnormalities and mitochondrial dysfunction. Overall, the metabolic analysis revealed tryptophan metabolic pathway variations in the prediabetic phase, even though the mitochondrial function remains unaffected. CONCLUSION: This study show that widespread methylations and impaired tryptophan metabolism occur in metabolic syndrome and are then followed by a decline in citric acid cycle intermediates, indicating mitochondrial dysfunction in diabetes.

Exercise training modulates the effects of lipoproteins on acetylcholine-induced endothelial calcium signaling in rat aortas.

Exercise training improves vascular endothelial functions, while oxidized low-density lipoproteins (oxLDLs) impede them. We proposed that exercise training might influence the endothelial sensitivity to lipoprotein-induced vascular changes. Male Wistar rats either exercised on a leveled treadmill for 8 weeks or remained sedentary as the control. The endothelial intracellular calcium level (EC [Ca(2+)](i)) in vitro was examined using dissected aortic segments treated with different lipoproteins, including native low-density lipoprotein (nLDL), various oxLDLs, and high-density lipoprotein (HDL). Our results indicated that i) none of the various lipoproteins directly evoked EC [Ca(2+)](i) elevation; ii) the acetylcholine-evoked EC [Ca(2+)](i) elevation in the control group was increased by nLDL and progressively suppressed by oxLDLs with increasing degrees of oxidation; iii) exercise training ameliorated the oxLDL-induced suppressive effects on acetylcholine-evoked EC [Ca(2+)](i) elevation; iv) HDL potentiated the acetylcholine-evoked EC [Ca(2+)](i) elevation in vessel segments from exercised rats but not those from control rats; and v) when HDL was present, the suppressive effects of extensively modified oxLDLs were reduced. Furthermore, comparing with the effects of various lipoproteins on EC calcium signaling, the lipoprotein effects on endothelium-dependent vasorelaxing response appeared to be similar but less pronounced. Taken together, one of the beneficial effects of exercise training on vascular functions might be to make blood vessels more resistant to oxLDLs and more sensitive to HDL.

Diabetes reduces aortic endothelial gap junctions in ApoE-deficient mice: simvastatin exacerbates the reduction.

We examined the endothelial gap junctions in diabetic hyperlipidemic mice. Male apolipoprotein E (apoE)-deficient mice were made diabetic by streptozotocin. Three weeks later, the animals were treated with simvastatin for 2 weeks. The expression of aortic gap junctions in the non-diabetic (n=10), untreated diabetic (n=10), and simvastatin-treated diabetic animals (n=6) was analyzed. There was a >4-fold increase in serum cholesterol level and >50% increase in plaque areas in the diabetic mice, regardless of simvastatin treatment. Western blotting of aortae showed reduced expression of connexin37 (Cx37) and Cx40 in the diabetic mice, which were further decreased in the simvastatin-treated diabetic mice. Immunoconfocal microscopy showed that endothelial gap junctions made of Cx37 and Cx40 were both reduced in the untreated diabetic mice compared with the non-diabetic mice (decrease: Cx37, 41%; Cx40, 42%; both p<0.01). The reduction was greater in the simvastatin-treated mice (decrease in treated diabetic vs non-diabetic: Cx37, 61%; Cx40, 79%; both p<0.01; decrease in treated diabetic vs untreated diabetic: Cx37, 34%; Cx40, 63%; both p<0.01). Cx37 and Cx40 were decreased in the endothelium of plaque surface. Cx43 appeared in the medial layer and inner layer of the intima. All three connexins were rarely expressed in monocytes/macrophages inside the plaques. In conclusion, in apoE-deficient mice, streptozotocin-induced diabetes is associated with downregulation of endothelial Cx37 and Cx40 gap junctions. Short-term treatment with simvastatin exacerbates the downregulation.