Enhanced liver but not muscle OXPHOS in diabetes and reduced glucose output by complex I inhibition.

Mitochondrial function is critical in energy metabolism. To fully capture how the mitochondrial function changes in metabolic disorders, we investigated mitochondrial function in liver and muscle of animal models mimicking different types and stages of diabetes. Type 1 diabetic mice were induced by streptozotocin (STZ) injection. The db/db mice were used as type 2 diabetic model. High-fat diet-induced obese mice represented pre-diabetic stage of type 2 diabetes. Oxidative phosphorylation (OXPHOS) of isolated mitochondria was measured with Clark-type oxygen electrode. Both in early and late stages of type 1 diabetes, liver mitochondrial OXPHOS increased markedly with complex IV-dependent OXPHOS being the most prominent. However, ATP, ADP and AMP contents in the tissue did not change. In pre-diabetes and early stage of type 2 diabetes, liver mitochondrial complex I and II-dependent OXPHOS increased greatly then declined to almost normal at late stage of type 2 diabetes, among which alteration of complex I-dependent OXPHOS was the most significant. In contrast, muscle mitochondrial OXPHOS in HFD, early-stage type 1 and 2 diabetic mice, did not change. In vitro, among inhibitors to each complex, only complex I inhibitor rotenone decreased glucose output in primary hepatocytes without cytotoxicity both in the absence and presence of oleic acid (OA). Rotenone affected cellular energy state and had no effects on cellular and mitochondrial reactive oxygen species production. Taken together, the mitochondrial OXPHOS of liver but not muscle increased in obesity and diabetes, and only complex I inhibition may ameliorate hyperglycaemia via lowering hepatic glucose production.

Development of Multimarker Diagnostic Models from Metabolomics Analysis for Gestational Diabetes Mellitus (GDM).

Although metabolomics are desirable to understand the pathophysiology of gestational diabetes mellitus (GDM), comprehensive metabolomic studies of GDM are rare. We aimed to offer a holistic view of metabolites alteration in GDM patients and investigate the possible multimarker models for GDM diagnosis. Biochemical parameters and perinatal data of 131 GDM cases and 138 controls were collected. Fasting serum samples at 75 g oral glucose tolerance test were used for metabolites by ultra performance liquid chromatography-quadrupole-time of flight-mass spectrometry, ultra performance liquid chromatography-triple triple-quadrupole-mass spectrometry and gas chromatography- time-of- flight mass spectrometry platforms. Significant changes were observed in free fatty acids, bile acids, branched chain amino acids, organic acids, lipids and organooxygen compounds between two groups. In receiver operating characteristic (ROC) analysis, different combinations of candidate biomarkers and metabolites in multimarker models achieved satisfactory discriminative abilities for GDM, with the values of area under the curve (AUC) ranging from 0.721 to 0.751. Model consisting of body mass index (BMI), retinol binding protein 4 (RBP4), n-acetylaspartic acid and C16:1 (cis-7) manifested the best discrimination [AUC 0.751 (95% CI: 0.693-0.809), p < 0.001], followed by model consisting of BMI, Cystatin C, acetylaspartic acid and 6,7-diketoLCA [AUC 0.749 (95% CI: 0.691-0.808), p < 0.001]. Metabolites alteration reflected disorders of glucose metabolism, lipid metabolism, amino acid metabolism, bile acid metabolism as well as intestinal flora metabolism in GDM state. Multivariate models combining clinical markers and metabolites have the potential to differentiate GDM subjects from healthy controls.

Inhibition of mitochondrial complex I improves glucose metabolism independently of AMPK activation.

Accumulating evidences showed metformin and berberine, well-known glucose-lowering agents, were able to inhibit mitochondrial electron transport chain at complex I. In this study, we aimed to explore the antihyperglycaemic effect of complex I inhibition. Rotenone, amobarbital and gene silence of NDUFA13 were used to inhibit complex I. Intraperitoneal glucose tolerance test and insulin tolerance test were performed in db/db mice. Lactate release and glucose consumption were measured to investigate glucose metabolism in HepG2 hepatocytes and C2C12 myotubes. Glucose output was measured in primary hepatocytes. Compound C and adenoviruses expressing dominant negative AMP-activated protein kinase (AMPK) α1/2 were exploited to inactivate AMPK pathway. Cellular NAD+ /NADH ratio was assayed to evaluate energy transforming and redox state. Rotenone ameliorated hyperglycaemia and insulin resistance in db/db mice. It induced glucose consumption and glycolysis and reduced hepatic glucose output. Rotenone also activated AMPK. Furthermore, it remained effective with AMPK inactivation. The enhanced glycolysis and repressed gluconeogenesis correlated with a reduction in cellular NAD+ /NADH ratio, which resulted from complex I suppression. Amobarbital, another representative complex I inhibitor, stimulated glucose consumption and decreased hepatic glucose output in vitro, too. Similar changes were observed while expression of NDUFA13, a subunit of complex I, was knocked down with gene silencing. These findings reveal mitochondrial complex I emerges as a key drug target for diabetes treatment. Inhibition of complex I improves glucose homoeostasis via non-AMPK pathway, which may relate to the suppression of the cellular NAD+ /NADH ratio.

Heterogeneity of insulin resistance and beta cell dysfunction in gestational diabetes mellitus: a prospective cohort study of perinatal outcomes.

BACKGROUND: Insulin resistance and beta cell dysfunction were reported to be responsible for gestational diabetes mellitus (GDM). However, little is known about the heterogeneity of these factors and its influences on perinatal outcomes. We investigated whether subtypes of insulin resistance and beta cell dysfunction in gestational diabetes mellitus have different impacts on perinatal outcomes. METHODS: In this prospective cohort study, we followed 554 pregnant women and glucose challenge test was performed at 24-28th weeks of their gestation. Women with plasma glucose ≥ 7.8 mmol/L would be included and advised to undergo the diagnostic 75-g, 3-h oral glucose tolerance test. According to indices of measuring insulin resistance or beta cell function were below the 25th percentile of women with normal glucose tolerance (NGT), women with GDM were defined as three subtypes: GDM with the beta cell dysfunction, GDM with the insulin resistance defect or GDM with both traits mentioned above (GDM-mixed). Perinatal outcomes were documented. RESULTS: The levels of prepregnancy and maternal BMI in the GDM-mix group were higher compared to women in the NGT group (23.2 ± 4.0 vs 20.8 ± 3.7 kg/m2, P < 0.001; 24.5 ± 4.3 vs 21.8 ± 3.4 kg/m2, P < 0.001, respectively). Furthermore, women in GDM-mix group more likely to be subjected to LGA (P = 0.008) adverse perinatal outcomes (P = 0.005), although these differences were normalized after adjusting age, prepregnancy and maternal BMI (GDM-mix vs. NGT: P = 0.141 for LGA and P = 0.186 for adverse outcomes). On the other hand, all perinatal outcomes were similar between other two GDM subgroups and NGT group. CONCLUSIONS: Women with GDM display respective characteristics on metabolism disorders and confer discriminating risks of adverse perinatal outcomes because of this heterogeneity.

[Effects of metformin therapy on serum CA125 levels and its related factors in type 2 diabetics].

OBJECTIVE: To explore the effects of metformin therapy on serum carbohydrate antigen 125 (CA125) levels and its related factors in type 2 diabetics with normal liver and kidney function. METHODS: Serum CA125 level was measured and compared for a total of 1 136 type 2 diabetics. They were assigned into groups according to gender, age and body mass in dex (BMI) and subgroups according to metformin dosing. Associations between CA125 and other variables were assessed with Spearman or Pearson's correlation and multiple stepwise regression analysis. RESULTS: The CA125 level of metformin group was significantly lower than that of non-metformin group (10.51(8.18, 13.80) vs 11.93(9.05, 15.52) U/ml, P < 0.01) . The CA125 levels of males aged 30-39 and 50-59 as well as females aged over 50 remarkably decreased after metformin dosing (all P < 0.05). The difference of CA125 level between metformin and non-metformin groups in patients with normal BMI and obese females reached statistical significance (all P < 0.05). The correlation analysis showed that serum CA125 level was positively associated with gender, HbA1c, glycated serum albumin, triglyceride, total cholesterol (P < 0.05), but negatively with metformin and creatinine (P < 0.01). Multiple stepwise regression analysis further revealed that GA, metformin dosing, gender and total glycerides were independent influencing factors of CA125 concentrations (all P < 0.05). CONCLUSION: Metformin dosing is an independent associated factor of serum CA125 levels reduction in type 2 diabetics, especially females.

Early elevated alkaline phosphatase increases the risk of large-for-gestational-age birth weight in pregnant women with normal glucose tolerance.

AIMS: The aim of this study was to assess the association between levels of alkaline phosphatase (ALP) in early pregnancy and the incidence of large-for-gestational-age (LGA) neonates in pregnant women without gestational diabetes mellitus. METHODS: A prospective cohort was carried out in 544 women and their biochemical parameters including serum ALP and demographic characteristics were collected in 13-16th gestational week. At 24-28th weeks of gestation, 50 g oral glucose challenge test and oral 75 g glucose tolerance test was performed. LGA was defined as birth weight ≥ 90th percentile for completed week of gestational age based on the sex-specific growth curves. Logistic regression and receiver operating characteristic analysis were utilized to identify independent risk factors and odds ratio among ALP quartiles for incidence of LGA. RESULTS: Women diagnosed as LGA held higher level of ALP than women in non-LGA group (P = 0.008). Moreover, ALP (odds ratio (OR) 1.05 [95% confidence interval (CI): 1.00, 1.10]) was the independent risk factors associated with LGA. Compared with ALP quartile 1, women in quartile 4 had more than 2.5-fold increased odds of LGA (OR 3.78, 95% CI: 1.10, 13.02), and the risk reached 4 times after adjusting several covariates (OR 4.15, 95% CI: 1.14,15.13). CONCLUSIONS: A significantly increased risk of LGA was associated with higher serum concentrations of ALP in pregnant women with NGT, even it is in normal reference range.

Elevated First-Trimester Total Bile Acid is Associated with the Risk of Subsequent Gestational Diabetes.

The aim of the current study is to assess whether total bile acid (TBA) level in first trimester pregnancy is associated with gestational diabetes mellitus (GDM). Biochemical parameters including serum TBA of 742 pregnant women were collected within 12 weeks of gestation and compared. At 24-28th weeks of gestation, 75 g oral glucose tolerance test (OGTT) was performed. The perinatal data of 330 women were collected. The results demonstrated women with GDM (n = 268) had higher first-trimester serum levels of TBA compared with healthy subjects (n = 474) (2.3 ± 1.4 µmol/L vs. 1.9 ± 1.0 µmol/L, P < 0.001). TBA was independently associated with GDM [adjusted odds ratio (AOR), 1.38; 95% confidence interval (CI), 1.18-1.61, P < 0.001]. Compared to the first category of TBA, women in the highest category had a marked increase in risk for GDM (AOR, 7.72; 95% CI, 3.22-18.50, P < 0.001). In conclusion, higher first-trimester TBA levels, even within normal range, may help indicate increased risk of GDM.

Metformin reduces serum CA199 levels in type 2 diabetes Chinese patients with time-effect and gender difference.

BACKGROUND: This study was designed to clarify the influence of metformin on serum carbohydrate antigen 199 (CA199) levels and its associated factors in Chinese type 2 diabetes mellitus (T2DM) patients. SUBJECTS AND METHODS: In total, 1,253 T2DM patients were enrolled, including a non-metformin group (n = 616), a short-term metformin group (at least 1 week to 2 years; n=325), and a long-term metformin group (≥ 2 years; n = 312). Their clinical and biochemical characteristics were collected and compared. After 1 year, the biochemical parameters were re-examined in 296 patients. Sex hormones were determined, and associations between CA199 and other variables were assessed. RESULTS: At baseline, the incidence of abnormal CA199 levels was 14.7%, 8.9%, and 4.7% in the non-metformin, short-term metformin, and long-term metformin groups, respectively. CA199 levels in females were significantly higher than in males (P < 0.01) and decreased significantly with the time of taking metformin (25.60 ± 13.68 U/mL in non-metformin controls vs. 17.62 ± 10.87 U/mL in the short-term group vs. 10.54 ± 8.14 U/mL in the long-term group; P = 0.000). The correlation and multiple stepwise regression analysis revealed that glycosylated hemoglobin, metformin, gender, total cholesterol, and follicle-stimulating hormone were independent impact factors on CA199 concentrations (all P < 0.05). Binary logistic regression revealed that the risk of abnormal CA199 concentrations of the total population with short-term metformin or long-term metformin treatment decreased 11% (odds ratio = 0.89; P = 0.001) and 30% (odds ratio = 0.70; P = 0.000), respectively, at baseline. After a 1-year follow-up, the incidence of high CA199 level decreased in both the short-term and the long-term metformin group compared with that of controls (P < 0.05). The extent of CA199 decrease in the long-term metformin group was the greatest (-17% vs. -4.9% in the short-term group vs. 3% in controls, P = 0.000), and the group's risk of high blood CA199 level was reduced 67% (odds ratio = 0.33; P = 0.023). The reduction in women was more apparent than that in men (-18% vs. -5%, P = 0.000). CONCLUSIONS: Metformin therapy reduced the CA199 level in Chinese T2DM patients, and its greatest decrease occurred in women with longer therapeutic time.

Polymorphism of organic cation transporter 2 improves glucose-lowering effect of metformin via influencing its pharmacokinetics in Chinese type 2 diabetic patients.

BACKGROUND AND OBJECTIVES: This study aimed to investigate how the organic cation transporter 2 nucleotide polymorphism at site 808 (G → T) affects metformin pharmacokinetics and its long-term anti-diabetic effect. METHODS: A total of 220 newly diagnosed type 2 diabetes patients taking oral metformin were recruited, genotyped and then divided into three groups by SLC22A2 genotypes (G/G, G/T, T/T). Nine patients in the GG genotype group, five patients in the GT genotype group and four patients in the TT genotype group were randomly selected for the metformin pharmacokinetic study. A randomized cohort study with 1-year follow-up was performed to clarify the metformin pharmacodynamics. RESULTS: After 1 year, the decrease in glycosylated hemoglobin (HbA1c) levels in subjects with the heterozygous variant genotype (GT) was significantly greater than in those with the wild-type homozygote (-2.2 % in GT vs. -1.1 % in GG, P < 0.05) after adjustment for baseline HbA1c levels, exercise and diet in each group. There were also differences in the pharmacokinetic parameters (95 % confidence interval) of metformin between these two groups [area under the concentration-time curve (AUC)0-∞ 19.7 (15.7-23.8) vs. 14.3 (11.7-16.9) µg h/L; renal clearance (CLr) 16.8 (8.5-25.0) vs. 34.1 (24.9-43.2) L/h; tubular secretion clearance (CLt) 8.1 (2.2-18.1) vs. 22.7 (15.5-29.8) L/h; all P < 0.05]. Multivariate analysis further revealed that the presence of T alleles and gender were independent influencing factors of urine excretion of metformin (P < 0.05). CONCLUSION: As well as gender, the glucose-lowering efficiency of metformin can be enhanced by SLC22A2 808G > T variants through the delay of its transportation and CLr in Chinese type 2 diabetes populations.

Berberine promotes glucose consumption independently of AMP-activated protein kinase activation.

Berberine is a plant alkaloid with anti-diabetic action. Activation of AMP-activated protein kinase (AMPK) pathway has been proposed as mechanism for berberine's action. This study aimed to examine whether AMPK activation was necessary for berberine's glucose-lowering effect. We found that in HepG2 hepatocytes and C2C12 myotubes, berberine significantly increased glucose consumption and lactate release in a dose-dependent manner. AMPK and acetyl coenzyme A synthetase (ACC) phosphorylation were stimulated by 20 µmol/L berberine. Nevertheless, berberine was still effective on stimulating glucose utilization and lactate production, when the AMPK activation was blocked by (1) inhibition of AMPK activity by Compound C, (2) suppression of AMPKα expression by siRNA, and (3) blockade of AMPK pathway by adenoviruses containing dominant-negative forms of AMPKα1/α2. To test the effect of berberine on oxygen consumption, extracellular flux analysis was performed in Seahorse XF24 analyzer. The activity of respiratory chain complex I was almost fully blocked in C2C12 myotubes by berberine. Metformin, as a positive control, showed similar effects as berberine. These results suggest that berberine and metformin promote glucose metabolism by stimulating glycolysis, which probably results from inhibition of mitochondrial respiratory chain complex I, independent of AMPK activation.