Optimization of the extract from flower of Citrus aurantium L. var. amara Engl. and its inhibition of lipid accumulation.

Flower of Citrus aurantium L. var. amara Engl. (CAVA) has been confirmed to have promising anti-obesity effects. However, the regulation of alkaloid extracts from flower of CAVA (Al) on lipid metabolism remain unknown. In this study, Al was optimized by ultrasound-assisted extraction using response surface methodology. The optimal conditions were ultrasonic time 72 min, ethanol concentration 78% and liquid/solid ratio 30 ml/g with the maximum alkaloid yield 5.66%. LC-MS assay indicated that the alkaloid compounds were enriched in Al after optimization. Nine alkaloid compounds were identified in Al by LC-MS assay and stachydrine, caffeine and cathine appeared as the major alkaloid compounds. Bioactivity assay showed that Al treatment significantly increased superoxide dismutase (SOD) activity, and reduced malonaldehyde (MDA) and reactive oxygen species (ROS) levels. Al administration also reversed oleic acid-induced hepatic steatosis in Hep G2 cells by inhibiting the expression of lipogenesis-signaling genes including fatty acid synthase (FAS), peroxisome proliferator-activated receptor subtype γ (PPARγ), uncoupling protein 2 (UCP2), and retinol binding protein (RBP4). However, OA-induced reduction of lipolysis-related gene carnitine palmitoyl transferase 1A (CPT1A) in Hep G2 cells was not improved by Al supplementation. Moreover, the increased SOD activity and decreased MDA and ROS contents were also observed in Caenorhabditis elegans by Al addition. Al intervention exhibited the ability to inhibit lipid accumulation in C. elegans by suppressing expression of lipid metabolism-related genes. These results suggested that the alkaloid extracts from the flower of CAVA showed great potential to regulate lipid metabolism. PRACTICAL APPLICATIONS: The extraction of alkaloid extracts from the flower of CAVA was optimized with a maximum yield of 5.66%. The regulatory effects and mechanisms of Al on lipid metabolism of Hep G2 cells and Caenorhabditis elegans were also investigated. More clinical studies are required to evaluate the potential of using alkaloids from the flower of CAVA as therapeutic agents against lipid metabolic disorders.

Expression and hormone regulation of UCP2 in goat uterus.

Pregnancy success is closely related to the molecular mechanisms that control energy metabolism balance. However, the mechanisms have not been fully understood. Uncoupling protein 2 (UCP2) plays a physiological role by regulating energy metabolism in numerous tissues. In this study, we determined the expression and hormone regulation of UCP2 in goat uterus. UCP2 is expressed in the luminal and glandular epithelia of goat uterus during early pregnancy, as revealed by in situ hybridization and immunohistochemistry conducted on pregnant goats. The signals were detected from day 0 (D0) to D30 of pregnancy, though weak on D16 (the adhesion period). The low levels of UCP2 on D16 were confirmed by RT-qPCR and western blot. In goat uterus and endometrial epithelial cells (EECs), UCP2 was up-regulated by progesterone and estrogen. In addition, after goat EECs were treated with genipin (an inhibitor of UCP2), not only UCP2 expression but also cell proliferation was inhibited. Collectively, UCP2 is dynamically expressed in goat uterus and can affect EEC proliferation, suggesting that it may participate in regulating the energy metabolism balance of goat uterus during early pregnancy.

Role and mechanism of FLNa and UCP2 in the development of cervical cancer.

Recent studies have reported that filamin A (FLNa) and uncoupling protein 2 (UCP2) are highly expressed in various types of cancer, but little is currently known about their roles in cervical cancer (CC). In the present study, immunohistochemical staining of paraffin sections of cervical tissues was performed in order to compare the differential expression of FLNa, UCP2, p16 and Ki67 between CC and high­grade intraepithelial neoplasia (HSIL). UCP2 and FLNa were knocked down in CC cell lines to investigate the effects on cell proliferation, cell cycle arrest, apoptosis, migration and invasion. In addition, the present study investigated the expression of cell­associated proteins [extracellular signal­regulated kinase (ERK), phosphorylated (p) ERK, protein kinase B (AKT), p­AKT and B­cell lymphoma­2 (Bcl­2)] and the mRNA levels of cellular proteins such as Ras, matrix metalloproteinase (MMP)­2 and MMP­9. FLNa and UCP2 expression levels were significantly higher in CC tissues than in HSIL tissues, with no significant differential expression of p16 or Ki67. UCP2 expression was significantly different in patients with clinical stage II or higher or lymph node metastasis compared with in other patients with cervical cancer. FLNa or UCP2 knockdown slowed or decreased SiHa and HeLa cell proliferation, migration and invasion, with no significant change in apoptosis, and downregulated the protein levels of p­ERK1/2, and the mRNA levels of Ras, MMP­2 and MMP­9. UCP2 knockdown arrested the cell cycle at the G2 phase in SiHa and HeLa cells, while FLNa knockdown arrested the cell cycle at the G2 phase in HeLa cells. The results of the present study revealed that FLNa and UCP2 play roles in the development and progression of CC via the Ras/MAPK/ERK signalling pathway. FLNa and UCP2 are superior to p16 and Ki67 for early prediction of CC, indicating that FLNa and UCP2 may be used for the early diagnosis of CC. UCP2 may be used to predict the prognosis of CC.

Fecal fermentation products of common bean-derived fiber inhibit C/EBPα and PPARγ expression and lipid accumulation but stimulate PPARδ and UCP2 expression in the adipogenesis of 3T3-L1 cells.

Clinical and animal studies have suggested efficacies of common bean (Phaseolus vulgaris) consumption on weight loss. Fermentation of common bean-derived dietary fiber by gut microbiota is proposed to mitigate obesity; however, the mechanism of action is unclear. The objective of this study was to investigate whether and how fecal fermentation of common bean-derived dietary fiber impacts adipogenesis in a cell model. Dietary fiber was generated by in vitro digestion of cooked, lyophilized common bean flour, followed by anaerobic fermentation with the use of fresh feces from healthy mice without antibiotics treatment. The murine 3T3-L1 cells were induced to differentiate in the presence of the fermentation products. Treatment of the fecal fermentation products inhibited adipocyte differentiation and lipid accumulation in a dose- and time-dependent manner. The fermentation products decreased (P<.05) protein levels of two key transcription factors for adipogenesis, CCAAT/enhancer binding protein α and peroxisome proliferator-activated receptor γ by 79-92% and 78-90%, respectively, and one of their downstream targets fatty acid binding protein 4 by 49-86% and 63-98% at protein and mRNA levels, respectively, during the time course. In contrast, the fermentation products increased (P<.05) levels of two proteins promoting energy expenditure, peroxisome proliferator-activated receptor δ (71-91%) on days 2 and 4 and mitochondrial uncoupling protein 2 (1.1-1.2 fold) on days 4-8. Altogether, fecal fermentation of dietary fiber derived from in vitro digestion of common bean temporally and dose-dependently inhibits adipogenesis and key adipogenic transactivators, but activates two energy expenditure proteins in 3T3-L1 cells.

Cellular and subcellular localization of uncoupling protein 2 in the human kidney.

The uncoupling protein-2 (UCP2) is an anion transporter that plays a key role in the control of intracellular oxidative stress. In animal models UCP2 downregulation has several pathological sequelae, particularly affecting the vasculature and the kidney. Specifically, in these models kidney damage is highly favored in the absence of UCP2 in the context of experimental hypertension. Confirmations of these data in humans awaits further information, as no data are yet available concerning the cell-type and subcellular expression in the human kidney. In the present study, we aimed to characterize the UCP2 protein distribution in human kidney biopsies. In humans UCP2 is mainly localized in proximal convoluted tubule cells, with an intracytoplasmic punctate staining. UCP2 positive puncta are often localized at the interface between the endoplasmic reticulum and the mitochondria. Glomerular structures do not express UCP2 at detectable levels. The expression of UCP2 in proximal tubular cells may explain their relative propensity to damage in pathological conditions including the hypertensive disease.

Response of skeletal muscle UCP2-expression during metabolic adaptation to caloric restriction.

BACKGROUND/OBJECTIVES: Spendthrift vs. thrifty individuals expend more energy and experience greater weight loss during caloric restriction (CR). Adaptive mechanisms in skeletal muscle, adipose tissue, and on hormone level modulate energy expenditure (EE) during weight loss. Metabolic mechanisms underlying the variability in EE during CR are unclear. The present study explored whether during long-term CR (i) gene expression changes in skeletal muscle and adipose tissue relate with the individual EE response and weight loss, and (ii) altered catecholamine and FGF21-concentrations are associated with measures of metabolic adaptation. SUBJECTS/METHODS: In a 10-week inpatient study, 24-h EE was measured before and after 6 weeks of 50% CR in 12 subjects using whole-room indirect calorimetry. Weight loss was assessed and repeated hormone measurements performed. Muscle and adipose tissue biopsies were taken before and after CR, and gene expression was assessed (RNA-Seq). Genes showing the most significant changes after CR were tested for association with EE and followed-up for further association with metabolic measures in a separate phenotyping study (n = 103). RESULTS: Muscle UCP2 showed the strongest change after CR (log2-fold change = -1.57, false discovery rate = 0.10) and was considered the best gene for exploration of metabolic adaptive processes. A greater decrease in UCP2-expression was associated with less weight loss (P = 0.03, r = 0.77) and relatively lower 24-h EE after CR (P = 0.001, r = -0.96). Post-CR changes in FGF21-plasma concentrations correlated with UCP2-expression change (P = 0.02, r = -0.89) and weight loss (P = 0.003, r = -0.83). In a separate metabolic phenotyping study, muscle UCP2-expression correlated with respiratory quotient and macronutrient oxidation. In adipose tissue, no candidate genes for metabolic exploration were found. CONCLUSIONS: Changes in muscle UCP2-expression reflect an inter-individual metabolic response to long-term CR and may influence EE and weight loss via modulation of substrate oxidation.

A differential expression of uncoupling protein-2 associates with renal damage in stroke-resistant spontaneously hypertensive rat/stroke-prone spontaneously hypertensive rat-derived stroke congenic lines.

OBJECTIVES: Uncoupling protein-2 (UCP2), a mitochondrial anion transporter involved in mitochondrial uncoupling, limiting reactive oxygen species formation, is significantly downregulated in kidneys of high-salt-fed stroke-prone spontaneously hypertensive rat (SHRSP), where it associates with increased renal damage occurrence. METHODS: We aimed at establishing whether UCP2 differential expression associates with renal damage in two stroke-resistant spontaneously hypertensive rat (SHRSR)/SHRSP-derived stroke congenic lines. For this purpose, SHRSR, SHRSP, and two reciprocal stroke congenic lines carrying the (D1Rat134-Mt1pa) segment of chromosome 1 were fed with Japanese style diet for 8 weeks. At 4, 6, and 8 weeks of Japanese diet, kidneys were removed and analyzed for UCP2 gene and protein expression [UCP2 maps within (D1Rat134-Mt1pa)]; nuclear factor kappa-light-chain-enhancer of activated B cells protein expression; oxidized total protein levels; mitochondrial function; gene expression of cubulin, megalin, and nephrin. At 6 and 8 weeks of Japanese diet, histological damage and percentage of high molecular weight urinary proteins excretion were assessed. RESULTS: Introgression of UCP2 in the SHRSP configuration within the SHRSR genome led to UCP2 downregulation upon Japanese diet, as compared with the SHRSR, with significantly reduced ATP levels, increased rate of inflammation, oxidative stress, renal damage, and excretion of high molecular weight proteins. The opposite phenomena were observed in the reciprocal congenic line, compared with the SHRSP. In vitro, high-NaCl medium led to UCP2 downregulation, increased apoptosis/necrosis, and reduced viability in primary renal proximal tubular epithelial cells isolated from SHRSP. Exposure of the SHRSP/proximal tubular epithelial cells to recombinant UCP2 rescued the high-salt-dependent deleterious effects. CONCLUSION: A differential UCP2 expression associates with different degree of renal damage upon Japanese diet in two SHRSR/SHRSP-derived stroke congenic lines through modulation of mitochondrial function, inflammation, and oxidative stress.