The BRCA1/BARD1-interacting protein OLA1 functions in centrosome regulation.

The breast and ovarian cancer-specific tumor suppressor BRCA1, along with its heterodimer partner BRCA1-associated RING domain protein (BARD1), plays important roles in DNA repair, centrosome regulation, and transcription. To explore further functions of BRCA1/BARD1, we performed mass spectrometry analysis and identified Obg-like ATPase 1 (OLA1) as a protein that interacts with the carboxy-terminal region of BARD1. OLA1 directly bound to the amino-terminal region of BRCA1 and γ-tubulin. OLA1 localized to centrosomes in interphase and to the spindle pole in mitotic phase, and its knockdown resulted in centrosome amplification and the activation of microtubule aster formation. OLA1 with a mutation observed in breast cancer cell line, E168Q, failed to bind BRCA1 and rescue the OLA1 knockdown-induced centrosome amplification. BRCA1 variant I42V also abrogated the binding of BRCA1 to OLA1. These findings suggest that OLA1 plays an important role in centrosome regulation together with BRCA1.

α-globulin-rich rice cultivar, low glutelin content-1 (LGC-1), decreases serum cholesterol concentration in exogenously hypercholesterolemic rats.

BACKGROUND: Rice α-globulin has been reported to have serum cholesterol-lowering activity in rats. However, it is still unclear whether α-globulin exerts this effect when taken as one of the dietary components. In the present study, we investigated the effect of two cultivars of rice, low glutelin content (LGC)-1 and LGC-Jun, on reducing serum cholesterol in exogenously hypercholesterolemic (ExHC) rats. LGC-1 is enriched in α-globulin (10.6 mg g-1 rice flour, which is an approximately 1.5 times higher α-globulin content than in Koshihikari a predominant rice cultivar in Japan), whereas LGC-Jun is a globulin-negative cultivar. METHODS: ExHC rats, the model strain of diet-induced hypercholesterolemia, were fed 50% LGC-1 or LGC-Jun and 0.5% cholesterol-containing diets for 2 weeks, followed by measurement of cholesterol metabolism parameters in serum and tissues. RESULTS: Serum cholesterol and non-high-density lipoprotein cholesterol levels were significantly lower in the LGC-1 group compared to the LGC-Jun group. Cholesterol intestinal absorption markers, hepatic and serum levels of campesterol and ß-sitosterol, and lymphatic cholesterol transport were not different between the two groups. Levels of 7α-hydroxycholesterol, an intermediate of bile acid synthesis, showed a downward trend in the livers of rats that were fed LGC-1 (P = 0.098). There was a significant decrease in the hepatic mRNA expression of Cyp7a1 (a synthetic enzyme for 7α-hydroxycholesterol) in the LGC-1 group compared to the LGC-Jun group. CONCLUSION: Dietary LGC-1 significantly decreased serum cholesterol levels in ExHC rats. The possible mechanism for the cholesterol-lowering activity of LGC-1 is partial inhibition of bile acid and cholesterol synthesis in the liver. © 2021 Society of Chemical Industry.

Inhibition of Niemann-Pick C1-Like 1 by Ezetimibe Reduces Dietary 5ß,6ß-Epoxycholesterol Absorption in Rats.

PURPOSE: Oxycholesterols (OCs) are produced from cholesterol by oxidation of the steroidal backbone and side-chain. OCs are present in blood and evidence suggests their involvement in disease development and progression. However, limited information is available regarding the absorption mechanisms and relative absorption rates of dietary OCs. Although ezetimibe is known to inhibit intestinal cholesterol absorption via Niemann-Pick C1-Like 1 (NPC1L1), whether it also inhibits dietary OC absorption is unclear. METHODS: We investigated the effects of ezetimibe on OC absorption in rats fed an OC-rich diet containing 10 different OCs. We collected lymphatic fluid using permanent cannulation of the thoracic duct and quantified OC levels. RESULTS: Ezetimibe treatment significantly reduced the apparent absorption of 5ß,6ß-epoxycholesterol (5,6ß-epoxy) and its levels in the proximal intestinal mucosa in OC-fed rats. Using in silico analyses, the binding energy of NPC1L1 N-terminal domain (NPC1L1-NTD) and 5,6ß-epoxy was found to be similar to that of NPC1L1-NTD and cholesterol, suggesting that polar uncharged amino acids located in the steroidal part of 5,6ß-epoxy were involved. CONCLUSION: Our results indicate that ezetimibe-mediated inhibition of dietary OC absorption varies depending on the specific OC, and only the absorption of 5,6ß-epoxy is significantly reduced.

Ezetimibe inhibits lymphatic transport of esterified cholesterol but not free cholesterol in thoracic lymph duct-cannulated rats.

PURPOSE: Ezetimibe has been shown to inhibit dietary cholesterol absorption in animal models and humans, but studies on lymphatic lipid transport have not yet been performed. Rats subjected to permanent lymph duct cannulation were used to investigate the effects of ezetimibe on lipid transport. METHODS: Rats were fed diets with and without ezetimibe (5.0 mg/kg), and their lymph was collected after feeding to quantify lymphatic lipid levels. Total cholesterol content in the intestinal mucosa was also measured. RESULTS: Rats that consumed ezetimibe had significantly lower lymphatic total cholesterol transport with the reduction of esterified cholesterol transport. According to the calculation based on cholesterol consumption, ezetimibe reduced the total cholesterol lymphatic recovery rate by 54 %. We also determined that ezetimibe significantly reduced the total cholesterol content in the intestinal mucosa. CONCLUSION: This is the first direct evidence that ezetimibe inhibits esterified but not free cholesterol lymphatic transport in thoracic duct-cannulated rats. The results also indicate that ezetimibe is not involved in the lymphatic transport of triacylglycerols, phospholipids, or α-tocopherol.

12α-Hydroxylated bile acid induces hepatic steatosis with dysbiosis in rats.

There is an increasing need to explore the mechanism of the progression of non-alcoholic fatty liver disease. Steroid metabolism is closely linked to hepatic steatosis and steroids are excreted as bile acids (BAs). Here, we demonstrated that feeding WKAH/HkmSlc inbred rats a diet supplemented with cholic acid (CA) at 0.5 g/kg for 13 weeks induced simple steatosis without obesity. Liver triglyceride and cholesterol levels were increased accompanied by mild elevation of aminotransferase activities. There were no signs of inflammation, insulin resistance, oxidative stress, or fibrosis. CA supplementation increased levels of CA and taurocholic acid (TCA) in enterohepatic circulation and deoxycholic acid (DCA) levels in cecum with an increased ratio of 12α-hydroxylated BAs to non-12α-hydroxylated BAs. Analyses of hepatic gene expression revealed no apparent feedback control of BA and cholesterol biosynthesis. CA feeding induced dysbiosis in cecal microbiota with enrichment of DCA producers, which underlines the increased cecal DCA levels. The mechanism of steatosis was increased expression of Srebp1 (positive regulator of liver lipogenesis) through activation of the liver X receptor by increased oxysterols in the CA-fed rats, especially 4ß-hydroxycholesterol (4ßOH) formed by upregulated expression of hepatic Cyp3a2, responsible for 4ßOH formation. Multiple regression analyses identified portal TCA and cecal DCA as positive predictors for liver 4ßOH levels. The possible mechanisms linking these predictors and upregulated expression of Cyp3a2 are discussed. Overall, our observations highlight the role of 12α-hydroxylated BAs in triggering liver lipogenesis and allow us to explore the mechanisms of hepatic steatosis onset, focusing on cholesterol and BA metabolism.

Up-regulation of P-glycoprotein expression in small intestine under chronic serotonin-depleted conditions in rats.

To investigate the role of serotonin (5-HT), an important neurotransmitter and hormone/paracrine agent in the small intestine, in the transport activity of P-glycoprotein (P-gp), the intestinal transport of quinidine, a P-gp substrate, was examined in 5-HT-depleted rats prepared by intraperitoneal administration of p-chlorophenylalanine, a specific inhibitor of tryptophan hydroxylase in 5-HT biosynthesis. In the in vitro transport study, quinidine transport across rat jejunum was significantly enhanced in both the secretory and absorptive directions under 5-HT-depleted conditions, although the secretory transport was still predominant. The electrophysiological study suggested that the quinidine transport via passive diffusion was enhanced presumably through a paracellular route. This might be due to looser tight junctions under 5-HT-depleted conditions. The voltage-clamp technique clearly indicated that the secretory transport of quinidine through the transcellular pathway was also enhanced by the depletion of 5-HT. Furthermore, 5-HT depletion increased verapamil-sensitive secretory transport of quinidine in rat jejunum. These results indicate that the secretory transport of quinidine via P-gp was significantly enhanced under 5-HT-depleted conditions. The level of ATP, an energy source for functioning P-gp, wet weight of jejunum, and total protein level in rat jejunal mucosa were not changed by 5-HT depletion, but the expression of P-gp in the brush-border membrane of rat jejunum was significantly induced, which is partly responsible for the enhancement of P-gp activity under the 5-HT-depleted condition.