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Objective: To study the preventive effects of Fructus Phyllanthiin (FP) alone and combined with tea polyphenols (FP-TP) or L-carnitine (FP-LC) on high-fat diet-induced obesity in rats. Methods: The experimental, model control and positive control groups were fed a high-fat diet while the negative control group was fed a normal diet. Meanwhile, the experimental group was gavaged with FP, FP-TP or FP-LC for seven weeks. At the end of the gavage period, body mass, perirenal fat mass and peritesticular fat mass were measured, and four items of blood lipid tests in serum and liver lipid levels were detected. The levels of serum leptin, resistin and lipocalin were measured, and the activities of related enzymes and total protein levels in the liver and mRNA expression in adipose tissues were measured to observe the effects of FP and its combination with TP or LC on obesity alleviation and protection against liver injury in experimentally obese rats. Results: The body mass and body fat content of the rats in the model control group increased significantly when compared with the negative control group (P < 0.001). The rats in the FP and FP-LC intervention groups showed significant decreases in body mass, and perirenal fat and peritesticular fat contents and indexes. The levels of total cholesterol (TC), triglyceride (TG) and low-density lipoprotein cholesterol (LDL-C) in the serum and liver decreased significantly, and the level of high-density lipoprotein cholesterol (HDL-C) increased significantly. Moreover, serum leptin and resistin levels significantly dropped, and lipocalin levels increased; the activities of alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT) and the content of malondialdehyde (MDA) in the liver decreased, and the activity of superoxide dismutase (SOD) increased; the mRNA expression of adenosine 5’-monophosphate-activated protein kinase (AMPK) α-2 was significantly up-regulated in adipose tissues. On the other hand, FP-TP intervention had no significant anti-obesity effect. Conclusion: FP can prevent high-fat diet-induced obesity in rats, being more effective when used alone than when combined with TP or LC.

CYP8B1 is the unique P450 enzyme with sterol 12-oxidation activity, playing an exclusive role in 12α-hydroxylating intermediates along the bile acid (BA) synthesis pathway. Despite the long history of BA metabolism studies, it is unclear whether CYP8B1 catalyzes 12α-hydroxylation of C 27 BAs, the key intermediates shuttling between mitochondria and peroxisomes. This work provides robust in vitro evidence that both microsomal and recombinant CYP8B1 enzymes catalyze the 12α-hydroxylation of dihydroxycoprostanic acid (DHCA) into trihydroxycoprostanic acid (THCA). On the one hand, DHCA 12α-hydroxylation reactivity is conservatively detected in liver microsomes of both human and preclinical animals. The reactivity of human tissue fractions conforms well with the selectivity of CYP8B1 mRNA expression, while the contribution of P450 enzymes other than CYP8B1 is excluded by reaction phenotyping in commercial recombinant enzymes. On the other hand, we prepared functional recombinant human CYP8B1 proteins according to a recently published protocol. Titration of the purified CYP8B1 proteins with either C4 (7α-hydroxy-4-cholesten-3-one) or DHCA yields expected blue shifts of the heme Soret peak (type I binding). The recombinant CYP8B1 proteins efficiently catalyze 12α-hydroxylation of both DHCA and C4, with K m of 3.0 and 1.9 μM and k cat of 3.2 and 2.6 min -1 , respectively. In summary, the confirmed role of CYP8B1 in 12α-hydroxylation of C 27 BAs has furnished the forgotten passageway in the BA synthesis pathway. The present finding might have opened a new window to consider the biology of CYP8B1 in glucolipid metabolism and to evaluate CYP8B1 inhibition as a therapeutic approach of crucial interest for metabolic diseases. Significance Statement Academic community has spent about 90 years interpreting the synthesis of bile acids. However, the 12α-hydroxylation of intermediates catalyzed by CYP8B1 is not completely mapped on the classic pathway, particularly for the C 27 bile acids, the pivotal intermediates shuttling between mitochondria and peroxisomes. This work discloses the forgotten 12α-hydroxylation pathway from dihydroxycoprostanic acid into trihydroxycoprostanic acid. The present finding may facilitate evaluating CYP8B1 inhibition as a therapeutic approach of crucial interest for metabolic diseases., (Copyright © 2024 American Society for Pharmacology and Experimental Therapeutics.)