SUMMARY While activation of beige thermogenesis is a promising approach for treatment of obesity-associated diseases, there are currently no known pharmacological means to induce beiging in humans. Intermittent fasting is an effective and natural strategy for weight control, but the mechanism for its efficacy is poorly understood. Here, we show that an every other day fasting (EODF) regimen selectively stimulates beige fat development within white adipose tissue, and dramatically ameliorates obesity, insulin resistance and hepatic steatosis. EODF treatment results in a shift in the gut microbiota composition leading to the elevation of the fermentation products acetate and lactate, and the selective upregulation of monocarboxylate transporter 1 expression in beige cells. Microbiota-depleted mice are resistance to EODF-induced beiging, while transplantation of the microbiota from EODF-treated mice to microbiota-depleted mice activates beiging and improves metabolic homeostasis. These findings provide a new gut microbiota-driven mechanism for activating adipose tissue browning and treating metabolic diseases.

Niemann-Pick C1-like 1 (NPC1L1) is required for cholesterol absorption. Intestinal NPC1L1 appears to be a target of ezetimibe, a cholesterol absorption inhibitor that effectively lowers plasma LDL-cholesterol in humans. However, human liver also expresses NPC1L1. Hepatic function of NPC1L1 was previously unknown, but we recently discovered that NPC1L1 localizes to the canalicular membrane of primate hepatocytes and that NPC1L1 facilitates cholesterol uptake in hepatoma cells. Based upon these findings, we hypothesized that hepatic NPC1L1 allows the retention of biliary cholesterol by hepatocytes and that ezetimibe disrupts hepatic function of NPC1L1. To test this hypothesis, transgenic mice expressing human NPC1L1 in hepatocytes (L1-Tg mice) were created. Hepatic overexpression of NPC1L1 resulted in a 10-to 20-fold decrease in biliary cholesterol concentration, but not phospholipid and bile acid concentrations. This decrease was associated with a 30%-60% increase in plasma cholesterol, mainly because of the accumulation of apoE-rich HDL. Biliary and plasma cholesterol concentrations in these animals were virtually returned to normal with ezetimibe treatment. These findings suggest that in humans, ezetimibe may reduce plasma cholesterol by inhibiting NPC1L1 function in both intestine and liver, and hepatic NPC1L1 may have evolved to protect the body from excessive biliary loss of cholesterol.

Although NPC1L1 is required for intestinal cholesterol absorption, data demonstrating mechanisms by which this protein facilitates the process are few. In this study, a hepatoma cell line stably expressing human NPC1L1 was established, and cholesterol uptake was studied. A relationship between NPC1L1 intracellular trafficking and cholesterol uptake was apparent. At steady state, NPC1L1 proteins localized predominantly to the transferrin-positive endocytic recycling compartment, where free cholesterol also accumulated as revealed by filipin staining. Interestingly, acute cholesterol depletion induced with methyl-␤-cyclodextrin stimulated relocation of NPC1L1 to the plasma membrane, preferentially to a newly formed "apical-like" subdomain. This translocation was associated with a remarkable increase in cellular cholesterol uptake, which in turn was dose-dependently inhibited by ezetimibe, a novel cholesterol absorption inhibitor that specifically binds to NPC1L1. These findings define a cholesterol-regulated endocytic recycling of NPC1L1 as a novel mechanism regulating cellular cholesterol uptake.Whole body cholesterol homeostasis is maintained through three major pathways: de novo synthesis, intestinal absorption, and biliary excretion. Mice lacking npc1l1 (Niemann-Pick C1-like 1) have a substantial reduction in intestinal cholesterol absorption and are resistant to high cholesterol diet-induced cholesterol accumulation (1-3). The phenotypes of npc1l1-null mice recapitulate the effect of ezetimibe (1, 2), a novel cholesterol absorption inhibitor (4 -6), indicating that NPC1L1 is in the ezetimibe inhibitory pathway. Although both the annexin-2/caveolin-1 complex and aminopeptidase N have been reported previously to be the direct target of ezetimibe (7, 8), caveoilin-1 knockout mice have a normal percentage of cholesterol absorption (9), and the physiological evidence for aminopeptidase N as the ezetimibe target has yet to be shown. On the other hand, ezetimibe was recently shown to specifically bind to NPC1L1 (10). All these data strongly support that NPC1L1 is the target of ezetimibe and resides within the cholesterol uptake pathway. However, the reconstitution of NPC1L1-dependent cholesterol transport in cultured cell systems has been unsuccessful, and tissue-specific cofactors were speculated to be needed (1), limiting further exploration of the molecular basis for cholesterol absorption.The NPC1L1 gene was initially identified to be a homolog of NPC1 (Niemann-Pick C1) and was predicted to be involved in intracellular cholesterol trafficking (11) based on the fact that mutations in the NPC1 gene result in a lipid storage disease, Niemann-Pick disease type C1 (12, 13). NPC1L1 is widely expressed in many human tissues, with the highest expression in the liver and small intestine (1, 3, 11). The expression pattern varies among species. Mouse and rat npc1l1 mRNAs are much more abundant in the small intestine than in the liver (1, 3). The reason for the different tissue expression patterns among species is unknown.T...

Summary Lipid droplet (LD) lipolysis in brown adipose tissue (BAT) is generally considered to be required for cold-induced nonshivering thermogenesis. Here we show that mice lacking BAT Comparative Gene Identification-58 (CGI-58), a lipolytic activator essential for the stimulated LD lipolysis, have normal thermogenic capacity and are not cold sensitive. Relative to littermate controls, these animals had higher body temperatures when they were provided food during cold exposure. The increase in body temperature in the fed, cold-exposed knockout mice was associated with increased energy expenditure and increased sympathetic innervation and browning of white adipose tissue (WAT). Mice lacking CGI-58 in both BAT and WAT were cold sensitive, but only in the fasted state. Thus, LD lipolysis in BAT is not essential for cold-induced nonshivering thermogenesis in vivo. Rather CGI-58-dependent LD lipolysis in BAT regulates WAT thermogenesis, and our data uncover an essential role of WAT lipolysis in fueling thermogenesis during fasting.