Conjugation of small interfering RNA (siRNA) to an asialoglycoprotein receptor ligand derived from N-acetylgalactosamine (GalNAc) facilitates targeted delivery of the siRNA to hepatocytes in vitro and in vivo. The ligands derived from GalNAc are compatible with solid-phase oligonucleotide synthesis and deprotection conditions, with synthesis yields comparable to those of standard oligonucleotides. Subcutaneous (SC) administration of siRNA−GalNAc conjugates resulted in robust RNAi-mediated gene silencing in liver. Refinement of the siRNA chemistry achieved a 5-fold improvement in efficacy over the parent design in vivo with a median effective dose (ED 50 ) of 1 mg/kg following a single dose. This enabled the SC administration of siRNA−GalNAc conjugates at therapeutically relevant doses and, importantly, at dose volumes of ≤1 mL. Chronic weekly dosing resulted in sustained dose-dependent gene silencing for over 9 months with no adverse effects in rodents. The optimally chemically modified siRNA−GalNAc conjugates are hepatotropic and long-acting and have the potential to treat a wide range of diseases involving liver-expressed genes.

Many favorable metabolic effects have been attributed to thermogenic activity of brown adipose tissue (BAT). Yet, time of day has rarely been considered in this field of research. Here, we show that a diurnal rhythm in BAT activity regulates plasma lipid metabolism. We observed a high-amplitude rhythm in fatty acid uptake by BAT that synchronized with the light/dark cycle. Highest uptake was found at the onset of the active period, which coincided with high lipoprotein lipase expression and low angiopoietin-like 4 expression by BAT. Diurnal rhythmicity in BAT activity determined the rate at which lipids were cleared from the circulation, thereby imposing the daily rhythm in plasma lipid concentrations. In mice as well as humans, postprandial lipid excursions were nearly absent at waking. We anticipate that diurnal BAT activity is an important factor to consider when studying the therapeutic potential of promoting BAT activity.

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The production and subsequent secretion of glucocorticoids by adrenocortical cells of the zona fasciculata is dependent on the availability of the steroidogenic precursor cholesterol. Unesterifi ed cholesterol is converted to glucocorticoids through a series of side-chain modifi cations by cytochrome P450 enzymes and hydroxysteroid dehydrogenases ( 1 ). The intramitochondrial transfer of unesterifi ed cholesterol by the enzyme steroidogenic acute regulatory protein is considered to be the rate-limiting step in the basal synthesis of glucocorticoids. In vitro studies using isolated adrenocortical cells have suggested that HDL and apoBcontaining lipoproteins are able to provide cholesterol as source for the synthesis of glucocorticoids ( 2-5 ). We and others have shown that under conditions where glucocorticoids are physiologically relevant (i.e., under stress), the exogenous uptake and intracellular processing of lipoprotein-associated cholesteryl esters becomes of crucial importance to maintain optimal adrenal glucocorticoid function in vivo. Probucol-induced depletion of plasma cholesterol associated with HDL and LDL in C57BL/6 wild-type mice is associated with a lower stress-induced glucocorticoid level ( 6 ). In addition, a defect in the hydrolysis of lipoproteinassociated cholesteryl esters in hormone-sensitive lipase knockout (KO) mice is associated with adrenocortical hypofunction ( 7 ). Furthermore, apolipoprotein A1 (APOA1) KO mice that virtually lack HDL particles and scavenger receptor BI (SR-BI) KO mice that exhibit an impaired uptake of cholesteryl esters from HDL show a parallel diminished adrenal glucocorticoid function ( 8-10 ). Combined, these fi ndings suggest that the uptake of HDL-cholesteryl esters by the adrenals is essential to maintain optimal glucocorticoid production in vivo.Abstract In vitro studies have suggested that HDL and apoB-containing lipoproteins can provide cholesterol for synthesis of glucocorticoids. Here we assessed adrenal glucocorticoid function in LCAT knockout (KO) mice to determine the specifi c contribution of HDL-cholesteryl esters to adrenal glucocorticoid output in vivo. LCAT KO mice exhibit an 8-fold higher plasma free cholesterol-to-cholesteryl ester ratio ( P < 0.001) and complete HDL-cholesteryl ester defi ciency. ApoB-containing lipoprotein and associated triglyceride levels are increased in LCAT KO mice as compared with C57BL/6 control mice (44%; P < 0.05). Glucocorticoidproducing adrenocortical cells within the zona fasciculata in LCAT KO mice are devoid of neutral lipids. However, adrenal weights and basal corticosterone levels are not significantly changed in LCAT KO mice. In contrast, adrenals of LCAT KO mice show compensatory up-regulation of genes involved in cholesterol synthesis (HMG-CoA reductase; 516%; P < 0.001) and acquisition (LDL receptor; 385%; P < 0.001) and a marked 40-50% lower glucocorticoid response to adrenocorticotropic hormone exposure, endotoxemia, or fasting ( P < 0.001 for all). In conclusion, our studies show that HDL-cholesteryl es...