Autophagy is an evolutionally conserved catabolic process that recycles nutrients upon starvation and maintains cellular energy homeostasis1–3. Its acute regulation by nutrient sensing signaling pathways is well described, but its longer-term transcriptional regulation is not. The nuclear receptors PPARα and FXR are activated in the fasted or fed liver, respectively4,5. Here we show that both regulate hepatic autophagy. Pharmacologic activation of PPARα reverses the normal suppression of autophagy in the fed state, inducing autophagic lipid degradation, or lipophagy. This response is lost in PPARα knockout (PPARα−/−) mice, which are partially defective in the induction of autophagy by fasting. Pharmacologic activation of the bile acid receptor FXR strongly suppresses the induction of autophagy in the fasting state, and this response is absent in FXR knockout (FXR−/−) mice, which show a partial defect in suppression of hepatic autophagy in the fed state. PPARα and FXR compete for binding to shared sites in autophagic gene promoters, with opposite transcriptional outputs. These results reveal complementary, interlocking mechanisms for regulation of autophagy by nutrient status.

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Neonatal cholestasis is a potentially life-threatening condition requiring prompt diagnosis. Mutations in several different genes can cause progressive familial intrahepatic cholestasis, but known genes cannot account for all familial cases. Here we report four individuals from two unrelated families with neonatal cholestasis and mutations in NR1H4, which encodes the farnesoid X receptor (FXR), a bile acid-activated nuclear hormone receptor that regulates bile acid metabolism. Clinical features of severe, persistent NR1H4-related cholestasis include neonatal onset with rapid progression to end-stage liver disease, vitamin K-independent coagulopathy, low-to-normal serum gamma-glutamyl transferase activity, elevated serum alpha-fetoprotein and undetectable liver bile salt export pump (ABCB11) expression. Our findings demonstrate a pivotal function for FXR in bile acid homeostasis and liver protection.

Glucose-6-phosphate dehydrogenase (G6PD) produces cellular NADPH, which is required for the biosynthesis of fatty acids and cholesterol. Although G6PD is required for lipogenesis, it is poorly understood whether G6PD in adipocytes is involved in energy homeostasis, such as lipid and glucose metabolism. We report here that G6PD plays a role in adipogenesis and that its increase is tightly associated with the dysregulation of lipid metabolism and insulin resistance in obesity. We observed that the enzymatic activity and expression levels of G6PD were significantly elevated in white adipose tissues of obese models, including db/db, ob/ob, and dietinduced obesity mice. In 3T3-L1 cells, G6PD overexpression stimulated the expression of most adipocyte marker genes and elevated the levels of cellular free fatty acids, triglyceride, and FFA release. Consistently, G6PD knockdown via small interfering RNA attenuated adipocyte differentiation with less lipid droplet accumulation. Surprisingly, the expression of certain adipocytokines such as tumor necrosis factor alpha and resistin was increased, whereas that of adiponectin was decreased in G6PD overexpressed adipocytes. In accordance with these results, overexpression of G6PD impaired insulin signaling and suppressed insulindependent glucose uptake in adipocytes. Taken together, these data strongly suggest that aberrant increase of G6PD in obese and/or diabetic subjects would alter lipid metabolism and adipocytokine expression, thereby resulting in failure of lipid homeostasis and insulin resistance in adipocytes.Adipocytes are highly specialized cell type that maintains whole-body energy homeostasis by regulating glucose and lipid metabolism (60, 61). For a long time, adipocytes have been considered as an energy depot that stores and mobilizes triglyceride, a major energy fuel. Recently, adipocytes are also being recognized as an endocrine tissue with the discoveries of several adipocyte-secreted molecules, including lipid metabolites and adipocytokines (55). Using these molecules, adipocytes are able to communicate with other tissues and organs to regulate lipid and glucose metabolism, energy balance, insulin action, and reproduction.To maintain lipid homeostasis, adipocytes carry out two reciprocal biochemical processes: lipogenesis and lipolysis. These two processes are tightly controlled by several hormones, lipid metabolites, and nutritional conditions such as feeding and fasting. With these signals, lipogenic transcription factors, including ADD1/SREBP1c, LXRs, and PPAR␥ actively participate in lipid metabolism in adipocytes. These transcription factors are highly expressed in adipose tissue and play a key role in adipocyte differentiation by coordinating lipogenic and adipocyte-specific gene expression (50). Because lipogenesis is tightly linked with adipogenesis, it is difficult to distinguish the two processes-lipogenesis and adipogenesis-during adipocyte differentiation.Malonyl coenzyme A (malonyl-CoA) and NADPH are considered to be essential elements in lipogen...

In this unit, we describe two protocols for analyzing cell cycle status using flow cytometry. The first is based on the simultaneous analysis of proliferation specific marker (Ki-67) and cellular DNA content, which discriminates resting/quiescent cell populations (G0 cell) and quantifies cell cycle distribution (G1, S or G2/M, respectively). The second is based on differential staining of DNA and RNA through co-staining of Hoechst 33342 and Pyronin Y, which is also useful to identify G0 cells from G1 cells. Along with these methods for analyzing cell cycle status, we outline the basics of two additional methods for cell proliferation assays and recent updates of newly-developed fluorophores, which allows multiplex analysis of cell cycle status, cell proliferation and a gene of interest using flow cytometry.