SUMMARY The molecular mechanisms involved in the development of obesity and related complications remain unclear. Here, we report that obese mice and human subjects have increased activity of neutrophil elastase (NE) and decreased serum levels of the NE inhibitor, α1-antitrypsin (A1AT, SerpinA1). NE null (Ela2−/−) mice and A1AT transgenic mice were resistant to high-fat diet (HFD)-induced bodyweight gain, insulin resistance, inflammation and fatty liver. NE inhibitor GW311616A reversed insulin resistance and bodyweight gain in HFD-fed mice. Compared with wild-type mice, Ela2−/− mice augmented circulating high molecular weight (HMW) adiponectin levels, phosphorylation of AMP-activated protein kinase (AMPK) and fatty acid oxidation (FAO) in the liver and brown adipose tissue (BAT), and uncoupling protein (UCP1) levels in the BAT. These data suggest that the A1AT-NE system regulates AMPK signaling, FAO and energy expenditure. The imbalance between A1AT and NE contributes to the development of obesity and related inflammation, insulin resistance and liver steatosis.

High affinity uptake of serum-derived low density lipoprotein (LDL) cholesterol is accomplished through the LDL receptor in the liver. In mammals, thyroid hormone depletion leads to decreased LDL receptor expression and elevated serum cholesterol. The clinical association in humans has been known since the 1920s; however, a molecular explanation has been lacking. LDL receptor levels are subject to negative feedback regulation by cellular cholesterol through sterol regulatory element-binding protein-2 (SREBP-2). Here we demonstrate that the SREBP-2 gene is regulated by thyroid hormone and that increased SREBP-2 nuclear protein levels in hypothyroid animals results in thyroid hormone-independent activation of LDL receptor gene expression and reversal of the associated hypercholesterolemia. This occurs without effects on other thyroid hormone-regulated genes. Thus, we propose that the decreased LDL receptor and increased serum cholesterol associated with hypothyroidism are secondary to the thyroid hormone effects on SREBP-2. These results suggest that hypercholesterolemia associated with hypothyroidism can be reversed by agents that directly increase SREBP-2. Additionally, these results indicate that mutations or drugs that lower nuclear SREBP-2 would cause hypercholesterolemia.The inverse correlation between serum levels of cholesterol and thyroid hormone (TH) 1 has been known from clinical observations that date back over 70 years (1). More recent studies indicate that low LDL-cholesterol uptake in hypothyroid patients is stimulated by thyroid hormone treatment (2), and studies in experimental animals have established that the levels of LDL receptor mRNA and protein in the liver are directly associated with serum thyroid hormone levels (3, 4). Although there is one report of activation of the LDL receptor promoter by the addition of thyroid hormone receptor (TR) and 3,3Ј,5-triiodo-L-thyronine (T3) to transfected HepG2 cells, there was no putative TR site in the promoter region under study, and direct TR binding was not evaluated (5). Even though these studies cannot rule out the existence of a functional binding site for TR, they do not address the possibility of a TR site at a remote location in the LDL receptor locus; they do suggest that the regulation by TR may be at least partly indirect.In contrast to the lack of defined TR sites in the LDL receptor promoter, there are well defined functional sites through which cholesterol regulates gene expression (6). The key site binds the sterol regulatory element-binding proteins (SREBPs) (7). There are three major SREBPs that are encoded by two genes (8). SREBP-1a and -1c are from overlapping mRNAs encoded by one gene and the single SREBP-2 is encoded by a separate gene. All three are synthesized and first inserted into membranes of the endoplasmic reticulum and nuclear envelope where they cannot directly influence gene expression (9). When the sterol level of a cell falls, a multistep maturation process is initiated by a sterol-dependent alteration in membrane traff...

Cholesterol 7-␣-hydroxylase (CYP7A1) is the key enzyme that commits cholesterol to the neutral bile acid biosynthesis pathway and is highly regulated. In the current studies, we have uncovered a role for the transcriptional co-activator PGC-1␣ in CYP7A1 gene transcription. PGC-1␣ plays a vital role in adaptive thermogenesis in brown adipose tissue and stimulates genes important to mitochondrial function and oxidative metabolism. It is also involved in the activation of hepatic gluconeogenesic gene expression during fasting. Because the mRNA for CYP7A1 was also induced in mouse liver by fasting, we reasoned that PGC-1␣ might be an important co-activator for CYP7A1. Here we show that PGC-1␣ and CYP7A1 are also co-induced in livers of mice in response to streptozotocin induced diabetes. Additionally, infection of cultured HepG2 cells with a recombinant adenovirus expressing PGC-1␣ directly activates CYP7A1 gene expression and increases bile acid biosynthesis as well. Furthermore, we show that PGC-1␣ activates the CYP7A1 promoter directly in transient transfection assays in cultured cells. Thus, PGC-1␣ is a key activator of CYP7A1 and bile acid biosynthesis and is likely responsible for the fasting and diabetes dependent induction of CYP7A1. PGC-1␣ has already been shown to be a critical activator of several other oxidative processes including adaptive thermogenesis and fatty acid oxidation. Our studies provide further evidence of the fundamental role played by PGC-1␣ in oxidative metabolism and define PGC-1␣ as a link between diabetes and bile acid metabolism. The CYP7A11 enzyme converts cholesterol into 7-␣-hydroxycholesterol, which is the first specific intermediate in the neutral bile acid biosynthesis pathway in the liver (1). This is a crucial enzyme in mammalian cholesterol metabolism as diversion into the bile acid pathway is the main route for eliminating excess cholesterol from the body. Because of its key role in cholesterol metabolism, the CYP7A1 enzyme and its gene have been studied as an important model for dietary regulation for several years. These studies have revealed that there is a significant amount of regulation at the level of transcription initiation (2); however, post-transcriptional mechanisms for control also occur (1, 3).The CYP7A1 promoter has been extensively evaluated by several groups and the proximal regions of both the mouse and rat promoters contain two direct repeat type elements that bind several nuclear receptors, some of which are indicated in Fig. 1. There is also a binding site for the monomeric orphan receptor LRH-1 that overlaps the DR-1 element. The DR-4 is a target site for the nuclear receptor LXR, which confers positive regulation by cholesterol to the CYP7A1 promoter in mice and rats (2). However, the DR-4 is not conserved in the human gene which is not subject to feed forward regulation by cholesterol (4).CYP7A1 is also activated in livers of fasted mice (5). Similarly, the transcriptional co-activator PGC-1␣ is induced by fasting in liver where it activates transcrip...

The current studies show FGF15 signaling decreases hepatic forkhead transcription factor 1 (FoxO1) activity through phosphatidylinositol (PI) 3-kinase-dependent phosphorylation. The bile acid receptor FXR (farnesoid X receptor) activates expression of fibroblast growth factor (FGF) 15 in the intestine, which acts through hepatic FGFR4 to suppress cholesterol-7␣ hydroxylase (CYP7A1) and limit bile acid production. Because FoxO1 activity and CYP7A1 gene expression are both increased by fasting, we hypothesized CYP7A1 might be a FoxO1 target gene. Consistent with recently reported results, we show CYP7A1 is a direct target of FoxO1. Additionally, we show that the PI 3-kinase pathway is key for both the induction of CYP7A1 by fasting and the suppression by FGF15. FGFR4 is the major hepatic FGF receptor isoform and is responsible for the hepatic effects of FGF15. We also show that expression of FGFR4 in liver was decreased by fasting, increased by insulin, and reduced by streptozotocin-induced diabetes, implicating FGFR4 as a primary target of insulin regulation. Because insulin and FGF both target the PI 3-kinase pathway, these observations suggest FoxO1 is a key node in the convergence of FGF and insulin signaling pathways and functions as a key integrator for the regulation of glucose and bile acid metabolism.Hepatic cholesterol is converted to bile acids, secreted into the gallbladder, and during a meal is released into the small intestine to enhance digestion and absorption of dietary lipids and fat-soluble vitamins. The majority of the bile acid pool (95%) is recycled back to the liver, whereas the remaining 5% is eliminated through fecal excretion (1, 2). This is an important route for the elimination of excess cholesterol and underscores the importance that bile acids play in regulating mammalian cholesterol metabolism. The initial and rate-controlling step in the classic pathway for cholesterol conversion into bile acids is catalyzed by cholesterol 7␣-hydroxylase (CYP7A1).2 CYP7A1 regulation is primarily transcriptional, and expression of its gene is dynamically regulated by hormones and metabolites (2-6). Importantly, bile acids themselves regulate CYP7A1 gene expression through a multicomponent negative feedback pathway. One of the molecular pathways for bile acid regulation is initiated by the activation of the farnesoid X receptor (FXR) responding directly to bile acid agonists (7). Ligand-activated FXR directly binds to a site in the promoter for the small heterodimer partner (SHP) gene and induces expression of SHP mRNA (8, 9). The translated SHP protein lacks the signature nuclear receptor zinc finger DNA binding domain but uses its conserved dimerization motif to form protein-protein contacts with DNA bound activators, usually other nuclear receptors, to inhibit or interfere with their activation potential (10, 11).The first identified target for SHP repression was the CYP7A1 promoter, and SHP was proposed to interfere with activation by the DNA-bound monomeric liver receptor homologue 1 (LRH-1) nucl...

Mice were subjected to different dietary manipulations to selectively alter expression of hepatic sterol regulatory elementbinding protein 1 (SREBP-1) or SREBP-2. mRNA levels for key target genes were measured and compared with the direct binding of SREBP-1 and -2 to the associated promoters using isoform specific antibodies in chromatin immunoprecipitation studies. A diet supplemented with Zetia (ezetimibe) and lovastatin increased and decreased nuclear SREBP-2 and SREBP-1, respectively, whereas a fasting/refeeding protocol dramatically altered SREBP-1 but had modest effects on SREBP-2 levels. Binding of both SREBP-1 and -2 increased on promoters for 3-hydroxy-3-methylglutaryl-CoA reductase, fatty-acid synthase, and squalene synthase in livers of Zetia/lovastatin-treated mice despite the decline in total SREBP-1 protein. In contrast, only SREBP-2 binding was increased for the low density lipoprotein receptor promoter. Decreased SREBP-1 binding during fasting and a dramatic increase upon refeeding indicates that the lipogenic "overshoot" for fatty-acid synthase gene expression known to occur during high carbohydrate refeeding can be attributed to a similar overshoot in SREBP-1 binding. SREBP co-regulatory protein recruitment was also increased/decreased in parallel with associated changes in SREBP binding, and there were clear distinctions for different promoters in response to the dietary manipulations. Taken together, these studies reveal that there are alternative molecular mechanisms for activating SREBP target genes in response to the different dietary challenges of Zetia/lovastatin versus fasting/refeeding. This underscores the mechanistic flexibility that has evolved at the individual gene/promoter level to maintain metabolic homeostasis in response to shifting nutritional states and environmental fluctuations. 3-Hydroxy-3-methylglutaryl (HMG)3 -CoA reductase catalyzes a critical early reaction in the biosynthetic pathway for isoprenoids and cholesterol and is subject to multivalent regulation to ensure optimal pathway activity (1). The molecular events targeted for regulation include primary transcriptional as well as translational and post-translational mechanisms (2). The principal transcriptional regulators for HMG-CoA reductase gene expression are the basic helix-loop-helix (bHLH) leucine zipper sterol regulatory element-binding proteins (SREBPs), which have unique features that distinguish them from other bHLH leucine zipper transcription factors. The first of these is the presence of two closely spaced membrane-spanning helices that bisect the coding sequence and target the SREBPs to the endoplasmic reticulum membrane. These are followed by a carboxyl-terminal domain that interacts with regulatory proteins and controls their trafficking, proteolytic activation, and membrane release (3, 4). SREBPs also have a signature tyrosine residue in the basic DNA-binding domain that is not present in any other bHLH proteins; this one amino acid is key for allowing specific recognition of both the canonical inv...