David DeSantis

Retinoic acid upregulates preadipocyte genes to block adipogenesis and suppress diet-induced obesity.

Retinoic acid (RA) protects mice from diet-induced obesity. The activity is mediated in part through activation of the nuclear receptors RA receptors (RARs) and peroxisome proliferator–activated receptor β/δ and their associated binding proteins cellular RA binding protein type II (CRABP-II) and fatty acid binding protein type 5 in adipocytes and skeletal muscle, leading to enhanced lipid oxidation and energy dissipation. It was also reported that RA inhibits differentiation of cultured preadipocytes. However, whether the hormone suppresses adipogenesis in vivo and how the activity is propagated remained unknown. In this study, we show that RA inhibits adipocyte differentiation by activating the CRABP-II/RARγ path in preadipose cells, thereby upregulating the expression of the adipogenesis inhibitors Pref-1, Sox9, and Kruppel-like factor 2 (KLF2). In turn, KLF2 induces the expression of CRABP-II and RARγ, further potentiating inhibition of adipocyte differentiation by RA. The data also indicate that RA suppresses adipogenesis in vivo and that the activity significantly contributes to the ability of the hormone to counteract diet-induced obesity.

The STRA6 receptor is essential for retinol-binding protein-induced insulin resistance but not for maintaining vitamin A homeostasis in tissues other than the eye.

Background: STRA6 transports retinol into cells and activates cell signaling. Results: Ablation of Stra6 does not impair vitamin A homeostasis in tissues other than the eye but protects mice against RBP-induced insulin resistance. Conclusion: One major function of STRA6 is to control cell signaling. Significance: The data point at a new function for vitamin A and its blood carrier RBP. The plasma membrane protein STRA6 is thought to mediate uptake of retinol from its blood carrier retinol-binding protein (RBP) into cells and to function as a surface receptor that, upon binding of holo-RBP, activates a JAK/STAT cascade. It was suggested that STRA6 signaling underlies insulin resistance induced by elevated serum levels of RBP in obese animals. To investigate these activities in vivo, we generated and analyzed Stra6-null mice. We show that the contribution of STRA6 to retinol uptake by tissues in vivo is small and that, with the exception of the eye, ablation of Stra6 has only a modest effect on retinoid homeostasis and does not impair physiological functions that critically depend on retinoic acid in the embryo or in the adult. However, ablation of Stra6 effectively protects mice from RBP-induced suppression of insulin signaling. Thus one biological function of STRA6 in tissues other than the eye appears to be the coupling of circulating holo-RBP levels to cell signaling, in turn regulating key processes such as insulin response.

Phosphoenolpyruvate carboxykinase (Pck1) helps regulate the triglyceride/fatty acid cycle and development of insulin resistance in mice

The aim of this study was to investigate the role of the cytosolic form of phosphoenolpyruvate carboxykinase (Pck1) in the development of insulin resistance. Previous studies have shown that the roles of Pck1 in white adipose tissue (WAT) in glyceroneogenesis and reesterification of free fatty acids (FFA) to generate triglyceride are vital for the prevention of diabetes. We hypothesized that insulin resistance develops when dysregulation of Pck1 occurs in the triglyceride/fatty acid cycle, which regulates lipid synthesis and transport between adipose tissue and the liver. We examined this by analyzing mice with a deletion of the PPARγ binding site in the promoter of Pck1 (PPARE−/−). This mutation reduced the fasting Pck1 mRNA expression in WAT in brown adipose tissue (BAT). To analyze insulin resistance, we performed hyperinsulinemic-euglycemic glucose clamp analyses. PPARE−/− mice were profoundly insulin resistant and had more FFA and glycerol released during the hyperinsulinemic-euglycemic clamp compared with wild-type mice (WT). Finally, we analyzed insulin secretion in isolated islets. We found a 2-fold increase in insulin secretion in the PPARE−/− mice at 16.7 mM glucose. Thus, the PPARE site in the Pck1 promoter is essential for maintenance of lipid metabolism and glucose homeostasis and disease prevention.

Erlotinib protects against LPS-induced Endotoxicity because TLR4 needs EGFR to signal

Significance The activation of nuclear factor κB (NFκB) in the normal inflammatory response is rapidly down regulated, whereas constitutive NFκB activation is a hallmark of cancer. We now reveal cross signaling between EGF receptor (EGFR) and Toll-like receptor 4 (TLR4). NFκB activation in response to EGF requires, in addition to EGFR, TLR4 and two downstream proteins. Conversely, EGFR is required for TLR4-mediated activation of NFκB in response to lipopolysaccharide (LPS). The LYN proto-oncogene (LYN) is required for NFκB activation in response to either ligand. In mice, the EGFR inhibitor erlotinib greatly reduces both cytokine expression and endotoxicity in response to LPS, suggesting that EGFR inhibitors may find use in treating septic shock. Several components of the canonical pathway of response to lipopolysaccharide (LPS) are required for the EGF-dependent activation of NFκB. Conversely, the ability of Toll-like Receptor 4 (TLR4) to activate NFκB in response to LPS is impaired by down regulating EGF receptor (EGFR) expression or by using the EGFR inhibitor erlotinib. The LYN proto-oncogene (LYN) is required for signaling in both directions. LYN binds to the EGFR upon LPS stimulation, and erlotinib impairs this association. In mice, erlotinib blocks the LPS-induced expression of tumor necrosis factor α (TNFα) and interleukin-6 (IL-6) and ameliorates LPS-induced endotoxity, revealing that EGFR is essential for LPS-induced signaling in vivo.

Alcohol-induced liver injury is modulated by Nlrp3 and Nlrc4 inflammasomes in mice.

Alcoholic liver disease (ALD) is characterized by increased hepatic lipid accumulation (steatosis) and inflammation with increased expression of proinflammatory cytokines. Two of these cytokines, interleukin-1β (IL-1β) and IL-18, require activation of caspase-1 via members of the NOD-like receptor (NLR) family. These NLRs form an inflammasome that is activated by pathogens and signals released through local tissue injury or death. NLR family pyrin domain containing 3 (Nlrp3) and NLR family CARD domain containing protein 4 (Nlrc4) have been studied minimally for their role in the development of ALD. Using mice with gene targeted deletions for Nlrp3 (Nlrp3−/−) and Nlrc4 (Nlrc4−/−), we analyzed the response to chronic alcohol consumption. We found that Nlrp3−/− mice have more severe liver injury with higher plasma alanine aminotransferase (ALT) levels, increased activation of IL-18, and reduced activation of IL-1B. In contrast, the Nlrc4−/− mice had similar alcohol-induced liver injury compared to C57BL/6J (B6) mice but had greatly reduced activation of IL-1β. This suggests that Nlrp3 and Nlrc4 inflammasomes activate IL-1β and IL-18 via caspase-1 in a differential manner. We conclude that the Nlrp3 inflammasome is protective during alcohol-induced liver injury.

Genetic resistance to liver fibrosis on A/J mouse chromosome 17.

BACKGROUND Because the histological and biochemical progression of liver disease is similar in alcoholic steatohepatitis (ASH) and nonalcoholic steatohepatitis (NASH), we hypothesized that the genetic susceptibility to these liver diseases would be similar. To identify potential candidate genes that regulate the development of liver fibrosis, we studied a chromosome substitution strain (CSS-17) that contains chromosome 17 from the A/J inbred strain substituted for the corresponding chromosome on the C57BL/6J (B6) genetic background. Previously, we identified quantitative trait loci (QTLs) in CSS-17, namely obesity-resistant QTL 13 and QTL 15 (Obrq13 and Obrq15, respectively), that were associated with protection from diet-induced obesity and hepatic steatosis on a high-fat diet. METHODS To test whether these or other CSS-17 QTLs conferred resistance to alcohol-induced liver injury and fibrosis, B6, A/J, CSS-17, and congenics 17C-1 and 17C-6 were either fed Lieber-DeCarli ethanol (EtOH)-containing diet or had carbon tetrachloride (CCl4 ) administered chronically. RESULTS The congenic strain carrying Obrq15 showed resistance from alcohol-induced liver injury and liver fibrosis, whereas Obrq13 conferred susceptibility to liver fibrosis. From published deep sequencing data for chromosome 17 in the B6 and A/J strains, we identified candidate genes in Obrq13 and Obrq15 that contained single-nucleotide polymorphisms (SNPs) in the promoter region or within the gene itself. NADPH oxidase organizer 1 (Noxo1) and NLR family, CARD domain containing 4 (Nlrc4) showed altered hepatic gene expression in strains with the A/J allele at the end of the EtOH diet study and after CCl4 treatment. CONCLUSIONS Aspects of the genetics for the progression of ASH are unique compared to NASH, suggesting that the molecular mechanisms for the progression of disease are at least partially distinct. Using these CSSs, we identified 2 candidate genes, Noxo1 and Nlrc4, which modulate genetic susceptibility in ASH.

Initial evaluation of hepatic T1 relaxation time as an imaging marker of liver disease associated with autosomal recessive polycystic kidney disease (ARPKD)

Autosomal recessive polycystic kidney disease (ARPKD) is a potentially lethal multi‐organ disease affecting both the kidneys and the liver. Unfortunately, there are currently no non‐invasive methods to monitor liver disease progression in ARPKD patients, limiting the study of potential therapeutic interventions. Herein, we perform an initial investigation of T1 relaxation time as a potential imaging biomarker to quantitatively assess the two primary pathologic hallmarks of ARPKD liver disease: biliary dilatation and periportal fibrosis in the PCK rat model of ARPKD. T1 relaxation time results were obtained for five PCK rats at 3 months of age using a Look–Locker acquisition on a Bruker BioSpec 7.0 T MRI scanner. Six three‐month‐old Sprague‐Dawley (SD) rats were also scanned as controls. All animals were euthanized after the three‐month scans for histological and biochemical assessments of bile duct dilatation and hepatic fibrosis for comparison. PCK rats exhibited significantly increased liver T1 values (mean ± standard deviation = 935 ± 39 ms) compared with age‐matched SD control rats (847 ± 26 ms, p = 0.01). One PCK rat exhibited severe cholangitis (mean T1 = 1413 ms), which occurs periodically in ARPKD patients. The observed increase in the in vivo liver T1 relaxation time correlated significantly with three histological and biochemical indicators of biliary dilatation and fibrosis: bile duct area percent (R = 0.85, p = 0.002), periportal fibrosis area percent (R = 0.82, p = 0.004), and hydroxyproline content (R = 0.76, p = 0.01). These results suggest that hepatic T1 relaxation time may provide a sensitive and non‐invasive imaging biomarker to monitor ARPKD liver disease. Copyright

Constitutive Activation of the Nlrc4 Inflammasome Prevents Hepatic Fibrosis and Promotes Hepatic Regeneration after Partial Hepatectomy

TThe molecular mechanisms responsible for the development of hepatic fibrosis are not fully understood. The Nlrc4 inflammasome detects cytosolic presence of bacterial components, activating inflammatory cytokines to facilitate clearance of pathogens and infected cells. We hypothesized that low-grade constitutive activation of the Nlrc4 inflammasome may lead to induced hepatocyte proliferation and prevent the development of hepatic fibrosis. The gene of Nlrc4 contains two single nucleotide polymorphisms (SNPs), one located within the Nlrc4 promoter and one contained within exon 5. These SNPs regulate Nlrc4 gene transcription and activation as measured through gene reporter assays and IL-1β secretion. The 17C-6 mice have increased IL-1β in plasma after chronic carbon tetrachloride (CCl4) administration compared to B6 mice. After two-thirds partial hepatectomy (2/3PH) 17C-6 mice have earlier restoration of liver mass with greater cyclin D1 protein and BrdU incorporation compared to B6 mice at several time points. These data reveal mild constitutive activation of the Nlrc4 inflammasome as the results of two SNPs, which leads to the stimulation of hepatocyte proliferation. The increased liver regeneration induces rapid liver mass recovery after hepatectomy and may prevent the development of hepatotoxin-induced liver fibrosis.

Role of Triglyceride/Fatty Acid Cycle in Development of Type 2 Diabetes

In United States, the prevalence of obesity has increased from 11.9% to 33.4% in men and from 16.6% to 36.5% in women from 1971 to 2006 (Austin, Spadano-Gasbarro et al. 2011). In general, obesity is caused by energy imbalance where energy intake exceeds energy expenditure for an extended period of time. The excess energy is stored in adipose tissue and results in enlarged fat cells and/or increased number of fat cells. Obesity is of great concern because a subset of the population has co-morbidities that develop as a consequence of obesity. Previous studies have shown that obesity is one of the risk factors linked to metabolic syndrome (Bray 2004). The World Health Organization (WHO) defines metabolic syndrome as a cluster of disorders that require the presence of diabetes, impaired glucose tolerance (IGT), impaired fasting glucose or insulin resistance and any two of the following abnormalities: central obesity, dyslipidemia (high triglycerides or low high-density lipoprotein (HDL) cholesterol concentration), elevated blood pressure, or micro albuminuria (Alberti and Zimmet 1998). Type 2 diabetes mellitus (T2DM) is well known to be a metabolic disease that is characterized by insulin resistance and impaired insulin secretion. Insulin resistance is a condition in which the peripheral tissues, such as muscle and adipose tissue, lose the ability to uptake plasma glucose efficiently at physiological concentrations of insulin. As a result, the pancreas secretes more insulin to compensate. Therefore, insulin resistance leads to high plasma concentrations of insulin and glucose, and results in T2DM. Moreover, hyperglycemia causes cardiovascular diseases and disease-specific complications such as blindness and kidney failure (Zimmet, Alberti et al. 2001). The increased prevalence of T2DM is strongly associated with obesity (Mokdad, Ford et al. 2003) and is becoming an increasing problem worldwide (King, Abdullaev et al. 1998; Cowie, Rust et al. 2006; Yoon, Lee et al. 2006). In just the United States, 23.6 million people over the age of 20 have diabetes, of which 90% to 95% have T2DM (Cowie, Rust et al. 2006). An overwhelming 25.9% of the US population over 20 years old has early stages of disease by exhibiting impaired fasting glucose levels (Cowie, Rust et al. 2006). Additionally, The International Diabetes Federation predicted that 333 million people will have diabetes by 2025 (King 1998; King, Abdullaev et al. 1998; King, Aubert et al. 1998). Therefore, it is very important to determine the causes of this disease and develop strategies for its prevention. The correlation between obesity and the prevalence of T2DM has been well established, but how does T2DM develop? In 1963, Randle et al proposed that lipids impaired insulin-

Abstract 2413: Diet-induced non-alcoholic steatohepatitis and hepatocellular carcinoma in chromosome substitution strains of mice

Hepatocellular Carcinoma (HCC) is the 5th most common cancer worldwide, with increased risk occurring in patients with hepatitis C and B, alcoholism, aflatoxin, and metabolic diseases. Animal models that target liver cells with chemical, physical or biological agents are useful for understanding molecular pathways, but do not capture the natural development of liver cancer through gene-environment interactions. By contrast, several inbred strains of mice have been used to study these interactions in diet-induced metabolic diseases. In particular, C57BL/6J (B6) inbred mice are susceptible to diet-induced obesity, lipid disorders and insulin resistance, whereas A/J mice produce no signs of these diet-induced conditions. In these inbred models, the action of many genes and gene-environment interactions more closely reflect the usual features in non-alcoholic steatohepatitis (NASH) and HCC pathogenesis. Previous studies showed that B6 males are susceptible to diet-induced obesity and non-alcoholic fatty liver disease (NAFLD). We therefore examined the long-term effects of a high fat (HF) diet versus a low fat (LF) diet in males from two inbred strains A/J and B6 to determine whether conditions associated with risk factors ultimately transition to HCC. On the HF diet, A/J males remained lean and were resistant to NASH, whereas B6 males were obese and showed histological and biochemical features of NASH, and in many cases NASH progressed to HCCs with molecular and histological features that were remarkably similar to those reported for the two major HCC classes in humans. On the LF diet, both A/J and B6 were resistant to NASH and HCC. Messenger RNA profiles of HCCs versus tumor-free livers implicated two signaling networks, one centered on Myc and the other on NFκB. Also, we tested whether long-term exposure to the HF diet would give the same response in Chromosome Substitution Strains (CSSs) as with the parental strains. The B6-ChrA/J CSS panel consists of 22 mouse strains in which each CSS consist of a single substituted chromosome from a donor strain (A/J) onto the background of a host strain (B6). We found that most CSSs were resistant to diet-induced obesity, matching their A/J parent, with a few resembling the obesity of the B6 parental strain. We selected two strains from the CSS panel, B6-Chr18A/J which shows diet-induced obesity, and B6-Chr5A/J, an obesity resistant strain, to test long-term HF diet effects. Although initially lean during short-term HF diet tests, B6-Chr5A/J eventually became obese and showed a strong susceptibility to NASH and HCC. By contrast, B6-Chr18A/J remained obese throughout the long-term study, but remarkably maintained features of a normal liver, showed a greatly reduced susceptibility to NASH, and complete resistance to HCC. These studies show that complex interactions between genetic and dietary factors modulate susceptibility to liver disease and cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2413. doi:10.1158/1538-7445.AM2011-2413