Using Stable Isotopes in Bone Marrow Derived Macrophage to Analyze Metabolism.

Using gas chromatography mass spectrometry (GC-MS) to analyze the citric acid cycle (CAC) and related intermediates (such as glutamate, glutamine, GABA, and aspartate) is an analytical approach to identify unexpected correlations between apparently related and unrelated pathways of energy metabolism. Intermediates can be as expressed as their absolute concentrations or relative ratios by using known amounts of added reference standards to the sample. GC-MS can also distinguish between heavy labeled molecules (2H- or 13C-labeled) and the naturally occurring most abundant molecules. Applications using tracers can also assess the turnover of specific metabolic pools under various physiological and pathological conditions as well as for pathway discovery. The following protocol is a relatively simple method that is not only sensitive for small concentrations of metabolic intermediates but can also be used in vivo or in vitro to determine the integrity of various metabolic pathways, such as flux changes within specific metabolite pools. We used this protocol to determine the role of phosphoenolpyruvate carboxykinase 1 (Pck1) gene in mouse macrophage cells to determine the percent contribution from a precursor of 13C labeled glucose into specific CAC metabolite pools.

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.

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-1ß secretion. The 17C-6 mice have increased IL-1ß 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.

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

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.

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.

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.

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.

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-1 ß (IL-1 ß ) 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-1 ß . This suggests that Nlrp3 and Nlrc4 inflammasomes activate IL-1 ß and IL-18 via caspase-1 in a differential manner. We conclude that the Nlrp3 inflammasome is protective during alcohol-induced liver injury.

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 ß/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.

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 PPARgamma 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.

Reduced milk triglycerides in mice lacking phosphoenolpyruvate carboxykinase in mammary gland adipocytes and white adipose tissue contribute to the development of insulin resistance in pups.

Obesity and type 2 diabetes are growing problems worldwide in adults and children. In this study, we focused on understanding the patterning of insulin resistance as a result of altered perinatal nutrition. We analyzed mice in which the binding site for PPARgamma was deleted from the promoter of the cytosolic phosphoenolpyruvate carboxykinase gene (Pck1) (PPARE(-/-)). We analyzed pups from dams with the same genotype as well as fostered and cross-fostered pups. Pck1 expression and triglyceride concentration in the milk were measured. The PPARE mutation reduced Pck1 expression in white adipose tissue (WAT) to 2.2% of wild type (WT) and reduced Pck1 expression in whole mammary gland tissue to 1% of WT. The female PPARE(-/-) mice had reduced lipid storage in mammary gland adipocytes and in WAT, resulting in a 40% reduction of milk triglycerides during lactation. Pups from PPARE(-/-) dams had insulin resistance as early as 14 d after birth, a condition that persisted into adulthood. WT pups fostered by PPARE(-/-) dams had lower body weights and plasma insulin concentrations compared with WT pups reared by WT dams. PPARE(-/-) pups fostered by WT dams had improved glucose clearance compared with pups raised by PPARE(-/-) dams. PPARE(+/-) and PPARE(-/-) dams also patterned newborn pups for reduced growth and insulin resistance in utero. Thus, the in utero environment and altered nutrition during the perinatal period cause epigenetic changes that persist into adulthood and contribute to the development of insulin resistance.

Diet-induced hepatocellular carcinoma in genetically predisposed mice.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer death worldwide, with approximately 70% of cases resulting from hepatitis B and C viral infections, aflatoxin exposure, chronic alcohol use or genetic liver diseases. The remaining approximately 30% of cases are associated with obesity, type 2 diabetes and related metabolic diseases, although a direct link between these pathologies and HCCs has not been established. We tested the long-term effects of high-fat and low-fat diets on males of two inbred strains of mice and discovered that C57BL/6J but not A/J males were susceptible to non-alcoholic steatohepatitis (NASH) and HCC on a high-fat but not low-fat diet. This strain-diet interaction represents an important model for genetically controlled, diet-induced HCC. Susceptible mice showed morphological characteristics of NASH (steatosis, hepatitis, fibrosis and cirrhosis), dysplasia and HCC. mRNA profiles of HCCs versus tumor-free liver showed involvement of two signaling networks, one centered on Myc and the other on NFkappaB, similar to signaling described for the two major classes of HCC in humans. miRNA profiles revealed dramatically increased expression of a cluster of miRNAs on the X chromosome without amplification of the chromosomal segment. A switch from high-fat to low-fat diet reversed these outcomes, with switched C57BL/6J males being lean rather than obese and without evidence for NASH or HCCs at the end of the study. A similar diet modification may have important implications for prevention of HCCs in humans.

Postsurgical rehabilitative management of avascular necrosis in the capitate.

OBJECTIVE: To discuss a case of avascular necrosis of the capitate and the follow-up postsurgical rehabilitation. CLINICAL FEATURES: A 41-year-old woman had severe left wrist pain. A short course of passive therapy was administered with no significant change. She was referred for additional diagnostic testing, including magnetic resonance imaging, which assisted in diagnosing the patient with avascular necrosis of the capitate. After undergoing surgical intervention, she was cleared to start an active rehabilitation program. She had significant loss of muscle strength and muscle atrophy and diminished active range of motion in the wrist and forearm. INTERACTION AND OUTCOME: The patient underwent surgical intervention consisting of an arthroplasty to the capitate. The follow-up rehabilitation techniques included the use of active and passive stretching, therapeutic putty, and light dumbbells. The patient was seen a total of 21 times during a 9-week time period. Outcome assessments were given intermittently during care to monitor progress. At the end of the program, she reported full function of the wrist with minimal to no pain. A 6-month follow-up showed improvements that were maintained with little pain. CONCLUSION: Avascular necrosis of the capitate is relatively rare. Advanced imaging should be used for diagnostic purposes. Surgical intervention may be warranted; however, it is recommended a postsurgical rehabilitative procedure be implemented.