• Palmitoleate Induces Hepatic Steatosis but Suppresses Liver Inflammatory Response in Mice

      Guo, Xin; Li, Honggui; Xu, Hang; Halim, Vera; Zhang, Weiyu; Wang, Huan; Ong, Kuok Teong; Woo, Shih-Lung; Walzem, Rosemary L.; Mashek, Douglas G.; et al. (2012-06-29)
      The interaction between fat deposition and inflammation during obesity contributes to the development of non-alcoholic fatty liver disease (NAFLD). The present study examined the effects of palmitoleate, a monounsaturated fatty acid (16⠶1n7), on liver metabolic and inflammatory responses, and investigated the mechanisms by which palmitoleate increases hepatocyte fatty acid synthase (FAS) expression. Male wild-type C57BL/6J mice were supplemented with palmitoleate and subjected to the assays to analyze hepatic steatosis and liver inflammatory response. Additionally, mouse primary hepatocytes were treated with palmitoleate and used to analyze fat deposition, the inflammatory response, and sterol regulatory element-binding protein 1c (SREBP1c) activation. Compared with controls, palmitoleate supplementation increased the circulating levels of palmitoleate and improved systemic insulin sensitivity. Locally, hepatic fat deposition and SREBP1c and FAS expression were significantly increased in palmitoleate-supplemented mice. These pro-lipogenic events were accompanied by improvement of liver insulin signaling. In addition, palmitoleate supplementation reduced the numbers of macrophages/Kupffer cells in livers of the treated mice. Consistently, supplementation of palmitoleate decreased the phosphorylation of nuclear factor kappa B (NF-κB, p65) and the expression of proinflammatory cytokines. These results were recapitulated in primary mouse hepatocytes. In terms of regulating FAS expression, treatment of palmitoleate increased the transcription activity of SREBP1c and enhanced the binding of SREBP1c to FAS promoter. Palmitoleate also decreased the phosphorylation of NF-κB p65 and the expression of proinflammatory cytokines in cultured macrophages. Together, these results suggest that palmitoleate acts through dissociating liver inflammatory response from hepatic steatosis to play a unique role in NAFLD.
    • Protection of Rat Cardiac Myocytes by Fructose-1,6-Bisphosphate and 2,3-Butanedione

      Wheeler, Thomas J.; Chien, Sufan; McNeil, Paul L.; Department of Cellular Biology and Anatomy; College of Graduate Studies (2012-04-27)
      Earlier studies by our group showed that fructose-1,6-bisphosphate (FBP) enhances the hypothermic preservation of rat cardiac myocytes and the functional recovery of animal hearts after hypothermic storage. However, the mechanisms involved were not clear. We extended the cardiomyocyte studies by testing whether the FBP effects were due to chelation of extracellular calcium, leading to lower intracellular levels. We also tested effects of 2,3-butanedione monoxime (BDM), pyruvate, and adenine nucleotide precursors. Cardiomyocytes were incubated in ischemic suspension at 3°C, and aliquots examined over 48 to 72 hours for retention of rod-shaped morphology, a measure of viability. Cytosolic Ca2+ levels were measured in some experiments. FBP at 5 mM reduced the death rate even when added after one or two days of incubation. It caused cytosolic calcium levels that were 33% lower than controls in freshly-isolated cells and 70% lower after one day of incubation. EGTA protected against cell death similarly to FBP. These results indicated that one of the mechanisms by which FBP exerts protective effects is through chelation of extracellular calcium. BDM was strongly protective and reduced cytosolic calcium by 30% after one day of incubation. As with FBP, BDM was effective when added after one or two days of incubation. BDM may be useful in combination with FBP in preserving heart tissue. Pyruvate, adenine, and ribose provided little or no protection during hypothermia.
    • Reduced-folate carrier (RFC) is expressed in placenta and yolk sac, as well as in cells of the developing forebrain, hindbrain, neural tube, craniofacial region, eye, limb buds and heart.

      Maddox, Dennis M; Manlapat, Anna K; Roon, Penny; Prasad, Puttur D; Ganapathy, Vadivel; Smith, Sylvia B; Department of Cellular Biology and Anatomy; Department of Obstetrics and Gynecology; Department of Biochemistry and Molecular Biology; Department of Ophthalmology (2003-10-29)
      BACKGROUND: Folate is essential for cellular proliferation and tissue regeneration. As mammalian cells cannot synthesize folates de novo, tightly regulated cellular uptake processes have evolved to sustain sufficient levels of intracellular tetrahydrofolate cofactors to support biosynthesis of purines, pyrimidines, and some amino acids (serine, methionine). Though reduced-folate carrier (RFC) is one of the major proteins mediating folate transport, knowledge of the developmental expression of RFC is lacking. We utilized in situ hybridization and immunolocalization to determine the developmental distribution of RFC message and protein, respectively. RESULTS: In the mouse, RFC transcripts and protein are expressed in the E10.0 placenta and yolk sac. In the E9.0 to E11.5 mouse embryo RFC is widely detectable, with intense signal localized to cell populations in the neural tube, craniofacial region, limb buds and heart. During early development, RFC is expressed throughout the eye, but by E12.5, RFC protein becomes localized to the retinal pigment epithelium (RPE). CONCLUSIONS: Clinical studies show a statistical decrease in the number of neural tube defects, craniofacial abnormalities, cardiovascular defects and limb abnormalities detected in offspring of female patients given supplementary folate during pregnancy. The mechanism, however, by which folate supplementation ameliorates the occurrence of developmental defects is unclear. The present work demonstrates that RFC is present in placenta and yolk sac and provides the first evidence that it is expressed in the neural tube, craniofacial region, limb buds and heart during organogenesis. These findings suggest that rapidly dividing cells in the developing neural tube, craniofacial region, limb buds and heart may be particularly susceptible to folate deficiency.
    • Rhabdomyosarcomas in Aging A/J Mice

      Sher, Roger B.; Cox, Gregory A.; Mills, Kevin D.; Sundberg, John P.; McNeil, Paul L.; Department of Cellular Biology and Anatomy; College of Graduate Studies (2011-08-10)
      Rhabdomyosarcomas (RSCs) are skeletal muscle neoplasms found in humans and domestic mammals. The A/J inbred strain developed a high frequency (between 70â 80%) of adult pleomorphic type (APT) RSC at >20 months of age while BALB/cByJ also develop RSC but less frequently. These neoplasms invaded skeletal muscle surrounding either the axial or proximal appendicular skeleton and were characterized by pleomorphic cells with abundant eosinophilic cytoplasm, multiple nuclei, and cross striations. The diagnosis was confirmed by detection of alpha-sarcomeric actin and myogenin in the neoplastic cells using immunocytochemistry. The A/J strain, but not the related BALB/c substrains, is also characterised by a progressive muscular dystrophy homologous to limb-girdle muscular dystrophy type 2B. The association between the development of RSC in similar muscle groups to those most severely affected by the progressive muscular dystrophy suggested that these neoplasms developed from abnormal regeneration of the skeletal muscle exacerbated by the dysferlin mutation. Transcriptome analyses of RSCs revealed marked downregulation of genes in muscular development and function signaling networks. Non-synonymous coding SNPs were found in Myl1, Abra, Sgca, Ttn, and Kcnj12 suggesting these may be important in the pathogenesis of RSC. These studies suggest that A strains of mice can be useful models for dissecting the molecular genetic basis for development, progression, and ultimately for testing novel anticancer therapeutic agents dealing with rhabdomyosarcoma.