Nina Mayorek

Diclofenac inhibits tumor growth in a murine model of pancreatic cancer by modulation of VEGF levels and arginase activity.

Background Diclofenac is one of the oldest anti-inflammatory drugs in use. In addition to its inhibition of cyclooxygenases (COX), diclofenac potently inhibits phospholipase A2 (PLA2), thus yielding a broad anti-inflammatory effect. Since inflammation is an important factor in the development of pancreatic tumors we explored the potential of diclofenac to inhibit tumor growth in mice inoculated with PANCO2 cells orthotopically. Methodology/Principal Findings We found that diclofenac treatment (30 mg/kg/bw for 11 days) of mice inoculated with PANC02 cells, reduced the tumor weight by 60%, correlating with increased apoptosis of tumor cells. Since this effect was not observed in vitro on cultured PANCO2 cells, we theorized that diclofenac beneficial treatment involved other mediators present in vivo. Indeed, diclofenac drastically decreased tumor vascularization by downregulating VEGF in the tumor and in abdominal cavity fluid. Furthermore, diclofenac directly inhibited vascular sprouting ex vivo. Surprisingly, in contrast to other COX-2 inhibitors, diclofenac increased arginase activity/arginase 1 protein content in tumor stroma cells, peritoneal macrophages and white blood cells by 2.4, 4.8 and 2 fold, respectively. We propose that the subsequent arginine depletion and decrease in NO levels, both in serum and peritoneal cavity, adds to tumor growth inhibition by malnourishment and poor vasculature development. Conclusion/Significance In conclusion, diclofenac shows pronounced antitumoral properties in pancreatic cancer model that can contribute to further treatment development. The ability of diclofenac to induce arginase activity in tumor stroma, peritoneal macrophages and white blood cells provides a tool to study a controversial issue of pro-and antitumoral effects of arginine depletion.

Enzymes of glucose and fatty acid metabolism in early and term human placenta.

The activity of enzymes involved in glycolysis, gluconeogenesis, and lipoenesis in early and term human placenta was determined. A high activity of pyruvate kinase was found, indicating high glycolytic potential. The activity of this enzyme tended to decrease with gestation. The presence of phosphoenolpyruvate carboxylase activity was detected, suggesting the possibility of gluconeogenesis in the placenta. Very low activity of enzymes involved in fatty acid synthesis was found, whereas the activity of the pentose shunt pathway enzymes, glucose-6-phosphogluconate dehydrogenases, was relatively high. This suggested a role of this pathway in the synthesis of lipids other than fatty acids in the placenta. The activities of enzymes in the human placenta and their changes during gestation where compared to previous observations on enzymes in rat placenta.

Sensitization to Insulin Induced by β,β′-Methyl-Substituted Hexadecanedioic Acid (MEDICA 16) in Obese Zucker Rats In Vivo

β,β′-methyl-substituted hexadecanedioic acid (MEDICA 16) consists of a nonmetabolizable long-chain fatty acid designed to probe the effect exerted by fatty acids on insulin sensitivity. The effect of MEDICA 16 was evaluated in insulin-resistant Zucker (fa/fa) rats in terms of liver, muscle, and adipose tissue response to clamped euglycemic hyperinsulinemia in vivo. Nontreated Zucker rats were insulin resistant, maintaining basal rates of total-body glucose disposal, glucose production in liver, free fatty acid (FFA) flux into plasma, and FFA reesterification in adipose tissue, irrespective of the insulin levels induced. MEDICA 16 treatment resulted in an insulin-induced decrease in hepatic glucose production, together with an insulin-induced increase in total-body glucose disposal. Intracellular reesterification of lipolysed FFA in adipose tissue was specifically activated by MEDICA 16, resulting in a pronounced decrease in FFA release, with a concomitant decrease in plasma FFA. In conclusion, MEDICA 16 treatment results in the sensitization of liver, muscle, and adipose tissue to insulin in an animal model for obesity-induced insulin resistance.

Suppression of FoxO1 activity by long-chain fatty acyl analogs.

OBJECTIVE Overactivity of the Forkhead transcription factor FoxO1 promotes diabetic hyperglycemia, dyslipidemia, and acute-phase response, whereas suppression of FoxO1 activity by insulin may alleviate diabetes. The reported efficacy of long-chain fatty acyl (LCFA) analogs of the MEDICA series in activating AMP-activated protein kinase (AMPK) and in treating animal models of diabesity may indicate suppression of FoxO1 activity. RESEARCH DESIGN AND METHODS The insulin-sensitizing and anti-inflammatory efficacy of a MEDICA analog has been verified in guinea pig and in human C-reactive protein (hCRP) transgenic mice, respectively. Suppression of FoxO1 transcriptional activity has been verified in the context of FoxO1- and STAT3-responsive genes and compared with suppression of FoxO1 activity by insulin and metformin. RESULTS Treatment with MEDICA analog resulted in total body sensitization to insulin, suppression of lipopolysaccharide-induced hCRP and interleukin-6–induced acute phase reactants and robust decrease in FoxO1 transcriptional activity and in coactivation of STAT3. Suppression of FoxO1 activity was accounted for by its nuclear export by MEDICA-activated AMPK, complemented by inhibition of nuclear FoxO1 transcriptional activity by MEDICA-induced C/EBPβ isoforms. Similarly, insulin treatment resulted in nuclear exclusion of FoxO1 and further suppression of its nuclear activity by insulin-induced C/EBPβ isoforms. In contrast, FoxO1 suppression by metformin was essentially accounted for by its nuclear export by metformin-activated AMPK. CONCLUSIONS Suppression of FoxO1 activity by MEDICA analogs may partly account for their antidiabetic anti-inflammatory efficacy. FoxO1 suppression by LCFA analogs may provide a molecular rational for the beneficial efficacy of carbohydrate-restricted ketogenic diets in treating diabetes.

Modulation of Insulin Secretion by Fatty Acyl Analogs

The secretagogue, the incretin-like, and the suppressive activities of long-chain fatty acids (LCFAs) in modulating insulin secretion in vivo and in cultured islets were simulated here by β,β′-tetramethyl-hexadecanedioic acid (M16) and α,α′-tetrachloro-tetradecanedioic acid (Cl-DICA). M16, but not Cl-DICA, serves as a substrate for ATP-dependent CoA thioesterification but is not further metabolized. M16, but not Cl-DICA, acted as a potent insulin secretagogue in islets cultured in basal but not high glucose. Short-term exposure to M16 or Cl-DICA resulted in activation of glucose- but not arginine-stimulated insulin secretion. Long-term exposure to M16, but not to Cl-DICA, resulted in suppression of glucose-, arginine-, and K+-stimulated insulin secretion; inhibition of glucose-induced proinsulin biosynthesis; and depletion of islets insulin. β-Cell mass and islet ATP content remained unaffected. Hence, nonmetabolizable LCFA analogs may highlight discrete LCFA metabolites and pathways involved in modulating insulin secretion, which could be overlooked due to the rapid turnover of natural LCFA.

The specificity of triacylglycerol synthesis for medium-chain fatty acids in rat and human adipose preparations

The availability of medium-chain fatty acids as substrates for triacylglycerol synthesis was studied in cultured and suspended rat adipocytes and in pieces of human adipose tissue. Octanoate was virtually excluded from glycerol 3-phosphate esterification while serving as a substrate for diacylglycerol esterification. This specificity was similar to that of cultured rat hepatocytes.

Germline DNA replication timing shapes mammalian genome composition

Abstract Mammalian DNA replication is a highly organized and regulated process. Large, Mb-sized regions are replicated at defined times along S-phase. Replication Timing (RT) is thought to play a role in shaping the mammalian genome by affecting mutation rates. Previous analyses relied on somatic RT profiles. However, only germline mutations are passed on to offspring and affect genomic composition. Therefore, germ cell RT information is necessary to evaluate the influences of RT on the mammalian genome. We adapted the RT mapping technique for limited amounts of cells, and measured RT from two stages in the mouse germline - primordial germ cells (PGCs) and spermatogonial stem cells (SSCs). RT in germline cells exhibited stronger correlations to both mutation rate and recombination hotspots density than those of RT in somatic tissues, emphasizing the importance of using correct tissues-of-origin for RT profiling. Germline RT maps exhibited stronger correlations to additional genetic features including GC-content, transposable elements (SINEs and LINEs), and gene density. GC content stratification and multiple regression analysis revealed independent contributions of RT to SINE, gene, mutation, and recombination hotspot densities. Together, our results establish a central role for RT in shaping multiple levels of mammalian genome composition.