Dominique Brees

Severe diabetes, age-dependent loss of adipose tissue, and mild growth deficiency in mice lacking Akt2/PKBβ

The serine/threonine kinase Akt/PKB plays key roles in the regulation of cell growth, survival, and metabolism. It remains unclear, however, whether the functions of individual Akt/PKB isoforms are distinct. To investigate the function of Akt2/PKBbeta, mice lacking this isoform were generated. Both male and female Akt2/PKBbeta-null mice exhibit mild growth deficiency and an age-dependent loss of adipose tissue or lipoatrophy, with all observed adipose depots dramatically reduced by 22 weeks of age. Akt2/PKBbeta-deficient mice are insulin resistant with elevated plasma triglycerides. In addition, Akt2/PKBbeta-deficient mice exhibit fed and fasting hyperglycemia, hyperinsulinemia, glucose intolerance, and impaired muscle glucose uptake. In males, insulin resistance progresses to a severe form of diabetes accompanied by pancreatic beta cell failure. In contrast, female Akt2/PKBbeta-deficient mice remain mildly hyperglycemic and hyperinsulinemic until at least one year of age. Thus, Akt2/PKBbeta-deficient mice exhibit growth deficiency similar to that reported previously for mice lacking Akt1/PKBalpha, indicating that both Akt2/PKBbeta and Akt1/PKBalpha participate in the regulation of growth. The marked hyperglycemia and loss of pancreatic beta cells and adipose tissue in Akt2/PKBbeta-deficient mice suggest that Akt2/PKBbeta plays critical roles in glucose metabolism and the development or maintenance of proper adipose tissue and islet mass for which other Akt/PKB isoforms are unable to fully compensate.

Increased Atherosclerosis in Hyperlipidemic Mice With Inactivation of ABCA1 in Macrophages

The ATP-binding cassette transporter A1 (ABCA1) encodes a membrane protein that promotes cholesterol and phospholipid efflux from cells. Mutations in ABCA1 lead to HDL deficiency and tissue accumulation of macrophages in patients with homozygous Tangier disease. In this study, we examined whether the complete absence of ABCA1 or selected inactivation in macrophages is accompanied by an increase in atherosclerotic lesion progression in hypercholesterolemic apolipoprotein E–deficient (apoE−/−) mice and LDLR receptor–deficient (LDLr−/−) mice. The absence of ABCA1 led to reduced plasma cholesterol levels in both the apoE−/− and LDLr−/− mice, along with severe skin xanthomatosis characterized by marked foamy macrophages and cholesterol ester accumulation. However, the complete absence of ABCA1 did not affect the development, progression, or composition of atherosclerotic lesions in either the LDLr−/− or the apoE−/− mice fed a chow or atherogenic diet. In contrast, bone marrow transplantation studies demonstrated that the selective inactivation of ABCA1 in macrophages markedly increased atherosclerosis and foam cell accumulation in apoE−/−. Taken together, these findings demonstrate that the complete absence of ABCA1 has a major impact on plasma lipoprotein homeostasis, and the proposed antiatherogenic effect resulting from ABCA1 deficiency is compensated by a less atherogenic profile. ABCA1 deficiency in macrophages, however, demonstrates the antiatherogenic properties of ABCA1 independent of plasma lipids and HDL levels.

ABCA1-Deficient Mice. Insights Into the Role of Monocyte Lipid Efflux in HDL Formation and Inflammation

Studies with ATP-binding cassette transporter (ABCA1)-deficient mice have been critical in demonstrating the relation between ABCA1 expression, cellular lipid efflux, and HDL metabolism. The phenotype of the ABCA1-deficient mouse parallels the phenotype observed in human Tangier disease, including substantial reductions in both apolipoprotein B and apolipoprotein AI with confounding affects on atherosclerosis.

Increased Cholesterol Deposition, Expression of Scavenger Receptors, and Response to Chemotactic Factors in Abca1-Deficient Macrophages

Objective—Studies in bone marrow transplanted from ATP-binding cassette transporter A1 (ABCA1)–deficient mice into normal mice provides direct evidence that the absence of leukocyte ABCA1 exerts a marked proatherogenic effect independent of changes in plasma lipids, suggesting that ABCA1 plays a key role in the regulation of cholesterol homeostasis and function of macrophages. Therefore, we examined whether the absence of ABCA1 affects the morphology, properties, and functional activities of macrophages that could be related to the development of atherosclerosis. Methods and Results—We conducted a series of experiments in macrophages isolated from Abca1-deficient and wild-type mice and compared several of their properties that are thought to be related to the development of atherosclerosis. Macrophages isolated from Abca1-deficient mice have an increase in cholesterol content, expression of scavenger receptors, and secretion of chemokines, growth factors, and cytokines, resulting in an increased ability to respond to a variety of chemotactic factors. Conclusion—Our studies indicate that the absence of ABCA1 leads to significant changes in the morphology, properties, and functional activities of macrophages. These changes, together with the proinflammatory condition present in ABCA1-deficient mice and increased reactivity of macrophages to chemotactic factors, play a key role in the development and progression of atherosclerosis.

Quantitative analyses and transcriptomic profiling of circulating messenger RNAs as biomarkers of rat liver injury

Serum aminotransferases have been the clinical standard for evaluating liver injury for the past 50‐60 years. These tissue enzymes lack specificity, also tracking injury to other tissues. New technologies assessing tissue‐specific messenger RNA (mRNA) release into blood should provide greater specificity and permit indirect assessment of gene expression status of injured tissue. To evaluate the potential of circulating mRNAs as biomarkers of liver injury, rats were treated either with hepatotoxic doses of D‐(+)‐galactosamine (DGAL) or acetaminophen (APAP) or a myotoxic dose of bupivacaine HCl (BPVC). Plasma, serum, and liver samples were obtained from each rat. Serum alanine aminotransferase and aspartate aminotransferase were increased by all three compounds, whereas circulating liver‐specific mRNAs were only increased by the hepatotoxicants. With APAP, liver‐specific mRNAs were significantly increased in plasma at doses that had no effect on serum aminotransferases or liver histopathology. Characterization of the circulating mRNAs by sucrose density gradient centrifugation revealed that the liver‐specific mRNAs were associated with both necrotic debris and microvesicles. DGAL treatment also induced a shift in the size of plasma microvesicles, consistent with active release of microvesicles following liver injury. Finally, gene expression microarray analysis of the plasma following DGAL and APAP treatment revealed chemical‐specific profiles. Conclusion: The comparative analysis of circulating liver mRNAs with traditional serum transaminases and histopathology indicated that the circulating liver mRNAs were more specific and more sensitive biomarkers of liver injury. Further, the possibility of identifying chemical‐specific transcriptional profiles from circulating mRNAs could open a range of possibilities for identifying the etiology of drug/chemical‐induced liver injury. HEPATOLOGY 2010

Metabolic Adaptive ALT Isoenzyme Response in Livers of C57/BL6 Mice Treated with Dexamethasone

Alanine aminotransferase (ALT) is used as an indicator of hepatocellular injury. Since ALT consists of two isoenzymes, a better understanding of ALT isoenzyme biology in response to compounds that cause metabolic adaptive versus hepatotoxic responses will allow for a more accurate assessment of the significance of an ALT increase. The purpose of this study was to characterize the ALT isoenzyme response in mice treated with 25 or 75 mg/kg of dexamethasone, which is known to induce a progluconeogenic state, for 24 or 72 hr. Those mice treated with 75 mg/kg for 72 hr showed an increase in total liver ALT activity. Western blot showed that there was an increase in ALT2 at both doses and time points and there was a concurrent increase in ALT2 ribonucleic acid at 24 and 72 hr. The ALT isoenzyme response assessed by an activity assay showed an increase in ALT2. The increases in liver ALT were associated with an increase in liver glycogen and there was no hepatocellular necrosis. There was an increase in total serum ALT activity, although serum isoenzymes were not evaluated. Thus, the authors demonstrated that dexamethasone induced increases in hepatic and serum ALT, which reflect a hepatocellular progluconeogenic metabolic adaptive response.

Pharmacological Effects of Nicotine on Norepinephrine Metabolism in Rat Brown Adipose Tissue: Relevance to Nicotinic Therapies for Smoking Cessation

In a two-year carcinogenicity study with administration of high doses of the partial nicotinic agonist varenicline (recently approved for smoking cessation), mediastinal hibernomas occurred in three male rats. To investigate potential mechanisms for partial and full nicotinic agonists to contribute to development of hibernomas, the effects of nicotine on rat brown adipose tissue (BAT) were studied. Male and female rats were administered nicotine at doses of 0, 0.3, and 1 mg/kg subcutaneously for fourteen days. Intrathoracic (mediastinal periaortic and mediastinal perithymic) BAT and interscapular BAT were examined microscopically, and determinations of uncoupling protein-1 (UCP-1) expression and norepinephrine (NE) content were made. Additionally, NE turnover was measured in mediastinal periaortic and perithymic BAT. Nicotine (1 mg/kg) administration resulted in decreased vacuolation only in mediastinal periaortic and mediastinal perithymic BAT of males and elevated UCP-1 in mediastinal periaortic BAT of males and females. Increased NE content occurred only in mediastinal periaortic BAT of males given 0.3 and 1 mg/kg doses, whereas NE turnover was decreased in both males and females given 1 mg/kg. Together, these data demonstrate that nicotine primarily affects mediastinal BAT in male rats, consistent with the gender and location of the hibernomas observed in the two-year carcinogenicity study.

Using Laser Scanning Cytometry to Measure PPAR-Mediated Peroxisome Proliferation and β Oxidation

Laser scanning cytometry (LSC) is a new technology that combines the properties and advantages of flow cytometry (FC) and immunohistochemistry (IHC), thus providing qualitative and quantitative information on protein expression with the additional perspective provided by cell and tissue localization. Formalin-fixed, paraffin embedded liver sections from rats exposed to a Peroxisome Proliferator Activated Receptor (PPAR) agonist were stained with antibodies against peroxisomal targeting signal-1 (PTS-1) (a highly conserved tripeptide contained within all peroxisomal enzymes), Acyl CoA oxidase (AOX) (the rate limiting enzyme of peroxisomal β oxidation), and catalase (an inducible peroxisomal antioxidant enzyme) to evaluate peroxisomal β oxidation, oxidative stress, and peroxisome proliferation. The LSC showed increased AOX, catalase, and PTS-1 expression in centrilobular hepatocytes that correlated favorably with the microscopic observation of centrilobular hepatocellular hypertrophy and with the palmitoyl CoA biochemical assay for peroxisomal β oxidation, and provided additional morphologic information about peroxisome proliferation and tissue patterns of activation. Therefore, the LSC provides qualitative and quantitative evaluation of peroxisome activity with similar sensitivity but higher throughput than the traditional biochemical methods. The additional benefits of the LSC include the direct correlation between histopathologic observations and peroxisomal alterations and the potential utilization of archived formalin-fixed tissues from a variety of organs and species.

24th Annual Meeting of the British Society of Toxicological Pathologists: Biomarkers of Safety and Pharmacology. Meeting Abstracts

Systemic inflammatory response syndrome (SIRS) is a ‘‘state of whole body inflammation’’ that can result in multiple organ dysfunction (MOD) circulatory collapse and even death. SIRS is considered as a self-defense mechanism to nonspecific insults that arise from chemical, necrotic (as a result of tissue damage), ischemic, or infectious stimuli that induce organ pathology. SIRS and MOD are complex processes that involve hemodynamic, humoral and cellular responses, complement activation, and cytokine cascade. SIRS and MOD develop in stages that are mediated in part through acute phase proteins, cytokine dysregulation, and hemodynamic events. In toxicology studies, safety evaluation of xenobiotics, pharmacologically active immune stimulants, and immunosuppressants can induce SIRS and MOD in rats, dogs, and nonhuman primates, and therefore, measurement of circulating mediators as biomarkers of SIRS and MOD will enable clinicians to avoid a potentially severe catastrophic event. Furthermore, chemically induced pathology resulting from events such as ischemia, extensive tissue damage and necrosis, and activation and release of stress hormones all can induce SIRS and MOD as a secondary toxic response. Whole-body SIRS is considered an adverse finding in preclinical toxicology studies. Therefore, knowledge and use of the appropriate inflammatory biomarkers that reflect this pathological process is quite useful both preclinically and clinically. Additionally, preclinical and clinical monitoring of biomarkers that are early predictors or reporters of SIRS are valuable to the toxicologist in hazard identification and risk assessment of novel therapeutics with the potential to cause a proinflammatory response.

Integrated Approach to the Assessment of Acetaminophen Hepatoxicity: Reduction of “Omics” and New Technology to Practice

The Safety Sciences group at Pfizer has developed an integrated strategy for the analysis of toxicological data to reduce early compound attrition. This strategy helps to identify screening approaches and biomarkers for assessing the potential toxicity of candidate drugs by determining if they are structurally or mechanistically similar to known hepatotoxicants. These approaches are highly dependent on the emerging knowledge of genes, proteins, and molecular events in the mediation of adverse effects. Understanding the biology of adverse events is facilitated by a plethora of new technologies and bioinformatics tools available to the safety scientist. Presented here is a case scenario in which acetaminophen (APAP)-induced hepatotoxicity was studied using predictive in silico, in vitro, and in vivo techniques, including structure-activity relationships, biochemical evaluations, quantitative immunohistochemistry, gene expression profiling, and proteomics. Using in silico databases to search chemical specific structure-activity relationships, it was predicted that the hydroxyaniline moiety of APAP has the ability to form a quinone imine, which in turn can react with hepatic proteins to potentially cause toxicity. In primary cultures of rat hepatocytes and intact rats APAP caused depletion of glycogen prior to membrane leakage/necrosis. Induction of CYP3A increased APAP toxicity in rat but not human hepatocytes. Treatment of human hepatocytes with the combination of APAP and barbiturates resulted in toxicity associated with inhibition of APAP conjugation, indicating drug-drug interaction due to compromised APAP glucuronidation. This response was potentiated in cells prepared from individuals deficient in UDPGT activity. In intact rats, the necrosis predicted in silico and in vitro and was confirmed via histopathology and enzyme chemistry. Insight into the mechanism of APAP-mediated hepatotoxicity was achieved by showing increased expression of Heat Shock Protein 70 (HSP-70) using laser scanning cytometry, which suggested abnormal protein folding. It was also shown using enzyme chemistry (glutathione reductase, glucose 6-phosphate dehydrogenase, GSH) that APAP caused depletion of the glutathione system and secondary production of oxidative species. In addition, using gene expression-based statistical models, the livers from rats exposed to acetaminophen were predicted to be responding to a compound that causes necrosis, and more specifically had gene expression profiles similar to carbon tetrachloride and acetaminophen. Potentially predictive serum biomarkers were developed using Proteomics (SELDI TOF-MS) conducted on serum of animals experienced different severity of centrilobular necrosis as determined by histopathology. Controls were separated in different cluster from treated animals and some increases in proteins were directly proportional to the degree of damage detected via histopathology. In conclusion, an integrated approach can be used to identify potential toxic liability of compounds using multiple technologies to cross validate the endpoints results in identification of most efficient screening tools, drug-drug interaction and potential biomarker that contribute to toxicological decision-making during drug developmental process.