Susanna S. T. Lee

Targeted disruption of the alpha isoform of the peroxisome proliferator-activated receptor gene in mice results in abolishment of the pleiotropic effects of peroxisome proliferators.

To gain insight into the function of peroxisome proliferator-activated receptor (PPAR) isoforms in rodents, we disrupted the ligand-binding domain of the alpha isoform of mouse PPAR (mPPAR alpha) by homologous recombination. Mice homozygous for the mutation lack expression of mPPAR alpha protein and yet are viable and fertile and exhibit no detectable gross phenotypic defects. Remarkably, these animals do not display the peroxisome proliferator pleiotropic response when challenged with the classical peroxisome proliferators, clofibrate and Wy-14,643. Following exposure to these chemicals, hepatomegaly, peroxisome proliferation, and transcriptional-activation of target genes were not observed. These results clearly demonstrate that mPPAR alpha is the major isoform required for mediating the pleiotropic response resulting from the actions of peroxisome proliferators. mPPAR alpha-deficient animals should prove useful to further investigate the role of this receptor in hepatocarcinogenesis, fatty acid metabolism, and cell cycle regulation.

Growth, Adipose, Brain, and Skin Alterations Resulting from Targeted Disruption of the Mouse Peroxisome Proliferator-Activated Receptor β(δ)

ABSTRACT To determine the physiological roles of peroxisome proliferator-activated receptor β (PPARβ), null mice were constructed by targeted disruption of the ligand binding domain of the murine PPARβ gene. Homozygous PPARβ-null term fetuses were smaller than controls, and this phenotype persisted postnatally. Gonadal adipose stores were smaller, and constitutive mRNA levels of CD36 were higher, in PPARβ-null mice than in controls. In the brain, myelination of the corpus callosum was altered in PPARβ-null mice. PPARβ was not required for induction of mRNAs involved in epidermal differentiation induced byO-tetradecanoylphorbol-13-acetate (TPA). The hyperplastic response observed in the epidermis after TPA application was significantly greater in the PPARβ-null mice than in controls. Inflammation induced by TPA in the skin was lower in wild-type mice fed sulindac than in similarly treated PPARβ-null mice. These results are the first to provide in vivo evidence of significant roles for PPARβ in development, myelination of the corpus callosum, lipid metabolism, and epidermal cell proliferation.

Role of CYP2E1 in the Hepatotoxicity of Acetaminophen

CYP2E1, a cytochrome P-450 that is well conserved across mammalian species, metabolizes ethanol and many low molecular weight toxins and cancer suspect agents. The cyp2e1 gene was isolated, and a mouse line that lacks expression of CYP2E1 was generated by homologous recombination in embryonic stem cells. Animals deficient in expression of the enzyme were fertile, developed normally, and exhibited no obvious phenotypic abnormalities, thus indicating that CYP2E1 has no critical role in mammalian development and physiology in the absence of external stimuli. When cyp2e1 knockout mice were challenged with the common analgesic acetaminophen, they were found to be considerably less sensitive to its hepatotoxic effects than wild-type animals, indicating that this P-450 is the principal enzyme responsible for the metabolic conversion of the drug to its active hepatotoxic metabolite.

Reduction of benzene metabolism and toxicity in mice that lack CYP2E1 expression

Transgenic CYP2E1 knockout mice (cyp2e1-/-) were used to investigate the involvement of CYP2E1 in the in vivo metabolism of benzene and in the development of benzene-induced toxicity. After benzene exposure, absence of CYP2E1 protein was confirmed by Western blot analysis of mouse liver samples. For the metabolism studies, male cyp2e1-/- and wild-type control mice were exposed to 200 ppm benzene, along with a radiolabeled tracer dose of [14C]benzene (1.0 Ci/mol) by nose-only inhalation for 6 hr. Total urinary radioactivity and all radiolabeled individual metabolites were reduced in urine of cyp2e1-/- mice compared to wild-type controls during the 48-hr period after benzene exposure. In addition, a significantly greater percentage of total urinary radioactivity could be accounted for as phenylsulfate conjugates in cyp2e1-/- mice compared to wild-type mice, indicating the importance of CYP2E1 in oxidation of phenol following benzene exposure in normal mice. For the toxicity studies, male cyp2e1-/-, wild-type, and B6C3F1 mice were exposed by whole-body inhalation to 0 ppm (control) or 200 ppm benzene, 6 hr/day for 5 days. On Day 5, blood, bone marrow, thymus, and spleen were removed for evaluation of micronuclei frequencies and tissue cellularities. No benzene-induced cytotoxicity or genotoxicity was observed in cyp2e1-/- mice. In contrast, benzene exposure resulted in severe genotoxicity and cytotoxicity in both wild-type and B6C3F1 mice. These studies conclusively demonstrate that CYP2E1 is the major determinant of in vivo benzene metabolism and benzene-induced myelotoxicity in mice.

Peroxisome proliferator-activated receptor-alpha regulates lipid homeostasis, but is not associated with obesity: studies with congenic mouse lines

Considerable controversy exists in determining the role of peroxisome proliferator-activated receptor-α (PPARα) in obesity. Two purebred congenic strains of PPARα-null mice were developed to study the role of this receptor in modulating lipid transport and storage. Weight gain and average body weight in wild-type and PPARα-null mice on either an Sv/129 or a C57BL/6N background were not markedly different between genotypes from 3 to 9 months of age. However, gonadal adipose stores were significantly greater in both strains of male and female PPARα-null mice. Hepatic accumulation of lipids was greater in both strains and sexes of PPARα-null mice compared with wild-type controls. Administration of the peroxisome proliferator WY-14643 caused hepatomegaly, alterations in mRNAs encoding proteins that regulate lipid metabolism, and reduced serum triglycerides in a PPARα-dependent mechanism. Constitutive differences in serum cholesterol and triglycerides in PPARα-null mice were found between genetic backgrounds. Results from this work establish that PPARα is a critical modulator of lipid homeostasis in two congenic mouse lines. This study demonstrates that disruption of the murine gene encoding PPARα results in significant alterations in constitutive serum, hepatic, and adipose tissue lipid metabolism. However, an overt, obese phenotype in either of the two congenic strains was not observed. In contrast to earlier published work, this study establishes that PPARα is not associated with obesity in mice.

Xenobiotic receptor knockout mice

Administration of certain foreign chemicals to animals elicits responses that are due to receptor-mediated activation of gene expression. Among the most well studied receptors are the Ah receptor (AHR) that binds 2,3,7,8-tetrachlorodibenzo-p-dioxin and related compounds and the peroxisome proliferator-activated receptors, PPARs, that mediate gene activation by the diverse group of peroxisome proliferators. These receptors may also have critical roles in development or physiological homeostasis in addition to their abilities to allow animals to interact with exogenous chemicals or xenobiotics. To explore the function of AHR and PPAR alpha and to determine whether they participate in the adverse effects of dioxins and peroxisome proliferators, gene knockout mice were developed.

Requirement of PPARα in maintaining phospholipid and triacylglycerol homeostasis during energy deprivation

The peroxisome proliferator-activated receptor α (PPARα) has been implicated as a key control of fatty acid catabolism during the cellular fasting. However, little is known regarding changes of individual fatty acids in hepatic triacylglycerol (TG) and phospholipid (PL) as a result of starvation. In the present work, the effects of 72 h fasting on hepatic TG and PL fatty acid profiles in PPARα-null (KO) mice and their wild-type (WT) counterparts were investigated. Our results indicated that mice deficient in PPARα displayed hepatomegaly and hypoketonemia following 72 h starvation. Histochemical analyses revealed that severe fatty infiltration was observed in the livers of KO mice under fasted conditions. Furthermore, 72 h fasting resulted in a 2.8-fold higher accumulation of hepatic TG in KO mice than in WT mice fasted for the same length of time. Surprisingly, the total hepatic PL contents in fasted KO mice decreased by 45%, but no significant change in hepatic PL content was observed in WT mice following starvation. Gas chromatographic analysis indicated that KO mice were deprived of arachidonic (20:4n-6) and docosahexaenoic (22:6n-3) acids during fasting. Taken together, these results show that PPARα plays an important role in regulation of fatty acid metabolism as well as phospholipid homeostasis during energy deprivation.

Immunohistochemical colocalization of type II angiotensin receptors with somatostatin in rat pancreas

Earlier studies indicate that binding sites of type II angiotensin (AT2) receptors are detected all over the pancreas, as well as in the pancreatic exocrine cell line AR4-2J. However, lack of corresponding functional AT2 receptor responses can be detected in the exocrine pancreas. The aim of present study is to determine the protein expression of AT2 receptors in the pancreas by probing with an AT2 receptor-specific antibody, and to examine the role of AT2 receptors in the regulation of pancreatic endocrine hormone release. In Western protein analysis of adult rat tissues, expression of AT2 receptor-immunoreactive bands of 56, 68, and 78 kDa was detected in the adrenal, kidney, liver, salivary glands, and pancreas. In adult rat pancreas, strong immunoreactivity was detected on cells that were located at the outer region of Langerhans islets. Immunohistochemical studies indicated that AT2 receptors colocalized with somatostatin-producing cells in the endocrine pancreas. Consistent with the findings in adult pancreas, abundant expression of AT2 receptors was also detected in immortalized rat pancreatic endocrinal cells lines RIN-m and RIN-14B. To examine the role of AT2 receptors on somatostatin secretion in the pancreas, angiotensin-stimulated somatostatin release from pancreatic RIN-14B cells was studied by an enzyme immunoassay in the absence or presence of various subtype-selective angiotensin analogues. There was a basal release of somatostatin from RIN-14B cells at a rate of 8.72 +/- 4.21 ng/10(6) cells (n = 7). Angiotensin II (1 nM-10 microM) stimulated a biphasic somatostatin release in a dose-dependent manner with an apparent EC50 value of 49.3 +/- 25.9 nM (n = 5), and reached maximal release at 1 microM angiotensin II (982 +/- 147.34% over basal secretion; n = 5). Moreover, the AT2 receptor-selective angiotensin analogue, CGP42112, was 1000 times more potent than the AT1 receptor-selective angiotensin analogue, losartan, in inhibiting angiotensin II-stimulated somatostatin release. These results suggest that angiotensin may modulate pancreatic hormone release via regulation of somatostatin secretion.

One-step expression and purification of single-chain variable antibody fragment using an improved hexahistidine tag phagemid vector.

Millions of candidate clones are commonly obtained following rounds of phage-displayed antibody library panning, and expression of those selected single-chain variable fragment (scFv) is required for secondary functional screening to identify positive clones. Large scale functional screening is often hampered by the time-consuming and labor-intensive subcloning of those candidate scFv clones into a bacterial expression vector carrying an affinity tag for scFv purification and detection. To overcome the limitations and to develop a multiplex approach, an improved hexahistidine tag phagemid vector was constructed for one-step scFv expression and purification. By using hexahistidine as an affinity tag, soluble scFvs can be rapidly and cost-effectively captured from Escherichia coli periplasmic extracts. For proof-of-concept, feasibility of the improved phagemid vector was examined against two scFvs, L17E4d targeting a cell surface antigen and L18Hh5 recognizing a monoclonal antibody (mAb). Using 1 ml of Ni-NTA agarose, 0.2-0.5 mg of soluble scFv was obtained from 1 L of bacteria culture, and the purified scFvs bound specifically to their target antigens with high affinity. Moreover, using two randomly selected hapten-specific scFv phage clones, it was demonstrated that the display of scFvs on phage surface was not affected by the hexahistidine affinity tag. These results suggest the improved phagemid vector allows the shuttle of phage-displayed antibody library panning and functional scFv production. Importantly, the improved phagemid vector can be easily adapted for multiplex screening.

Cross-reactivity of antibody against SARS-coronavirus nucleocapsid protein with IL-11.

Abstract Infection of SARS-associated coronavirus (SARS-CoV) induced a strong anti-nucleocapsid (anti-N) antibody response. However, the pathophysiological significance of the anti-N antibodies in SARS pathogenesis is largely unknown. To profile the anti-N antibodies, a phage-displayed scFv library was prepared from mice immunized with heat-inactivated SARS-CoV-infected Vero E6 cell lysate. Specific anti-N scFvs were isolated by panning against a recombinant nucleocapsid protein and reactivity was confirmed with phage-ELISA. Sequence analysis indicated that two of the isolated anti-N scFv clones were identical and displayed a high homology with an scFv specific for interleukin 11 (IL-11), an anti-inflammatory cytokine derived from bone marrow stroma cells. In a neutralization assay, IL-11-induced STAT 3 phosphorylation in rat intestinal epithelial IEC-18 cells was completely suppressed by the anti-N scFv clone L9N01.