Two of the fundamental issues in diabetes research are whether beta precursor cells populate islets of adult mammals and the identity of the signals leading to their differentiation. In this study, we sought to answer these questions by using a double transgenic mouse model (RipCre-ER/ZAP mice) that harbors one transgene which has a rat insulin promoter linked to a tamoxifen-dependent Cre-recombinase (RipCre-ER). The Cre-oestrogen receptor (ER) fusion gene is expressed in the cells with an active insulin promoter (Rip), but excluded from the nucleus. The second transgene is comprised of the beta-actin promoter linked to a floxed reporter gene encoding human placental alkaline phosphatase (PLAP). In these double transgenic mice, injection of Tamoxifen results in the activation of Cre-recombinase, which leads to the deletion of two loxP sites and to the expression of a reporter gene PLAP. The expression of the transgene was specific for beta cells. Due to the dose of TM used, only 30% of the islet bet cells expressed PLAP. We found that islets of RipCre-ER/ZAP control mice contained ß cells that expressed PLAP (PLAP+IN+), ß cells that lacked PLAP expression (PLAP+IN) and also PLAP+ cells that did not express insulin (PLAP+IN). PLAP+IN cells did not proliferate and expressed transcription factors characteristic of cells in the early stage of endocrine cell differentiation. To determine the fate of the PLAP+INcells, RipCre-ER/ZAP mice that received TM were later injected with streptozotocin (STZ), a beta cell toxin that partially ablated the resident population of beta cells. Animals were examined following the end of the treatment. It was found that PLAP+IN cells of STZ treated mice were mitotically active and differentiated into insulin expressing cells (PLAP+IN+). Our results strongly suggest the presence of a population of ß precursor cells in adult control mice. Those ß precursor cells replicate and initiate insulin synthesis following islet injury, partially replenishing the depleted beta cell population in STZ-treated animals. These islet-resident ß precursor cells could become an important source of new beta cells for therapy in type I diabetes.

Mast cell activation through its high affinity IgE receptor has been extensively studied and is thought to play an important role in allergic diseases and parasite rejection. However, mast cells can release mediators in response to a variety of stimuli independent of IgE antibodies, including certain neuropeptides, and in particular substance P, suggesting a role for mast cells in neurogenic inflammation. We therefore took a combined in vitro and in vivo approach to elucidate the mediator production, gene expression, and biological significance of mast cell interactions with substance P. Using in vitro derived bone marrow mast cells, we found distinct activation profiles when we compared substance P and IgE/Ag stimulation. Specifically, different patterns of mediators release occurred, which were dependent on the nature of the stimulus, the phenotypic characteristics of the mast cells, and presence or absence of calcium in the incubation medium. We also examined the gene expression pattern in response to substance P using microarray technology. We found that calcium had a profound influence on the number of genes expressed in response to substance P. In the presence of calcium, more genes were upregulated, and of particular interest were several genes involved in signal transduction (Ramp2, rhoGAP1, Map2k3) and inflammatory response (MCPT-4, MCPT-7, IL-1 β, CCL3, CCL4, CCL7). We utilized normal (+/+), mast cell-deficient KitW/KitW-v , and mast cell-deficient KitW/KitW-v mice that had undergone local and selective mast cell reconstitution to investigate the role of mast cells and mast cell mediators in substance P-induced gastric inflammation in vivo. We found that substance P elicited mast cell-dependent neutrophil infiltration. Interestingly, unlike IgE and antigen responses in the stomach, substance Pelicited inflammation did not require TNF-α production by mast cells for the recruitment of neutrophils. However, we did demonstrate a role for leukotriene production by mast cells in this response. These studies provide the first in vivo evidence of differential mast cell mediator production. We also examined the gene expression pattern induced by substance P in mast cell deficient and normal mice. We found an upregulation of certain genes that were dependent on the presence of mast cells; including certain inflammatory response genes (Adora1, CXCL4, CXCL5, CXCR3) and signal transduction molecules (Rac1, Grk6) that could potentially play a role in substance P-induced gastric inflammation. Activation of mast cells by substance P determines a distinct activation profile, which is reflected in a specific pattern of in vitro mediator release, an in vivo characteristic reaction during gastric inflammation and distinctive patterns of gene expression in cultured mast cells and stomach tissues. Our findings suggest a complex and unique pattern of responses of mast cells to the neuropeptide substance P and offer insights into the unique contribution that mast cells may make to neurogenic inflammation.

ApoB, MTP and lipids are essential for apoB-lipoprotein assembly and secretion. Deficiency in any of these three factors affects lipoprotein production. The main function of apoB-lipoproteins is to deliver fat to other tissues for normal physiologic activities; but their excess accumulation in the plasma induces atherosclerosis and an array of metabolic complications such as Type 2 diabetes and obesity. It is evident that approaches to reduce apoB-lipoprotein production would help treat dyslipidemias. Although the exact mechanism of apoB-lipoprotein assembly and secretion is unknown, it is believed that microsomal triglyceride transfer protein (MTP) transfers neutral lipids onto nascent apoB while it is co-translationally translocated into the endoplasmic reticulum (ER) to form primordial lipoproteins. The goal of this study was to obtain structure and function of MTP to elucidate the role of different lipid transfer activities of MTP in the biosynthesis of apoB-lipoproteins. We used two independent approaches. In the first approach, we used evolutionarily diverse MTP orthologs that differ in their lipid transfer properties. Second, we used missense mutations reported in human that display different disease phenotype to elucidate the importance of different amino acids, α-helices and β-sheets in the MTP molecule that are crucial for different lipid transfer properties. Our evolutionary studies indicate that MTP evolved as a phospholipid transfer protein and acquired triglyceride transfer activity during a transition from invertebrate to vertebrate. Comparative in vitro studies revealed that Drosophila MTP (dMTP) could transfer phospholipids but does not transfer triglyceride. In contrast, human MTP (hMTP) transfers both phospholipids and triglycerides. The importance and exact role of these lipid transfer activities in apoB-lipoprotein assembly is not known. We set out to find the physiological gains endowed on MTP as it acquires triglyceride transfer activities. To delineate in vivo roles of these transfer activities, we expressed these orthologs in liver-specific MTP deficient mice that have low plasma and high hepatic lipids. MTP orthologs reduced hepatic lipids and partially restored plasma lipids in these mice. dMTP mice produced apoB100/apoB48 VLDL and phospholipid-rich apoB48 HDL lipoproteins. Lower plasma lipids in dMTP mice, compared to hMTP were due to increased degradation of apoB. hMTP increased association of triglycerides with apoB in the endoplasmic reticulum membranes, inhibited proteasomal degradation, and formed triglyceride-rich VLDL. These studies demonstrate that MTP’s phospholipid transfer activity prevents hepatosteatosis and maintains low plasma lipids by synthesizing triglyceride-poor VLDL and phospholipid-rich HDL particle. We posit that triglyceride transfer activity of MTP is dispensable and its specific inhibition could be used to lower plasma lipids. Our missense mutation studies show that complete loss of phospholipid and triglyceride transfer activities abolish apoB secretion. This is consistent with the absence of apoB-lipoproteins in abetalipoproteinemia. Analysis of missense mutations found in hypobetalipoproteinemia showed that these mutants lacked phospholipid transfer activity and exhibited partial triglyceride transfer activity. These mutants supported secretion of apoB-lipoproteins but to a lower extent compared to normal MTP. Thus partial loss of triglyceride transfer activity and selective loss of phospholipid transfer activity in MTP could support apoB-lipoprotein assembly and secretion but to lesser extent thereby resulting in a less severe phenotype in individuals resembling heterozygous hypobetalipoproteinemia which is primarily caused by mutations in apoB rather than abetalipoproteinemia. Therefore, hypobetalipoproteinemia phenotype could be due to inefficient assembly and secretion of apoB-lipoproteins. It is likely that genetic screening for mutations in MTP besides apoB could potentially explain hypobetalipoproteinemia in subjects that do not carry mutations in apoB. In short, our evolutionary and mutation studies suggest that both phospholipid and triglyceride transfer activities of MTP are required for optimum apoB-lipoprotein assembly. MTP has been a therapeutic target to lower hyperlipidemia; however available drugs globally inhibit MTP leading to adverse events. Recognition of different roles for phospholipid and triglyceride transfer activities suggested that these activities could be separated and targeted for differential inhibition. Domain specific antagonists rather than global inhibition of MTP could help identify better therapeutic approaches to combat hyperlipidemia, obesity and atherosclerosis.

Cognitive impairments are the best determinants of functional outcome in schizophrenia but there is currently no effective procognitive treatment available. Efforts to improve cognition in schizophrenia are limited, in part because the mechanisms of the cognitive deficits are unclear. The discoordination hypothesis postulates that the underlying pathophysiological mechanism of the cognitive deficits is the aberrant coordination of neural activity within and between the neural networks that subserve cognition. We examined the discoordination hypothesis in the neonatal ventral hippocampal lesion (NVHL) rats, an established neurodevelopmental animal model of schizophrenia. To study the association between abnormal neural synchrony and cognitive impairment in NVHL rats, we recorded local field potentials and assessed cognitive control using the two-frame place avoidance task. Local field potentials were recorded from the hippocampus and the prefrontal cortex. Adult NVHL rats were impaired in performing the two-frame task compared to the control rats. The decreased inter-hippocampal synchrony in the NVHL rats also correlated with their poor performance in the two-frame task. We evaluated therapeutic options to improve cognitive performance in the NVHL rats by targeting abnormal synchrony. We first examined the effects of early intervention by giving cognitive training at adolescent period (P35). Adolescent NVHL and control rats learned the two-frame task without any difficulty. Adolescent cognitive training prevented adult cognitive control impairment in adult NVHL rats, and the benefit generalized to different tasks such as alternation T-maze task and the conflict avoidance task. Adult NVHL rats that had received adolescent cognitive training had increased inter-hippocampal synchrony while performing the two-frame task and this increase in synchrony was normalized to the level of the control rats. This again demonstrates that improved performance in the two-maze task correlated with increased inter-hippocampal synchrony in NVHL rats. We also examined the effects of olanzapine and ethosuxmide drug treatments in adult NVHL rats. Both olanzapine and ethosuximide treatments hypersynchronized the inter-hippocampal and the inter-hippocampus-prefrontal networks but only ethosuxmide treatment increased inter-prefrontal synchrony in NVHL rats. We have established a direct association between abnormal neural synchrony and cognitive control impairment in the NVHL rats by demonstrating the following: 1) NVHL rats have decreased long-range inter-hippocampal synchrony that correlates with poor cognitive control performance in the two-frame task; 2) early cognitive training prevents adult cognitive control impairment and normalizes the long-range inter-hippocampal synchrony to the level of the control rats; 3) targeting synchrony with drug treatments can change cognitive performance levels in the adult NVHL rats. These data demonstrate that targeting synchrony abnormality can offer tremendous promise for improving intellectual competence in people at risk for schizophrenia.

Posttraumatic stress disorder (PTSD) can be a sequela of a traumatic event that elicits an extremely fearful reaction, and is accompanied by re-experiencing of the trauma, numbing or avoidance behavior, and persistent hyperarousal. A signature feature of PTSD is the recurrent, involuntary, and intrusive recollection of a traumatic memory, colloquially referred to as flashbacks, which occur outside of the original experience. Whether an aberrant stress response is a contributing factor to eliciting these intrusive memories, our rodent studies suggest that the response to a stressful experience can activate previously acquired memories in a context that is unrelated to the original learning situation. This observation, which we call 'out-of-context activation of memory' (OCAM), may be a useful model with which to study how an extreme stress response can influence unrelated memories, and that the subsequent modification of these unrelated memories may interfere with normal functioning and contribute to the behavioral alterations and disturbances that characterize disorders such as PTSD. Our previous work reported that after a swim-stress experience, the expression of an unrelated memory was enhanced. The learning environment and the swim environment had no physically identifiable common feature, and yet swim-stress, conducted one day after learning a left/right (L/R) discrimination task, enhanced the subsequent recall of the L/R discrimination task. In addition, swim-stress induced a stable memory to become susceptible to amnestic treatments such as propranolol and electro-convulsive shock. Taken together, this evidence suggests that the stress response to an adverse situation can modify stable, unrelated memories. The goal of my work was to evaluate and characterize the limitations of a stress-induced modification of stable, unrelated memories. There four main findings: (1) the level of physiological stress was not a common feature between the learning and swim-stress experience, (2) corticosteroids play a necessary but not sufficient role in enhancing the expression of a stable, unrelated memory, (3) swim-stress can enhance the expression of a stable, unrelated memory within a time-limited window of up to at least one week after learning, and (4) swim-stress does not enhance memories that are dependent on the hippocampus for its expression.

During retinal development, multipotent retinal progenitor cells (RPCs) give rise to successive and overlapping waves of postmitotic neurons and Müller glia. The expression of specific transcription factors is well-known to direct cell fate specification from RPCs. However, the cell-extrinsic factors governing RPC differentiation are less understood. The extracellular matrix (ECM) protein Cellular Communication Network Factor 2 (CCN2), also known as Connective Tissue Growth Factor (CTGF), is a context-dependent mediator of angiogenesis, cytoskeletal remodeling, ECM stiffness, cell motility, proliferation, apoptosis, and adhesion. Recent studies suggest CCN2 is also a critical regulator of embryonic retinal development as well as the development of the retinal vasculature. Evidence further suggests retinogenesis is mediated by a Yes-associated protein (YAP)-CCN2 axis. However, the relevance of CCN2 and YAP-CCN2 regulation in postnatal retinal development and visual function are unclear. We aimed to address these questions by characterizing CCN2-dependent, retinal cell type-specific gene regulation and employing novel mouse models to interrogate CCN2 functions and the role of YAP-CCN2 regulation in the retina. By employing single-cell transcriptional profiling of CCN2-/- and CCN2+/+ embryonic retinas, we found CCN2 regulated ECM gene transcription in a cell type-dependent manner and facilitated retinal ganglion cell (RGC) specification. We also found retina-specific, mosaic CCN2 deletion permitted development of major retinal cell types and blood vessels yet resulted in selective loss of YAP expression by Müller glia and age-dependent visual dysfunction as measured by electroretinography. These findings indicate CCN2 is a critical regulator of RGC specification and implicate Müller glia-specific YAP-CCN2 regulation as essential for visual function.

Entorhinal cortex dysfunction has been implicated early in Alzheimer’s disease, which is characterized by changes in tau protein and in the cleaved fragments of amyloid precursor protein (APP). This thesis presents work to address basic questions about entorhinal cortex pathophysiology using a high-resolution functional magnetic resonance imaging (fMRI) variant that can map metabolic defects in patients and mouse models of disease in the basal state. Recent work has demonstrated that the entorhinal cortex is divided into functionally distinct regions, the medial entorhinal cortex (MEC) and the lateral entorhinal cortex (LEC). We exploited the high-resolution capabilities of this fMRI variant, cerebral blood volume (CBV) imaging, to ask whether preclinical Alzheimer’s disease differentially affected these subregions. Next, we imaged three mouse models of disease to clarify the role that tau and APP play in driving entorhinal cortex dysfunction and to determine whether the entorhinal cortex can act as a source of dysfunction observed in synaptically linked cortical areas. We found that the LEC was differentially affected by tau and APP in preclinical disease, that LEC dysfunction could spread to the parietal cortex during preclinical disease and that APP expression potentiated tau toxicity in driving LEC dysfunction. Taken together, these findings help to explain regional vulnerability in Alzheimer’s disease.

Alzheimer’s disease (AD) is a chronic progressive neurodegenerative disorder, and is the leading cause of dementia. Histopathologically, AD is characterized by two major lesions: extracellular deposits of amyloid as diffuse and neuritic plaques composed of amyloid beta (Aβ) peptide, and intraneuronal neurofibrillary tangles (NFTs) composed of abnormally hyperphosphorylated tau protein. Alongside Aβ plaques and NFTs, impairments in neurogenesis and synaptic plasticity, and profound neurodegeneration are also major features of AD. As yet, there is no effective treatment for AD, and currently the five FDA approved drugs, available for AD treatment, provide only mild symptomatic benefit for a limited period of time. During the last two decades, several clinical trials targeting the AD pathology at moderate to advanced stages of the disease have failed to meet the primary endpoint. Thus, a paradigm shift has occurred, and increasingly early AD pathology is being targeted to develop successful treatment. On similar lines, this thesis evaluated the effects of early disease mechanisms-based treatments in AD transgenic mouse models. Studies described in this thesis employed two therapeutic strategies: (1) a pro-neurogenic modality (neurotrophic factor small-molecule mimetic) and, (2) reducing neuronal network dysfunction (in the form of epileptic activity), at early stages of AD pathology, and evaluated their effects at early, moderate, and advanced stages of the disease. The Specific Aim 1 of this thesis evaluated the effects of early neural regeneration-based treatment with a ciliary neurotrophic factor (CNTF) small-molecule mimetic, Peptide 021 (P021; Ac-DGGLAG-NH2), in a transgenic mouse model of familial AD, i.e., 3xTg-AD mouse that harbors mutated human amyloid β precursor protein (APP), tau, and presenilin 1 genes. The chronic treatment with compound P021, started at early stages of AD pathology, ameliorated dentate gyrus neurogenesis deficit, synaptic and dendritic loss, and cognitive impairment at moderate-to-severe stages of the disease in 3xTg-AD mice. P021 treatment also robustly decreased the tau pathology and mildly attenuated the Aβ pathology both at moderate and severe stages of the disease. This disease-modifying effect of compound P021 was found to be mediated via increased brain derived neurotrophic factor (BDNF) expression-induced decrease in glycogen synthase kinase-3-β (GSK3β) activity. Thus, Specific Aim 1 of this thesis demonstrated the beneficial effect of early, chronic treatment with a pro-neurogenic modality at moderate-to-severe stages of the disease pathology in a transgenic mouse model of AD. The Specific Aim 2 of this thesis analyzed the effects of early neural regeneration-based treatment with the compound P021, on AD in the context of Down syndrome (DS) in Ts65Dn mice. DS, caused by trisomy 21, is a well-established genetic cause of early-onset AD. The Ts65Dn mice carry a trisomy that is analogous to DS trisomy, including a triple copy of the APP gene. The prenatal to early postnatal treatment with compound P021 ameliorated developmental delay during the early postnatal period and AD-like cognitive impairment in adult life in Ts65Dn mice. P021 treatment also increased the expression of synaptic plasticity markers. Thus, Specific Aim 2 of this thesis showed the beneficial effect of treatment with a pro-neurogenic modality, administered only during the early stages, at moderate-to advanced stages of cognitive impairment in a trisomic mouse model of DS. The Specific Aim 3 of this thesis probed neuronal network dysfunction, in the form of epileptic activities, as a biomarker for early AD pathology in 3xTg-AD mice. Furthermore, it evaluated the effect of targeting early AD pathology on neuronal network dysfunction in these mice. Increased audiogenic seizure susceptibility and hippocampal CA3 hypersynchronous network activity was found at ~ 3 weeks of age (prior to Aβ plaque deposition, neurofibrillary pathology, and cognitive impairment) in 3xTg-AD mice. Both audiogenic seizure susceptibility and hippocampal CA3 hypersynchronous network activity in 3xTg-AD mice were attenuated either by passive immunization with anti-human APP/Aβ antibody (6E10) or by metabotropic glutamate receptor 5 (mGluR5) blockade with the selective antagonist, 2-methyl-6-(phenylethynyl)pyridine hydrochloride (MPEP). Remarkably, hypersynchronous network activity positively correlated with intraneuronal human APP/Aβ expression in hippocampal CA3 region. Thus, Specific Aim 3 of this thesis identified neuronal network dysfunction, in the form of epileptic activities, as a potential biomarker of early disease pathology in a transgenic mouse model of AD. Furthermore, it showed the beneficial effect of targeting the early AD pathology to suppress neuronal network dysfunction. Overall, this thesis revealed the beneficial effects of targeting early disease pathology in AD transgenic mice. By employing a pro-neurogenic modality and by evaluating the neuronal network dysfunction in experimental mouse models of AD and DS, this thesis identified potential drug targets and therapeutic strategies for early treatment of AD.

Cellular plasma membrane is composed of phospholipid bilayer. Our understanding about the plasma membrane has been reshaped during the last two decades. According to the recent model, lipids and proteins are heterogeneously distributed in the plasma membrane forming the membrane microdomains. Microdomains serve as the cellular signaling platform in the plasma membrane. Phosphatidylcholine (PC) and sphingomyelin (SM) are the two major lipids in the microdomains of cellular plasma membrane. Phosphatidylcholine and sphingomyelin compositions within the microdomains determine the bio-physical properties of the plasma membrane. Here, we have evaluated the effect of Lysophosphatidylcholine acyltransferase (LPCAT), which is involved in phosphatidylcholine (PC) remodeling, and serine palmitoyltransferase (SPT), which is the key enzyme in sphingolipid biosynthesis, in the intestinal plasma membrane. Lysophosphatidylcholine acyltransferase 3 (Lpcat3) is involved in phosphatidylcholine (PC) remodeling in the small intestine and liver. We investigated lipid metabolism in inducible intestine-specific and liver-specific Lpcat3 gene knockout mice. We produced Lpcat3-Flox/villinCre-ERT2 mice, which were treated with tamoxifen (at day 1, 3, 5, and 7) to delete Lpcat3 specifically in the intestine. At day 9 after the treatment, we found that Lpcat3 deficiency in enterocytes significantly reduced polyunsaturated PCs in the enterocyte plasma membrane and reduced Niemann-Pick C1-like 1 (NPC1L1), CD36, ATP-binding cassette transporter 1 (ABCA1), and ABCG8 levels on the membrane, thus significantly reduced lipid absorption, cholesterol secretion through apoB-dependent and apoB-independent pathways, and plasma triglyceride, cholesterol, and phospholipid levels, as well as body weight. Moreover, Lpcat3 deficiency does not cause significant lipid accumulation in the small intestine. We also utilized adeno-associated virus (AAV)-Cre to deplete Lpcat3 in the liver. We found that the liver deficiency only reduces plasma triglyceride but not other lipid levels. Furthermore, there is no significant lipid accumulation in the liver. Importantly, small intestine Lpcat3 deficiency has much bigger effect on plasma lipid levels than that of the liver deficiency. Thus, inhibition of small intestine Lpcat3 might constitute a novel approach for treating hyperlipidemia. Serine palmitoyltransferase (SPT) is the first and rate-limiting enzyme of the de novo biosynthetic pathway of sphingolipids. It has been demonstrated that bioactive sphingolipids are implicated in proliferation, differentiation, cellular growth and apoptosis. Here, we have investigated the role of SPT subunit 2 (Sptlc2) on intestinal functions, again using intestinespecific gene knock out approach. We have generated Sptlc2-Flox/villin-Cre-ERT2 mice which were treated with tamoxifen (at day 1, 3, and 5) to ablate Sptlc2 specifically in the intestine. At day 6 after the tamoxifen treatment, we found that Sptlc2 deficiency in intestine significantly decreased the body weight. The number of Paneth and goblet cells were greatly reduced in both small intestine and colon of Sptlc2 deficient mice compared to the controls. We found that Sptlc2 deficiency significantly reduced sphingolipid levels in the plasma membrane of the small intestine and colon. The Sptlc2 ablation dramatically suppressed mucin 2 (Muc2) expression in the colon and significantly increased plasma lipopolysaccharides (LPS) levels. All intestinal Sptlc2 deficient mice died at 7-10 days interval after tamoxifen treatment; however, multiple low absorbable antibiotics and dexamethasone supplementation rescued around 70% of the lethality. This study has evaluated the relationship between intestinal sphingolipids and the maintenance of the intestinal barrier functions. The Sptlc2 deficient mice could be used as a model for human inflammatory bowel diseases. This study will shed light on the development of new modality for the therapeutic interventions for intestinal inflammatory diseases.

Microsomal triglyceride transfer protein (MTP) is a target to reduce plasma lipids because of its indispensable role in triglyceride-rich lipoprotein biosynthesis. Genetic ablation/chemical inhibition of MTP in western diet fed mice and monkeys decreased plasma triglycerides and cholesterol while increasing plasma transaminases (ALT and AST), which are indicators of hepatic injury. MTP deficiency also demonstrated significant accumulations of triglycerides and free cholesterol in the liver. Subcellular localization studies revealed free cholesterol and triglyceride accretion in the endoplasmic reticulum (ER) and mitochondria. Reductions in hepatic free cholesterol attenuated plasma transaminase elevations, indicating that free cholesterol, and not triglyceride, is related to MTP inhibitor linked adverse events. Further, there was a significant positive correlation between free cholesterol and plasma transaminase concentrations. Mechanistic studies revealed increased free cholesterol in the ER and mitochondria, which activated ER and oxidative stress pathways, respectively, without causing inflammation or cell death. Alleviation of hepatic ER stress by molecular chaperones or free cholesterol lowering therapies blunted changes in plasma ALT/AST levels in MTP deficient animals. In the absence of cell death as a mechanism for plasma transaminase release in MTP deficient animals and cells, we identified increased transcription of the GPT/GOT1 genes, the genes that code for the ALT1 and AST1 proteins, respectively. We identified IRE1α, an ER stress signal transducer, as a positive effector of GPT and GOT1 transcription. Signaling cascades activated by IRE1α led to the association of the cJun transcription factor with the GPT/GOT1 promoters, which mediated their transcriptional up-regulation. Increased transcription was associated with augmented cellular synthesis of ALT1 and AST1 and subsequent release into the extracellular environment as evidenced by metabolic labeling studies. Therefore, our studies indicate that increased hepatic free cholesterol as a result of MTP inhibition/ablation elicits an integrated sub-cellular response involving transcriptional up-regulation of GPT/GOT1 via activation of Ire1α and cJun. This response can be avoided by co-administering cellular triglyceride/free cholesterol lowering agents to MTP inhibitor therapy. These studies provide mechanistic insight into the toxicities associated with MTP deficiency and may shed some light into the cause of increased plasma transaminases in other models of hepatic steatosis.