Browsing by Subject "Mice, Knockout"
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Item Functional Analysis of Liver Receptor Homolog-1 and Farnesoid X Receptor in Enterohepatic Physiology(2008-05-13) Lee, Youn-Koung; Kliewer, StevenLiver receptor homolog-1 (LRH-1), an orphan nuclear receptor, and farnesoid X receptor (FXR), a bile acid receptor, are both highly expressed in liver and intestine, where they regulate bile acid homeostasis. To gain further insight into their biological actions, we investigated their function in vivo using gain-of-function and loss-of-function strategies. For LRH-1, three different experimental approaches were used. First, we generated and analyzed mice deficient for LRH-1 in either hepatocytes or intestinal epithelium. These tissue-specific knockout mice had altered expression of a large number of genes involved in bile acid metabolism. Furthermore, there was a marked change in the composition of the bile acid pool in mice lacking LRH-1 in hepatocytes. In a second experimental approach, a constitutively-active form of LRH-1 (VP16LRH-1) was expressed in the intestine of transgenic mice. The intestines of these mice were profoundly enlarged due to alterations in pathways controlling proliferation and apoptosis. In a third experimental approach, the effect of LRH-1 on early developmental decisions was examined in Xenopus laevis. In animal cap explant assays, LRH-1 induced early molecular markers of endoderm differentiation. Taken together, the Xenopus laevis and mouse studies reveal the diverse roles that LRH-1 plays during development and in adult physiology, including effects on endoderm formation, intestinal proliferation, and bile acid homeostasis. FXR regulates bile acid homeostasis through actions in both liver and intestine. In studies designed to probe for additional actions, we found that FXR has an important role in preventing the overgrowth of bacteria in the small intestine. In summary, these studies reveal the diverse processes regulated by the nuclear receptors LRH-1 and FXR and their profound impact on enterohepatic physiology.Item The Physiological Function of Endothelin-2 in Mice.(2009-06-15) Chang, Inik; Yanagisawa, MasashiIn order to directly explore the physiological function of ET-2, we generated constitutive, tissue-specific and systemically inducible knockout mice. Global ET-2 deficient mice exhibited severe growth retardation and juvenile lethality. Despite normal milk intake, they suffered from an apparent internal starvation characterized by hypoglycemia, ketonemia, and increased expression of starvation-induced genes in liver. Based on its abundant expression in the gut, I hypothesized that intestinal function of ET-2 is essential for the growth and survival of mice. However, unexpectedly, the intestine was morphologically and functionally normal in the global mutant mice. Moreover, intestine-specific ET-2 deficient mice showed no detectable abnormalities in growth and survival. Instead, I observed that colonic ET-2 has a protective role in epithelial cell injury. Global ET-2 knockout mice were profoundly hypothermic, even at ambient temperatures. Despite the severe hypothermia, DIO2 and UCP-1 failed to increase in brown adipose tissue in ET-2 knockouts. Housing these mice in a warm environment significantly extended the median life span. As temperature regulation is controlled by the central nervous system (CNS), I examined the phenotype in neuron-specific ET-2 knockout mice. However, the mutant mice displayed normal core body temperature, suggesting that ET-2 is not playing a role in CNS-regulated body temperature. ET-2 expression is clearly detected in the lung, with a sharp and transient increase soon after birth. The emphysematous structural change, which is associated with an increase of total lung capacity, resulted in chronic hypoxemia, hypercapnia, and increased erythropoietin synthesis. Finally, to rule out effects of ET-2 during embryonic development, I used the Cre-loxP system to delete ET-2 in neonatal and adult mice, and found that these mice fully reproduced the phenotype previously observed in global knockouts. Together, these findings reveal that ET-2 is critical for growth and survival of postnatal mice by playing important roles in energy homeostasis, thermoregulation, and maintenance of lung morphology and function. My studies rule out ET-2 function in the intestine and brain as being responsible for these phenotypes. However, the dramatic effects of the lung are newly discovered as a potential candidate tissue for critical ET-2 action and lung ET-2 function deserves further investigation.Item Requirement of a High-Flux Metabolic State for Mouse Embryonic Stem Cell Self-Renewal(2010-11-02T18:10:36Z) Alexander, Peter Barton; McKnight, StevenUnbiased profiling of global metabolite levels has revealed that cultured mouse embryonic stem (ES) cells exist in a unique metabolic state. Metabolites fluctuating dramatically in response to ES cell differentiation include purine nucleotides, acetyl-CoA, the amino acid threonine, and folic acid derivatives. These altered metabolic pathways, collectively known as the high-flux backbone (HFB) of metabolism, are surmised to be responsible for the rapid proliferation of this cell type. In particular, the amino acid threonine is shown here to be critical for mouse ES cell self-renewal. Gene and protein expression analysis has revealed that the enzyme threonine dehydrogenase (TDH) has the potential to play a major role in the establishment of HFB metabolism. TDH breaks down threonine into glycine and acetyl-CoA, molecules which are used to drive purine biosynthesis and ATP production, respectively. Using multiple approaches, we show here that TDH is strongly expressed both in ES cells and in the inner cell mass of the mouse blastocyst. Identification of potent and specific small molecule inhibitors has made possible the targeted elimination of the TDH enzyme in mouse ES cells. Using these compounds, we have determined that metabolic flux through this pathway is essential for ES cell selfrenewal. TDH inhibition is shown to cause an alteration in the cell’s metabolic state that results in increased autophagic activity and cell death. This study also reports on the generation of TDH conditional knockout mice, which will enable further elucidation of the role of HFB metabolism in adult and developing animals.Item Voltage-Gated Sodium Channel Activity in Mouse Skeletal Muscle Fibers: Normal Gating and Defects Associated with Periodic Paralysis Mutants(2010-11-02T18:11:14Z) Fu, Yu; Cannon, StephenMutations in SCN4A, the gene encoding the skeletal muscle Na+ channel (NaV1.4) α-subunit, cause several disorders related to skeletal muscle excitability. The functional consequences of these NaV1.4 mutations have been extensively characterized in heterologous expression systems. These studies have significantly advanced our understanding of the pathophysiology of these disorders. The in vivo functional consequences on channel activity, however, have yet to be defined. Animal models are now available in genetically engineered mice, which provide an opportunity to examine channel function in mature skeletal muscle. We optimized a two-electrode voltage clamp protocol to improve the fidelity of Na+ current recording from acutely dissociated intact muscle fibers. Computer simulation, incorporating measured capacitance and ionic current densities, was used to confirm sufficient voltage control and distortion-free Na+ currents. The gating properties of endogenous Na+ currents were measured and compared between two mouse strains, C57BL/6 and 129-E. The most dramatic finding was a hyperpolarized shift in the voltage dependence of activation (-25 mV) and fast inactivation (-18 mV) as compared to the studies in HEK293 cells expressing NaV1.4 plus the accessory β1-subunit. A possible contribution from NaV1.5 channels in the mouse muscle preparation was excluded by RT-PCR and TTX sensitivity. There was no significant difference in voltage dependence of fast gating between C57BL/6 and 129-E. The entry rate into slow inactivation was slower for Na+ channel in 129-E fibers; while the recovery from slow inactivation was similar between two mouse stains. Two NaV1.4 missense mutations associated with divergent clinical phenotypes - NaV1.4-M1592V in hyperkalemic periodic paralysis (HyperPP) and NaV1.4-R663H (homolog of human R669H) in hypokalemic periodic paralysis (HypoPP) - were characterized with voltage-clamp recordings in fully differentiated fibers from knock-in mutant mice. The NaV1.4-M1592V mutation produced gain-of-function defects, with the major changes being a slightly increased persistent current and moderately disrupted slow inactivation. In contrast, the HypoPP knock-in mutant R663H resulted in loss-of-function changes, due to an enhancement of inactivation, both fast and slow, and impaired activation. These observations provide important validation of prior findings using heterologous expression systems and yield quantitative information on the severity of the gating defects in mammalian skeletal muscles.