Effect of ApoA4 on SERPINA3 mediated by nuclear receptors NR4A1 and NR1D1 in hepatocytes.

ApoA4 exerts anti-inflammatory effects, but the mechanism remains unclear. SERPINA3 is a member of the serine proteinase inhibitor gene family, and has been shown to be involved in anti-inflammation and associated with a number of human diseases. In this study, we revealed that ApoA4 stimulates the gene expression of SERPINA3 in mouse hepatocytes both in vivo and in vitro, in a dose- and time-dependent manner. The transcriptional response of SERPINA3 to ApoA4 is regulated through the binding of ApoA4 with nuclear receptors NR4A1 and NR1D1 on the SERPINA3 promoter, which was verified with ChIP, Luciferase activity assay and RNA interference-mediated NR4A1 or NR1D1 gene knockdown. These data suggests that ApoA4 transcriptionally induced SERPINA3 expression via NR1D1 and NR4A1. Our findings may throw light on the function of ApoA4 in inflammatory responses and acute-phase reactions, as well as the development of SERPINA3 relative diseases.

Energy homeostasis in apolipoprotein AIV and cholecystokinin-deficient mice.

Apolipoprotein AIV (ApoAIV) and cholecystokinin (CCK) are well-known satiating signals that are stimulated by fat consumption. Peripheral ApoAIV and CCK interact to prolong satiating signals. In the present study, we hypothesized that ApoAIV and CCK control energy homeostasis in response to high-fat diet feeding. To test this hypothesis, energy homeostasis in ApoAIV and CCK double knockout (ApoAIV/CCK-KO), ApoAIV knockout (ApoAIV-KO), and CCK knockout (CCK-KO) mice were monitored. When animals were maintained on a low-fat diet, ApoAIV/CCK-KO, ApoAIV-KO, and CCK-KO mice had comparable energy intake and expenditure, body weight, fat mass, fat absorption, and plasma parameters relative to the controls. In contrast, these KO mice exhibited impaired lipid transport to epididymal fat pads in response to intraduodenal infusion of dietary lipids. Furthermore, ApoAIV-KO mice had upregulated levels of CCK receptor 2 (CCK2R) in the small intestine while ApoAIV/CCK-KO mice had upregulated levels of CCK2R in the brown adipose tissue. After 20 wk of a high-fat diet, ApoAIV-KO and CCK-KO mice had comparable body weight and fat mass, as well as lower energy expenditure at some time points. However, ApoAIV/CCK-KO mice exhibited reduced body weight and adiposity relative to wild-type mice, despite having normal food intake. Furthermore, ApoAIV/CCK-KO mice displayed normal fat absorption and locomotor activity, as well as enhanced energy expenditure. These observations suggest that mice lacking ApoAIV and CCK have reduced body weight and adiposity, possibly due to impaired lipid transport and elevated energy expenditure.

Apolipoprotein A-IV reduces hepatic gluconeogenesis through nuclear receptor NR1D1.

We showed recently that apoA-IV improves glucose homeostasis by enhancing pancreatic insulin secretion in the presence of elevated levels of glucose. Therefore, examined whether apolipoprotein A-IV (apoA-IV) also regulates glucose metabolism through the suppression of hepatic gluconeogenesis. The ability of apoA-IV to lower gluconeogenic gene expression and glucose production was measured in apoA-IV(-/-) and wild-type mice and primary mouse hepatocytes. The transcriptional regulation of Glc-6-Pase and phosphoenolpyruvate carboxykinase (PEPCK) by apoA-IV was determined by luciferase activity assay. Using bacterial two-hybrid library screening, NR1D1 was identified as a putative apoA-IV-binding protein. The colocalization and interaction between apoA-IV and NR1D1 were confirmed by immunofluorescence, in situ proximity ligation assay, and coimmunoprecipitation. Enhanced recruitment of NR1D1 and activity by apoA-IV to Glc-6-Pase promoter was verified with ChIP and a luciferase assay. Down-regulation of apoA-IV on gluconeogenic genes is mediated through NR1D1, as illustrated in cells with NR1D1 knockdown by siRNA. We found that apoA-IV suppresses the expression of PEPCK and Glc-6-Pase in hepatocytes; decreases hepatic glucose production; binds and activates nuclear receptor NR1D1 and stimulates NR1D1 expression; in cells lacking NR1D1, fails to inhibit PEPCK and Glc-6-Pase gene expression; and stimulates higher hepatic glucose production and higher gluconeogenic gene expression in apoA-IV(-/-) mice. We conclude that apoA-IV inhibits hepatic gluconeogenesis by decreasing Glc-6-Pase and PEPCK gene expression through NR1D1. This novel regulatory pathway connects an influx of energy as fat from the gut (and subsequent apoA-IV secretion) with inhibition of hepatic glucose production.

The role of apolipoprotein A-IV in regulating glucagon-like peptide-1 secretion.

Both glucagon-like peptide-1 (GLP-1) and apolipoprotein A-IV (apoA-IV) are produced from the gut and enhance postprandial insulin secretion. This study investigated whether apoA-IV regulates nutrient-induced GLP-1 secretion and whether apoA-IV knockout causes compensatory GLP-1 release. Using lymph-fistula-mice, we first determined lymphatic GLP-1 secretion by administering apoA-IV before an intraduodenal Ensure infusion. apoA-IV changed neither basal nor Ensure-induced GLP-1 secretion relative to saline administration. We then assessed GLP-1 in apoA-IV-/- and wild-type (WT) mice administered intraduodenal Ensure. apoA-IV-/- mice had comparable lymph flow, lymphatic triglyceride, glucose, and protein outputs as WT mice. Intriguingly, apoA-IV-/- mice had higher lymphatic GLP-1 concentration and output than WT mice 30 min after Ensure administration. Increased GLP-1 was also observed in plasma of apoA-IV-/- mice at 30 min. apoA-IV-/- mice had comparable total gut GLP-1 content relative to WT mice under fasting, but a lower GLP-1 content 30 min after Ensure administration, suggesting that more GLP-1 was secreted. Moreover, an injection of apoA-IV protein did not reverse the increased GLP-1 secretion in apoA-IV-/- mice. Finally, we assessed gene expression of GLUT-2 and the lipid receptors, including G protein-coupled receptor (GPR) 40, GPR119, and GPR120 in intestinal segments. GLUT-2, GPR40 and GPR120 mRNAs were unaltered by apoA-IV knockout. However, ileal GPR119 mRNA was significantly increased in apoA-IV-/- mice. GPR119 colocalizes with GLP-1 in ileum and stimulates GLP-1 secretion by sensing OEA, lysophosphatidylcholine, and 2-monoacylglycerols. We suggest that increased ileal GPR119 is a potential mechanism by which GLP-1 secretion is enhanced in apoA-IV-/- mice.

Is apolipoprotein A-IV rate limiting in the intestinal transport and absorption of triglyceride?

Apolipoprotein A-IV (apoA-IV) is synthesized by the intestine and secreted when dietary fat is absorbed and transported into lymph associated with chylomicrons. We have recently demonstrated that loss of apoA-IV increases chylomicron size and delays its clearance from the blood. There is still uncertainty, however, about the precise role of apoA-IV on the transport of dietary fat from the intestine into the lymph. ApoA-IV knockout (KO) mice do not have a gross defect in dietary lipid absorption, as measured by oral fat tolerance and fecal fat measurements. Here, using the in vivo lymph fistula mouse model, we show that the cumulative secretion of triglyceride (TG) into lymph in apoA-IV KO mice is very similar to that of wild-type (WT) mice. However, the apoA-IV KO mice do have subtle changes in TG accumulation in the intestinal mucosa during a 6-h continuous, but not bolus, infusion of lipid. There are no changes in the ratio of esterified to free fatty acids in the intestinal mucosa of the apoA-IV KO, however. When we extended these findings, by giving a higher dose of lipid (6 µmol/h) and for a longer infusion period (8 h), we found no effect of apoA-IV KO on intestinal TG absorption. This higher lipid infusion most certainly stresses the intestine, as we see a drastically lower absorption of TG (in both WT and KO mice); however, the loss of A-IV does not exacerbate this effect. This supports our hypothesis that apoA-IV is not required for TG absorption in the intestine. Our data suggest that the mechanisms by which the apoA-IV KO intestine responds to intestinal lipid may not be different from their WT counterparts. We conclude that apoA-IV is not required for normal lymphatic transport of TG.

c-Fos facilitates the acquisition and extinction of cocaine-induced persistent changes.

Development of drug addiction involves persistent neurobiological changes. The dopamine D1 receptor is involved in mediating cocaine-induced neuroadaptation, yet the underlying intracellular mechanisms remain unclear. We examined a potential role of the immediate early gene Fos, which is robustly and rapidly induced by cocaine via D1 receptors, in mediating cocaine-induced persistent neurobiological changes by creating and analyzing a mouse in which Fos is primarily disrupted in D1 receptor-expressing neurons in the brain. We show that the expression levels of several transcription factors, neurotransmitter receptors, and intracellular signaling molecules induced by repeated cocaine administration are altered in Fos-deficient brains. Dendritic remodeling of medium spiny neurons induced by repeated exposure to cocaine is blunted in the mutant mice. The mutant mice exhibit attenuated behavioral sensitization after repeated exposure to cocaine and more persistent memory of cocaine-induced conditioned place preference. Our findings indicate that c-Fos produced in D1 receptor-expressing neurons integrates mechanisms to facilitate both the acquisition and extinction of cocaine-induced persistent changes.

Cocaine-induced intracellular signaling and gene expression are oppositely regulated by the dopamine D1 and D3 receptors.

Repeated exposure to cocaine can induce neuroadaptations in the brain. One mechanism by which persistent changes occur involves alterations in gene expression mediated by the dopamine receptors. Both the dopamine D1 and D3 receptors have been shown to mediate gene expression changes. Moreover, the D1 and D3 receptors are also coexpressed in the same neurons, particularly in the nucleus accumbens and also caudoputamen (CPu). Little is known however, whether these two receptors coordinately regulate gene expression after cocaine administration and the underlying mechanisms. We have used various gene mutant mice to address this issue. We show that extracellular signal-regulated kinase (ERK) activation and c-fos induction in the CPu in response to acute cocaine administration is mediated by the D1 receptor and inhibited by the D3 receptor. Moreover, ERK activation mediates acute cocaine-induced expression of Fos family genes, including c-fos, fosB and fra2. Interestingly, dynorphin, neogenin, and synaptotagmin VII, genes that possess cAMP-response element binding protein and AP-1 transcription complex-binding consensus sequences in their promoters, are also oppositely regulated by the D1 and D3 receptors after repeated exposure to cocaine. Furthermore, such regulation depends on proper ERK activation and c-fos function. These results suggest that the D1 and D3 receptors elicit opposite regulation of target gene expression by regulating ERK activation and c-fos induction after acute and chronic cocaine treatment.

Repeated cocaine administration induces gene expression changes through the dopamine D1 receptors.

Drug addiction involves compulsive drug-seeking and drug-taking despite known adverse consequences. The enduring nature of drug addiction suggests that repeated exposure to abused drugs leads to stable alterations that likely involve changes in gene expression in the brain. The dopamine D1 receptor has been shown to mediate the long-term behavioral effects of cocaine. To examine how the persistent behavioral effects of cocaine correlate with underlying changes in gene expression, we have used D1 receptor mutant and wild-type mice to identify chronic cocaine-induced gene expression changes mediated via the D1 receptors. We focused on the caudoputamen and nucleus accumbens, two key brain regions that mediate the long-term effects of cocaine. Our analyses demonstrate that repeated cocaine administration induces changes in the expression of 109 genes, including those encoding the stromal cell-derived factor I, insulin-like growth factor binding protein 6, sigma 1 receptor, regulators of G-protein signaling protein 4, Wnt1 responsive Cdc42 homolog, Ca2+/calmodulin-dependent protein kinase II alpha subunit, and cyclin D2, via the D1 receptors. Moreover, the seven genes contain AP-1 binding sites in their promoter regions. These results suggest that genes encoding certain extracellular factors, membrane receptors and modulators, and intracellular signaling molecules, among others, are regulated by cocaine via the D1 receptor, and these AP-1 transcription complex-regulated genes might contribute to persistent cocaine-induced behavioral changes.

ABCG5/G8 deficiency in mice reduces dietary triacylglycerol and cholesterol transport into the lymph.

The adenosine triphosphate-binding cassette (ABC) transporter G5/G8 is critical in protecting the body from accumulating dietary plant sterols. Expressed in the liver and small intestine, it transports plant sterols into the biliary and intestinal lumens, thus promoting their excretion. The extent to which G5/G8 regulates cholesterol absorption remains unclear. G5/G8 is also implicated in reducing the absorption of dietary triacylglycerols (TAG) by unknown mechanisms. We hypothesized that G5/G8 suppresses the production of chylomicrons, and its deficiency would enhance the absorption of both dietary TAG and cholesterol. The aim of this study was to investigate the effects of G5/G8 deficiency on lipid uptake and secretion into the lymph under steady-state conditions. Surprisingly, compared with wild-type mice (WT) (n = 9), G5/G8 KO (n = 13) lymph fistula mice given a continuous intraduodenal infusion of [3H]-TAG and [14C]-cholesterol showed a significant (P < 0.05) reduction in lymphatic transport of both [(3)H]-TAG and [(14)C]-cholesterol, concomitant with a significant (P < 0.05) increase of [(3)H]-TAG and [(14)C]-cholesterol accumulated in the intestinal lumen. There was no difference in the total amount of radiolabeled lipids retained in the intestinal mucosa between the two groups. G5/G8 KO mice given a bolus of TAG showed reduced intestinal TAG secretion compared with WT, suggesting an independent role for G5/G8 in facilitating intestinal TAG transport. Our data demonstrate that G5/G8 deficiency reduces the uptake and secretion of both dietary TAG and cholesterol by the intestine, suggesting a novel role for the sterol transporter in the formation and secretion of chylomicrons.

C-fos-deficient mouse hippocampal CA3 pyramidal neurons exhibit both enhanced basal and kainic acid-induced excitability.

Excitotoxicity is a process in which glutamate or other excitatory amino acids induce neuronal cell death. To address whether and how c-fos is involved in neuronal excitotoxicity, we previously generated mice in which the c-fos expression is eliminated specifically in the hippocampus. We found that these mutant mice exhibit increased kainic acid (KA)-induced seizure severity, increased neuronal excitability as measured by electroencephalogram, and increased neuronal cell death compared to wild-type control mice. To further assess the role of c-fos in regulating neuronal excitability at a cellular level, we performed hippocampal slice recording in the current study. We found that c-fos-deficient CA3 pyramidal neurons exhibit both enhanced basal and KA-induced excitability compared to normal control neurons. Our results suggest that c-fos regulates CA3 neuronal excitability.

C-fos regulates neuropeptide Y expression in mouse dentate gyrus.

Excitotoxicity by which excitatory amino acid induces neuronal cell death may underlie mechanisms of neurodegenerative diseases. We previously found that c-fos is critically involved in neuronal excitability and survival. Mice that carry hippocampal mutations of c-fos exhibited hyper-excitability, hyper-excitotoxicity and higher mortality in kainic acid (KA)-induced seizures compared to wild-type mice. To further understand the neuroprotective signal transduction pathways regulated by c-fos in the hippocampal formation, we identified 172 genes that are either regulated by KA or are differentially expressed in wild-type and hippocampal-specific c-fos mutant mice using cDNA microarrays. One gene encodes the neuropeptide Y (NPY). We confirmed that c-fos regulates the expression of NPY by using immunohistochemistry. We found that c-fos is critical in up-regulation of NPY expression in the granule cell layer of dentate gyrus in response to KA administration. As NPY is an important endogenous anti-epileptic agent, our result is consistent with a hypothesis that the neuroprotective function of c-fos is mediated in part by regulation of NPY expression.

Overexpression of apolipoprotein C-III decreases secretion of dietary triglyceride into lymph.

Abstract Apolipoprotein C-III (apoC-III) is not only predominantly synthesized by the liver but also by the small intestine. Because apoC-III is secreted from the intestine on the chylomicron along with lipid absorption, we questioned whether apoC-III might play a role in intestinal lipid absorption and/or transport. Using both wild-type (WT) and apoC-III transgenic (apoC-III Tg) mice, we showed that apoC-III Tg mice have decreased lymphatic lipid transport compared with WT mice in response to an intraduodenal infusion of radiolabeled lipid. This is associated with accumulation of radiolabeled lipids in the luminal compartment of the apoC-III Tg mice, indicating delayed lipid uptake from the lumen. The total amount of radioactive lipids in the mucosal compartment did not differ between apoC-III Tg and WT mice, but the lipid distribution analysis indicated a predominance of free fatty acids and monoacylglycerol in the mucosa of apoC-III Tg mice, implying impaired esterification capacity. Thus, the mechanisms underlying the reduced lymphatic lipid transport in apoC-III Tg mice involve both a delayed lipid uptake into enterocytes, as well as impaired esterification to form triglyceride in the mucosa. These data document a novel role for apoC-III in the uptake, re-esterification, and lymphatic transport of dietary lipids in the intestine.

Dopamine D(1) and D(3) receptors oppositely regulate NMDA- and cocaine-induced MAPK signaling via NMDA receptor phosphorylation.

Development of drug addiction involves complex molecular changes in the CNS. The mitogen-activated protein kinase (MAPK) signaling pathway plays a key role in mediating neuronal activation induced by dopamine, glutamate, and drugs of abuse. We previously showed that dopamine D(1) and D(3) receptors play different roles in regulating cocaine-induced MAPK activation. Although there are functional and physical interactions between dopamine and glutamate receptors, little is known regarding the involvement of D(1) and D(3) receptors in modulating glutamate-induced MAPK activation and underlying mechanisms. In this study, we show that D(1) and D(3) receptors play opposite roles in regulating N-methyl-d-aspartate (NMDA) -induced activation of extracellular signal-regulated kinase (ERK) in the caudate putamen (CPu). D(3) receptors also inhibit NMDA-induced activation of the c-Jun N-terminal kinase and p38 kinase in the CPu. NMDA-induced activation of the NMDA-receptor R1 subunit (NR1), Ca(2+)/calmodulin-dependent protein kinase II and the cAMP-response element binding protein (CREB), and cocaine-induced CREB activation in the CPu are also oppositely regulated by dopamine D(1) and D(3) receptors. Finally, the blockade of NMDA-receptor reduces cocaine-induced ERK activation, and inhibits phosphorylation of NR1, Ca(2+)/calmodulin-dependent protein kinase II, and CREB, while inhibiting ERK activation attenuates cocaine-induced CREB phosphorylation in the CPu. These results suggest that dopamine D(1) and D(3) receptors oppositely regulate NMDA- and cocaine-induced MAPK signaling via phosphorylation of NR1.

Endonuclease G is required for early embryogenesis and normal apoptosis in mice.

Endonuclease G (EndoG) is a nuclear-encoded mitochondrial protein reported to be important for both nuclear DNA fragmentation during apoptosis and mitochondrial DNA replication. To evaluate the in vivo function of EndoG, we have investigated the effects of EndoG deficiency in cells and mice. We found that EndoG homozygous mutant embryos die between embryonic days 2.5 and 3.5. Mitochondrial DNA copy numbers in ovulated oocytes from EndoG heterozygous mutant and wild-type mice are similar, suggesting that EndoG is involved in a cellular function unrelated to mitochondrial DNA replication. Interestingly, we found that cells from EndoG heterozygous mutant mice exhibit increased resistance to both tumor necrosis factor alpha- and staurosporine-induced cell death. Moreover, spontaneous cell death of spermatogonia in EndoG heterozygous mutant mice is significantly reduced compared with wild-type mice. DNA fragmentation is also reduced in EndoG+/- thymocytes and splenocytes compared with wild-type cells, as well as in EndoG+/- thymus in vivo compared with that of the wild-type mice, on activation of apoptosis. These findings indicate that EndoG is essential during early embryogenesis and plays a critical role in normal apoptosis and nuclear DNA fragmentation.