Molecular markers of brain cholesterol homeostasis are unchanged despite a smaller brain mass in a mouse model of cholesteryl ester storage disease.

Lysosomal acid lipase (LAL), encoded by the gene LIPA, facilitates the intracellular processing of lipids by hydrolyzing cholesteryl esters and triacylglycerols present in newly internalized lipoproteins. Loss-of-function mutations in LIPA result in cholesteryl ester storage disease (CESD) or Wolman disease when mutations cause complete loss of LAL activity. Although the phenotype of a mouse CESD model has been extensively characterized, there has not been a focus on the brain at different stages of disease progression. In the current studies, whole-brain mass and the concentrations of cholesterol in both the esterified (EC) and unesterified (UC) fractions were measured in Lal-/- and matching Lal+/+ mice (FVB-N strain) at ages ranging from 14 up to 280 days after birth. Compared to Lal+/+ controls at 50, 68-76, 140-142, and 230-280 days of age, Lal-/- mice had brain weights that averaged approximately 6%, 7%, 18%, and 20% less, respectively. Brain EC levels were higher in the Lal-/- mice at every age, being elevated 27-fold at 230-280 days. Brain UC concentrations did not show a genotypic difference at any age. The elevated brain EC levels in the Lal-/- mice did not reflect EC in residual blood. An mRNA expression analysis for an array of genes involved in the synthesis, catabolism, storage, and transport of cholesterol in the brains of 141-day old mice did not detect any genotypic differences although the relative mRNA levels for several markers of inflammation were moderately elevated in the Lal-/- mice. The possible sites of EC accretion in the central nervous system are discussed.

Tonic prime-boost of STING signalling mediates Niemann-Pick disease type C.

The classic mode of STING activation is through binding the cyclic dinucleotide 2'3'-cyclic GMP-AMP (cGAMP), produced by the DNA sensor cyclic GMP-AMP synthase (cGAS), which is important for the innate immune response to microbial infection and autoimmune disease. Modes of STING activation that are independent of cGAS are much less well understood. Here, through a spatiotemporally resolved proximity labelling screen followed by quantitative proteomics, we identify the lysosomal membrane protein Niemann-Pick type C1 (NPC1) as a cofactor in the trafficking of STING. NPC1 interacts with STING and recruits it to the lysosome for degradation in both human and mouse cells. Notably, we find that knockout of Npc1 'primes' STING signalling by physically linking or 'tethering' STING to SREBP2 trafficking. Loss of NPC1 protein also 'boosts' STING signalling by blocking lysosomal degradation. Both priming and boosting of STING signalling are required for severe neurological disease in the Npc1-/- mouse. Genetic deletion of Sting1 (the gene that encodes STING) or Irf3, but not that of Cgas, significantly reduced the activation of microglia and relieved the loss of Purkinje neurons in the cerebellum of Npc1-/- mice, leading to improved motor function. Our study identifies a cGAS- and cGAMP-independent mode of STING activation that affects neuropathology and provides a therapeutic target for the treatment of Niemann-Pick disease type C.

Delineation of metabolic responses of Npc1-/-nih mice lacking the cholesterol-esterifying enzyme SOAT2 to acute treatment with 2-hydroxypropyl-ß-cyclodextrin.

Lipids present in lipoproteins cleared from the circulation are processed sequentially by three major proteins within the late endosomal/lysosomal (E/L) compartment of all cells: lysosomal acid lipase (LAL), Niemann-Pick (NPC) C2 and NPC1. When all three of these proteins are functioning normally, unesterified cholesterol (UC) exits the E/L compartment and is used in plasma membrane maintenance and various pathways in the endoplasmic reticulum including esterification by sterol O-acyltransferase 2 (SOAT2) or SOAT1 depending partly on cell type. Mutations in either NPC2 or NPC1 result in continual entrapment of UC and glycosphingolipids leading to neurodegeneration, pulmonary dysfunction, splenomegaly and liver damage. To date, the most effective agent for promoting release of entrapped UC in nearly all organs of NPC1-deficient mice and cats is 2-hydroxypropyl-ß-cyclodextrin (2HPßCD). The cytotoxic nature of the liberated UC triggers various defenses including suppression of sterol synthesis and increased esterification. The present studies, using the Npc1-/-nih mouse model, measured the comparative quantitative importance of these two responses in the liver versus the spleen of Npc1-/-: Soat2+/+ and Npc1-/-: Soat2-/- mice in the 24 h following a single acute treatment with 2HPßCD. In the liver but not the spleen of both types of mice suppression of synthesis alone or in combination with increased esterification provided the major defense against the rise in unsequestered cellular UC content. These findings have implications for systemic 2HPßCD treatment in NPC1 patients in view of the purportedly low levels of SOAT2 activity in human liver.

Ontogenesis and Modulation of Intestinal Unesterified Cholesterol Sequestration in a Mouse Model of Niemann-Pick C1 Disease.

BACKGROUND: Mutations in the NPC1 gene result in sequestration of unesterified cholesterol (UC) and glycosphingolipids in most tissues leading to multi-organ disease, especially in the brain, liver, lungs, and spleen. Various data from NPC1-deficient mice suggest the small intestine (SI) is comparatively less affected, even in late stage disease. METHODS: Using the Npc1nih mouse model, we measured SI weights and total cholesterol (TC) levels in Npc1-/- versus Npc1+/+ mice as a function of age, and then after prolonged ezetimibe-induced inhibition of cholesterol absorption. Next, we determined intestinal levels of UC and esterified cholesterol (EC), and cholesterol synthesis rates in Npc1-/- and Npc1+/+ mice, with and without the cholesterol-esterifying enzyme SOAT2, following a once-only subcutaneous injection with 2-hydroxypropyl-ß-cyclodextrin (2HPßCD). RESULTS: By ~ 42 days of age, intestinal TC levels averaged ~ 2.1-fold more (mostly UC) in the Npc1-/- versus Npc1+/+ mice with no further increase thereafter. Chronic ezetimibe treatment lowered intestinal TC levels in the Npc1-/- mice by only ~ 16%. In Npc1-/- mice given 2HPßCD 24 h earlier, UC levels fell, EC levels increased (although less so in mice lacking SOAT2), and cholesterol synthesis was suppressed equally in the Npc1-/-:Soat2+/+ and Npc1-/-:Soat2-/- mice. CONCLUSIONS: The low and static levels of intestinal UC sequestration in Npc1-/- mice likely reflect the continual sloughing of cells from the mucosa. This sequestration is blunted by about the same extent following a single acute treatment with 2HPßCD as it is by a prolonged ezetimibe-induced block of cholesterol absorption.

1,25-Dihydroxyvitamin D3 enhances glucose-stimulated insulin secretion in mouse and human islets: a role for transcriptional regulation of voltage-gated calcium channels by the vitamin D receptor.

AIM: Vitamin D deficiency in rodents negatively affects glucose-stimulated insulin secretion (GSIS) and human epidemiological studies connect poor vitamin D status with type 2 diabetes. Previous studies performed primarily in rat islets have shown that vitamin D can enhance GSIS. However the molecular pathways linking vitamin D and insulin secretion are currently unknown. Therefore, experiments were undertaken to elucidate the transcriptional role(s) of the vitamin D receptor (VDR) in islet function. METHODS: Human and mouse islets were cultured with vehicle or 1,25-dihydroxyvitamin-D3 (1,25D3) and then subjected to GSIS assays. Insulin expression, insulin content, glucose uptake and glucose-stimulated calcium influx were tested. Microarray analysis was performed. In silico analysis was used to identify VDR response elements (VDRE) within target genes and their activity was tested using reporter assays. RESULTS: Vdr mRNA is abundant in islets and Vdr expression is glucose-responsive. Preincubation of mouse and human islets with 1,25D3 enhances GSIS and increases glucose-stimulated calcium influx. Microarray analysis identified the R-type voltage-gated calcium channel (VGCC) gene, Cacna1e, which is highly upregulated by 1,25D3 in human and mouse islets and contains a conserved VDRE in intron 7. Results from GSIS assays suggest that 1,25D3 might upregulate a variant of R-type VGCC that is resistant to chemical inhibition. CONCLUSION: These results suggest that the role of 1,25D3 in regulating calcium influx acts through the R-Type VGCC during GSIS, thereby modulating the capacity of beta cells to secrete insulin.

ACSS2 promotes systemic fat storage and utilization through selective regulation of genes involved in lipid metabolism.

Acetyl-CoA synthetase 2 (ACSS2) is a conserved nucleocytosolic enzyme that converts acetate to acetyl-CoA. Adult mice lacking ACSS2 appear phenotypically normal but exhibit reduced tumor burdens in mouse models of liver cancer. The normal physiological functions of this alternate pathway of acetyl-CoA synthesis remain unclear, however. Here, we reveal that mice lacking ACSS2 exhibit a significant reduction in body weight and hepatic steatosis in a diet-induced obesity model. ACSS2 deficiency reduces dietary lipid absorption by the intestine and also perturbs repartitioning and utilization of triglycerides from adipose tissue to the liver due to lowered expression of lipid transporters and fatty acid oxidation genes. In this manner, ACSS2 promotes the systemic storage or metabolism of fat according to the fed or fasted state through the selective regulation of genes involved in lipid metabolism. Thus, targeting ACSS2 may offer a therapeutic benefit for the treatment of fatty liver disease.

Niemann-Pick C1-deficient mice lacking sterol O-acyltransferase 2 have less hepatic cholesterol entrapment and improved liver function.

Cholesteryl esters are generated at multiple sites in the body by sterol O-acyltransferase (SOAT) 1 or SOAT2 in various cell types and lecithin cholesterol acyltransferase in plasma. Esterified cholesterol and triacylglycerol contained in lipoproteins cleared from the circulation via receptor-mediated or bulk-phase endocytosis are hydrolyzed by lysosomal acid lipase within the late endosomal/lysosomal (E/L) compartment. Then, through the successive actions of Niemann-Pick C (NPC) 2 and NPC 1, unesterified cholesterol (UC) is exported from the E/L compartment to the cytosol. Mutations in either NPC1 or NPC2 lead to continuing entrapment of UC in all organs, resulting in multisystem disease, which includes hepatic dysfunction and in some cases liver failure. These studies investigated primarily whether elimination of SOAT2 in NPC1-deficient mice impacted hepatic UC sequestration, inflammation, and transaminase activities. Measurements were made in 7-wk-old mice fed a low-cholesterol chow diet or one enriched with cholesterol starting 2 wk before study. In the chow-fed mice, NPC1:SOAT2 double knockouts, compared with their littermates lacking only NPC1, had 20% less liver mass, 28% lower hepatic UC concentrations, and plasma alanine aminotransferase and aspartate aminotransferase activities that were decreased by 48% and 36%, respectively. mRNA expression levels for several markers of inflammation were all significantly lower in the NPC1 mutants lacking SOAT2. The existence of a new class of potent and selective SOAT2 inhibitors provides an opportunity for exploring if suppression of this enzyme could potentially become an adjunctive therapy for liver disease in NPC1 deficiency. NEW & NOTEWORTHY In Niemann-Pick type C1 (NPC1) disease, the entrapment of unesterified cholesterol (UC) in the endosomal/lysosomal compartment of all cells causes multiorgan disease, including neurodegeneration, pulmonary dysfunction, and liver failure. Some of this sequestered UC entered cells initially in the esterified form. When sterol O-acyltransferase 2, a cholesterol esterifying enzyme present in enterocytes and hepatocytes, is eliminated in NPC1-deficient mice, there is a reduction in their hepatomegaly, hepatic UC content, and cellular injury.

Normalization of Hepatic Homeostasis in the Npc1nmf164 Mouse Model of Niemann-Pick Type C Disease Treated with the Histone Deacetylase Inhibitor Vorinostat.

Niemann-Pick type C (NP-C) disease is a fatal genetic lipidosis for which there is no Food and Drug Administration (FDA)-approved therapy. Vorinostat, an FDA-approved inhibitor of histone deacetylases, ameliorates lysosomal lipid accumulation in cultured NP-C patient fibroblasts. To assess the therapeutic potential of histone deacetylase inhibition, we pursued these in vitro observations in two murine models of NP-C disease. Npc1nmf164 mice, which express a missense mutation in the Npc1 gene, were treated intraperitoneally, from weaning, with the maximum tolerated dose of vorinostat (150 mg/kg, 5 days/week). Disease progression was measured via gene expression, liver function and pathology, serum and tissue lipid levels, body weight, and life span. Transcriptome analyses of treated livers indicated multiple changes consistent with reversal of liver dysfunction that typifies NP-C disease. Significant improvements in liver pathology and function were achieved by this treatment regimen; however, NPC1 protein maturation and levels, disease progression, weight loss, and animal morbidity were not detectably altered. Vorinostat concentrations were >200 µm in the plasma compartment of treated animals but were almost 100-fold lower in brain tissue. Apolipoprotein B metabolism and the expression of key components of lipid homeostasis in primary hepatocytes from null (Npc1-/-) and missense (Npc1nmf164 ) mutant mice were altered by vorinostat treatment, consistent with a response by these cells independent of the status of the Npc1 locus. These results suggest that HDAC inhibitors have utility to treat visceral NP-C disease. However, it is clear that improved blood-brain barrier penetration will be required to alleviate the neurological symptoms of human NP-C disease.

Impact of physiological levels of chenodeoxycholic acid supplementation on intestinal and hepatic bile acid and cholesterol metabolism in Cyp7a1-deficient mice.

Mice deficient in cholesterol 7α-hydroxylase (Cyp7a1) have a diminished bile acid pool (BAP) and therefore represent a useful model for investigating the metabolic effects of restoring the pool with a specific BA. Previously we carried out such studies in Cyp7a1(-/-) mice fed physiological levels of cholic acid (CA) and achieved BAP restoration, along with an increased CA enrichment, at a dietary level of just 0.03% (w/w). Here we demonstrate that in Cyp7a1(-/-) mice fed chenodeoxycholic acid (CDCA) at a level of 0.06% (w/w), the BAP was restored to normal size and became substantially enriched with muricholic acid (MCA) (>70%), leaving the combined contribution of CA and CDCA to be <15%. This resulted in a partial to complete reversal of the main changes in cholesterol and BA metabolism associated with Cyp7a1 deficiency such as an elevated rate of intestinal sterol synthesis, an enhanced level of mRNA for Cyp8b1 in the liver, and depressed mRNA levels for Ibabp, Shp and Fgf15 in the distal small intestine. When Cyp7a1(-/-) and matching Cyp7a1(+/+) mice were fed a diet with added cholesterol (0.2%) (w/w), either alone, or also containing CDCA (0.06%) (w/w) or CA (0.03%) (w/w) for 18days, the hepatic total cholesterol concentrations (mg/g) in the Cyp7a1(-/-) mice were 26.9±3.7, 16.4±0.9 and 47.6±1.9, respectively, vs. 4.9±0.4, 5.0±0.7 and 6.4±1.9, respectively in the corresponding Cyp7a1(+/+) controls. These data affirm the importance of using moderate levels of dietary BA supplementation to elicit changes in hepatic cholesterol metabolism through shifts in BAP size and composition.

Hepatic entrapment of esterified cholesterol drives continual expansion of whole body sterol pool in lysosomal acid lipase-deficient mice.

Cholesteryl ester storage disease (CESD) results from loss-of-function mutations in LIPA, the gene that encodes lysosomal acid lipase (LAL). Hepatomegaly and deposition of esterified cholesterol (EC) in multiple organs ensue. The present studies quantitated rates of synthesis, absorption, and disposition of cholesterol, and whole body cholesterol pool size in a mouse model of CESD. In 50-day-old lal(-/-) and matching lal(+/+) mice fed a low-cholesterol diet, whole animal cholesterol content equalled 210 and 50 mg, respectively, indicating that since birth the lal(-/-) mice sequestered cholesterol at an average rate of 3.2 mg·day(-1)·animal(-1). The proportion of the body sterol pool contained in the liver of the lal(-/-) mice was 64 vs. 6.3% in their lal(+/+) controls. EC concentrations in the liver, spleen, small intestine, and lungs of the lal(-/-) mice were elevated 100-, 35-, 15-, and 6-fold, respectively. In the lal(-/-) mice, whole liver cholesterol synthesis increased 10.2-fold, resulting in a 3.2-fold greater rate of whole animal sterol synthesis compared with their lal(+/+) controls. The rate of cholesterol synthesis in the lal(-/-) mice exceeded that in the lal(+/+) controls by 3.7 mg·day(-1)·animal(-1). Fractional cholesterol absorption and fecal bile acid excretion were unchanged in the lal(-/-) mice, but their rate of neutral sterol excretion was 59% higher than in their lal(+/+) controls. Thus, in this model, the continual expansion of the body sterol pool is driven by the synthesis of excess cholesterol, primarily in the liver. Despite the severity of their disease, the median life span of the lal(-/-) mice was 355 days.

Histone deacetylase 9 represses cholesterol efflux and alternatively activated macrophages in atherosclerosis development.

OBJECTIVE: Recent genome-wide association studies revealed that a genetic variant in the loci corresponding to histone deacetylase 9 (HDAC9) is associated with large vessel stroke. HDAC9 expression was upregulated in human atherosclerotic plaques in different arteries. The molecular mechanisms how HDAC9 might increase atherosclerosis is not clear. APPROACH AND RESULTS: In this study, we show that systemic and bone marrow cell deletion of HDAC9 decreased atherosclerosis in LDLr(-/-) (low density lipoprotein receptor) mice with minimal effect on plasma lipid concentrations. HDAC9 deletion resulted upregulation of lipid homeostatic genes, downregulation of inflammatory genes, and polarization toward an M2 phenotype via increased accumulation of total acetylated H3 and H3K9 at the promoters of ABCA1 (ATP-binding cassette transporter), ABCG1, and PPAR-γ (peroxisome proliferator-activated receptor) in macrophages. CONCLUSIONS: We conclude that macrophage HDAC9 upregulation is atherogenic via suppression of cholesterol efflux and generation of alternatively activated macrophages in atherosclerosis.

Hearing loss is an early consequence of Npc1 gene deletion in the mouse model of Niemann-Pick disease, type C.

Niemann-Pick disease, type C1 (NPC1) is a rare lysosomal lipidosis that is most often the result of biallelic mutations in NPC1, and is characterized by a fatal neurological degeneration. The pathophysiology is complex, and the natural history of the disease is poorly understood. Recent findings from patients with NPC1 and hearing loss suggest that multiple steps along the auditory pathway are affected. The current study was undertaken to determine the auditory phenotype in the Npc1 (nih) mutant mouse model, to extend analyses to histologic evaluation of the inner ear, and to compare our findings to those reported from human patients. Auditory testing revealed a progressive high-frequency hearing loss in Npc1 (-/-) mice that is present as early as postnatal day 20 (P20), well before the onset of overt neurological symptoms, with evidence of abnormalities involving the cochlea, auditory nerve, and brainstem auditory centers. Distortion product otoacoustic emission amplitude and auditory brainstem response latency data provided evidence for a disruption in maturational development of the auditory system in Npc1 (-/-) mice. Anatomical study demonstrated accumulation of lysosomes in neurons, hair cells, and supporting cells of the inner ear in P30 Npc1 (-/-) mice, as well as increased numbers of inclusion bodies, myelin figures, and swollen nerve endings in older (P50-P70) mutant animals. These findings add unique perspective to the pathophysiology of NPC disease and suggest that hearing loss is an early and sensitive marker of disease progression.

Sustained and selective suppression of intestinal cholesterol synthesis by Ro 48-8071, an inhibitor of 2,3-oxidosqualene:lanosterol cyclase, in the BALB/c mouse.

The small intestine plays a fundamentally important role in regulating whole body cholesterol balance and plasma lipoprotein composition. This is articulated through the interplay of a constellation of genes that ultimately determines the net amount of chylomicron cholesterol delivered to the liver. Major advances in our insights into regulation of the cholesterol absorption pathway have been made using genetically manipulated mouse models and agents such as ezetimibe. One unresolved question is how a sustained pharmacological inhibition of intestinal cholesterol synthesis in vivo may affect cholesterol handling by the absorptive cells. Here we show that the lanosterol cyclase inhibitor, Ro 48-8071, when fed to BALB/c mice in a chow diet (20 mg/day/kg body weight), leads to a rapid and sustained inhibition (>50%) of cholesterol synthesis in the whole small intestine. Sterol synthesis was also reduced in the large intestine and stomach. In contrast, hepatic cholesterol synthesis, while markedly suppressed initially, rebounded to higher than baseline rates within 7 days. Whole body cholesterol synthesis, fractional cholesterol absorption, and fecal neutral and acidic sterol excretion were not consistently changed with Ro 48-8071 treatment. There were no discernible effects of this agent on intestinal histology as determined by H&E staining and the level of Ki67, an index of proliferation. The mRNA expression for multiple genes involved in intestinal cholesterol regulation including NPC1L1 was mostly unchanged although there was a marked rise in the mRNA level for the PXR target genes CYP3A11 and CES2A.

A functional, genome-wide evaluation of liposensitive yeast identifies the "ARE2 required for viability" (ARV1) gene product as a major component of eukaryotic fatty acid resistance.

The toxic subcellular accumulation of lipids predisposes several human metabolic syndromes, including obesity, type 2 diabetes, and some forms of neurodegeneration. To identify pathways that prevent lipid-induced cell death, we performed a genome-wide fatty acid sensitivity screen in Saccharomyces cerevisiae. We identified 167 yeast mutants as sensitive to 0.5 mm palmitoleate, 45% of which define pathways that were conserved in humans. 63 lesions also impacted the status of the lipid droplet; however, this was not correlated to the degree of fatty acid sensitivity. The most liposensitive yeast strain arose due to deletion of the "ARE2 required for viability" (ARV1) gene, encoding an evolutionarily conserved, potential lipid transporter that localizes to the endoplasmic reticulum membrane. Down-regulation of mammalian ARV1 in MIN6 pancreatic ß-cells or HEK293 cells resulted in decreased neutral lipid synthesis, increased fatty acid sensitivity, and lipoapoptosis. Conversely, elevated expression of human ARV1 in HEK293 cells or mouse liver significantly increased triglyceride mass and lipid droplet number. The ARV1-induced hepatic triglyceride accumulation was accompanied by up-regulation of DGAT1, a triglyceride synthesis gene, and the fatty acid transporter, CD36. Furthermore, ARV1 was identified as a transcriptional of the protein peroxisome proliferator-activated receptor α (PPARα), a key regulator of lipid homeostasis whose transcriptional targets include DGAT1 and CD36. These results implicate ARV1 as a protective factor in lipotoxic diseases due to modulation of fatty acid metabolism. In conclusion, a lipotoxicity-based genetic screen in a model microorganism has identified 75 human genes that may play key roles in neutral lipid metabolism and disease.

Role of calcium and EPAC in norepinephrine-induced ghrelin secretion.

Ghrelin is an orexigenic hormone secreted principally from a distinct population of gastric endocrine cells. Molecular mechanisms regulating ghrelin secretion are mostly unknown. Recently, norepinephrine (NE) was shown to enhance ghrelin release by binding to ß1-adrenergic receptors on ghrelin cells. Here, we use an immortalized stomach-derived ghrelin cell line to further characterize the intracellular signaling pathways involved in NE-induced ghrelin secretion, with a focus on the roles of Ca(2+) and cAMP. Several voltage-gated Ca(2+) channel (VGCC) family members were found by quantitative PCR to be expressed by ghrelin cells. Nifedipine, a selective L-type VGCC blocker, suppressed both basal and NE-stimulated ghrelin secretion. NE induced elevation of cytosolic Ca(2+) levels both in the presence and absence of extracellular Ca(2+). Ca(2+)-sensing synaptotagmins Syt7 and Syt9 were also highly expressed in ghrelin cell lines, suggesting that they too help mediate ghrelin secretion. Raising cAMP with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine also stimulated ghrelin secretion, although such a cAMP-mediated effect likely does not involve protein kinase A, given the absence of a modulatory response to a highly selective protein kinase A inhibitor. However, pharmacological inhibition of another target of cAMP, exchange protein-activated by cAMP (EPAC), did attenuate both basal and NE-induced ghrelin secretion, whereas an EPAC agonist enhanced basal ghrelin secretion. We conclude that constitutive ghrelin secretion is primarily regulated by Ca(2+) influx through L-type VGCCs and that NE stimulates ghrelin secretion predominantly through release of intracellular Ca(2+). Furthermore, cAMP and its downstream activation of EPAC are required for the normal ghrelin secretory response to NE.

The Xbp1s/GalE axis links ER stress to postprandial hepatic metabolism.

Postprandially, the liver experiences an extensive metabolic reprogramming that is required for the switch from glucose production to glucose assimilation. Upon refeeding, the unfolded protein response (UPR) is rapidly, though only transiently, activated. Activation of the UPR results in a cessation of protein translation, increased chaperone expression, and increased ER-mediated protein degradation, but it is not clear how the UPR is involved in the postprandial switch to alternate fuel sources. Activation of the inositol-requiring enzyme 1 (IRE1) branch of the UPR signaling pathway triggers expression of the transcription factor Xbp1s. Using a mouse model with liver-specific inducible Xbp1s expression, we demonstrate that Xbp1s is sufficient to provoke a metabolic switch characteristic of the postprandial state, even in the absence of caloric influx. Mechanistically, we identified UDP-galactose-4-epimerase (GalE) as a direct transcriptional target of Xbp1s and as the key mediator of this effect. Our results provide evidence that the Xbp1s/GalE pathway functions as a novel regulatory nexus connecting the UPR to the characteristic postprandial metabolic changes in hepatocytes.

Carboxylesterases are uniquely expressed among tissues and regulated by nuclear hormone receptors in the mouse.

Carboxylesterases (CES) are a well recognized, yet incompletely characterized family of proteins that catalyze neutral lipid hydrolysis. Some CES have well-defined roles in xenobiotic clearance, pharmacologic prodrug activation, and narcotic detoxification. In addition, emerging evidence suggests other CES may have roles in lipid metabolism. Humans have six CES genes, whereas mice have 20 Ces genes grouped into five isoenzyme classes. Perhaps due to the high sequence similarity shared by the mouse Ces genes, the tissue-specific distribution of expression for these enzymes has not been fully addressed. Therefore, we performed studies to provide a comprehensive tissue distribution analysis of mouse Ces mRNAs. These data demonstrated that while the mouse Ces family 1 is highly expressed in liver and family 2 in intestine, many Ces genes have a wide and unique tissue distribution defined by relative mRNA levels. Furthermore, evaluating Ces gene expression in response to pharmacologic activation of lipid- and xenobiotic-sensing nuclear hormone receptors showed differential regulation. Finally, specific shifts in Ces gene expression were seen in peritoneal macrophages following lipopolysaccharide treatment and in a steatotic liver model induced by high-fat feeding, two model systems relevant to disease. Overall these data show that each mouse Ces gene has its own distinctive tissue expression pattern and suggest that some CES may have tissue-specific roles in lipid metabolism and xenobiotic clearance.

Cyclodextrin mediates rapid changes in lipid balance in Npc1-/- mice without carrying cholesterol through the bloodstream.

An injection of 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD) to mice lacking Niemann Pick type C (NPC) protein results in delayed neurodegeneration, decreased inflammation, and prolonged lifespan. Changes in sterol balance observed in Npc1(-/-) mice 24 h after HP-ß-CD administration suggest that HP-ß-CD facilitates the release of accumulated lysosomal cholesterol, the molecular hallmark of this genetic disorder. Current studies were performed to evaluate the time course of HP-ß-CD effects. Within 3 h after HP-ß-CD injection, decreases in cholesterol synthesis rates and increases in cholesteryl ester levels were detected in tissues of Npc1(-/-) mice. The levels of RNAs for target genes of sterol-sensing transcription factors were altered by 6 h in liver, spleen, and ileum. Despite the cholesterol-binding capacity of HP-ß-CD, there was no evidence of increased cholesterol in plasma or urine of treated Npc1(-/-) mice, suggesting that HP-ß-CD does not carry sterol from the lysosome into the bloodstream for ultimate urinary excretion. Similar changes in sterol balance were observed in cultured cells from Npc1(-/-) mice using HP-ß-CD and sulfobutylether-ß-CD, a variant that can interact with sterol but not facilitate its solubilization. Taken together, our results demonstrate that HP-ß-CD works in cells of Npc1(-/-) mice by rapidly liberating lysosomal cholesterol for normal sterol processing within the cytosolic compartment.

Delineation of biochemical, molecular, and physiological changes accompanying bile acid pool size restoration in Cyp7a1(-/-) mice fed low levels of cholic acid.

Cholesterol 7α-hydroxylase (CYP7A1) is the initiating and rate-limiting enzyme in the neutral pathway that converts cholesterol to primary bile acids (BA). CYP7A1-deficient (Cyp7a1(-/-)) mice have a depleted BA pool, diminished intestinal cholesterol absorption, accelerated fecal sterol loss, and increased intestinal cholesterol synthesis. To determine the molecular and physiological effects of restoring the BA pool in this model, adult female Cyp7a1(-/-) mice and matching Cyp7a1(+/+) controls were fed diets containing cholic acid (CA) at modest levels [0.015, 0.030, and 0.060% (wt/wt)] for 15-18 days. A level of just 0.03% provided a CA intake of ~12 µmol (4.8 mg) per day per 100 g body wt and was sufficient in the Cyp7a1(-/-) mice to normalize BA pool size, fecal BA excretion, fractional cholesterol absorption, and fecal sterol excretion but caused a significant rise in the cholesterol concentration in the small intestine and liver, as well as a marked inhibition of cholesterol synthesis in these organs. In parallel with these metabolic changes, there were marked shifts in intestinal and hepatic expression levels for many target genes of the BA sensor farnesoid X receptor, as well as genes involved in cholesterol transport, especially ATP-binding cassette (ABC) transporter A1 (ABCA1) and ABCG8. In Cyp7a1(+/+) mice, this level of CA supplementation did not significantly disrupt BA or cholesterol metabolism, except for an increase in fecal BA excretion and marginal changes in mRNA expression for some BA synthetic enzymes. These findings underscore the importance of using moderate dietary BA levels in studies with animal models.

Ghrelin directly stimulates glucagon secretion from pancreatic alpha-cells.

Previous work has demonstrated that the peptide hormone ghrelin raises blood glucose. Such has been attributed to ghrelin's ability to enhance GH secretion, restrict insulin release, and/or reduce insulin sensitivity. Ghrelin's reported effects on glucagon have been inconsistent. Here, both animal- and cell-based systems were used to determine the role of glucagon in mediating ghrelin's effects on blood glucose. The tissue and cell distribution of ghrelin receptors (GHSR) was evaluated by quantitative PCR and histochemistry. Plasma glucagon levels were determined following acute acyl-ghrelin injections and in pharmacological and/or transgenic mouse models of ghrelin overexpression and GHSR deletion. Isolated mouse islets and the α-cell lines αTC1 and InR1G9 were used to evaluate ghrelin's effects on glucagon secretion and the role of calcium and ERK in this activity. GHSR mRNA was abundantly expressed in mouse islets and colocalized with glucagon in α-cells. Elevation of acyl-ghrelin acutely (after sc administration, such that physiologically relevant plasma ghrelin levels were achieved) and chronically (by slow-releasing osmotic pumps and as observed in transgenic mice harboring ghrelinomas) led to higher plasma glucagon and increased blood glucose. Conversely, genetic GHSR deletion was associated with lower plasma glucagon and reduced fasting blood glucose. Acyl-ghrelin increased glucagon secretion in a dose-dependent manner from mouse islets and α-cell lines, in a manner requiring elevation of intracellular calcium and phosphorylation of ERK. Our study shows that ghrelin's regulation of blood glucose involves direct stimulation of glucagon secretion from α-cells and introduces the ghrelin-glucagon axis as an important mechanism controlling glycemia under fasting conditions.