Oral aversion to dietary sugar, ethanol and glycerol correlates with alterations in specific hepatic metabolites in a mouse model of human citrin deficiency.

Mice carrying simultaneous homozygous mutations in the genes encoding citrin, the mitochondrial aspartate-glutamate carrier 2 (AGC2) protein, and mitochondrial glycerol-3-phosphate dehydrogenase (mGPD), are a phenotypically representative model of human citrin (a.k.a., AGC2) deficiency. In this study, we investigated the voluntary oral intake and preference for sucrose, glycerol or ethanol solutions by wild-type, citrin (Ctrn)-knockout (KO), mGPD-KO, and Ctrn/mGPD double-KO mice; all substances that are known or suspected precipitating factors in the pathogenesis of human citrin deficiency. The double-KO mice showed clear suppressed intake of sucrose, consuming less with progressively higher concentrations compared to the other mice. Similar observations were made when glycerol or ethanol were given. The preference of Ctrn-KO and mGPD-KO mice varied with the different treatments; essentially no differences were observed for sucrose, while an intermediate intake or similar to that of the double-KO mice was observed for glycerol and ethanol. We next examined the hepatic glycerol 3-phosphate, citrate, citrulline, lysine, glutamate and adenine nucleotide levels following forced enteral administration of these solutions. A strong correlation between the simultaneous increased hepatic glycerol 3-phosphate and decreased ATP or total adenine nucleotide content and observed aversion of the mice during evaluation of their voluntary preferences was found. Overall, our results suggest that the aversion observed in the double-KO mice to these solutions is initiated and/or mediated by hepatic metabolic perturbations, resulting in a behavioral response to increased hepatic cytosolic NADH and a decreased cellular adenine nucleotide pool. These findings may underlie the dietary predilections observed in human citrin deficient patients.

Metabolomic analysis reveals hepatic metabolite perturbations in citrin/mitochondrial glycerol-3-phosphate dehydrogenase double-knockout mice, a model of human citrin deficiency.

The citrin/mitochondrial glycerol-3-phosphate dehydrogenase (mGPD) double-knockout mouse displays phenotypic attributes of both neonatal intrahepatic cholestasis and adult-onset type II citrullinemia, making it a suitable model of human citrin deficiency. In the present study, we investigated metabolic disturbances in the livers of wild-type, citrin (Ctrn) knockout, mGPD knockout, and Ctrn/mGPD double-knockout mice following oral sucrose versus saline administration using metabolomic approaches. By using gas chromatography/mass spectrometry and capillary electrophoresis/mass spectrometry, we found three general groupings of metabolite changes in the livers of the double-knockout mice following sucrose administration that were subsequently confirmed using liquid chromatography/mass spectrometry or enzymatic methods: a marked increase of hepatic glycerol 3-phosphate, a generalized decrease of hepatic tricarboxylic acid cycle intermediates, and alterations of hepatic amino acid levels related to the urea cycle or lysine catabolism including marked increases in citrulline and lysine. Furthermore, concurrent oral administration of sodium pyruvate with sucrose ameliorated the hyperammonemia induced by sucrose, as had been shown previously, as well as almost completely normalizing the hepatic metabolite perturbations found. Overall, we have identified additional metabolic disturbances in double-KO mice following oral sucrose administration, and provided further evidence for the therapeutic use of sodium pyruvate in our mouse model of citrin deficiency.

Decreased insulin secretion and accumulation of triglyceride in beta cells overexpressing a dominant-negative form of AMP-activated protein kinase.

Adenosine 5' -monophosphate-activated protein kinase (AMPK) has been implicated in the regulation of energy metabolism, although its role in the pancreatic beta cells remains unclear. In the present, we have overexpressed a dominant negative form of AMPKalpha1 subunit (Asp57Ala) tagged with c-myc epitope (AMPKalpha1-DN) in INS-1D cells with an adenoviral vector. After 48 h of adenoviral infection, overexpression of AMPKalpha1-DN in INS-1D cells was confirmed by Western blot analysis with anti-c-myc antibody. Phosphorylation of the Thr172 in AMPKalpha1/alpha2 subunit was progressively decreased in parallel with increasing number of adenoviral titers. Glucose-stimulated insulin secretion in response to 30 mmol/L glucose was decreased in INS-1D cells overexpressing AMPKalpha1-DN as compared to control cells infected with adeno- LacZ vector. Neither cellular insulin content nor insulin mRNA level was changed between the two groups. Phosphorylation of acetyl-CoA carboxylase (ACC), a down-stream substrate of AMPK, was decreased, indicating that ACC activity was increased, due to the decreased AMPK activity. In fact, intracellular triglyceride content was increased as compared to control cells. The beta-oxidation of palmitate was decreased at 30 mmol/L glucose. Insulin secretion in response to potassium chloride or glibenclamide was also decreased as compared to control cells. In conclusion, suppression of AMPK activity in beta-cells inhibited insulin secretion in response to glucose, potassium chloride or glibenclamide without altering insulin content. Accumulation of triglyceride subsequent to the activation of ACC by suppression of AMPK activity, was suggested to be, at least in part, responsible for the impaired insulin secretion through so-called lipotoxicity mechanism.

A role for suppressed incisor cuspal morphogenesis in the evolution of mammalian heterodont dentition.

Changes in tooth shape have played a major role in vertebrate evolution with modification of dentition allowing an organism to adapt to new feeding strategies. The current view is that molar teeth evolved from simple conical teeth, similar to canines, by progressive addition of extra "cones" to form progressively complex multicuspid crowns. Mammalian incisors, however, are neither conical nor multicuspid, and their evolution is unclear. We show that hypomorphic mutation of a cell surface receptor, Lrp4, which modulates multiple signaling pathways, produces incisors with grooved enamel surfaces that exhibit the same molecular characteristics as the tips of molar cusps. Mice with a null mutation of Lrp4 develop extra cusps on molars and have incisors that exhibit clear molar-like cusp and root morphologies. Molecular analysis identifies misregulation of Shh and Bmp signaling in the mutant incisors and suggests an uncoupling of the processes of tooth shape determination and morphogenesis. Incisors thus possess a developmentally suppressed, cuspid crown-like morphogenesis program similar to that in molars that is revealed by loss of Lrp4 activity. Several mammalian species naturally possess multicuspid incisors, suggesting that mammals have the capacity to form multicuspid teeth regardless of location in the oral jaw. Localized loss of enamel may thus have been an intermediary step in the evolution of cusps, both of which use Lrp4-mediated signaling.

A novel method of mouse ex utero transplantation of hepatic progenitor cells into the fetal liver.

Avoiding the limitations of the adult liver niche, transplantation of hepatic stem/progenitor cells into fetal liver is desirable to analyze immature cells in a hepatic developmental environment. Here, we established a new monitor tool for cell fate of hepatic progenitor cells transplanted into the mouse fetal liver by using ex utero surgery. When embryonic day (ED) 14.5 hepatoblasts were injected into the ED14.5 fetal liver, the transplanted cells expressed albumin abundantly or alpha-fetoprotein weakly, and contained glycogen in the neonatal liver, indicating that transplanted hepatoblasts can proliferate and differentiate in concord with surrounding recipient parenchymal cells. The transplanted cells became mature in the liver of 6-week-old mice. Furthermore, this method was applicable to transplantation of hepatoblast-like cells derived from mouse embryonic stem cells. These data indicate that this unique technique will provide a new in vivo experimental system for studying cell fate of hepatic stem/progenitor cells and liver organogenesis.

Citrin/mitochondrial glycerol-3-phosphate dehydrogenase double knock-out mice recapitulate features of human citrin deficiency.

Citrin is the liver-type mitochondrial aspartate-glutamate carrier that participates in urea, protein, and nucleotide biosynthetic pathways by supplying aspartate from mitochondria to the cytosol. Citrin also plays a role in transporting cytosolic NADH reducing equivalents into mitochondria as a component of the malate-aspartate shuttle. In humans, loss-of-function mutations in the SLC25A13 gene encoding citrin cause both adult-onset type II citrullinemia and neonatal intrahepatic cholestasis, collectively referred to as human citrin deficiency. Citrin knock-out mice fail to display features of human citrin deficiency. Based on the hypothesis that an enhanced glycerol phosphate shuttle activity may be compensating for the loss of citrin function in the mouse, we have generated mice with a combined disruption of the genes for citrin and mitochondrial glycerol 3-phosphate dehydrogenase. The resulting double knock-out mice demonstrated citrullinemia, hyperammonemia that was further elevated by oral sucrose administration, hypoglycemia, and a fatty liver, all features of human citrin deficiency. An increased hepatic lactate/pyruvate ratio in the double knock-out mice compared with controls was also further elevated by the oral sucrose administration, suggesting that an altered cytosolic NADH/NAD(+) ratio is closely associated with the hyperammonemia observed. Microarray analyses identified over 100 genes that were differentially expressed in the double knock-out mice compared with wild-type controls, revealing genes potentially involved in compensatory or downstream effects of the combined mutations. Together, our data indicate that the more severe phenotype present in the citrin/mitochondrial glycerol-3-phosphate dehydrogenase double knock-out mice represents a more accurate model of human citrin deficiency than citrin knock-out mice.

Cytoplasmic sequestration of cyclin D1 associated with cell cycle withdrawal of neuroblastoma cells.

The regulation of D-type cyclin-dependent kinase activity is critical for neuronal differentiation and apoptosis. We recently showed that cyclin D1 is sequestered in the cytoplasm and that its nuclear localization induces apoptosis in postmitotic primary neurons. Here, we further investigated the role of the subcellular localization of cyclin D1 in cell cycle withdrawal during the differentiation of N1E-115 neuroblastoma cells. We show that cyclin D1 became predominantly cytoplasmic after differentiation. Targeting cyclin D1 expression to the nucleus induced phosphorylation of Rb and cdk2 kinase activity. Furthermore, cyclin D1 nuclear localization promoted differentiated N1E-115 cells to reenter the cell cycle, a process that was inhibited by p16(INK4a), a specific inhibitor of D-type cyclin activity. These results indicate that cytoplasmic sequestration of cyclin D1 plays a role in neuronal cell cycle withdrawal, and suggests that the abrogation of machinery involved in monitoring aberrant nuclear cyclin D1 activity contributes to neuronal tumorigenesis.

Twist is required for establishment of the mouse coronal suture.

Cranial sutures are the growth centres of the skull, enabling expansion of the skull to accommodate rapid growth of the brain. Haploinsufficiency of the human TWIST gene function causes the craniosynostosis syndrome, Saethre-Chotzen syndrome (SCS), in which premature fusion of the coronal suture is a characteristic feature. Previous studies have indicated that Twist is expressed in the coronal suture during development, and therefore that it may play an important role in development and maintenance of the suture. The Twist-null mouse is lethal before the onset of osteogenesis, and the heterozygote exhibits coronal suture synostosis postnatally. In this study we investigated the function of Twist in the development of the mouse coronal suture, by inhibiting Twist synthesis using morpholino antisense oligonucleotides in calvarial organ culture. Decreased Twist production resulted in a narrow sutural space and fusion of bone domains within 48 h after the addition of the morpholino oligonucleotides. Proliferation activity in the sutural cells was decreased, and the expression of osteogenic marker genes such as Runx2 and Fgfr2 was up-regulated in the developing bone domain within 4 h. These results suggest that during establishment of the suture area, Twist is required for the regulation of sutural cell proliferation and osteoblast differentiation.

Dynamic interaction of p220(NPAT) and CBP/p300 promotes S-phase entry.

Cajal bodies contain cyclin E/cdk2 and the substrate p220(NPAT) to regulate the transcription of histones, which is essential for cell proliferation, however, recent mouse knockout studies indicate that cyclin E and cdk2 are dispensable for these events. Because the CBP/p300 histone acetyltransferase are also known to be involved in cell proliferation, we examined the molecular and functional interactions of p220(NPAT) with the CBP/p300 at the G1/S boundary as cell cycle regulators. The subnuclear localization of p220(NPAT) and CBP/p300 proteins showed that their foci partially overlapped in a cell cycle dependent manner. Overexpression of p220(NPAT) and CBP/p300 cooperatively enhanced G1/S transition and DNA synthesis even without cdk2 phosphorylation site. Finally, molecular alignment analysis indicated that p220(NPAT) contains several potential substrate sites for CBP/p300. Overall, our findings demonstrate that p220(NPAT) and CBP/p300 form a transient complex at the G1/S boundary to play cooperative roles to promote the S-phase entry.

In situ tissue engineering of periodontal tissues by seeding with periodontal ligament-derived cells.

The feasibility of an in situ tissue-engineering method employing cell-based therapy with autologous periodontal ligament-derived cells was investigated. Periodontal ligament cells were obtained from six beagle dogs. Periodontal fenestration defects (6 x 4 mm) were created bilaterally at a location 6 mm apical to the marginal alveolar crest in the maxillary canines. Alkaline phosphatase-positive periodontal ligament cells (3 x 10(5) cells) were seeded onto a collagen sponge scaffold just before implantation. One defect was filled with the cell-scaffold construct, and another was left empty as the control. All animals were killed 4 weeks after surgery, and specimens were evaluated histomorphometrically. All the histomorphometrical data were analyzed by three-way analysis of variance with the Bonferroni multiple comparisons test. Regeneration of apical tissue was faster than that of coronal and isolated tissues on the control side (apical > coronal > isolated; p < 0.0001). On the other hand, on the cell-seeded side, regeneration of the cementum was observed uniformly on the root surface. Our data suggest that the seeded cells induced cementum regeneration on the root surface, indicating the potential of in situ tissue engineering using autologous cells for the regeneration of periodontal tissues.

Role of uncoupling protein-2 up-regulation and triglyceride accumulation in impaired glucose-stimulated insulin secretion in a beta-cell lipotoxicity model overexpressing sterol regulatory element-binding protein-1c.

Triglyceride (TG) accumulation in pancreatic beta-cells is thought to be associated with impaired insulin secretory response to glucose (lipotoxicity). To better understand the mechanism of the impaired insulin secretory response to glucose in beta-cell lipotoxicity, we overexpressed a constitutively active form of the sterol regulatory element-binding protein- 1c (SREBP-1c), a master transcriptional factor of lipogenesis, in INS-1 cells with an adenoviral vector. This treatment was associated with strong activation of transcription of the genes involved in fatty acid biosynthesis, increased cellular TG content, severely blunted glucose-stimulated insulin secretion, and enhanced expression of the uncoupling protein-2 (UCP-2), which supposedly dissipates the mitochondrial electrochemical potential. To decrease the up-regulated UCP-2 expression, small interfering RNA for UCP-2 was used. Introduction of the small interfering RNA increased the ATP/ADP ratio and partially rescued the glucose-stimulated insulin secretion in the cells overexpressing SREBP-1c, but did not affect the cellular TG content. Next, the effect of the AMP-activated protein kinase (AMPK) agonist, 5-amino-4-imidazolecarboxamide riboside, was examined in the lipotoxicity model. Exposure of the cells with lipotoxicity to 5-amino-4-imidazolecarboxamide riboside increased free fatty acid oxidation, partially reversed the TG accumulation, phosphorylated AMPK and acetyl-coenzyme A carboxylase, and improved the impaired glucose-stimulated insulin secretion. These results suggest that UCP-2 down-regulation and AMPK activation could be candidate targets for releasing beta-cells from lipotoxicity.

Role of cyclin D1 cytoplasmic sequestration in the survival of postmitotic neurons.

Cyclin D-dependent kinases phosphorylate the retinoblastoma (Rb) protein and play a critical role in neuronal cell cycle control and apoptosis. Here we show that cyclin D1 became predominantly cytoplasmic as primary cortical progenitor cells underwent cell cycle withdrawal and terminal differentiation. Furthermore, ectopically expressed cyclin D1 sequestered in the cytoplasm of postmitotic neurons, whereas it efficiently entered the nucleus of proliferating progenitor cells. Cytoplasmic cyclin D1 were complexed with cyclin-dependent kinase 4 (CDK4), and also with CDK inhibitors, p27(Kip)(I) or p21(Cip)(I), which positively regulate assembly and nuclear accumulation of the cyclin D1-CDK4 complex. Although overexpression of p21(Cip)(I) promoted cyclin D1 nuclear localization, inhibition of either glycogen synthase kinase 3beta- or CRM1-mediated cyclin D1 nuclear export did not, suggesting that the inhibition of its nuclear import, rather than the acceleration of nuclear export, contributes to cytoplasmic sequestration of cyclin D1 in postmitotic neurons. In differentiated progenitor cells, nuclear localization of ectopic cyclin D1 induced apoptosis, and the DNA-damaging compound camptothecin caused nuclear accumulation of endogenous cyclin D1, accompanied by Rb phosphorylation. These results indicate that nuclear accumulation of cyclin D1 is inhibited in postmitotic neurons and suggest a role of its subcellular localization in neuronal death and survival.

Distinct recruitment of E2F family members to specific E2F-binding sites mediates activation and repression of the E2F1 promoter.

The activity of E2F transcription factors plays a crucial role in mammalian cell-cycle progression and is controlled by physical association with the pocket proteins (pRb and its related p107 and p130). The E2F1 promoter, which contains two overlapping E2F-binding sites, is activated at the G1/S transition in an E2F-dependent manner. Mutational experiments have shown that the distal E2F-binding site on the E2F1 promoter is required for transcriptional repression in the G0 phase, whereas the proximal E2F-binding site contributes to transcriptional activation at the G1/S boundary. Consistent with these results, chromatin immunoprecipitation assays have revealed that the E2F4/p130 repressor complex specifically binds to the distal E2F-binding site, whereas E2F1 and E2F3 activators preferentially bind to the proximal E2F-binding site. The assays also showed that the specific binding of E2F4/p130 complex to the distal site was dramatically impaired by a mutation introduced into the contiguous repression site (cell Cycle gene Homology Region; CHR). Taken together, these findings indicate that the two E2F-binding sites play distinct roles in the regulation of E2F1 transcription by interacting with different sets of E2F members and cooperating with the contiguous repressor element.

Exposure of neonatal rats to diethylstilbestrol affects the expression of genes involved in ovarian differentiation.

Exposing neonatal rats with the synthetic estrogen, diethylstilbestrol (DES), induces morphological and functional abnormalities in the adult ovary. We examined the events that lead to this condition using female rats that were exposed to DES for the first five days after birth. The expression of steroidogenic acute regulatory protein (StAR) and cholesterol side-chain cleavage cytochrome P450 (P450scc), which are both required for steroidogenesis in the theca/interstitial region was markedly reduced. The expression of Mullerian inhibiting substance (MIS) was transiently increased in small growing follicles in the ovary of DES-treated rats at postnatal day 7 (P7), and the expression profile in the ovary differed between DES- and vehicle oil-treated rats at P14 and P21. The expression of the transcription factor, steroidogenic factor-1 (SF-1), reduced in theca/interstitial cells, but increased in granulosa cells of primary follicles. These results indicate that altered steroidogenesis and MIS production are mechanisms through which DES induces abnormal ovarian development, and support the notion that androgens and MIS are both critical factors in regulating early ovarian differentiation.

Disappearance of epidermal growth factor receptor is essential in the fusion of the nasal epithelium.

Abstract Epidermal growth factor (EGF) and receptor (-R) signaling pathway is required for epithelial cell growth and differentiation such as the degeneration of the medial edge epithelial cells during the fusion process of secondary palate formation. As epithelial fusion takes place during primary palate formation, we investigated the involvement of the EGF-R in fusion of the medial (MNP) and lateral (LNP) nasal prominences of the mouse embryo was examined. Immunoreactivity of EGF-R was investigated in embryonic day 10 embryos (32-37 somite stages). The EGF-R immunoreactivity was observed in the nasal epithelia of the presumptive fusion area before fusion. It became undetectable just prior to the fusion and faintly reappeared at the time of the fusion. In contrast, the non-fusing epithelial cells of the nasal groove maintained the immunoreactivity throughout these stages. In order to elucidate whether the EGF/EGF-R signaling pathway was involved in nasal epithelial fusion, EGF solution was injected into the exocoelum of explanted mouse embryos, and the embryos were cultured for 18-24 h by whole embryo culture (WEC). This exogenous EGF inhibited fusion of nasal prominences in 66.7-81.5% of the embryos. Treatment with EGF for 4-14 h showed that exogenous EGF disturbed the EGF-R disappearance and normal alteration of epithelial cell morphology in the fusion area. These results suggest that temporal disappearance of the EGF/EGF-R signaling from presumptive fusion of the nasal prominences is required for morphological change of the epithelial cells leading to the fusion of MNP and LNP.

Glucose metabolism and glutamate analog acutely alkalinize pH of insulin secretory vesicles of pancreatic beta-cells.

We studied acute changes of secretory vesicle pH in pancreatic beta-cells with a fluorescent pH indicator, lysosensor green DND-189. Fluorescence was decreased by 0.66 +/- 0.10% at 149 +/- 16 s with 22.2 mM glucose stimulation, indicating that vesicular pH was alkalinized by approximately 0.016 unit. Glucose-responsive pH increase was observed when cytosolic Ca2+ influx was blocked but disappeared when an inhibitor of glycolysis or mitochondrial ATP synthase was present. Glutamate dimethyl ester (GME), a plasma membrane-permeable analog of glutamate, potentiated glucose-stimulated insulin secretion at 5 mM without changing cellular ATP content or cytosolic Ca2+ concentration ([Ca2+]). Application of GME at basal glucose concentration decreased DND-189 fluorescence by 0.83 +/- 0.19% at 38 +/- 2 s. These results indicated that the acutely alkalinizing effect of glucose on beta-cell secretory vesicle pH was dependent on glucose metabolism but independent of modulations of cytosolic [Ca2+]. Moreover, glutamate derived from glucose may be one of the mediators of this alkalinizing effect of glucose, which may have potential relevance to the alteration of secretory function by glutamate.

Globular adiponectin protected ob/ob mice from diabetes and ApoE-deficient mice from atherosclerosis.

The adipocyte-derived hormone adiponectin has been shown to play important roles in the regulation of energy homeostasis and insulin sensitivity. In this study, we analyzed globular domain adiponectin (gAd) transgenic (Tg) mice crossed with leptin-deficient ob/ob or apoE-deficient mice. Interestingly, despite an unexpected similar body weight, gAd Tg ob/ob mice showed amelioration of insulin resistance and beta-cell degranulation as well as diabetes, indicating that globular adiponectin and leptin appeared to have both distinct and overlapping functions. Amelioration of diabetes and insulin resistance was associated with increased expression of molecules involved in fatty acid oxidation such as acyl-CoA oxidase, and molecules involved in energy dissipation such as uncoupling proteins 2 and 3 and increased fatty acid oxidation in skeletal muscle of gAd Tg ob/ob mice. Moreover, despite similar plasma glucose and lipid levels on an apoE-deficient background, gAd Tg apoE-deficient mice showed amelioration of atherosclerosis, which was associated with decreased expression of class A scavenger receptor and tumor necrosis factor alpha. This is the first demonstration that globular adiponectin can protect against atherosclerosis in vivo. In conclusion, replenishment of globular adiponectin may provide a novel treatment modality for both type 2 diabetes and atherosclerosis.

Mouse GLI3 regulates Fgf8 expression and apoptosis in the developing neural tube, face, and limb bud.

The zinc finger transcription factor GLI3 is considered a repressor of vertebrate Hedgehog (Hh) signaling. In humans, the absence of GLI3 function causes Greig cephalopolysyndactyly syndrome, affecting the development of the brain, eye, face, and limb. Because the etiology of these malformations is not well understood, we examined the phenotype of mouse Gli3-/- mutants as a model to investigate this. We observed an up-regulation of Fgf8 in the anterior neural ridge, isthmus, eye, facial primordia, and limb buds of mutant embryos, sites coinciding with the human disease. Intriguingly, endogenous apoptosis was reduced in Fgf8-positive areas in Gli3-/- mutants. Since SHH is thought to be involved in Fgf8 regulation, we compared Fgf8 expression in Shh-/- and Gli3-/-;Shh-/- mutant embryos. Whereas Fgf8 expression was almost absent in Shh-/- mutants, it was up-regulated in Gli3-/-;Shh-/- double mutants, suggesting that SHH is not required for Fgf8 induction, and that GLI3 normally represses Fgf8 independently of SHH. In the limb bud, we provide evidence that ectopic expression of Gremlin in Gli3-/- mutants might contribute to a decrease in apoptosis. Together, our data reveal that GLI3 limits Fgf8-expression domains in multiple tissues, through a mechanism that may include the induction or maintenance of apoptosis.

Role of mitochondrial NADH shuttle system in acute amylase secretion by acetylcholine from mouse pancreatic acinar cells.

Using the mice that lack mitochondrial glycerol-3-phosphate dehydrogenase (mGPDH), a rate limiting enzyme of the glycerol-phosphate NADH shuttle, we investigated the role of the NADH shuttle system in amylase secretion in response to acetylcholine (ACh) in pancreatic acinar cells. The pancreatic acinar cells of mGPDH-deficient mice were not different in histology and immunohistochemistry from those of wild-type mice. In both types of pancreatic acinar cells from wild-type and mGPDH-deficient mice, ACh similarly potentiated amylase secretion, measured in 30 minutes after the ACh stimulation. A 30 minutes pre-treatment of wild-type cells with aminooxyacetate (AOA), an inhibitor of aspartate aminotransferases of the malate-aspartate NADH shuttle, did not change the rate of ACh-induced amylase secretion, measured in the following 30 minutes. In also mGPDH-deficient cells treated with AOA, thus in this situation all mitochondrial NADH shuttles being dysfunctioning, ACh induced amylase release in a similar amount to that in AOA-untreated cells. The basal levels of intracellular Ca2+ concentration ([Ca2+]i), the ACh-stimulated levels of [Ca2+]i and Ca2+ oscillation patterns in response to ACh were similar in wild-type and mGPDH-deficient cells, and the AOA-treatment did not affect these [Ca2+]i responses. The levels of intracellular concentration of ATP before and during stimulation with ACh were similar in wild-type and mGPDH-defficient cells. In only AOA-treated mGPDH-deficient cells, the level of ATP decreased after the ACh stimulation. These results suggest that acute response of amylase secretion to ACh from mouse pancreatic acinar cells does not require simultaneous functioning of the mitochondrial NADH shuttle system, although the supply of intracellular ATP decreases during the ACh stimulation.