Pivotal role of inter-organ aspartate metabolism for treatment of mitochondrial aspartate-glutamate carrier 2 (citrin) deficiency, based on the mouse model.

Previous studies using citrin/mitochondrial glycerol-3-phosphate (G3P) dehydrogenase (mGPD) double-knockout mice have demonstrated that increased dietary protein reduces the extent of carbohydrate-induced hyperammonemia observed in these mice. This study aimed to further elucidate the mechanisms of this effect. Specific amino acids were initially found to decrease hepatic G3P, or increase aspartate or citrulline levels, in mGPD-knockout mice administered ethanol. Unexpectedly, oral glycine increased ammonia in addition to lowering G3P and increasing citrulline. Subsequently, simultaneous glycine-plus-sucrose (Gly + Suc) administration led to a more severe hyperammonemic state in double-KO mice compared to sucrose alone. Oral arginine, ornithine, aspartate, alanine, glutamate and medium-chain triglycerides all lowered blood ammonia following Gly + Suc administration, with combinations of ornithine-plus-aspartate (Orn + Asp) or ornithine-plus-alanine (Orn + Ala) suppressing levels similar to wild-type. Liver perfusion and portal vein-arterial amino acid differences suggest that oral aspartate, similar to alanine, likely activated ureagenesis from ammonia and lowered the cytosolic NADH/NAD+ ratio through conversion to alanine in the small intestine. In conclusion, Gly + Suc administration induces a more severe hyperammonemic state in double-KO mice that Orn + Asp or Orn + Ala both effectively suppress. Aspartate-to-alanine conversion in the small intestine allows for effective oral administration of either, demonstrating a pivotal role of inter-organ aspartate metabolism for the treatment of citrin deficiency.

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.

Evaluation of the efficacy and safety of lixisenatide add-on treatment to basal insulin therapy among T2DM patients with different body mass indices from GetGoal trials.

BACKGROUND: Using patient data from the GetGoal-Duo1, -L, and L-Asia trials, the objectives of this study were to evaluate and compare the impact of lixisenatide once-daily add-on treatment to basal insulin therapy ±oral antidiabetic drugs (OADs) among type 2 diabetes (T2DM) patients subdivided into groups, based on their baseline body mass indices (BMI). METHODS: Data of patients treated with lixisenatide were extracted from the modified intent-to-treat populations of the trials. Patients were subdivided into 4 groups based on baseline BMI category (BMIs <25, 25-<30, 30-<35, and ≥35 kg/m(2)). At the unadjusted data level, efficacy and safety endpoints were evaluated and compared among study cohorts. Additionally, multivariable regression analyses were used to specify key patient characteristics and then assess the adjusted outcomes. RESULTS: Of the 662 T2DM patients, the mean changes in HbA1c (-0.63 to -0.73 %, p = 0.88) and FPG levels (-3.9 to 3.2 mg/dL, p = 0.60) were not significantly different among the different BMI groups. The proportions of T2DM patients that achieved HbA1c <7 % ranged between 34.7 and 46.8 %. After adjusted for patient characteristics, T2DM patients in the lowest BMI group relative to those in the highest BMI group had a smaller reduction in HbA1c during the trial periods (difference: 0.32 %, confidence interval: 0.10, 0.53, p = 0.005) and were less likely to achieve HbA1c <7 %. CONCLUSIONS: The findings of this analysis of the GetGoal clinical trials suggest that lixisenatide may be a good treatment option for optimizing glycemic control in patients unable to achieve their HbA1c target on basal insulin therapy ±OADs, regardless of BMI category.

Mechanism for increased hepatic glycerol synthesis in the citrin/mitochondrial glycerol-3-phosphate dehydrogenase double-knockout mouse: Urine glycerol and glycerol 3-phosphate as potential diagnostic markers of human citrin deficiency.

The mitochondrial aspartate-glutamate carrier isoform 2 (citrin) and mitochondrial glycerol-3-phosphate dehydrogenase (mGPD) double-knockout mouse has been a useful model of human citrin deficiency. One of the most prominent findings has been markedly increased hepatic glycerol 3-phosphate (G3P) following oral administration of a sucrose solution. We aimed to investigate whether this change is detectable outside of the liver, and to explore the mechanism underlying the increased hepatic G3P in these mice. We measured G3P and its metabolite glycerol in plasma and urine of the mice under various conditions. Glycerol synthesis from fructose was also studied using the liver perfusion system. The citrin/mGPD double-knockout mice showed increased urine G3P and glycerol under normal, fed conditions. We also found increased plasma glycerol under fasted conditions, while oral administration of different carbohydrates or ethanol led to substantially increased plasma glycerol. Fructose infusion to the perfused liver of the double-knockout mice augmented hepatic glycerol synthesis, and was accompanied by a concomitant increase in the lactate/pyruvate (L/P) ratio. Co-infusion of either pyruvate or phenazine methosulfate, a cytosolic oxidant, with fructose corrected the high L/P ratio, leading to reduced glycerol synthesis. Overall, these findings suggest that hepatic glycerol synthesis is cytosolic NADH/NAD(+) ratio-dependent and reveal a likely regulatory mechanism for hepatic glycerol synthesis following a high carbohydrate load in citrin-deficient patients. Therefore, urine G3P and glycerol may represent potential diagnostic markers for human citrin deficiency.

[Glucagon-like peptide-1 receptor agonists].

Recently, the number of diabetic patients with obesity has increased by changes in life-style including food and physical exercise. Appearance of incretin-related drugs has given us more options for treating type 2 diabetes, and they are evaluated in regard to realizing appropriately controlled glycemic status. One of incretin-related drugs, glucagon-like peptide-1 receptor agonist (GLP-1RA), possesses pleiotropic actions to pancreatic ß/α cells and other targets, and is highly expected from the clinical aspect. Specifically, the long-acting GLP-1RAs lower fasting glucose levels, and the short-acting GLP-1RAs lower post-prandial glucose levels. By optimally employing these drugs, better glycemic management should be enabled.

One-year real-life efficacy of sitagliptin revealed importance of concomitant pioglitazone use in Japanese patients with type 2 diabetes mellitus.

AIMS: Dipeptidyl-peptidase 4 inhibitors have become one of the most popular antidiabetic drugs. However, what kind of combinations with other drugs were advantageous was not known. Here, we tried to elucidate it in a real-life clinical setting. METHODS: We retrospectively studied efficacies of sitagliptin in 87 Japanese patients with type 2 diabetes mellitus for 52 weeks. We divided subjects into excellent, effective and unresponsive subgroups according to glycemic responses to sitagliptin. RESULTS: In the excellent and effective groups the minimum HbA1c values were attained at 16 weeks while HbA1c levels in the unresponsive group kept increasing during the study period. There was a significant difference in the baseline HbA1c values between the excellent and unresponsive groups (p=0.02). Interestingly, the mean doses of pioglitazone were highest in the excellent group and lowest in the unresponsive group (p=0.02). When we compared the effective and unresponsive groups, the mean doses of sulfonylureas were constantly higher in the effective group than in the unresponsive group (p=0.05). CONCLUSIONS: These data suggest that the baseline HbA1c value can be a factor that predicts the extent of HbA1c reduction and reveal a possibility that the concomitant use of pioglitazone augments glycemic responsiveness to sitagliptin.

Predominant microvessel proliferation in coronary stent restenotic tissue in patients with diabetes: insights from optical coherence tomography image analysis.

BACKGROUND: Coronary optical coherence tomography (OCT) enables virtual depiction of histological findings of in-stent restenotic tissue. The aim of this study was to investigate the microvessel proliferation within in-stent restenotic tissue and the influence of diabetes mellitus (DM). METHODS: We examined 54 in-stent restenotic coronary artery lesions (stenotic area>50%) from 50 consecutive patients including 28 with DM (56%) and 9 insulin-treated DM patients (18%); who underwent coronary time-domain OCT imaging with automatic pull back (1mm/s, 20 frames/s). Microvessels were defined as low-signal cavities with a diameter of 50-150 microns and a trajectory parallel to the lumen recognized on 3 consecutive cross-sectional OCT image frames. The microvessel index was calculated as the number of frames with microvessel/total number of frames × 100. Patients were stratified into 3 groups: 1) without microvessels, 2) with a low (< median value) microvessel index, 3) with a high microvessel index. RESULTS: Microvessels were detected in 566 frames (3.1%) from 26 lesions (48%) in 24 patients (48%). A greater incidence of DM and higher serum glucose levels were observed in the high microvessel index group (DM: 42% vs 58% vs 83%, p=0.049; serum glucose level: 118.2 ± 44.6 vs 122.6 ± 31.0 vs 172.8 ± 63.1mg/dL, p=0.03 between low and high microvessel index group, p=0.005 between no microvessel and high microvessel index group). CONCLUSIONS: Microvessel formation may be a unique pathophysiological factor of in-stent restenoses in patients with DM.

Effects of supplementation on food intake, body weight and hepatic metabolites in the citrin/mitochondrial glycerol-3-phosphate dehydrogenase double-knockout mouse model of human citrin deficiency.

The C57BL/6:Slc23a13(-/-);Gpd2(-/-) double-knockout (a.k.a., citrin/mitochondrial glycerol 3-phosphate dehydrogenase double knockout or Ctrn/mGPD-KO) mouse displays phenotypic attributes of both neonatal intrahepatic cholestasis (NICCD) and adult-onset type II citrullinemia (CTLN2), making it a suitable model of human citrin deficiency. In the present study, we show that when mature Ctrn/mGPD-KO mice are switched from a standard chow diet (CE-2) to a purified maintenance diet (AIN-93M), this resulted in a significant loss of body weight as a result of reduced food intake compared to littermate mGPD-KO mice. However, supplementation of the purified maintenance diet with additional protein (from 14% to 22%; and concomitant reduction or corn starch), or with specific supplementation with alanine, sodium glutamate, sodium pyruvate or medium-chain triglycerides (MCT), led to increased food intake and body weight gain near or back to that on chow diet. No such effect was observed when supplementing the diet with other sources of fat that contain long-chain fatty acids. Furthermore, when these supplements were added to a sucrose solution administered enterally to the mice, which has been shown previously to lead to elevated blood ammonia as well as altered hepatic metabolite levels in Ctrn/mGPP-KO mice, this led to metabolic correction. The elevated hepatic glycerol 3-phosphate and citrulline levels after sucrose administration were suppressed by the administration of sodium pyruvate, alanine, sodium glutamate and MCT, although the effect of MCT was relatively small. Low hepatic citrate and increased lysine levels were only found to be corrected by sodium pyruvate, while alanine and sodium glutamate both corrected hepatic glutamate and aspartate levels. Overall, these results suggest that dietary factors including increased protein content, supplementation of specific amino acids like alanine and sodium glutamate, as well as sodium pyruvate and MCT all show beneficial effects on citrin deficiency by increasing the carbohydrate tolerance of Ctrn/mGPD-KO mice, as observed through increased food intake and maintenance of body weight.

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.

Distinct association of serum FGF21 or adiponectin levels with clinical parameters in patients with type 2 diabetes.

Fibroblast growth factor 21 (FGF21) has been identified as a novel metabolic regulator. This cross-sectional study was performed to clarify how serum FGF21 levels were associated with clinical parameters in Japanese subjects with type 2 diabetes (n=139). Anthropometric and blood biochemical parameters, uses of drugs for diabetes, hypertension and dyslipidemia were examined regarding associations with fasting serum FGF21 concentrations. FGF21 levels were 6-times higher in those subjects taking fibrates. However, a use of thiazolidinediones did not affect serum FGF21 levels while it induced higher serum adiponectin levels. In univariate analyses, FGF21 levels showed associations with a use of fibrates, triglyceride levels, creatinine levels, waist circumference, and BMI. Multiple regression analyses adjusted for age, gender and BMI showed that a use of fibrates, triglyceride levels and creatinine levels were strong contributors to serum FGF21 levels. In contrast, a use of thiazolidinediones, HDL-cholesterol levels and fasting insulin levels were strong contributors to serum adiponectin levels. This study revealed that serum FGF21 levels were biochemical indicators correlating to a set of essential metabolic parameters, which was distinct from that correlating to serum adiponectin levels in subjects with type 2 diabetes.

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.

Combined in silico and in vivo analyses reveal role of Hes1 in taste cell differentiation.

The sense of taste is of critical importance to animal survival. Although studies of taste signal transduction mechanisms have provided detailed information regarding taste receptor calcium signaling molecules (TRCSMs, required for sweet/bitter/umami taste signal transduction), the ontogeny of taste cells is still largely unknown. We used a novel approach to investigate the molecular regulation of taste system development in mice by combining in silico and in vivo analyses. After discovering that TRCSMs colocalized within developing circumvallate papillae (CVP), we used computational analysis of the upstream regulatory regions of TRCSMs to investigate the possibility of a common regulatory network for TRCSM transcription. Based on this analysis, we identified Hes1 as a likely common regulatory factor, and examined its function in vivo. Expression profile analyses revealed that decreased expression of nuclear HES1 correlated with expression of type II taste cell markers. After stage E18, the CVP of Hes1(-/) (-) mutants displayed over 5-fold more TRCSM-immunoreactive cells than did the CVP of their wild-type littermates. Thus, according to our composite analyses, Hes1 is likely to play a role in orchestrating taste cell differentiation in developing taste buds.

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.

Induction of ABCA1 by overexpression of hormone-sensitive lipase in macrophages.

Initial step toward the reverse-cholesterol transport is cholesterol efflux that is mediated by the ATP-binding cassette transporter A1 (ABCA1). However, it is unknown how the cholesteryl ester (CE) hydrolysis induces the expression of the ABCA1 gene. Overexpression of hormone-sensitive lipase (HSL) increased the hydrolysis of CE and stimulated the expression of ABCA1 gene at the transcriptional level in RAW 264.7 macrophages. The stimulatory effects of the HSL overexpression and cholesterol loading on the ABCA1 promoter activity were additive. Mutational analyses of the promoter of ABCA1 identified the responsible element as the direct repeat-4 (DR-4) that binds LXR/RXR heterodimers. In conclusion, stimulation of hydrolysis of CE in macrophages induces the expression of ABCA1 gene primarily via the LXR-dependent pathway and can be useful for the prevention of atherosclerosis.

Impaired meningeal development in association with apical expansion of calvarial bone osteogenesis in the Foxc1 mutant.

Loss of function of the mouse forkhead/winged helix transcription factor Foxc1 induces congenital hydrocephalus and impaired skull bone development due to failure of apical expansion of the bone. In this study we investigated meningeal development in the congenital hydrocephalus (ch) mouse with spontaneous loss of function mutant of Foxc1, around the period of initiation of skull bone apical expansion. In situ hybridization of Runx2 revealed active apical expansion of the frontal bone begins between embryonic day 13.5 and embryonic day 14.5 in the wild type, whereas expansion was inhibited in the mutant. Ultrastructural analysis revealed that three layers of the meninges begin to develop at E13.5 in the basolateral site of the head and subsequently progress to the apex in wild type. In ch homozygotes, although three layers were recognized at first at the basolateral site, cell morphology and structure of the layers became abnormal except for the pia mater, and arachnoidal and dural cells never differentiated in the apex. We identified meningeal markers for each layer and found that their expression was down-regulated in the mutant arachnoid and dura maters. These results suggest that there is a close association between meningeal development and the apical growth of the skull bones.

Lrp4 modulates extracellular integration of cell signaling pathways in development.

The extent to which cell signaling is integrated outside the cell is not currently appreciated. We show that a member of the low-density receptor-related protein family, Lrp4 modulates and integrates Bmp and canonical Wnt signalling during tooth morphogenesis by binding the secreted Bmp antagonist protein Wise. Mouse mutants of Lrp4 and Wise exhibit identical tooth phenotypes that include supernumerary incisors and molars, and fused molars. We propose that the Lrp4/Wise interaction acts as an extracellular integrator of epithelial-mesenchymal cell signaling. Wise, secreted from mesenchyme cells binds to BMP's and also to Lrp4 that is expressed on epithelial cells. This binding then results in the modulation of Wnt activity in the epithelial cells. Thus in this context Wise acts as an extracellular signaling molecule linking two signaling pathways. We further show that a downstream mediator of this integration is the Shh signaling pathway.

Experimental induction of palate shelf elevation in glutamate decarboxylase 67-deficient mice with cleft palate due to vertically oriented palatal shelf.

BACKGROUND: Gamma-aminobutyric acid is an inhibitory neurotransmitter, synthesized by two isoforms of glutamate decarboxylase (GAD), GAD65 and -67. Unexpectedly, inactivation of GAD67 induces cleft palate in mice. Reduction of spontaneous tongue movement resulting from decreased motor nerve activity has been related to the development of cleft palate in GAD67(-/-) fetuses. In the present study, development of cleft palate was examined histologically and manipulated with culture of the maxilla and partial resection of fetal tongue. METHODS: GAD67(-/-) mice and their littermates were used. Histological examination and immunohistochemistry were performed conventionally. Organ culture of the maxilla was carried out as reported previously. Fetuses were maintained alive under anesthesia and tips of their tongues were resected. RESULTS: Elevation of palatal shelves, the second step of palate formation, was not observed in GAD67(-/-) mice. In wild-type mice, GAD67 and gamma-aminobutyric acid were not expressed in the palatal shelves, except in the medial edge epithelium. During 2 days of culture of maxillae dissected from E13.5-E14.0 GAD67(-/-) fetuses, elevation and fusion of the palatal shelves were induced. When E13.5-15.5 mutant fetuses underwent partial tongue resection, the palatal shelves became elevated within 30 min. CONCLUSIONS: These results suggest that the potential for palate formation is maintained in the palatal shelves of GAD67(-/-) fetuses, but it is obstructed by other, probably neural, factors, resulting in cleft palate.

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.

Glucokinase and IRS-2 are required for compensatory beta cell hyperplasia in response to high-fat diet-induced insulin resistance.

Glucokinase (Gck) functions as a glucose sensor for insulin secretion, and in mice fed standard chow, haploinsufficiency of beta cell-specific Gck (Gck(+/-)) causes impaired insulin secretion to glucose, although the animals have a normal beta cell mass. When fed a high-fat (HF) diet, wild-type mice showed marked beta cell hyperplasia, whereas Gck(+/-) mice demonstrated decreased beta cell replication and insufficient beta cell hyperplasia despite showing a similar degree of insulin resistance. DNA chip analysis revealed decreased insulin receptor substrate 2 (Irs2) expression in HF diet-fed Gck(+/-) mouse islets compared with wild-type islets. Western blot analyses confirmed upregulated Irs2 expression in the islets of HF diet-fed wild-type mice compared with those fed standard chow and reduced expression in HF diet-fed Gck(+/-) mice compared with those of HF diet-fed wild-type mice. HF diet-fed Irs2(+/-) mice failed to show a sufficient increase in beta cell mass, and overexpression of Irs2 in beta cells of HF diet-fed Gck(+/-) mice partially prevented diabetes by increasing beta cell mass. These results suggest that Gck and Irs2 are critical requirements for beta cell hyperplasia to occur in response to HF diet-induced insulin resistance.