A DNA methylation site within the KLF13 gene is associated with orexigenic processes based on neural responses and ghrelin levels.

We investigated five methylation markers recently linked to body mass index, for their role in the neuropathology of obesity. In neuroimaging experiments, our analysis involving 23 participants showed that methylation levels for the cg07814318 site, which lies within the KLF13 gene, correlated with brain activity in the claustrum, putamen, cingulate gyrus and frontal gyri, some of which have been previously associated to food signaling, obesity or reward. Methylation levels at cg07814318 also positively correlated with ghrelin levels. Moreover, expression of KLF13 was augmented in the brains of obese and starved mice. Our results suggest the cg07814318 site could be involved in orexigenic processes, and also implicate KLF13 in obesity. Our findings are the first to associate methylation levels in blood with brain activity in obesity-related regions, and further support previous findings between ghrelin, brain activity and genetic differences.

High Variability of Insulin Sensitivity in Closely Related Obese Mouse Inbred Strains.

Obesity is one of several risk factors for insulin resistance and type 2 diabetes. Here we examined males of 6 obese mouse inbred lines derived from the Berlin Fat Mouse (BFM) outbred population with respect to insulin sensitivity and factors of the metabolic syndrome with focus on the skeletal muscle as a major target of insulin dependent glucose uptake.Males were kept on a rodent standard diet and several approaches were carried out to address insulin sensitivity, adiposity and lipids in the serum. Transcript and protein levels of several genes in the insulin signalling pathway were measured. 2 of the lines, BFMI860-12 and in particular BFMI861-S1, showed a markedly reduced insulin sensitivity already at the age of 20 weeks. BFMI861-S1 mice also displayed elevated liver triglyceride levels as a sign of lipid overload and ectopic fat storage. The analysis of the insulin signalling pathway in skeletal muscle provided evidence for low insulin receptor (INSR) and normal glucose 4 transporter (GLUT4) protein amounts in BFMI861-S1 mice, while BFMI860-12 mice showed increased INSR and very low GLUT4 protein amounts. Interestingly, the sublines BFMI860-S2 and BFMI861-S2, which are highly related to the former 2 lines, respectively, were inconspicuously insulin sensitive. The expected few genetic differences among the BFMI lines facilitate the identification of causal genetic variation. This study identified 2 mouse lines with different impairments of insulin signalling. These lines resemble useful models for studying mechanisms leading to the pathophysiology of the metabolic syndrome, in particular insulin resistance.

Diet-dependent alterations of hepatic Scd1 expression are accompanied by differences in promoter methylation.

Obesity and alterations of lipid homeostasis are hallmarks of the metabolic syndrome and largely influenced by the dietary conditions of the individual. Although heritability is considered to be a major risk factor, the almost 40 candidate genes identified by genome-wide association studies (GWAS) so far account for only 5-10% of the observed variance in BMI in human subjects. Alternatively, diet-induced changes of epigenetic gene regulation might be involved in disturbed lipid homeostasis and weight development. The aim of this study was to investigate how a high-carbohydrate diet (HCD; 70 kcal% from carbohydrates, 10 kcal% from fat) or a high-fat diet (HFD; 20 kcal% from carbohydrates, 60 kcal% from fat) affects hepatic expression of genes involved in fatty acid metabolism and if these alterations are correlated to changes in promoter methylation. Expression of stearoyl-CoA desaturase 1 (Scd1) was lower in livers from HFD-fed C57BL/6 J mice compared to HCD-fed animals and correlated inversely with the degree of DNA methylation at 2 distinct, adjacent CpG sites in the Scd1 promoter. In contrast, expression of transcription factors peroxisome proliferator activated receptor alpha and gamma (Ppara, Pparg), and sterol regulatory element binding transcription factor 1 (Srebf1) was not affected. The degree of hepatic Scd1 promoter methylation at these CpG sites correlated positively to fat mass and serum leptin levels, whereas serum ghrelin levels were inversely correlated with methylation at both CpG sites. Taken together, hepatic expression of Scd1 is differentially affected by carbohydrate- and lipid content of the diet. These differences in Scd1 expression are associated with altered promoter methylation, indicating that diets affect lipid metabolism in the liver via epigenetic mechanisms.

Estrogen deficiency aggravates insulin resistance and induces ß-cell loss and diabetes in female New Zealand obese mice.

In several rodent strains such as the New Zealand Obese (NZO) mouse, the incidence of obesity-associated diabetes mellitus is much higher in males than in females. In the present study, we investigated the effects of ovariectomy on glucose homeostasis in female NZO mice in order to elucidate the mechanism of their diabetes resistance. NZO females were ovariectomized at the age of 4 weeks, received a high-fat diet and body weight, body fat, glucose and insulin tolerance were investigated in comparison to sham-operated mice. In a second experiment, operated mice were fed a carbohydrate-free diet up to the age of 19 weeks before they received the high-fat diet. In comparison with a sham-operated control group, ovariectomized female NZO mice exhibited similar body weights, a reduced glucose tolerance, developed significantly higher blood glucose levels, lost insulin producing ß-cells, which finally resulted in a diabetes prevalence of 73% at the age of 16 weeks vs. 25% in controls. Similar to male NZO mice, ovariectomized females presented a more severe insulin resistance in the insulin tolerance test than sham-operated controls. Furthermore, the more severe insulin resistance in ovariectomized mice preceded the development of diabetes and pancreatic insulin depletion that was caused by a dietary regimen of carbohydrate restriction and subsequent re-exposure. In summary our data demonstrate that estrogen protects NZO females from ß-cell loss and obesity-associated diabetes mellitus. This effect is due to a reduced insulin resistance and possibly also to a reduced sensitivity of ß-cells to glucolipotoxic conditions.

Dissociation of lipotoxicity and glucotoxicity in a mouse model of obesity associated diabetes: role of forkhead box O1 (FOXO1) in glucose-induced beta cell failure.

AIMS/HYPOTHESIS: Carbohydrate-free diet prevents hyperglycaemia and beta cell destruction in the New Zealand Obese (NZO) mouse model. Here we have used a sequential dietary regimen to dissociate the effects of obesity and hyperglycaemia on beta cell function and integrity, and to study glucose-induced alterations of key transcription factors over 16 days. METHODS: Mice were rendered obese by feeding a carbohydrate-free diet for 18 weeks. Thereafter, a carbohydrate-containing diet was given. Plasma glucose, plasma insulin and total pancreatic insulin were determined, and forkhead box O1 protein (FOXO1) phosphorylation and the transcription factors pancreatic and duodenal homeobox 1 (PDX1), NK6 homeobox 1 protein (NKX6.1) and v-maf musculoaponeurotic fibrosarcoma oncogene family, protein A (avian) (MAFA) were monitored by immunohistochemistry for 16 days. RESULTS: Dietary carbohydrates produced a rapid and continuous increase in plasma glucose in NZO mice between day 2 and 16 after the dietary challenge. Hyperglycaemia caused a dramatic dephosphorylation of FOXO1 at day 2, followed by a progressive depletion of insulin stores. The loss of beta cells was triggered by apoptosis (detectable at day 8), associated with reduction of crucial transcription factors (PDX1, NKX6.1 and MAFA). Incubation of isolated islets from carbohydrate-restricted NZO mice or MIN6 cells with palmitate and glucose for 48 h resulted in a dephosphorylation of FOXO1 and thymoma viral proto-oncogene 1 (AKT) without changing the protein levels of both proteins. CONCLUSIONS/INTERPRETATION: The dietary regimen dissociates the effects of obesity (lipotoxicity) from those of hyperglycaemia (glucotoxicity) in NZO mice. Obese NZO mice are unable to compensate for the carbohydrate challenge by increasing insulin secretion or synthesising adequate amounts of insulin. In response to the hyperglycaemia, FOXO1 is dephosphorylated, leading to reduced levels of beta cell-specific transcription factors and to apoptosis of the cells.

Diet dependence of diabetes in the New Zealand Obese (NZO) mouse: total fat, but not fat quality or sucrose accelerates and aggravates diabetes.

BACKGROUND: Obesity and diabetes in mice can be modified by dietary variables. Here we systematically analysed the effect of the sucrose and fat content and of the fat quality in New Zealand Obese mice, a mouse model of the metabolic syndrome. RESULTS: Male NZO mice fed a semi-purified diet with sucrose exhibited an identical weight gain and diabetes incidence as controls without sucrose. In contrast, mice on a chow diet gained weight more slowly and developed diabetes approximately 10 weeks later than those on the semi-purified diet (energy density 3.05 vs. 3.85 kcal/g; fibre content 12.9 vs. 4.7%). In a second experimental series, neither the fat content (10 vs. 40% of the total energy) nor the quality of the fat (lard, safflower oil, or fish oil) of semi-purified diets modified weight gain. However, diabetes started approximately 2 weeks earlier and appeared more severe (blood glucose 30 vs. 20 mmol/l at week 13) in the high-fat diet group (energy density 4.58 kcal/g; fibre content 5.7%). CONCLUSIONS: Obesity in NZO mice develops independent of the dietary sucrose or fat content, and of the fat quality. However, the dietary fat content accelerates the onset of diabetes without enhancing adiposity. In contrast, chow diet exerts an anti-adipogenic/anti-diabetogenic effect that appears to be due to its lower caloric density and/or its higher fibre content.

Essential role of glucose transporter GLUT3 for post-implantation embryonic development.

Deletion of glucose transporter gene Slc2a3 (GLUT3) has previously been reported to result in embryonic lethality. Here, we define the exact time point of growth arrest and subsequent death of the embryo. Slc2a3(-/-) morulae and blastocysts developed normally, implanted in vivo, and formed egg-cylinder-stage embryos that appeared normal until day 6.0. At day 6.5, apoptosis was detected in the ectodermal cells of Slc2a3(-/-) embryos resulting in severe disorganization and growth retardation at day 7.5 and complete loss of embryos at day 12.5. GLUT3 was detected in placental cone, in the visceral ectoderm and in the mesoderm of 7.5-day-old wild-type embryos. Our data indicate that GLUT3 is essential for the development of early post-implanted embryos.

Deletion of glucose transporter GLUT8 in mice increases locomotor activity.

Transport of glucose into neuronal cells is predominantly mediated by the glucose transporters GLUT1 and GLUT3. In addition, GLUT8 is expressed in some regions of the brain. By in situ hybridization we detected GLUT8-mRNA in hippocampus, thalamus, and cortex. However, its cellular and physiological function is still unknown. Thus, GLUT8 knockout (Slc2a8 -/-) mice were used for a screening approach in the modified hole board (mHB) behavioral test to analyze the role of GLUT8 in the central nervous system. Slc2a8 -/- mice showed increased mean velocity, total distance traveled and performed more turns in the mHB test. This hyperactivity of Slc2a8 -/- mice was confirmed by monitoring locomotor activity in the home cage and voluntary activity in a running wheel. In addition, Slc2a8 -/- mice showed increased arousal as indicated by elevated defecation, reduced latency to the first defecation and a tendency to altered grooming. Furthermore, the mHB test gave evidence that Slc2a8 -/- mice exhibit a reduced risk assessment because they performed less rearings in an unprotected area and showed significantly reduced latency to stretched body posture. Our data suggest that behavioral alterations of Slc2a8 -/- mice are due to dysfunctions in neuronal processes presumably as a consequence of defects in the glucose metabolism.

Development of diabetes in obese, insulin-resistant mice: essential role of dietary carbohydrate in beta cell destruction.

AIMS/HYPOTHESIS: The role of dietary carbohydrate in the pathogenesis of type 2 diabetes is still a subject of controversial debate. Here we analysed the effects of diets with and without carbohydrate on obesity, insulin resistance and development of beta cell failure in the obese, diabetes-prone New Zealand Obese (NZO) mouse. MATERIALS AND METHODS: NZO mice were kept on a standard diet (4% [w/w] fat, 51% carbohydrate, 19% protein), a high-fat diet (15, 47 and 17%, respectively) and a carbohydrate-free diet in which carbohydrate was exchanged for fat (68 and 20%, respectively). Body composition and blood glucose were measured over a period of 22 weeks. Glucose tolerance tests and euglycaemic-hyperinsulinaemic clamps were performed to analyse insulin sensitivity. Islet morphology was assessed by immunohistochemistry. RESULTS: Mice on carbohydrate-containing standard or high-fat diets developed severe diabetes (blood glucose >16.6 mmol/l, glucosuria) due to selective destruction of pancreatic beta cells associated with severe loss of immunoreactivity of insulin, glucose transporter 2 (GLUT2) and musculoaponeurotic fibrosarcoma oncogene homologue A (MafA). In contrast, mice on the carbohydrate-free diet remained normoglycaemic and exhibited hyperplastic islets in spite of a morbid obesity associated with severe insulin resistance and a massive accumulation of macrophages in adipose tissue. CONCLUSIONS/INTERPRETATION: These data indicate that the combination of obesity, insulin resistance and the inflammatory response of adipose tissue are insufficient to cause beta cell destruction in the absence of dietary carbohydrate.

Coincidence of different structures of mucosa-associated lymphoid tissue (MALT) in the respiratory tract of children: no indications for enhanced mucosal immunostimulation in sudden infant death syndrome (SIDS).

Mucosa-associated lymphoid tissue (MALT) is the principal inductive site for mucosal immune responses that are capable of T and B cell responses and antigen-specific responses. In previous independent studies different structures of MALT, e.g. bronchus-, larynx- and nose-associated lymphoid tissue (BALT, LALT, NALT) have been described separately in various frequencies in the human respiratory tract over life spans. Because upper respiratory tract infections are common in infants, dysregulations of mucosal immune responses might be seriously involved in the aetiology of sudden infant death syndrome (SIDS). In the present study the coincidental occurrence of the three different MALT structures in the respiratory tract within the same patients were studied, and cases of SIDS and children who had died from different traumatic and natural causes of death (non-SIDS) were compared. First, the frequency of BALT and LALT in 46 children (35 SIDS, 11 non-SIDS) with or without NALT were examined. A tendency was found of a coincidence of respiratory MALT structures. In 50 additional cases of infant death (30 SIDS, 20 non-SIDS) from the multi-centric German Study on Sudden Infant Death Syndrome (GeSID) where death had occurred in the first year of life, the coincidence was evaluated. A coincidental occurrence of BALT, LALT and NALT or BALT and LALT (each about 30%) was found in both groups, whereby the coincidence in SIDS and the control patients did not differ. Interestingly, the children with coincidental MALT were strikingly older, supporting the hypothesis of respiratory MALT formation via environmental stimulation over time.

Effects of two fluoridation measures on erosion progression in human enamel and dentine in situ.

The aim of the present study was to evaluate the effects of fluoride on erosive mineral loss in human enamel and dentine using a cyclic de- and remineralisation model in situ. The study was a three-treatment (5 days each) crossover design involving 4 (enamel) or 6 (dentine) healthy volunteers. Samples were recessed in palatal mouth appliances and worn day and night except during meals and were demineralised extraorally with 0.05 M citric acid (pH 2.3) for 6 x 5 min daily. Fluoridation was performed with toothpaste (SnF2/Olaflur; 0.14% F-) for 3 x 5 min daily (toothpaste fluoridation) or with toothpaste in combination with a mouthrinse (SnF2/Olaflur; 0.025% F-) for 3 x 5 min daily and with a gel (NaF/Olaflur, 1.25% F-) on days 1 and 3 instead of the toothpaste (intensive fluoridation). In the control group no fluoridation was performed. Mineral loss (microm) was determined with the use of longitudinal microradiography. In enamel, mineral loss was 40.7 +/- 15.1 microm in the control group, 18.3 +/- 12.4 microm after toothpaste fluoridation and 5.0 +/- 12.2 microm after intensive fluoridation. The respective values for dentine were 49.0 +/- 15.4, 35.0 +/- 15.5 and 19.8 +/- 12.0 microm. All differences were statistically significant (p < or = 0.001). The results indicate that intensive fluoridation is effective in preventing enamel and dentine from mineral loss even under severely erosive conditions.

GLUT11, but not GLUT8 or GLUT12, is expressed in human skeletal muscle in a fibre type-specific pattern.

Nine novel sugar transporter-like proteins have been discovered in the past 5 years. The mRNA for three of these, the glucose transporters (GLUT) GLUT8, GLUT11 and GLUT12, have been detected in human skeletal muscle. In the present study, we examined the pattern of expression and localization of the GLUT isoforms 8, 11 and 12 in human skeletal muscle using an immunohistochemical approach. Biopsies of human skeletal muscle from sedentary or trained healthy adults, from fetal muscle (24 weeks of gestation), from obese type-2 diabetic subjects, and from patients suffering from polymyositis or amyotrophic lateral sclerosis (ALS) were studied. GLUT8 and 12 immunoreactivity was below detection level in both developing and adult muscle fibres. GLUT11 immunoreactivity, however, was present in slow-twitch muscle fibres, but not in fast twitch fibres. Since, in contrast, GLUT4 was expressed in all investigated muscle fibres, the pattern of expression of GLUT11 differs from that of GLUT4, suggesting a specialized function for GLUT11 with a regulation independent from that of GLUT4. Obesity, type-2 diabetes, training, conditions of de- and reinnervation (ALS) and regeneration (polymyositis) failed to induce GLUT8 or -12 expression. Likewise, the fibre type-dependent pattern of GLUT11 immunoreactivity was unaltered. However, some slow muscle fibres lose their GLUT11 immunoreactivity under regeneration. Our results indicate that GLUT11 immunoreactivity, in contrast to that of GLUT4, is expressed exclusively in slow-twitch muscle fibres and is unaffected by physiological and pathophysiological conditions except in primary myopathy. GLUT8 and GLUT12 do not appear to be of importance in human muscle under physiological and pathophysiological conditions.

Nitrate-consuming processes in a petroleum-contaminated aquifer quantified using push-pull tests combined with 15N isotope and acetylene-inhibition methods.

Nitrate consumption in aquifers may result from several biogenic and abiotic processes such as denitrification, assimilatory NO3- reduction, dissimilatory NO3- reduction to ammonium (DNRA), or abiotic NO3- (or NO2-) reduction. The objectives of this study were to investigate the fate of NO3- in a petroleum-contaminated aquifer, and to assess the feasibility of using single-well push-pull tests (PPTs) in combination with 15N isotope and C2H2 inhibition methods for the quantification of processes contributing to NO3- consumption. Three consecutive PPTs were performed in a monitoring well of a heating oil-contaminated aquifer in Erlen, Switzerland. For each test, we injected 500 l of test solution containing 0.5 mM Br- as conservative tracer and either 0.5 mM unlabeled NO3- or approximately 0.3 mM 15N-labeled NO3- as reactant. Test solutions were sparged during preparation and injection with either N2, Ar or 10% C2H2 in Ar. After an initial incubation period of 1.5-3.2 h, we extracted the test solution/groundwater mixtures from the same location and measured concentrations of relevant species including Br-, NO3-, NO2-, N2O, N2, and NH4+. In addition, we determined the 15N contents of N2, N2O, NH4+, and suspended biomass from 15N/14N isotope-ratio measurements. Average total test duration was 50.5 h. First-order rate coefficients (k) were computed from measured NO3- consumption, N2-15N production and N2O-15N production. From measured NO3- consumption we obtained nearly identical estimates of k for all PPTs with small 95% confidence intervals, indicating good reproducibility and accuracy for the tests. Estimates of k from N2-15N production and N2O-15N production indicated that denitrification accounted for only 46-49% of observed NO3- consumption. Production of N2-15N in the presence of C2H2 was observed during one of the tests, which may be an indicator for abiotic NO3- reduction. Moreover, 15N isotope analyses confirmed occurrence of assimilatory NO3- reduction (0.58 at.% 15N in suspended biomass) and to a smaller extent DNRA (up to 4 at.% 15N in NH4+). Our results indicated that the combination of PPTs, 15N-isotope and C2H2 inhibition methods provided improved information on denitrification as well as alternative fates of NO3- in this aquifer.

Characterisation of the mouse diabetes susceptibilty locus Nidd/SJL: islet cell destruction, interaction with the obesity QTL Nob1, and effect of dietary fat.

AIMS/HYPOTHESIS: The diabetes susceptibility locus Nidd/SJL was identified in an outcross of New Zealand obese (NZO) and lean Swiss/Jackson Laboratory mouse strain (SJL) mice. Here we characterise its effects in a NZO x F1(SJLxNZO) backcross population raised on high-fat or standard diet, and describe its interaction with the obesity quantitative trait locus (QTL) Nob1. METHODS: NZO x F1(SJLxNZO) backcross mice were raised on a normal or high fat diet and were monitored (body weight, blood glucose, serum insulin) for 22 weeks. Genotypes of polymorphic markers were determined by PCR, and linkage analysis was done. Pancreas morphology was assessed by conventional staining and immunohistochemistry of insulin. RESULTS: In backcross mice raised on a high-fat diet, Nidd/SJL produced hyperglycaemia (maximum likelihood of the odds (LOD) score 9.9), hypoinsulinaemia, reduction of islet-cell volume, and loss of beta cells. No effect was observed on body weight and serum insulin concentrations before the onset of hyperglycaemia. The development of diabetes in carriers of Nidd/SJL was markedly accelerated and aggravated by the obesity/hyperinsulinaemia QTL Nob1; together, these loci were responsible for approximately 90% of the diabetes observed in the backcross population. When raised on a standard diet, Nidd/SJL carriers exhibited a fivefold higher prevalence of diabetes, but Nob1 failed to enhance the effect of Nidd/SJL. CONCLUSION/INTERPRETATION: Diabetes in this obese mouse model is the result of an interaction of genes responsible for obesity/insulin resistance (e.g. Nob1) and islet cell failure ( Nidd/SJL). The combined diabetogenic effects of Nidd/SJL and Nob1 were markedly enhanced by a high-fat diet, whereas that of Nidd/SJL alone was independent of the dietary fat content.

Comparison of psychiatric ICD-10 diagnoses in Denmark and Germany.

A dataset of psychiatric ICD-10 diagnoses from the Danish case register concerning psychiatric hospitals was compared with a sample of psychiatric diagnoses from 27 psychiatric hospitals in Germany. The comparison shows a higher proportion of F1 diagnoses in the German dataset and a difference in the coding of alcohol dependence and harmful use. Some further differences in the groups F0-F6 are demonstrated and some of them are discussed. The most frequent diagnoses found in both datasets but in different sequence are alcohol dependence syndrome and paranoid schizophrenia and, in third place, adjustment disorder. Various aspects of the problem of rarely used diagnoses are discussed.

Reduced sperm count and normal fertility in male mice with targeted disruption of the ADP-ribosylation factor-like 4 (Arl4) gene.

The ADP-ribosylation factor-like protein 4 (ARL4) is a 22-kDa GTP-binding protein which is abundant in testes of pubertal and adult rodents but absent in testes from prepubertal animals. During testis development, ARL4 expression starts at day 16 when the spermatogenesis proceeds to the late pachytene. In the adult testis, the ARL4 protein was detected in pre- and postmeiotic cells, spermatocytes, and spermatides, but not in spermatogonia and mature spermatozoa. Mouse Arl4-null mutants generated by targeted disruption of the Arl4 gene were viable and grew normally; male as well as female Arl4(-/-) mice were fertile. However, inactivation of the Arl4 gene resulted in a significant reduction of testis weight and sperm count by 30 and 60%, respectively, without reduction of litter size or frequency. It is suggested that the disruption of Arl4 produces a moderate retardation of germ cell development, possibly at the stage of meiosis.

Mouse ARF-related protein 1: genomic organization and analysis of its promoter.

ARF-related protein 1 (ARFRP1) is a membrane-associated GTPase which inhibits the ARF/Sec7-dependent activation of phospholipase D. We have recently shown that deletion of Arfrp1 in mice results in increased apoptosis of mesodermal cells during gastrulation, leading to early embryonic lethality. Here we describe the organization of the Arfrp1 gene and of its promoter region. The Arfrp1 gene spans approximately 7 kb and contains 8 exons. The proximal 5'-flanking regions of mouse and human ARFRP1 lack a TATA box and a CAAT box, are highly GC-rich and contain potential transcription factor binding sites. Interestingly, sequence analysis of human ARFRP1 showed its 5'-flanking region contains the first exon of another gene (DJ583P15.3 in the ensembl data base; www.ensembl.org) on the opposite strand. Promoter analysis revealed that the intergenic region between both genes (54 bp) exhibits bidirectional promoter activity. However, deletion analysis demonstrated that transcription of both genes is regulated by different cis-elements. Mutational analysis and electrophoretic mobility shift assays indicated that two short cRel- and cEts1-like elements in the 5'-flanking region of Arfrp1 (-76 to -53 and -45 to -23) are critical for the regulation of Arfrp1 expression.

Embryonic lethality caused by apoptosis during gastrulation in mice lacking the gene of the ADP-ribosylation factor-related protein 1.

ADP-ribosylation factor (ARF)-related protein 1 (ARFRP1) is a membrane-associated GTPase with significant similarity to the family of ARFs. We have recently shown that ARFRP1 interacts with the Sec7 domain of the ARF-specific guanine nucleotide exchange factor Sec7-1/cytohesin and inhibits the ARF/Sec7-dependent activation of phospholipase D in a GTP-dependent manner. In order to further analyze the function of ARFRP1, we cloned the mouse Arfrp1 gene and generated Arfrp1 null-mutant mice by gene targeting in embryonic stem cells. Heterozygous Arfrp1 mutants developed normally, whereas homozygosity for the mutant allele led to embryonic lethality. Cultured homozygous Arfrp1 null-mutant blastocysts were indistinguishable from wild-type blastocysts. In vivo, they implanted and formed egg cylinder stage embryos that appeared normal until day 5. Between embryonic days 6 and 7, however, apoptotic cell death of epiblast cells occurred in the embryonic ectoderm during gastrulation, as was shown by histological analysis combined with terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling. Epiblast cells that would normally differentiate to mesodermal cells detached from the ectodermal cell layer and were dispersed into the proamniotic cavity. In contrast, the development of extraembryonic structures appeared unaffected. Our results demonstrate that ARFRP1 is necessary for early embryonic development during gastrulation.

Mouse GLUT8: genomic organization and regulation of expression in 3T3-L1 adipocytes by glucose.

Glucose transporter 8 (GLUT8) is a class III sugar transport facilitator predominantly expressed in testis and insulin-regulated tissues. Here we describe its genomic organization, the identification of its promoter region, and the regulation of its expression in 3T3-L1 cells. The mouse Glut8 gene spans approximately 9 kb, consists of 10 exons, and is highly similar to the human GLUT6 gene. Its 5'-flanking region exhibits promoter activity when fused with a luciferase reporter construct and expressed in HEK-293T cells. A deletion analysis indicated that the critical promoter elements are located in a region between -381 and the transcription start. This region comprises a CAAT box and consensus binding sites for the transcription factors SRY and NF1 that were highly conserved in the mouse and in the human sequence. In 3T3-L1 cells, GLUT8 mRNA levels increased markedly during the differentiation of cells. In contrast to GLUT1, expression of GLUT8 mRNA was significantly reduced by glucose deprivation and by prolonged hypoxia. The present data suggest that the function of GLUT8 is related to the adipocyte-like phenotype of 3T3-L1 cells, and that its expression is controlled by the metabolism of the adipocyte.

HIV-1 Nef associated PAK and PI3-kinases stimulate Akt-independent Bad-phosphorylation to induce anti-apoptotic signals.

A highly conserved signaling property of Nef proteins encoded by human or simian immunodeficiency virus is the binding and activation of a PAK kinase whose function is unclear. Here we show that Nef-mediated p21-activated kinase (PAK) activation involves phosphatidylinositol 3-kinase, which acts upstream of PAK and is bound and activated by Nef similar to the manner of Polyoma virus middle T antigen. The Nef-associated phosphatidylinositol-3-PAK complex phosphorylated the pro-apoptotic Bad protein without involving the protein kinase B-Akt kinase, which is generally believed to inactivate Bad by serine phosphorylation. Consequently, Nef, but not a Nef mutant incapable of activating PAK, blocked apoptosis in T cells induced by serum starvation or HIV replication. Nef anti-apoptotic effects are likely a crucial mechanism for viral replication in the host and thus in AIDS pathogenesis.