Guidelines on severity assessment and classification of genetically altered mouse and rat lines.

Genetic alterations can unpredictably compromise the wellbeing of animals. Thus, more or less harmful phenotypes might appear in the animals used in research projects even when they are not subjected to experimental treatments. The severity classification of suffering has become an important issue since the implementation of Directive 2010/63/EU on the protection of animals used for scientific purposes. Accordingly, the breeding and maintenance of genetically altered (GA) animals which are likely to develop a harmful phenotype has to be authorized. However, a determination of the degree of severity is rather challenging due to the large variety of phenotypes. Here, the Working Group of Berlin Animal Welfare Officers (WG Berlin AWO) provides field-tested guidelines on severity assessment and classification of GA rodents. With a focus on basic welfare assessment and severity classification we provide a list of symptoms that have been classified as non-harmful, mild, moderate or severe burdens. Corresponding monitoring and refinement strategies as well as specific housing requirements have been compiled and are strongly recommended to improve hitherto applied breeding procedures and conditions. The document serves as a guide to determine the degree of severity for an observed phenotype. The aim is to support scientists, animal care takers, animal welfare bodies and competent authorities with this task, and thereby make an important contribution to a European harmonization of severity assessments for the continually increasing number of GA rodents.

The Aachen Minipig: Phenotype, Genotype, Hematological and Biochemical Characterization, and Comparison to the Göttingen Minipig.

BACKGROUND: The pig is one of the most frequently used large animal models for biomedical research, especially in the field of translational research and surgical models. While standard livestock breeds are used in short-term and acute studies, minipig breeds are the preferred breeds in long-term and chronic studies due to their limited growth and body weight. OBJECTIVE: In consideration of the 3R principle (refinement, reduction, replacement) and the increasing demand, the aim of this study was to generate a new, robust, non-specific-pathogen-free minipig breed, the Aachen minipig. METHODS: Phenotype, genotype, and hematological as well as clinical chemistry parameters were characterized, and reference values of the Aachen minipig were generated and compared to the values in the commonly used Göttingen minipig. Organ weights of the heart, kidney, liver, lung, spleen, and brain were determined using a laboratory balance. Blood samples were collected for hematology and clinical chemistry. Assessment of genetic diversity was performed by microsatellite markers. Nasal swabs were collected from 11 individual minipigs representing 6 races for DNA extraction. DNA was quantified and the identity and origin of the Aachen minipigs at the genomic level was determined by microsatellites. RESULTS: The Aachen minipig established here is based on the Mini-LEWE breed and consists of the Vietnamese potbelly pig, the Schwäbisch Hällisch Landpig, the German Landrace, and the Minnesota minipig. Relative organ weights (lung, heart, kidneys, brain), hematology (hemoglobin, hematocrit, platelet count, mean corpuscular hemoglobin concentration, segmented neutrophils, lymphocytes, eosinophils, basophils), and clinical chemistry parameters (sodium, calcium, chloride, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, gamma-glutamyl transferase, lactate dehydrogenase, triglycerides, blood urea nitrogen, creatinine, total bilirubin, total protein, creatine kinase) of the Aachen minipigs and the Göttingen minipigs were not significantly different. Significant differences where only seen in relative organ weights (liver, spleen), hematology (red blood cell count, mean corpuscular volume, mean corpuscular hemoglobin, white blood cell count, banded neutrophils, monocytes), and clinical chemistry parameters (inorganic phosphorus, potassium, glucose, cholesterol, albumin, amylase). CONCLUSION: The Aachen minipig is a suitable model for research due to its similarity to other minipig breeds, especially the Göttingen minipig. The reference values established in this study may be used for the comparison of scientific data and encourage the use of the Aachen minipig as an animal model for biomedical research.

Skeletal muscle mitochondrial uncoupling prevents diabetes but not obesity in NZO mice, a model for polygenic diabesity.

Induction of skeletal muscle (SM) mitochondrial stress by expression of uncoupling protein 1 (UCP1) in mice results in a healthy metabolic phenotype associated with increased secretion of FGF21 from SM. Here, we investigated whether SM mitochondrial uncoupling can compensate obesity and insulin resistance in the NZO mouse, a polygenic diabesity model. Male NZO mice were crossed with heterozygous UCP1 transgenic (tg) mice (mixed C57BL/6/CBA background) and further backcrossed to obtain F1 and N2 offspring with 50 and 75 % NZO background, respectively. Male F1 and N2 progeny were fed a high-fat diet ad libitum for 20 weeks from weaning. Blood glucose was reduced, and diabetes (severe hyperglycemia >300 mg/dl) was fully prevented in both F1- and N2-tg progeny compared to a diabetes prevalence of 15 % in F1 and 42 % in N2 wild type. In contrast, relative body fat content and plasma insulin were decreased, and glucose tolerance was improved, in F1-tg only. Both F1 and N2-tg showed decreased lean body mass. Accordingly, induction of SM stress response including FGF21 expression and secretion was similar in both F1 and N2-tg mice. In white adipose tissue, expression of FGF21 target genes was enhanced in F1 and N2-tg mice, whereas lipid metabolism genes were induced in F1-tg only. There was no evidence for induction of browning in either UCP1 backcross. We conclude that SM mitochondrial uncoupling induces FGF21 expression and prevents diabetes in mice with a 50-75 % NZO background independent of its effects on adipose tissue.

Porphyromonas gingivalis Suppresses Differentiation and Increases Apoptosis of Osteoblasts From New Zealand Obese Mice.

BACKGROUND: Metabolic syndrome (MetS), a complex cluster of risk factors for chronic diseases such as cardiovascular disease, is observed to be increasingly associated with periodontal disease. However, the fundamental contribution of periodontal bacteria to periodontal bone loss in patients with MetS remains unclear. The aim of the present study is to analyze the effect of Porphyromonas gingivalis on differentiation of primary osteoblasts from New Zealand obese (NZO) mice, a model for MetS, compared with C57 Black 6 JAX (C57BL/6J) mice osteoblasts. METHODS: Primary calvarial osteoblasts, isolated from 3-day-old NZO and control C57BL/6J mice, were stimulated with P. gingivalis. Proliferation was quantified by 5-bromo-2'-deoxyuridine incorporation. Cell cycle and early and late apoptosis were measured by flow cytometry. Gene expression was determined by real-time polymerase chain reaction (RT-PCR). RESULTS: Twelve hours after P. gingivalis stimulation, NZO osteoblasts showed significantly decreased proliferation (P ≤0.01) with increased G2 cell cycle phase compared with normal osteoblasts. Flow cytometry analysis demonstrated significant (P ≤0.01) increase of early apoptotic cells (annexin V positive) and late apoptosis (caspase 3 activity) in NZO cells compared with control cells at 3 and 6 hours after stimulation. No significant lactate dehydrogenase release was found after P. gingivalis stimulation. RT-PCR data showed significantly suppressed expression (P ≤0.01) of collagen 1, osteocalcin, and Runt-related transcription factor 2 in NZO cells compared with normal osteoblasts. CONCLUSIONS: The present data demonstrate that P. gingivalis downregulates proliferation and promotes apoptosis in primary NZO osteoblasts, unlike C57BL/6J osteoblasts. Also, suppressed osteoblastic marker expression in NZO cells may contribute to pathogenesis of periodontitis, suggesting a similar process in patients with MetS.

An interval of the obesity QTL Nob3.38 within a QTL hotspot on chromosome 1 modulates behavioral phenotypes.

A region on mouse distal chromosome 1 (Chr. 1) that is highly enriched in quantitative trait loci (QTLs) controlling neural and behavioral phenotypes overlaps with the peak region of a major obesity QTL (Nob3.38), which we identified in an intercross of New Zealand Obese (NZO) mice with C57BL/6J (B6). By positional cloning we recently identified a microdeletion within this locus causing the disruption of Ifi202b that protects from adiposity by suppressing expression of 11ß-Hsd1. Here we show that the Nob3.38 segment also corresponds with the QTL rich region (Qrr1) on Chr. 1 and associates with increased voluntary running wheel activity, Rota-rod performance, decreased grip strength, and anxiety-related traits. The characterization of a subcongenic line carrying 14.2 Mbp of Nob3.38 with a polymorphic region of 4.4 Mbp indicates that the microdeletion and/or other polymorphisms in its proximity alter body weight, voluntary activity, and exploration. Since 27 out of 32 QTL were identified in crosses with B6, we hypothesized that the microdeletion and or adjacent SNPs are unique for B6 mice and responsible for some of the complex Qrr1-mediated effects. Indeed, a phylogenic study of 28 mouse strains revealed a NZO-like genotype for 22 and a B6-like genotype for NZW/LacJ and 4 other C57BL strains. Thus, we suggest that a Nob3.38 interval (173.0-177.4 Mbp) does not only modify adiposity but also neurobehavioral traits by a haplotype segregating with C57BL strains.

Role of KCNQ channels in skeletal muscle arteries and periadventitial vascular dysfunction.

KCNQ channels have been identified in arterial smooth muscle. However, their role in vasoregulation and chronic vascular diseases remains elusive. We tested the hypothesis that KCNQ channels contribute to periadventitial vasoregulation in peripheral skeletal muscle arteries by perivascular adipose tissue and that they represent novel targets to rescue periadventitial vascular dysfunction. Two models, spontaneously hypertensive rats and New Zealand obese mice, were studied using quantitative polymerase chain reaction, the patch-clamp technique, membrane potential measurements, myography of isolated vessels, and blood pressure telemetry. In rat Gracilis muscle arteries, anticontractile effects of perivascular fat were inhibited by the KCNQ channel blockers XE991 and linopirdine but not by other selective K(+) channel inhibitors. Accordingly, XE991 and linopirdine blocked noninactivating K(+) currents in freshly isolated Gracilis artery smooth muscle cells. mRNAs of several KCNQ channel subtypes were detected in those arteries, with KCNQ4 channels being dominant. In spontaneously hypertensive rats, the anticontractile effect of perivascular fat in Gracilis muscle arteries was largely reduced compared with Wistar rats. However, the vasodilator effects of KCNQ channel openers and mRNA expression of KCNQ channels were normal. Furthermore, KCNQ channel openers restored the diminished anticontractile effects of perivascular fat in spontaneously hypertensive rats. Moreover, KCNQ channel openers reduced arterial blood pressure in both models of hypertension independent of ganglionic blockade. Thus, our data suggest that KCNQ channels play a pivotal role in periadventitial vasoregulation of peripheral skeletal muscle arteries, and KCNQ channel opening may be an effective mechanism to improve impaired periadventitial vasoregulation and associated hypertension.

Pathophysiology and genetics of obesity and diabetes in the New Zealand obese mouse: a model of the human metabolic syndrome.

The New Zealand Obese (NZO) mouse is one of the most thoroughly investigated polygenic models for the human metabolic syndrome and type 2 diabetes. It presents the main characteristics of the disease complex, including early-onset obesity, insulin resistance, dyslipidemia, and hypertension. As a consequence of this syndrome, a combination of lipotoxicity and glucotoxicity produces beta-cell failure and apoptosis resulting in hypoinsulinemia and diabetic hyperglycemia. With NZO as a breeding partner, several adipogenic and diabetogenic gene variants have been identified by hypothesis-free positional cloning (Tbc1d1, Zfp69) or by combining genetic screens and candidate gene approaches (Pctp, Abcg1, Nmur2, Lepr). This chapter summarizes the present knowledge of the NZO strain and describes its pathophysiology as well as the known underlying genetic defects.

Loss of function of Ifi202b by a microdeletion on chromosome 1 of C57BL/6J mice suppresses 11ß-hydroxysteroid dehydrogenase type 1 expression and development of obesity.

Nob3 is a major obesity quantitative trait locus (QTL) identified in an intercross of New Zealand Obese (NZO) mice with C57BL/6J (B6), and by introgression of its 38 Mbp peak region into B6 (B6.NZO-Nob3.38). B6.NZO-Nob3.38 mice carrying the NZO allele exhibited markedly increased body weight, fat mass, lean mass and a lower energy expenditure, than the corresponding B6 allele carriers. For positional cloning of the responsible obesity gene, five additional congenic lines (RCS) were generated and characterized, allowing to define a critical genomic interval comprising 43 genes. mRNA profiling and western blotting indicated that Ifi202b, a member of the Ifi200 family of interferon inducible transcriptional modulators, was expressed in NZO-allele carriers but was undetectable in tissues of homozygous B6-allele carriers due to a microdeletion, including the first exon and the 5'-flanking region of Ifi202b in B6. Transcriptome analysis of adipose tissue of RCS revealed a marked induction of 11ß-hydroxysteroid dehydrogenase type 1 (11ß-Hsd1) expression in mice expressing Ifi202b. Furthermore, siRNA-mediated Ifi202b suppression in 3T3-L1 adipocytes resulted in a significant inhibition of 11ß-Hsd1 expression, whereas an adenoviral-mediated overexpression of Ifi202b increased 11ß-Hsd1 mRNA levels. Expression of human IFI orthologues was significantly increased in visceral adipose tissue of obese subjects. We suggest that the disruption of Ifi202b in B6 is responsible for the effects of the obesity QTL Nob3, and that Ifi202b modulates fat accumulation through expression of adipogenic genes such as 11ß-Hsd1.

The GTPase ARFRP1 controls the lipidation of chylomicrons in the Golgi of the intestinal epithelium.

The uptake and processing of dietary lipids by the small intestine is a multistep process that involves several steps including vesicular and protein transport. The GTPase ADP-ribosylation factor-related protein 1 (ARFRP1) controls the ARF-like 1 (ARL1)-mediated Golgi recruitment of GRIP domain proteins which in turn bind several Rab-GTPases. Here, we describe the essential role of ARFRP1 and its interaction with Rab2 in the assembly and lipidation of chylomicrons in the intestinal epithelium. Mice lacking Arfrp1 specifically in the intestine (Arfrp1(vil-/-)) exhibit an early post-natal growth retardation with reduced plasma triacylglycerol and free fatty acid concentrations. Arfrp1(vil-/-) enterocytes as well as Arfrp1 mRNA depleted Caco-2 cells absorbed fatty acids normally but secreted chylomicrons with a markedly reduced triacylglycerol content. In addition, the release of apolipoprotein A-I (ApoA-I) was dramatically decreased, and ApoA-I accumulated in the Arfrp1(vil-/-) epithelium, where it predominantly co-localized with Rab2. The release of chylomicrons from Caco-2 was markedly reduced after the suppression of Rab2, ARL1 and Golgin-245. Thus, the GTPase ARFRP1 and its downstream proteins are required for the lipidation of chylo-microns and the assembly of ApoA-I to these particles in the Golgi of intestinal epithelial cells.

Role of medium- and short-chain L-3-hydroxyacyl-CoA dehydrogenase in the regulation of body weight and thermogenesis.

Dysregulation of fatty acid oxidation plays a pivotal role in the pathophysiology of obesity and insulin resistance. Medium- and short-chain-3-hydroxyacyl-coenzyme A (CoA) dehydrogenase (SCHAD) (gene name, hadh) catalyze the third reaction of the mitochondrial ß-oxidation cascade, the oxidation of 3-hydroxyacyl-CoA to 3-ketoacyl-CoA, for medium- and short-chain fatty acids. We identified hadh as a putative obesity gene by comparison of two genome-wide scans, a quantitative trait locus analysis previously performed in the polygenic obese New Zealand obese mouse and an earlier described small interfering RNA-mediated mutagenesis in Caenorhabditis elegans. In the present study, we show that mice lacking SCHAD (hadh(-/-)) displayed a lower body weight and a reduced fat mass in comparison with hadh(+/+) mice under high-fat diet conditions, presumably due to an impaired fuel efficiency, the loss of acylcarnitines via the urine, and increased body temperature. Food intake, total energy expenditure, and locomotor activity were not altered in knockout mice. Hadh(-/-) mice exhibited normal fat tolerance at 20 C. However, during cold exposure, knockout mice were unable to clear triglycerides from the plasma and to maintain their normal body temperature, indicating that SCHAD plays an important role in adaptive thermogenesis. Blood glucose concentrations in the fasted and postprandial state were significantly lower in hadh(-/-) mice, whereas insulin levels were elevated. Accordingly, insulin secretion in response to glucose and glucose plus palmitate was elevated in isolated islets of knockout mice. Therefore, our data indicate that SCHAD is involved in thermogenesis, in the maintenance of body weight, and in the regulation of nutrient-stimulated insulin secretion.

Role of zinc finger transcription factor zfp69 in body fat storage and diabetes susceptibility of mice.

Type 2 diabetes is a polygenic disease resulting from a combination of different disease alleles reflecting obesity, insulin resistance, and hyperglycemia. Using a positional cloning strategy with different inbred strains of mice, we mapped a disease locus for obesity-associated diabetes on chromosome 4. We analyzed all genes in this region and identified distinct differences in the expression levels of the transcription factor Zfp69. The expression of this gene mediated diabetes progression in a leptin-deficient congenic mouse line. The animals developed a disease pattern of hyperglycemia, reduced gonadal fat mass, and increased plasma and liver triglycerides, resembling a potential defect in triglyceride storage . In order to elucidate the impact of the human ortholog of Zfp69 in the development of type 2 diabetes, we tested its mRNA expression in human white adipose tissue. Consistent with the mouse data, mRNA-expression was significantly higher in diabetic subjects than in unaffected controls.

New function for an old enzyme: NEP deficient mice develop late-onset obesity.

BACKGROUND: According to the World Health Organization (WHO) there is a pandemic of obesity with approximately 300 million people being obese. Typically, human obesity has a polygenetic causation. Neutral endopeptidase (NEP), also known as neprilysin, is considered to be one of the key enzymes in the metabolism of many active peptide hormones. METHODOLOGY/PRINCIPAL FINDINGS: An incidental observation in NEP-deficient mice was a late-onset excessive gain in body weight exclusively from a ubiquitous accumulation of fat tissue. In accord with polygenetic human obesity, mice were characterized by deregulation of lipid metabolism, higher blood glucose levels, with impaired glucose tolerance. The key role of NEP in determining body mass was confirmed by the use of the NEP inhibitor candoxatril in wild-type mice that increased body weight due to increased food intake. This is a peripheral and not a central NEP action on the switch for appetite control, since candoxatril cannot cross the blood-brain barrier. Furthermore, we demonstrated that inhibition of NEP in mice with cachexia delayed rapid body weight loss. Thus, lack in NEP activity, genetically or pharmacologically, leads to a gain in body fat. CONCLUSIONS/SIGNIFICANCE: In the present study, we have identified NEP to be a crucial player in the development of obesity. NEP-deficient mice start to become obese under a normocaloric diet in an age of 6-7 months and thus are an ideal model for the typical human late-onset obesity. Therefore, the described obesity model is an ideal tool for research on development, molecular mechanisms, diagnosis, and therapy of the pandemic obesity.

Altered GLUT4 trafficking in adipocytes in the absence of the GTPase Arfrp1.

The GTPase ADP-ribosylation factor related protein 1 (ARFRP1) controls the recruitment of proteins such as golgin-245 to the trans-Golgi. ARFRP1 is highly expressed in adipose tissues in which the insulin-sensitive glucose transporter GLUT4 is processed through the Golgi to a specialized endosomal compartment, the insulin-responsive storage compartment from which it is translocated to the plasma membrane in response to a stimulation of cells by insulin. In order to examine the role of ARFRP1 for GLUT4 targeting, subcellular distribution of GLUT4 was investigated in adipose tissue specific Arfrp1 knockout (Arfrp1(ad)(-/-)) mice. Immunohistochemical and ultrastructural studies of brown adipocytes demonstrated an abnormal trans-Golgi in Arfrp1(ad)(-/-) adipocytes. In addition, in Arfrp1(ad)(-/-) adipocytes GLUT4 protein accumulated at the plasma membrane rather than being sequestered in an intracellular compartment. A similar missorting of GLUT4 was produced by siRNA-mediated knockdown of Arfrp1 in 3T3-L1 adipocytes which was associated with significantly elevated uptake of deoxyglucose under basal conditions. Thus, Arfrp1 appears to be involved in sorting of GLUT4.

The ARF-like GTPase ARFRP1 is essential for lipid droplet growth and is involved in the regulation of lipolysis.

ADP-ribosylation factor (ARF)-related protein 1 (ARFRP1) is a GTPase regulating protein trafficking between intracellular organelles. Here we show that mice lacking Arfrp1 in adipocytes (Arfrp1(ad-/-)) are lipodystrophic due to a defective lipid droplet formation in adipose cells. Ratios of mono-, di-, and triacylglycerol, as well as the fatty acid composition of triglycerides, were unaltered. Lipid droplets of brown adipocytes of Arfrp1(ad-/-) mice were considerably smaller and exhibited ultrastructural alterations, such as a disturbed interaction of small lipid-loaded particles with the larger droplets, suggesting that ARFRP1 mediates the transfer of newly formed small lipid particles to the large storage droplets. SNAP23 (synaptosomal-associated protein of 23 kDa) associated with small lipid droplets of control adipocytes but was located predominantly in the cytosol of Arfrp1(ad-/-) adipocytes, suggesting that lipid droplet growth is defective in Arfrp1(ad-/-) mice. In addition, levels of phosphorylated hormone-sensitive lipase (HSL) were elevated, and association of adipocyte triglyceride lipase (ATGL) with lipid droplets was enhanced in brown adipose tissue from Arfrp1(ad-/-) mice. Accordingly, basal lipolysis was increased after knockdown of Arfrp1 in 3T3-L1 adipocytes. The data indicate that disruption of ARFRP1 prevents the normal enlargement of lipid droplets and produces an activation of lipolysis.

Positional cloning of zinc finger domain transcription factor Zfp69, a candidate gene for obesity-associated diabetes contributed by mouse locus Nidd/SJL.

Polygenic type 2 diabetes in mouse models is associated with obesity and results from a combination of adipogenic and diabetogenic alleles. Here we report the identification of a candidate gene for the diabetogenic effect of a QTL (Nidd/SJL, Nidd1) contributed by the SJL, NON, and NZB strains in outcross populations with New Zealand Obese (NZO) mice. A critical interval of distal chromosome 4 (2.1 Mbp) conferring the diabetic phenotype was identified by interval-specific congenic introgression of SJL into diabetes-resistant C57BL/6J, and subsequent reporter cross with NZO. Analysis of the 10 genes in the critical interval by sequencing, qRT-PCR, and RACE-PCR revealed a striking allelic variance of Zfp69 encoding zinc finger domain transcription factor 69. In NZO and C57BL/6J, a retrotransposon (IAPLTR1a) in intron 3 disrupted the gene by formation of a truncated mRNA that lacked the coding sequence for the KRAB (Krüppel-associated box) and Znf-C2H2 domains of Zfp69, whereas the diabetogenic SJL, NON, and NZB alleles generated a normal mRNA. When combined with the B6.V-Lep(ob) background, the diabetogenic Zfp69(SJL) allele produced hyperglycaemia, reduced gonadal fat, and increased plasma and liver triglycerides. mRNA levels of the human orthologue of Zfp69, ZNF642, were significantly increased in adipose tissue from patients with type 2 diabetes. We conclude that Zfp69 is the most likely candidate for the diabetogenic effect of Nidd/SJL, and that retrotransposon IAPLTR1a contributes substantially to the genetic heterogeneity of mouse strains. Expression of the transcription factor in adipose tissue may play a role in the pathogenesis of type 2 diabetes.

Characterization of Nob3, a major quantitative trait locus for obesity and hyperglycemia on mouse chromosome 1.

New Zealand obese (NZO) mice present a metabolic syndrome of obesity, insulin resistance, and diabetes. To identify chromosomal segments associated with these traits, we intercrossed NZO mice with the lean and diabetes-resistant C57BL/6J (B6) strain. Obesity and hyperglycemia in the (NZO x B6)F2 intercross population were predominantly due to a broad quantitative trait locus (QTL) on chromosome 1 (Nob3; logarithm of the odds score 16.1, 16.0, 4.0 for body weight, body fat, and blood glucose, respectively), producing a difference between genotypes of 12.7 or 5.2 g of body weight and 12.0 or 4.0 g of body fat in females or males, respectively. In addition, significant QTL on chromosomes 3 and 13 and suggestive QTL on chromosomes 4, 6, 9, 12, 14, and 19 contributed to the obese phenotype. Distal chromosome 5 was significantly linked with plasma cholesterol (LOD score 10.7). Introgression of two segments of Nob3 into B6 confirmed the adipogenic effect of the QTL and suggested the presence of at least one causal gene. Haplotype mapping reduced the critical region of the distal part of the QTL to 31 Mbp containing the potential candidates Nr1i3, Apoa2, Atp1a2, Prox1, and Hsd11b1. We conclude that obesity and hyperglycemia of NZO is to a large part caused by variant genes located in Nob3 on chromosome 1. Since these exert robust effects on a B6 background, the QTL Nob3 is a prime target for identification of a novel diabesity gene.

Tbc1d1 mutation in lean mouse strain confers leanness and protects from diet-induced obesity.

We previously identified Nob1 as a quantitative trait locus for high-fat diet-induced obesity and diabetes in genome-wide scans of outcross populations of obese and lean mouse strains. Additional crossbreeding experiments indicated that Nob1 represents an obesity suppressor from the lean Swiss Jim Lambert (SJL) strain. Here we identify a SJL-specific mutation in the Tbc1d1 gene that results in a truncated protein lacking the TBC Rab-GTPase-activating protein domain. TBC1D1, which has been recently linked to human obesity, is related to the insulin signaling protein AS160 and is predominantly expressed in skeletal muscle. Knockdown of TBC1D1 in skeletal muscle cells increased fatty acid uptake and oxidation, whereas overexpression of TBC1D1 had the opposite effect. Recombinant congenic mice lacking TBC1D1 showed reduced body weight, decreased respiratory quotient, increased fatty acid oxidation and reduced glucose uptake in isolated skeletal muscle. Our data strongly suggest that mutation of Tbc1d1 suppresses high-fat diet-induced obesity by increasing lipid use in skeletal muscle.

Neuronal functions, feeding behavior, and energy balance in Slc2a3+/- mice.

Homozygous deletion of the gene of the neuronal glucose transporter GLUT3 (Slc2a3) in mice results in embryonic lethality, whereas heterozygotes (Slc2a3+/-) are viable. Here, we describe the characterization of heterozygous mice with regard to neuronal function, glucose homeostasis, and, since GLUT3 might be a component of the neuronal glucose-sensing mechanism, food intake and energy balance. Levels of GLUT3 mRNA and protein in brain were reduced by 50% in Slc2a3+/- mice. Electrographic features examined by electroencephalographic recordings give evidence for slightly but significantly enhanced cerebrocortical activity in Slc2a3+/- mice. In addition, Slc2a3+/- mice were slightly more sensitive to an acoustic startle stimulus (elevated startle amplitude and reduced prepulse inhibition). However, systemic behavioral testing revealed no other functional abnormalities, e.g., in coordination, reflexes, motor abilities, anxiety, learning, and memory. Furthermore, no differences in body weight, blood glucose, and insulin levels were detected between wild-type and Slc2a3+/- littermates. Food intake as monitored randomly or after intracerebroventricular administration of 2-deoxyglucose or d-glucose, or food choice for carbohydrates/fat was not affected in Slc2a3+/- mice. Taken together, our data indicate that, in contrast to Slc2a1, a single allele of Slc2a3 is sufficient for maintenance of neuronal energy supply, motor abilities, learning and memory, and feeding behavior.

Targeted disruption of Slc2a8 (GLUT8) reduces motility and mitochondrial potential of spermatozoa.

GLUT8 is a class 3 sugar transport facilitator which is predominantly expressed in testis and also detected in brain, heart, skeletal muscle, adipose tissue, adrenal gland, and liver. Since its physiological function in these tissues is unknown, we generated a Slc2a8 null mouse and characterized its phenotype. Slc2a8 knockout mice appeared healthy and exhibited normal growth, body weight development and glycemic control, indicating that GLUT8 does not play a significant role for maintenance of whole body glucose homeostasis. However, analysis of the offspring distribution of heterozygous mating indicated a lower number of Slc2a8 knockout offspring (30.5:47.3:22.1%, Slc2a8(+/+), Slc2a8(+/-), and Slc2a8(-/-) mice, respectively) resulting in a deviation (p=0.0024) from the expected Mendelian distribution. This difference was associated with lower ATP levels, a reduced mitochondrial membrane potential and a significant reduction of sperm motility of the Slc2a8 knockout in comparison to wild-type spermatozoa. In contrast, number and survival rate of spermatozoa were not altered. These data indicate that GLUT8 plays an important role in the energy metabolism of sperm cells.

High-fat, carbohydrate-free diet markedly aggravates obesity but prevents beta-cell loss and diabetes in the obese, diabetes-susceptible db/db strain.

OBJECTIVE: We have previously reported that a high-fat, carbohydrate-free diet prevents diabetes and beta-cell destruction in the New Zealand Obese (NZO) mouse strain. Here we investigated the effect of diets with and without carbohydrates on obesity and development of beta-cell failure in a second mouse model of type 2 diabetes, the db/db mouse. RESULTS: When kept on a carbohydrate-containing standard (SD; with (w/w) 5.1, 58.3, and 17.6% fat, carbohydrates and protein, respectively) or high-fat diet (HFD; 14.6, 46.7 and 17.1%), db/db mice developed severe diabetes (blood glucose >20 mmol/l, weight loss, polydipsia and polyurea) associated with a selective loss of pancreatic beta-cells, reduced GLUT2 expression in the remaining beta-cells, and reduced plasma insulin levels. In contrast, db/db mice kept on a high-fat, carbohydrate-free diet (CFD; with 30.2 and 26.4% (w/w) fat or protein) did not develop diabetes and exhibited near-normal, hyperplastic islets in spite of a morbid obesity (fat content >60%) associated with hyperinsulinaemia. CONCLUSION: These data indicate that in genetically different mouse models of obesity-associated diabetes, obesity and dietary fat are not sufficient, and dietary carbohydrates are required, for beta-cell destruction.