Biomedical subjects

K Walder

Publications and source records attributed to K Walder.

At least 19 recordsLinked to original sources

PubMed2005

The mitochondrial rhomboid protease PSARL is a new candidate gene for type 2 diabetes.

AIMS/HYPOTHESIS: This study aimed to identify genes that are expressed in skeletal muscle, encode proteins with functional significance in mitochondria, and are associated with type 2 diabetes. METHODS: We screened for differentially expressed genes in skeletal muscle of Psammomys obesus (Israeli sand rats), and prioritised these on the basis of genomic localisation and bioinformatics analysis for proteins with likely mitochondrial functions. RESULTS: We identified a mitochondrial intramembrane protease, known as presenilins-associated rhomboid-like protein (PSARL) that is associated with insulin resistance and type 2 diabetes. Expression of PSARL was reduced in skeletal muscle of diabetic Psammomys obesus, and restored after exercise training to successfully treat the diabetes. PSARL gene expression in human skeletal muscle was correlated with insulin sensitivity as assessed by glucose disposal during a hyperinsulinaemic-euglycaemic clamp. In 1,031 human subjects, an amino acid substitution (Leu262Val) in PSARL was associated with increased plasma insulin concentration, a key risk factor for diabetes. Furthermore, this variant interacted strongly with age to affect insulin levels, accounting for 5% of the variation in plasma insulin in elderly subjects. CONCLUSIONS/INTERPRETATION: Variation in PSARL sequence and/or expression may be an important new risk factor for type 2 diabetes and other components of the metabolic syndrome.

Amino Acid Sequence↗

PubMed2004

Identification and characterization of a lymphocytic Rho-GTPase effector: rhotekin-2.

Rhotekin belongs to the group of proteins containing a Rho-binding domain that are target peptides (effectors) for the Rho-GTPases. We previously identified a novel cDNA with homology to human rhotekin and in this study we cloned and characterized the coding region of this novel 12-exon gene. The ORF encodes a 609 amino-acid protein comprising a Class I Rho-binding domain and pleckstrin homology (PH) domain. Cellular cDNA expression of this new protein, designated Rhotekin-2 (RTKN2), was shown in the cytosol and nucleus of CHO cells. Using bioinformatics and RTPCR we identified three major splice variants, which vary in both the Rho-binding and PH domains. Real-time PCR studies showed exclusive RTKN2 expression in pooled lymphocytes and further purification indicated sole expression in CD4(pos) T-cells and bone marrow-derived B-cells. Gene expression was increased in quiescent T-cells but negligible in activated proliferating cells. In malignant samples expression was absent in myeloid leukaemias, low in most B-cell malignancies and CD8(pos) T-cell malignancies, but very high in CD4(pos)/CD8(pos) T-lymphoblastic lymphoma. As the Rho family is critical in lymphocyte development and function, RTKN2 may play an important role in lymphopoiesis.

Amino Acid Sequence↗

PubMed2003

ORP3 splice variants and their expression in human tissues and hematopoietic cells.

ORP3 is a member of the newly described family of oxysterol-binding protein (OSBP)-related proteins (ORPs). We previously demonstrated that this gene is highly expressed in CD34(+) hematopoietic progenitor cells, and deduced that the "full-length" ORP3 gene comprises 23 exons and encodes a predicted protein of 887 amino acids with a C-terminal OSBP domain and an N-terminal pleckstrin homology domain. To further characterize the gene, we cloned ORP3 cDNA from PCR products and identified multiple splice variants. A total of eight isoforms were demonstrated with alternative splicing of exons 9, 12, and 15. Isoforms with an extension to exon 15 truncate the OSBP domain of the predicted protein sequence. In human tissues there was specific isoform distribution, with most tissues expressing varied levels of isoforms with the complete OSBP domain; while only whole brain, kidney, spleen, thymus, and thyroid expressed high levels of the isoforms associated with the truncated OSBP domain. Interestingly, the expression in cerebellum, heart, and liver of most isoforms was negligible. These data suggest that differential mRNA splicing may have resulted in functionally distinct forms of the ORP3 gene.

Alternative Splicing↗

PubMed2003

Obesity and diabetes gene discovery approaches.

New treatments are currently required for the common metabolic diseases obesity and type 2 diabetes. The identification of physiological and biochemical factors that underlie the metabolic disturbances observed in obesity and type 2 diabetes is a key step in developing better therapeutic outcomes. The discovery of new genes and pathways involved in the pathogenesis of these diseases is critical to this process, however identification of genes that contribute to the risk of developing these diseases represents a significant challenge as obesity and type 2 diabetes are complex diseases with many genetic and environmental causes. A number of diverse approaches have been used to discover and validate potential new targets for obesity and diabetes. To date, DNA-based approaches using candidate gene and genome-wide linkage analysis have had limited success in identifying genomic regions or genes involved in the development of these diseases. Recent advances in the ability to evaluate linkage analysis data from large family pedigrees using variance components based linkage analysis show great promise in robustly identifying genomic regions associated with the development of obesity and diabetes. RNA-based technologies such as cDNA microarrays have identified many genes differentially expressed in tissues of healthy and diseased subjects. Using a combined approach, we are endeavouring to focus attention on differentially expressed genes located in chromosomal regions previously linked with obesity and/or diabetes. Using this strategy, we have identified Beacon as a potential new target for obesity and diabetes.

Amino Acid Sequence↗

PubMed2003

The Taq IA and Ser311 Cys polymorphisms in the dopamine D2 receptor gene and obesity.

Dopamine D2 receptors (DRD2) in the central nervous system are involved in the regulation of feeding. It remains to be elucidated if mutations in the DRD2 gene contribute to the development of obesity. The aim of the present study was to investigate whether the Taq IA and Ser311Cys polymorphisms in the DRD2 gene are associated with obesity in Nauruan and Australian subjects. Subjects were selected based on extremes of the body mass index (BMI) distribution. Two groups of Australian women were selected. The leanest group had a mean BMI of 22.5 kg/m2 (range: 20.3-24.3) and the heaviest group had a mean of 36.1 kg/m2 (32.5-44.1). Four groups of Nauruan subjects were selected. Leanest men had a mean BMI of 33.0 kg/m2 (28.4-36.9), heaviest men had a mean of 52.8 kg/m2 (46.5-69.2), leanest women had a mean of 34.8 kg/m2 (28.2-41.8) and heaviest women had a mean of 55.1 kg/m2 (49.3-73.8). Subjects were genotyped for the Taq IA and Ser311Cys polymorphisms using polymerase chain reaction (PCR) restriction fragment length polymorphism analysis and allelic discrimination Taqman PCR respectively. Leanest and heaviest groups were examined for differences in genotype frequency. Taq IA and Ser311Cys genotype frequencies did not differ significantly between leanest and heaviest Nauruan groups, or between leanest and heaviest Australians. Haplotype frequencies of these polymorphisms did not differ between leanest and heaviest groups. The Taq IA and Ser311Cys polymorphisms in the DRD2 gene are unlikely to be common causes of obesity in these populations.

Adult↗

PubMed2002

Calpain 3 gene expression in skeletal muscle is associated with body fat content and measures of insulin resistance.

OBJECTIVE: To investigate whether skeletal muscle gene expression of calpain 3 is related to obesity and insulin resistance. DESIGN: Cross-sectional studies in 27 non-diabetic human subjects and in Psammomys obesus, a polygenic animal model of obesity and type 2 diabetes. MEASUREMENTS: Expression of CAPN3 in skeletal muscle was measured using Taqman fluorogenic PCR. In the human subjects, body composition was assessed by DEXA and insulin sensitivity was measured by euglycemic-hyperinsulinemic clamp. In Psammomys obesus, body composition was determined by carcass analysis, and substrate oxidation rates, physical activity and energy expenditure were measured by whole-body indirect calorimetry. RESULTS: In human subjects, calpain 3 gene expression was negatively correlated with total (P = 0.022) and central abdominal fat mass (P = 0.034), and with blood glucose concentration in non-obese subjects (P = 0.017). In Psammomys obesus, calpain 3 gene expression was negatively correlated with circulating glucose (P = 0.013) and insulin (P = 0.034), and with body fat mass (P = 0.049). Indirect calorimetry revealed associations between calpain 3 gene expression and carbohydrate oxidation (P = 0.009) and energy expenditure (P = 0.013). CONCLUSION/INTERPRETATION: Lower levels of expression of calpain 3 in skeletal muscle were associated with reduced carbohydrate oxidation and elevated circulating glucose and insulin concentrations, and also with increased body fat and in particular abdominal fat. Therefore, reduced expression of calpain 3 in both humans and Psammomys obesus was associated with phenotypes related to obesity and insulin resistance.

Adipose Tissue↗

PubMed2002

Elevated hypothalamic beacon gene expression in Psammomys obesus prone to develop obesity and type 2 diabetes.

OBJECTIVE: To investigate hypothalamic beacon gene expression at various developmental stages in genetically selected diabetes-resistant and diabetes-prone Psammomys obesus. In addition, effects of dietary energy composition on beacon gene expression were investigated in diabetes-prone P. obesus. METHODS: Hypothalamic beacon gene expression was measured using Taqman fluorogenic PCR in 4-, 8- and 16-week-old animals from each genetically selected line. RESULTS: Expression of beacon was elevated in the diabetes-prone compared with diabetes-resistant P. obesus at 4 weeks of age despite no difference in body weight between the groups. At 8 weeks of age, hypothalamic beacon gene expression was elevated in diabetes-prone animals fed a high-energy diet, and was correlated with serum insulin concentration. CONCLUSION: P. obesus with a genetic predisposition for the development of obesity and type 2 diabetes have elevated hypothalamic beacon gene expression at an early age. Overexpression of beacon may contribute to the development of obesity and insulin resistance in these animals.

Aging↗

PubMed2001

AFLP fingerprinting of the human genome.

Elucidation of the genetic basis of complex traits and diseases in humans includes the use of genome-wide association studies that depend on the analysis of a large number of diallelic markers. We describe the application of the amplified fragment length polymorphism (AFLP) technique as an efficient approach for rapidly identifying and scoring multiple variants in the human genome. Using a commercially available kit, we found that AFLP yields reproducible DNA fingerprints consisting of 42-132 fragments, 8% of which show variability between individuals. These variant markers appear to be from different chromosomes, and the majority of them is diallelic. Based on the information obtained in this study, it is possible to approximate the minimum number of selective AFLP primer combinations needed to approach a desired coverage density of all chromosomes. To our knowledge, this is the first study showing the general applicability of AFLP in humans and providing a constructive guide for the design of genomic studies in Homo sapiens with this robust methodology.

DNA Fingerprinting↗

PubMed2001

Effects of beacon administration on energy expenditure and substrate utilisation in Psammomys obesus (Israeli sand rats).

OBJECTIVE: To investigate whether beacon administration affects substrate utilisation, physical activity levels or energy expenditure in Psammomys obesus. DESIGN: Pairs of age- and sex-matched Psammomys obesus were randomly assigned to either beacon-treated (15 microg/day for 7 days (i.c.v.)) or control (i.c.v. saline) groups. MEASUREMENTS: Indirect calorimetry on day 0 and day 7 to measure oxygen consumption and carbon dioxide production, which were used to calculate fat oxidation, carbohydrate oxidation and total energy expenditure. Physical activity in the calorimeter was measured using an infrared beam system. Food intake and body weight were measured daily. RESULTS: The administration of beacon significantly increased body weight compared to saline-treated control animals. This body weight gain was primarily due to increased body fat content. Average daily food intake tended to be higher in beacon-treated Psammomys obesus, but no effect of beacon administration on substrate oxidation, activity or energy expenditure was detected. CONCLUSION: The effects of beacon on body weight are due to increased food intake, with no detectable effect on nutrient partitioning, physical activity or energy expenditure.

Adipose Tissue↗

PubMed2000

A polymorphism in the gene encoding CART is not associated with obesity in Pima Indians.

Cocaine- and amphetamine-regulated transcript (CART) is a hypothalamic peptide that may be involved in the regulation of food intake. We screened the human CART gene and identified a novel C-->G substitution in the 3'-untranslated region of exon 3. This polymorphism was genotyped in a total of 68 Pima Indians with extremes of BMI. The frequencies for alleles C and G were 0.76 and 0.24, respectively. There was no evidence of an association between the genotype frequency at this variant and extremes of BMI in this group of Pima Indians. The results of this study do not suggest a significant role for this variant in exon 3 of CART as a determinant of obesity in Pima Indians.

Adult↗

PubMed2000

An autosomal genomic scan for loci linked to plasma leptin concentration in Pima Indians.

OBJECTIVE: To identify chromosomal regions linked to plasma leptin concentrations. DESIGN: Autosomal genome-wide scan, including 516 microsatellite markers. Sib-pair (Haseman-Elston) and variance components methods were used to assess genetic linkage. SUBJECTS: 770 Pima Indians comprising 239 nuclear families (for a total of 1199 sibling-pairs). MEASUREMENTS: Plasma leptin concentrations and body mass index (BMI), adjusted for age and sex. RESULTS: The strongest evidence for linkage with plasma leptin concentration was on chromosome 6p logarithm of odds (LOD) = 2.1 by variance components analysis). There was no evidence for linkage to BMI in this region. Additional regions with marginal evidence for linkage to plasma leptin concentration (LOD > or =1.0) were detected on chromosomes 3, 11, 13, 15 and 16. CONCLUSIONS: The results suggest that a locus on chromosome 6p influences plasma leptin concentrations. Replication studies are needed to exclude the possibility that linkage has been falsely detected.

Adolescent↗

PubMed2000

Novel polymorphisms in the neuropeptide-Y Y5 receptor associated with obesity in Pima Indians.

OBJECTIVE: To investigate whether the neuropeptide Y receptor 5 gene (NPY5R) is associated with obesity in humans. DESIGN: The NPY5R gene was screened for polymorphisms by direct sequencing in two groups of Pima Indians, selected for extremes of body mass index (BMI). Genotype frequencies were analyzed for association with BMI extreme. SUBJECTS: Full-heritage Pima Indians, non-diabetic and not first degree relatives. Obese group: 19 M/24 F, BMI = 49+/-7 kg/m2 (mean+/-s.d.) age = 24+/-2 y, lean group: 16 M/16 F, BMI = 23+/-2 kg/m2, age = 27+/-3 y. MEASUREMENTS: Initially, the entire gene (proximal promoter, exon 1A, coding sequence, 5' and 3' UTRs) was sequenced in a subset of 20 individuals. No variants were found in the coding sequence, however three novel single nucleotide polymorphisms were detected in the non-coding regions: (1) a C-->T transition located within the promoter 28 bp upstream of the exon 1A transcription start site; (2) a T-->C transition 94 bp downstream of the stop codon; and (3) a G-->A transition 432 bp downstream of the stop codon. The polymorphisms were then screened in all 75 subjects. RESULTS: The polymorphisms had mean heterozygosities of 0.34-0.50 and were in strong linkage disequilibrium (P<0.001). Genotype frequencies differed significantly in lean and obese Pimas for P2 (P=0.04) and for a triple haplotype (P=0.02, Bonferroni corrected). CONCLUSION: Considering the importance of this gene in regulation of body weight, the association of these polymorphisms with extremes of BMI in Pima Indians indicates that NPY5R, or a locus nearby, may contribute to susceptibility to obesity in this population.

Adult↗

PubMed2000

Impact of obesity and leptin treatment on adipocyte gene expression in Psammomys obesus.

We examined the effects of leptin treatment on the expression of key genes in adipocyte metabolism in Psammomys obesus (P. obesus), a polygenic rodent model of obesity. Lean and obese P. obesus were given three daily intraperitoneal injections of either saline or leptin (total of 45 mg/kg per day) for 7 days. In lean animals, leptin treatment led to reductions in food intake, body weight and fat mass. Pair-fed animals matched for the reduction in food intake of the lean leptin-treated animals demonstrated similar reductions in body weight and fat mass. In obese P. obesus, leptin treatment failed to have any effect on body weight or body fat mass, indicating leptin resistance. Lipoprotein lipase, hormone-sensitive lipase and peroxisome proliferator activated receptor gamma 2 mRNA levels were significantly reduced in lean leptin-treated animals, whereas pair-fed animals were similar to lean controls. Uncoupling protein 2 and glycerol phosphate acyltransferase were also reduced in the lean leptin-treated animals, but not significantly so. Obese animals did not show any gene expression changes after leptin treatment. In conclusion, high circulating concentrations of leptin in lean P. obesus resulted in decreased gene expression of a number of key lipid enzymes, independent of changes in food intake, body weight and fat mass. These effects of leptin were not found in obese P. obesus.

Adipocytes↗

PubMed2000

Beacon: a novel gene involved in the regulation of energy balance.

The hypothalamus plays a major role in the control of energy balance via the coordination of several neuropeptides and their receptors. We used a unique polygenic animal model of obesity, Psammomys obesus, and performed differential display polymerase chain reaction on hypothalamic mRNA samples to identify novel genes involved in obesity. In this study, we describe a novel gene that encodes a small protein we have termed "beacon." Beacon mRNA gene expression in the hypothalamus was positively correlated with percentage of body fat. Intracerebroventricular infusion of beacon resulted in a dose-dependent increase in food intake and body weight and an increase in hypothalamic expression of neuropeptide Y (NPY). Simultaneous infusion of beacon and NPY significantly potentiated the orexigenic response and resulted in rapid body weight gain. These data suggest a role for beacon in the regulation of energy balance and body weight homeostasis that may be mediated, at least in part, through the NPY pathway.

Adipose Tissue↗

PubMed1999

Human uncoupling proteins and obesity.

Uncoupling protein (UCP) 2 and UCP3 are newly discovered proteins that can uncouple ATP production from mitochondrial respiration, thereby dissipating energy as heat and affecting energy metabolism efficiency. In contrast to UCP1, which is only present in brown adipose tissue, UCP2 has a wide tissue distribution, whereas UCP3 is expressed predominantly in skeletal muscle. Some evidence of a role for UCPs in modulating metabolic rate was provided by linkage and association studies. Furthermore, UCP3 gene expression was found to correlate negatively with body mass index and positively with sleeping metabolic rate in Pima Indians. Treatment with thyroid hormone increases expression of the UCP2 and UCP3 genes. Other regulators of UCP2 and UCP3 gene expression are beta3-adrenergic agonists and glucocorticoids. Surprisingly, fasting has a stimulatory effect on UCP2 and UCP3 mRNA levels, possibly explained by the effects of free fatty acid on UCP2 and UCP3 gene expression.

Adenosine Triphosphate↗

PubMed1999

Leptin resistance in a polygenic, hyperleptinemic animal model of obesity and NIDDM: Psammomys obesus.

OBJECTIVE: To investigate the effects of leptin administration to Psammomys obesus, a polygenic animal model of obesity and type 2 diabetes mellitus. DESIGN: Longitudinal intervention study utilising three separate leptin treatment protocols lasting 7-14 d. MEASUREMENTS: Body weight and food intake were measured daily, body fat and muscle content were estimated by carcass analysis on completion of the study. Blood glucose, plasma insulin, leptin, triglycerides and cholesterol were measured at baseline and twice each week during the study. RESULTS: Relatively high doses of leptin were required to significantly reduce food intake and body fat content in lean Psammomys obesus, but had no discernible effect on their obese littermates. CONCLUSION: As a species, Psammomys obesus appear to be relatively insensitive to the effects of leptin administration, compared with other rodents. Obese Psammomys obesus are leptin resistant relative to their lean littermates.

Animals↗

PubMed1999

A novel polymorphism in the proximal UCP3 promoter region: effect on skeletal muscle UCP3 mRNA expression and obesity in male non-diabetic Pima Indians.

OBJECTIVE: UCP2 and UCP3 are newly discovered uncoupling proteins, which are thought to underlie the variability in energy metabolism in humans. Mutations in the UCP2 and/or UCP3 gene have been associated with sleeping metabolic rate. Recently we reported that skeletal muscle UCP3 mRNA expression was positively correlated with sleeping metabolic rate in Pima Indians. To study whether genetic variation in the promoter region of UCP3 contributed to the variation in expression of UCP3, we screened part of the proximal promoter region for polymorphisms. METHODS: In the first part of the study, the proximal promoter region of UCP3 was screened by direct sequencing in 24 non-diabetic Pima Indians (range body mass index (BMI): 18-47 kg/m2) (Schrauwen et al. Diabetes 1999; 48: 146-149) and skeletal muscle UCP3 mRNA expression was measured by RT-PCR. In the second part of the study, we typed the polymorphism found in the first part of the study in 67 Pima Indians (32 males, 35 females) from the upper and lower extremes of the BMI distribution. RESULTS: We identified a novel C to T substitution in the UCP3 promoter, 6bp upstream of the putative TATA signal, and 55bp upstream of the transcription starting site. Among 18 male subjects, skeletal muscle UCP3 mRNA expression was significantly higher in the C/T & T/T group compared to the C/C homozygotes (P<0.02). However, in the group of 67 Pima Indians genotype frequencies were not different in the obese and lean groups. CONCLUSION: We identified a novel polymorphism in the proximal promoter region of UCP3, which was associated with increased skeletal muscle expression of UCP3 in male non-diabetic Pima Indians. Considering the suggested role of UCP3 in energy metabolism, this polymorphism might be of physiological importance in the regulation of energy balance.

Amino Acid Substitution↗

PubMed1998

Dysregulation of leptin in response to fasting in insulin-resistant Psammomys obesus (Israeli sand rats).

Leptin is thought to play a significant role in energy balance as an afferent signal to the hypothalamus that reflects body fat content. In addition, leptin may also act as an acute sensor of energy balance independent of body fat mass, since ob gene expression and plasma leptin concentrations are decreased in lean animals and humans in response to short-term caloric deprivation. However, in obese animals and humans, the acute response of leptin to fasting is less clear. We investigated the effects of a 24-hour fast on circulating plasma leptin concentrations in lean and obese Psammomys obesus (Israeli sand rats). In the lean, insulin-sensitive group (n = 25) a 24-hour fast caused a 44% decrease in plasma leptin, whereas in the obese, insulin-resistant group (n = 24) plasma leptin increased by 18% after fasting (P < .003). There was no difference between the two groups regarding the effect of a 24-hour fast on body weight, blood glucose, or plasma insulin. Within the insulin-resistant group, there was no difference in the response of leptin to fasting between hyperglycemic and normoglycemic animals. We conclude that there is a dysregulation of leptin in response to acute caloric deprivation in obese, insulin-resistant but not in lean, insulin-sensitive P obesus.

Animals↗