Genetic variation in SH3-domain GRB2-like (endophilin)-interacting protein 1 has a major impact on fat mass.

OBJECTIVE: The SH3-domain GRB2-like (endophilin)-interacting protein 1 (SGIP1) gene has been shown to be differentially expressed in the hypothalamus of lean versus obese Israeli sand rats (Psammomys obesus), and is suspected of having a role in regulating food intake. The purpose of this study was to assess the role of genetic variation in SGIP1 in human disease. SUBJECTS: We performed single-nucleotide polymorphism (SNP) genotyping in a large family pedigree cohort from the island of Mauritius. The Mauritius Family Study (MFS) consists of 400 individuals from 24 Indo-Mauritian families recruited from the genetically homogeneous population of Mauritius. We measured markers of the metabolic syndrome, including diabetes and obesity-related phenotypes such as fasting plasma glucose, waist:hip ratio, body mass index and fat mass. RESULTS: Statistical genetic analysis revealed associations between SGIP1 polymorphisms and fat mass (in kilograms) as measured by bioimpedance. SNP genotyping identified associations between several genetic variants and fat mass, with the strongest association for rs2146905 (P=4.7 × 10(-5)). A strong allelic effect was noted for several SNPs where fat mass was reduced by up to 9.4% for individuals homozygous for the minor allele. CONCLUSIONS: Our results show association between genetic variants in SGIP1 and fat mass. We provide evidence that variation in SGIP1 is a potentially important determinant of obesity-related traits in humans.

Cross-species replication of a resistin mRNA QTL, but not QTLs for circulating levels of resistin, in human and baboon.

Resistin has been associated with inflammation and risk for cardiovascular disease. We previously reported evidence of a QTL on chromosome 19p13 affecting the abundance of resistin (RETN) mRNA in the omental adipose tissue of baboons (L0D score 3.8). In this study, whole genome transcription levels were assessed in human lymphocyte samples from 1240 adults participating in the San Antonio Family Heart Study, using the Sentrix Human-6 Expression Beadchip. Lymphocytes were surveyed, as it has been proposed that their expression levels may reflect those in harder to ascertain tissues, such as adipose tissue, that are thought to be more directly relevant to disease procesn was conducted to detect loci affecting RETN mRNA levels. We obtained significant evidence for a QTL influencing the RETN expression (LOD score 10.7) on chromosome 19p. This region is orthologous/homologous to the region previously localized on baboon chromosome 19. The strongest positional candidate gene in this region is the structural gene for resistin, itself. We also found evidence for a QTL influencing resistin protein levels (LOD score 5.3) on chromosome 14q. This differs from our previously reported QTL on chromosome 18 in baboons. The different QTLs for circulating protein suggests that post-translational processing and turnover may be influenced by different or multiple genes in baboons and humans. The parallel findings of a cis-eQTL for RETN mRNA in baboon omental tissue and human lymphocytes lends support to the strategy of using lymphocyte gene expression levels as a surrogate for gene expression levels in other tissues.

Effect of retinoic acid on cellular content and human parathyroid hormone activation of cyclic adenosine 3':5'-monophosphate-dependent protein kinase isoenzymes in clonal rat osteogenic sarcoma cells.

Pretreatment with 10(-8) M retinoic acid for 4 days caused changes in three distinct components of the parathyroid hormone (PTH)-stimulated cyclic adenosine 3':5'-monophosphate response in a clonal rat osteogenic sarcoma cell line, UMR 106-06: the amplitude of the cyclic adenosine 3':5'-monophosphate response to PTH was moderately increased after pretreatment with retinoic acid; while the cellular content of the two isoenzymes of the cyclic adenosine 3':5'-monophosphate-dependent protein kinase was approximately equal in control cells, retinoic acid pretreatment was associated with a marked increase in the ratio of type II to type I holoenzyme activity. This change might be due to a decrease in the type I holoenzyme as suggested by immunofluorescence detection of decreased type I regulatory subunit in fixed cells together with the relative decrease in type I holoenzyme determined biochemically; there was a marked alteration of the pattern of PTH-stimulated protein kinase isoenzyme activation from predominantly type I isoenzyme in control cells to almost exclusively type II isoenzyme in retinoic acid-treated cells. Growth inhibition by submaximal amounts of PTH and retinoic acid when added together was greater than that for either agent alone.

Ob (obese) gene expression and leptin levels in Psammomys obesus.

In this study we investigated ob gene expression and plasma leptin levels in Psammomys obesus (the Israeli Sand Rat), a polygenic animal model of obesity and non-insulin-dependent diabetes mellitus. The ob gene was expressed exclusively in adipocytes of Psammomys obesus. DNA sequencing revealed a high degree of homology with other species (90% with mouse, 88% with rat and 79% with human). No ob gene sequence differences were found between lean and obese Psammomys obesus, and the codon 105 mutation found in ob/ob mice was not detected. Ob gene expression in Psammomys obesus correlated with body weight (r = 0.436, p < 0.001), percent body fat (r = 0.645, p < 0.001) and plasma insulin concentration (r = 0.651, p < 0.001). This is the first time that ob gene expression has been shown to increase steadily over a continuous wide range of body weight or plasma insulin in an animal model of obesity. Ob gene expression was significantly elevated in obese compared with lean Psammomys obesus (p < 0.05). No significant difference in ob gene expression was found between the four adipose tissue depots tested. Psammomys obesus plasma leptin levels correlated with body weight (r = 0.36, p < 0.05), percent body fat (r = 0.702, p < 0.01) and plasma insulin concentration (r = 0.735, p < 0.001). Plasma leptin concentrations were significantly increased in insulin-resistant animals independent of body weight. These results show that Psammomys obesus is an excellent animal model in which to study the ob gene and leptin, and confirm the importance of insulin as a significant factor in the regulation of leptin and ob gene expression.

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.

The acute effect of fat on insulin secretion.

Previous studies suggest that the rate of rise of the plasma glucose-dependent insulinotropic peptide (GIP) concentration, rather than the steady state level achieved, may be the stimulus of the increased insulin secretion that occurs when fat is ingested with carbohydrate. To test this hypothesis six normal men were given a 5-g iv bolus dose of glucose 15 min after a carbohydrate meal with or without fat. At the time of the iv glucose injection after the fat-containing meal, the rate of rise of plasma GIP was maximum, but the level was only 40% of the achieved by 30 min. Plasma GIP did not change after the meal without fat. After the fat meal, peak insulin and C-peptide levels in response to iv glucose were 60% greater than those after carbohydrate alone despite similar peak blood glucose levels. The calculated insulin clearance was not altered by the fat meal. We conclude that glucose-stimulated insulin secretion is increased early after fat ingestion, possibly due to a rise in GIP or other incretins.

Serum leptin levels are associated with bone mass in nonobese women.

Both serum leptin and bone mineral density are positively correlated with body fat, generating the hypothesis that leptin may be a systemic and/or local regulator of bone mass. We investigated 214 healthy, nonobese Australian women aged 20-91 yr. Bone mineral content, projected bone area, and body fat mass were measured by dual energy x-ray absorptiometry and fasting serum leptin levels by RIA. Associations between bone mineral content (adjusted for age, body weight, body fat mass, and bone area) and the natural logarithm of serum leptin concentrations were analyzed by multiple regression techniques. There was a significant positive association at the lateral spine, two proximal femur sites (Ward's triangle and trochanter), and whole body (partial r(2) = 0.019 to 0.036; all P < 0.05). Similar trends were observed at the femoral neck and posterior-anterior-spine. With bone mineral density the dependent variable (adjusted for age, body weight, and body fat mass), the association with the natural logarithm of leptin remained significant at the lateral spine (partial r(2) = 0.030; P = 0.011), was of borderline significance at the proximal femur sites (partial r(2) = 0.012 to 0.017; P = 0.058 to 0.120), and was not significant at the other sites. Our results demonstrate an association between serum leptin levels and bone mass consistent with the hypothesis that circulating leptin may play a role in regulating bone mass.

Associations between the leptin receptor gene and adiposity in middle-aged Caucasian males from the HERITAGE family study.

Linkage and association studies between three exonic polymorphisms in the leptin receptor gene and body composition variables in the HERITAGE Family Study were undertaken. Polymorphisms K109R, Q223R, and K656N have been analyzed with body mass index (BMI), sum of height skinfolds (SF8), fat mass (FM), percent body fat (%FAT), fat free mass, and plasma leptin level. Single-point linkage analysis and covariance analysis across genotypes were performed, by race, on phenotypes adjusted for age and sex. Blacks (88 parents; 231 adult offspring) from 115 nuclear families (72-119 sibpairs) and Caucasians (192 parents; 330 adult offspring) from 99 nuclear families (319-364 sibpairs) were used for these analyses. In Caucasians, BMI and FM showed suggestive linkages with K109R (P = 0.02 and P = 0.05, respectively) and associations with Q223R (P = 0.005 and P = 0.03, respectively). In blacks, no statistically significant linkage or association was observed. In Caucasians, associations with Q223R were observed in parents, but not in offspring, for BMI, FM, and %FAT (0.04< or =P< or =0.0001). Males, not females, showed differences across genotypes for the same phenotypes plus SF8 and leptin (0.03< or = P< or =0.0002). Carriers of the R223 allele showed higher values than noncarriers for BMI (+4 U, P = 0.0001), SF8 (+30 mm, P = 0.01), FM (+7 kg, P = 0.0004), %FAT (+5%, P = 0.0002), and leptin (+4 ng/mL, P = 0.0006). These results indicate a significant effect of leptin receptor on adiposity in middle-aged Caucasian males.

Ingestion of different types of fat in the evening meal does not affect metabolic responses to a standard breakfast.

High-fat diets produce insulin resistance, but it is not known how quickly changes become evident or whether different types of fat produce different responses. The aim of this study was to determine whether a high-fat evening meal affected glucose response to a standard breakfast 12 h later. On six occasions eight weight-stable subjects consumed a standard evening meal (109 g carbohydrate, 27 g protein, 6 g fat, and 10.6 g fiber) that was either unsupplemented or supplemented with 41 g fat (safflower oil, olive oil, butter, or medium-chain triglyceride) or 75 g carbohydrate followed by a standard breakfast. Glucose, insulin, and free fatty acid responses were measured over 3 h. There were no differences in metabolic responses to the standard breakfast after the different evening meals, indicating that a single high-fat meal has no deleterious effects on carbohydrate metabolism 12 h later, regardless of the type of fat ingested.

Concurrent ingestion of fat and reduction in starch content impairs carbohydrate tolerance to subsequent meals.

We examined the effect of breakfasts of different macronutrient compositions on blood glucose and insulin levels after a standard lunch fed 4 h later. Fat-containing breakfasts were compared with a breakfast of equal carbohydrate and protein content and another with higher carbohydrate but equal calorie content. Concurrent ingestion of fat resulted in a large impairment of glucose tolerance following the standard lunch 4 h later. There was no significant difference in glucose and insulin if the fat eaten was butter or peanut butter. When the fat breakfast was compared with the isocaloric larger carbohydrate meal, the effect of fat on carbohydrate tolerance at lunch was even greater. Part of the reason for this additional difference is an improved glucose tolerance at lunch due to the higher carbohydrate content of the breakfast. Concurrent ingestion of fat and reduction of starch content have a persistent effect on carbohydrate tolerance to subsequent meals.

Prediction of glycemic response to mixed meals in noninsulin-dependent diabetic subjects.

Recent studies suggest the glycemic response of different mixed meals cannot be predicted from the glycemic index (GI) of individual carbohydrate foods. Postprandial glucose levels following five different mixed meals in six noninsulin-dependent diabetic volunteers were therefore assessed. Each meal comprised 50% carbohydrate, 30% fat, and 20% protein, varying only in type of carbohydrate. The carbohydrate exchanged in each meal (potato, white bread, rice, spaghetti, or lentils and barley) contributed 37% of total meal calories. The correlation between predicted glucose response and postprandial glucose area was highly significant; estimated meal GI was virtually proportional to the actual mean glycemic response. These results demonstrate that the relative glycemic effects of mixed meals can be predicted from the GI of their carbohydrate components, again stressing the importance of type of carbohydrate in regulating postprandial blood-glucose levels.

Second-meal effect: low-glycemic-index foods eaten at dinner improve subsequent breakfast glycemic response.

The effects of the glycemic index (GI) of carbohydrate eaten the previous night on the glycemic response to a standard test meal eaten subsequently in the morning (breakfast) was studied. On separate evenings normal subjects ate low- or high-GI test meals of the same nutrient composition. The dinners consisted of single foods in two experiments and mixed meals containing several foods in the third. The differences between the observed glycemic responses to low- and high-GI dinners were predicted by their GIs. The glycemic responses to breakfast were significantly lower on mornings after low-GI dinners than after high-GI dinners. Eating, at dinner, foods with different fiber contents but the same GI had no effect on postbreakfast glycemia. We conclude that the GI predicts the difference between glycemic responses of mixed dinner meals; breakfast carbohydrate tolerance is improved when low-GI foods are eaten the previous evening.

Resistant starch in the diet increases breath hydrogen and serum acetate in human subjects.

The colonic fermentation of two diets differing in amounts of resistant starch (RS) was studied. High- and low-RS diets were fed to eight healthy subjects in three meals for 1 d. Breath hydrogen and two blood samples were collected over a 28-h period. The high-RS diet provided 59.1 +/- 4.7 g (mean +/- SE) RS and the low-RS diet provided 5.2 +/- 0.4 g RS. Breath hydrogen and the average total serum acetate were significantly higher during the high-RS diet than during the low-RS diet: 34.1 +/- 4.7 and 23.9 +/- 3.9 ppm (P < 0.001) and 169.1 +/- 12.8 and 118 +/- 6.6 mumol/L (P < 0.01), respectively. Butyrate and propionate were also detected in serum samples. Although not statistically significant, there was a trend (P = 0.087) for butyrate to increase with the high-RS diet. Subjects reported greater gastrointestinal symptoms during the high-RS diet. These results suggest that RS may have effects comparable with those of some fermentable dietary fibers.

Metabolic effects of a low-glycemic-index diet.

Six healthy male volunteers underwent 2-wk metabolically controlled high-glycemic-index (GI) and low-GI diets in random order. Over the low-GI diet significant reductions were seen in serum fructosamine (7.0 +/- 1.0%, p less than 0.01), 12-h blood glucose profile (37 +/- 7%, p less than 0.01), and total serum cholesterol (15 +/- 3%, p less than 0.01). As a measure of insulin secretion, 24-h urinary C-peptide levels were 32 +/- 10% lower (p less than 0.05) after the low-GI than after the high-GI diet. Lower C-peptide levels were maintained after a standard carbohydrate challenge after the low-GI diet despite higher blood glucose levels. Differences in blood glucose were not seen after a 5-g intravenous glucose challenge. These results are of interest with respect to the effect that prolonged postprandial reductions in nutrient fluxes and insulin secretion may have on carbohydrate and lipid metabolism and renal function.

Muscle glycogen storage after prolonged exercise: effect of the frequency of carbohydrate feedings.

We reported previously that intake of carbohydrate foods with a high glycemic index (GI) produced greater glycogen storage and greater postprandial glucose and insulin responses during 24 h of postexercise recovery than did intake of low-GI carbohydrate foods. In the present study we examined the importance of the greater incremental glucose and insulin concentrations on glycogen repletion by comparing intake of large carbohydrate meals ("gorging") with a pattern of frequent, small, carbohydrate snacks ("nibbling"), which simulates the flattened glucose and insulin responses after low-GI carbohydrate meals. Eight well-trained triathletes [x +/- SEM: 25.6 +/- 1.5 y of age, weighing 70.2 +/- 1.9 kg, and with a maximal oxygen uptake (VO2max) of 4.2 +/- 0.2 L/min] undertook an exercise trial (2 h at 75% VO2max followed by four 30-s sprints) to deplete muscle glycogen on two occasions, 1 wk apart For 24 h after each trial, subjects rested and consumed the same diet composed exclusively of high-GI carbohydrate foods, providing 10 g carbohydrate/kg body mass. The "gorging" trial provided the food as four large meals of equal carbohydrate content eaten at 0, 4, 8, and 20 h of recovery, whereas in the "nibbling" trial each of the meals was divided into four snacks and fed at hourly intervals (0-11, 20-23 h). However, there was no significant difference in muscle glycogen storage between the two groups over the 24 h (gorging: 74.1 +/- 8.0 mmol/kg wet wt; nibbling: 94.5 +/- 14.6 mmol/kg wet wt). The results of this study suggest that there is no difference in postexercise glycogen storage over 24 h when a high-carbohydrate diet is fed as small frequent snacks or as large meals, and that a mechanism other than lowered blood glucose and insulin concentrations needs to be sought to explain the reduced rate of glycogen storage after consumption of low-GI carbohydrate foods.

Potential regulators of feeding behavior in anorexia nervosa.

We recruited 10 patients with anorexia nervosa and 6 age- and height-matched control subjects. Basal and postprandial concentrations of glucose, insulin, cholesterol, amino acids, gastrin, and pancreatic polypeptide (PP) were measured in response to a standard mixed meal. The only satiety signal that was significantly different between the anorectic group and the control group was PP (P less than 0.001). Tryptophan-LNAA and tyrosine-LNAA ratios were not significantly different in the two groups; however, there was a trend toward a lower tryptophan-LNAA ratio in the anorectic group. Gastrin concentrations were significantly decreased in the anorectic group (P less than 0.001) as were basal insulin concentrations (P less than 0.05). Decreased gastrin concentrations may play a role in the gastric symptoms associated with anorexia nervosa. Previous findings that PP release is diminished in obesity, together with the present findings of PP increase in anorexia nervosa, suggest that this peptide may play a role in appetite control mechanisms.

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.

Leptin inhibits insulin binding in isolated rat adipocytes.

Leptin is secreted from adipose tissue, and is thought to act as a 'lipostat', signalling the body fat levels to the hypothalamus resulting in adjustments to food intake and energy expenditure to maintain body weight homeostasis. In addition, plasma leptin concentrations have been shown to be related to insulin sensitivity independent of body fat content, suggesting that the hyperleptinemia found in obesity could contribute to the insulin resistance. We investigated the effects of leptin on insulin binding by isolated adipocytes. Adipocytes isolated from Sprague-Dawley rats exhibited a dose-dependent reduction in the uptake of 125I-labelled insulin when incubated with various concentrations of exogenous leptin. For example, addition of 50 nM leptin reduced total insulin binding in isolated adipocytes by 19% (P < 0.05). Analysis of displacement curve binding data suggested that leptin reduced maximal insulin binding in a dose-dependent manner, but had no significant effect on the affinity of insulin for its binding site. We conclude that leptin directly inhibited insulin binding by adipocytes, and the role of leptin in the development of insulin resistance in obese individuals requires further investigation.

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.

Role of N-6 and N-3 fatty acids in the dietary treatment of metabolic disorders.

The impetus to examine the effects on n-3 PUFA was generated from epidemiologic studies of a traditional population largely dependent on the marine environment. These studies and clinical interventions support a recommendation to increase the consumption of n-3 fatty acids, particularly in communities that currently have a low n-3 fatty acid intake. If we are to be guided by the diet of other traditional hunter-gatherer populations, dependent on both terrestrial and marine sources of food, it is clear that we may need to reappraise the importance of long chain PUFA of the n-6 series such as arachidonic acid. Recent studies on such populations indicate that elevated arachidonic acid levels, when associated with elevated n-3 PUFA, are likely not to be harmful to health.