Biomedical subjects
R L Leibel
Publications and source records attributed to R L Leibel.
Molecular mapping of mouse chromosomes 4 and 6: use of a flow-sorted Robertsonian chromosome.
The development of dense genetic maps of mammalian chromosomes is facilitated when chromosome-specific libraries are used as a source of genetic markers. To saturate the genetic maps of mouse chromosomes 4 and 6, we have made use of fluorescent-activated chromosome sorting to purify a 4:6 Robertsonian chromosome from a cell line harboring the Rb(4:6)2Bnr translocation. After staining with chromomycin A3 and Hoechst 33528, this chromosome was separated from the other mouse chromosomes. DNA was isolated from the fraction containing the Robertsonian chromosome and subcloned into the insertion vector lambda gt10, generating a library with 4.6 x 10(5) independent phage. A total of 19 single-copy sequences were used to type the progeny of a C57BL/6J x Mus spretus backcross that had previously been typed for loci on chromosomes 4 and 6. Approximately 70% of the clones in the library mapped to either chromosome 4 or 6 as assessed by genetic mapping and by use of a somatic cell hybrid panel. Simple sequence repeats have also been isolated from this library. Further characterization of these microsatellites should accelerate efforts to map mouse chromosomes 4 and 6 using PCR. In addition, flow sorting of Robertsonian chromosomes suggests a general approach for making chromosome-specific libraries in mouse.
A clinical perspective on peptides and food intake.
Viewed from a clinical perspective, it is difficult to generate enthusiasm for the likelihood of finding a peptide that could be helpful in the treatment of obesity. A deeper understanding of obesity, as will emerge from molecular biology, is more likely to point the way to a useful peptide than further evaluations of the clinical dilemmas posed by obesity. However, a clinical perspective may be useful in pointing the way to a system that needs to be examined by molecular biology and also to inject caution in the evaluation of early findings when peptides are used in treatment.
Energy intake required to maintain body weight is not affected by wide variation in diet composition.
Diets rich in fat may promote obesity by leading to a greater deposition of adipose-tissue triglycerides than do isoenergetic diets with less fat. This possibility was examined by a retrospective analysis of the energy needs of 16 human subjects (13 adults, 3 children) fed liquid diets of precisely known composition with widely varied fat content, for 15-56 d (33 +/- 2 d, mean +/- SE). Subjects lived in a metabolic ward and received fluid formulas with different fat and carbohydrate content, physical activity was kept constant, and precise data were available on energy intake and daily body weight. Isoenergetic formulas contained various percentages of carbohydrate as cerelose (low, 15%; intermediate, 40% or 45%; high, 75%, 80%, or 85%), a constant 15% of energy as protein (as milk protein), and the balance of energy as fat (as corn oil). Even with extreme changes in the fat-carbohydrate ratio (fat energy varied from 0% to 70% of total intake), there was no detectable evidence of significant variation in energy need as a function of percentage fat intake.
Fat as fuel and metabolic signal.
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Effects of systemic growth hormone (GH) administration on regional adipose tissue in children with non-GH-deficient short stature.
Chronic administration of exogenous GH to GH-deficient children is associated with a selective depletion of the abdominal sc fat depot and a resultant relative increase in gluteal, relative to abdominal, adipocyte lipid content. In GH-deficient children, the degree of this change in relative lipid content per adipocyte appears to be correlated with decreases in sensitivity of abdominal subcutaneous fat to the antilipolytic action of insulin. We studied abdominal and gluteal sc adipose tissue from 10 children with short stature (height less than 5% ile, growth velocity less than 5 cm/yr, bone age delayed at least 2 yr), who were not GH deficient based upon provocative testing (non-GH-deficient short stature) 1) before beginning and 2) after 3 months of therapy with exogenous GH (Humatrope, 0.1 mg/kg sc 3 times/week). In abdominal and gluteal adipocytes, we measured lipid content, rates of reesterification of fatty acids released by ongoing lipolysis and rates of in vitro lipolysis and lipogenesis in response to insulin, adenosine, and various adrenoreceptor agonists. These biochemical measures were correlated with measures of statural growth and adipose tissue distribution in each subject. We found that GH therapy was associated with a significant reduction in abdominal adipocyte size (0.48 microgram +/- 0.08 lipid per cell prior to therapy vs. 0.43 microgram +/- 0.08 lipid per cell after therapy, P less than 0.05) and a significant increase in responsiveness of gluteal sc adipose tissue to the lipogenic actions of insulin. The significant correlations of changes in abdominal adipocyte volume with changes in regional adipose tissue insulin sensitivity that were noted in GH-deficient children were not noted in this subject population, perhaps due to effects of endogenous GH on pretreatment insulin responsiveness of adipose tissue. These data reaffirm that GH has site-specific effects on regional adipose tissue depots.
Rat obesity gene fatty (fa) maps to chromosome 5: evidence for homology with the mouse gene diabetes (db).
The autosomal recessive mutations fa (rat) and db (mouse) cause obesity syndromes that develop early and ultimately become severe. Although both fa/fa rats and db/db mice have been studied extensively as models of human obesity and diabetes, the molecular bases of these phenotypes remain unknown. We have mapped fa in 50 fa/fa (obese) offspring of a (13M x Brown Norway) F1 fa/+ intercross relative to two molecular markers, Ifa and Glut-1, which flank db on mouse chromosome 4 and which are located on rat chromosome 5. Ifa and Glut-1 are linked to fa, with a gene order, Ifa-fa-Glut-1, that is identical to that for the region around db in the mouse genome. These results place fa on rat chromosome 5 and suggest that db and fa are mutations in homologous genes.
Molecular mapping of obesity genes.
Advances in molecular genetics have made it possible to clone mutant genes from mammals. This capability should facilitate efforts to determine the genetic factors that control food intake and body composition. In order to identify these genetic factors, we have been making use of mouse mutations that cause obesity. The basic premise of this approach is to take advantage of the mouse as a genetic system for the analysis of genetically complex disorders and to then apply that information to the study of human disease. This paper reviews: (1) current concepts concerning the control of body weight in man and other mammals; (2) the biologic characteristics of the mouse obesity mutations; (3) our progress in the use of positional cloning techniques to clone the mouse obese (ob) and diabetes (db) genes; (4) an approach to polygenic obesity in mice; and (5) the possible relevance of the mouse obesity mutations to human obesity.
Food deprivation and hypothalamic neuropeptide gene expression: effects of strain background and the diabetes mutation.
We have used a novel method to identify genes expressed in the hypothalamus which may be potentially involved in controlling food intake and energy metabolism. We assumed that food deprivation, a powerful stimulus of food intake, would stimulate the activity of neural pathways involved in feeding behavior which should be reflected in an increase in the synthesis of any relevant neuropeptide and its messenger RNA. A study of 5 neuropeptides in 5 strains of mice has identified neuropeptide Y (NPY) as a gene whose expression in the hypothalamus is controlled by nutritional status, suggesting that hypothalamic NPY neurons are a link in the neural network regulating feeding behavior and energy metabolism. In addition, we have studied the effect of the diabetes mutation on neuropeptide gene expression during fasting and refeeding. Our findings suggest that abnormal NPY and enkephalin gene expression in the hypothalamus may be two important determinants of the expression of the diabetes mutation.
Food deprivation and age modulate neuropeptide gene expression in the murine hypothalamus and adrenal gland.
We have developed a rapid and simple method for quantitative Northern blot analysis of rare messenger RNA species from single mouse hypothalami and adrenals. This technique has allowed us to study the effects of food deprivation on neuropeptide gene expression in the mouse hypothalamus and adrenal gland. The potential modulatory effects of sex and age were also investigated. Food deprivation induced a two-fold increase in the amount of hypothalamic neuropeptide Y (NPY) mRNA, but did not increase NPY mRNA in the brainstem. Age had a significant effect on levels of NPY messenger RNA levels in the hypothalamus. However, there were no gender-associated effects. Sexually immature females (6 weeks old) had higher levels of NPY expression than mature females (9 weeks old). In contrast, in the adrenal gland, increasing maturity was associated with higher levels of NPY mRNA. As in the hypothalamus, fasting caused approximately two-fold increases in NPY mRNA over levels in the ad libitum fed state for both mature and immature mice. Thus, hypothalamic NPY neurons are responsive to nutritional deprivation and developmental status, suggesting that NPY neurons may be important in energy homeostasis.
Molecular mapping of the mouse ob mutation.
The mouse ob mutation has been mapped relative to a series of RFLPs among the progeny of three separate mouse crosses: an intraspecific backcross, an intraspecific intercross, and an interspecific intercross. Genotypic assignment at the ob locus was made by making use of measurements of body mass index and the plasma concentrations of glucose and insulin. These data have suggested that the development of diabetes in these animals is a consequence of unlinked polygenes. There was also evidence that unlinked Mus spretus alleles can diminish the obesity of ob/ob mice. From these data we have mapped several markers on chromosome 6 with the following order: cen-Cola-2-Met-ob-Cpa-Tcrb. The homologs of markers that flank ob map to human chromosome 7q, suggesting that if there is a human homologue of ob, it maps to 7q31.
Molecular genetic linkage maps of mouse chromosomes 4 and 6.
We have generated a moderate resolution genetic map of mouse chromosomes 4 and 6 utilizing a (C57BL/6J x Mus spretus) F1 x Mus spretus backcross with RFLPs for 31 probes. The map for chromosome 4 covers 77 cM and details a large region of homology to human chromosome 1p. The map establishes the breakpoints in the mouse 4-human 1p region of homology to a 2-cM interval between Ifa and Jun in mouse and to the interval between JUN and ACADM in human. The map for mouse chromosome 6 spans a 65-cM region and contains a large region of homology to human 7q. These maps also provide chromosomal assignment and order for a number of previously unmapped probes. The maps should allow the rapid regional assignment of new markers to mouse chromosomes 4 and 6. In addition, knowledge of the gene order in mouse may prove useful in determining the gene order of the homologous regions in human.
Effects of early pregnancy on regional adipose tissue metabolism.
Adipose tissue metabolism was studied in needle biopsies from femoral and abdominal subcutaneous depots, in 12 healthy young women, during early (9-11 weeks) pregnancy, and 6 weeks after a legal abortion. Both during pregnant and non-pregnant conditions, a higher lipoprotein lipase (LPL) activity was seen in the femoral compared to the abdominal region, but the LPL activity was not influenced by early pregnancy. Rates of fatty acid esterification and acylglyceride synthesis were not different between regions, nor affected by pregnancy. The stimulatory effect of norepinephrine (10(-7) M) on lipolysis was significantly greater in the abdominal than in the femoral region in both the pregnant and non-pregnant condition. This difference was apparently due to higher alpha-adrenergic activity in the femoral region. Pregnancy per se had no effect on lipolytic response to norepinephrine. These findings indicate that lipid accumulation is favoured in the femoral region in young women both during pregnant and non-pregnant conditions.
Genetic analysis of complex disorders. Molecular mapping of obesity genes in mice and humans.
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Metabolic and hemodynamic responses to endogenous and exogenous catecholamines in formerly obese subjects.
Formerly or reduced-obese (RO) subjects exhibit diminished systemic energy requirements for weight maintenance. Based on studies in animals and humans, there is reason to believe that the autonomic nervous system (ANS) plays an important role in coordinate regulation of energy metabolism and may be altered in the RO state. We have, therefore, investigated aspects of autonomic physiology in obese (OB), RO, and never obese (NO) subjects subjected to insulin-mediated hypoglycemia and incremental intravenous infusions of epinephrine. Total catecholamine secretion during insulin-induced hypoglycemia was significantly lower in the RO. The RO showed increased sensitivity to in vivo lipolytic and cardiovascular effects of endogenous and infused catecholamines. In these studies, OB subjects responded more like the NO subjects than did the RO. The RO display characteristic changes in autonomic status that may play a role in their enhanced energy efficiency.
Heart rate variability as a measure of autonomic function during weight change in humans.
Changes in autonomic function were studied during experimentally induced weight changes in seven subjects. A spectral analysis of heart rate variability (HRV) was used to evaluate autonomic activity during weight change. With a 10% increase in body weight above the usual or starting weight, there was a decline in parasympathetic power accompanied by a rise in mean heart rate. Heart rate declined during weight reduction, but the power of HRV did not change significantly. Because heart rate power at the frequency of respiratory rate can be affected by respiratory rate, three additional subjects were tested at a constant respiratory rate. Weight increases in this group also led to a decline in the power of HRV at a frequency attributable to the parasympathetic nervous system. Such a parasympathetic effect of weight increase may be one mechanism for the arrhythmias and other cardiac alterations that accompany obesity.
Regional differences in adrenoreceptor status of adipose tissue in adults and prepubertal children.
The relative anatomical distribution of adipose tissue in central (abdominal) vs. peripheral (extremity) depots is highly correlated with the risk of adiposity-related morbidities, such as hypertension, cardiovascular disease, and diabetes mellitus. In adults, comparisons of the functional status of plasma membrane adrenergic receptors indicate that abdominal adipocytes are more responsive to the lipolytic action of beta 1-adrenergic agonists, while gluteal adipocytes are more responsive to the antilipolytic action of alpha 2-adrenergic agonists. To determine whether such regional differences in adipocyte adrenoreceptor status are present before puberty, we obtained needle biopsy samples of abdominal and gluteal sc adipose tissue in the post-absorptive state from 13 prepubertal children and 47 adults of varying body compositions (obese vs. lean). Lipolysis rates were measured in the basal state and in the presence of 10(-7) M norepinephrine (a mixed alpha- and beta-adrenergic agonist) and 10(-7) M isoproterenol (a beta-adrenergic agonist). In children, there were no significant regional differences in either the basal rate of lipolysis or the responses to adrenergic lipolytic and antilipolytic stimuli. In lean and obese adults, gluteal sc adipose tissue was strikingly more responsive to antilipolytic alpha-adrenergic stimulation (P less than 0.0001) and less responsive to lipolytic beta-adrenergic stimuli (P less than 0.005) compared to abdominal tissue. Abdominal sc adipocytes from children had a significantly lower rate of basal lipolysis (P less than 0.01) and were more responsive to alpha 2-adrenergic (antilipolytic) stimuli (P less than 0.05) than abdominal adipocytes in adults. These results suggest that peripubertal endocrine changes may mediate the striking regional differences in adrenoreceptor status of adult adipose tissue, and that a decrease in the preponderance of alpha 2-receptors (antilipolytic) in abdominal adipose tissue may account in part for the relative loss of central vs. peripheral fat that occurs during puberty.
Clinical review 28: A biological basis for human obesity.
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