The biology of white adipocyte proliferation.

Expanded adipose tissue mass increases the risk for many clinical conditions including diabetes, hypertension, coronary atherosclerotic heart disease, and some forms of cancer. Therefore, it is imperative that we understand the mechanisms by which fat pads expand. The enlargement of fat cells during the development of obesity has been previously hypothesized to be a triggering factor for the proliferation of new fat cells. There is now a preponderance of evidence that adipose tissue is a source of growth factors such as IGF-I, IGF binding proteins, TNF alpha, angiotensin II, and MCSF that are capable of stimulating proliferation. The relative importance of these autocrine/paracrine factors in the normal control of preadipocyte proliferation is unknown. In addition, the proliferative response of preadipocytes to the paracrine milieu is undoubtedly modulated by neural inputs to fat tissue and/or serum factors. Together, these multiple regulatory controls orchestrate overall and region-specific adipose tissue cellularity responses associated with the development of hyperplastic obesity. Both in vivo and in vitro studies are needed to understand the complex, interacting physiological mechanisms by which growth of this important organ is regulated.

Differences in metabolic and hormonal milieu in diabetic- and alcohol-induced ketoacidosis.

PURPOSE: Diabetic ketoacidosis (DKA) and alcoholic ketoacidosis (AKA) are two medical emergencies characterized by elevated total ketone body concentration. We aimed to determine differences in pathogenesis of ketoacidosis and its metabolic consequences by comparing both at presentation and during treatment, the different metabolic products and hormones involved in the ketoacidotic state. MATERIALS AND METHODS: We studied 12 patients with DKA and 8 patients with AKA. On admission and every 4 hours for 24 hours during treatment, samples were drawn for determination of serum ketone bodies, lactate and pyruvate, insulin, and counterregulatory hormones (glucagon, cortisol, growth hormone, and catecholamines). RESULTS: At presentation, with a similar beta-hydroxybutyrate concentration, patients with DKA had a higher plasma glucose (32 mmol/L vs. 6.6 mmol/L), lower beta-hydroxybutyrate/acetoacetate ratio (3:1 vs. 7:1), and a lower lactate/pyruvate ratio (11:1 vs. 19:1) than patients with AKA (all, P < .01). The mean time to resolve ketoacidosis in patients with AKA (6 +/- 1 hour) was significantly shorter than in patients with DKA (16 +/- 2 hours). At presentation, the mean insulin concentration in patients with DKA and AKA were similarly decreased (7.8 +/- 2 and 10.3 +/- 3 microU/mL, P = not significant [NS]). The mean glucagon level before therapy was 203 +/- 15 pg/mL and 188 +/- pg/mL for patients with DKA and AKA, respectively (P = NS). Levels of cortisol, growth hormone, and epinephrine at presentation and during the first 8 hours of treatment were higher in patients with DKA; however, the difference in these values did not reach statistical significance. During therapy, levels of counterregulatory hormones declined at similar rates and returned to normal values after resolution of ketoacidosis. CONCLUSIONS: Our results indicate that, in addition to a history of diabetes or alcoholism, patients with DKA and AKA differ in their metabolic parameters more than in their hormonal profile. The metabolic profile of DKA is characterized by a higher plasma glucose concentration, and lower beta-hydroxybutyrate to acetoacetate and lactate to pyruvate ratios compared with patients with AKA. The initial hormonal profile in both ketoacidotic states is characterized by similarly decreased insulin levels and elevated levels of counterregulatory hormones.

Expressions of leptin and insulin-like growth factor-I are highly correlated and region-specific in adipose tissue of growing rats.

OBJECTIVE: Anatomically distinct adipose tissue regions differ in their predominant modality of growth (i.e., cellular hypertrophy vs. hyperplasia). We examined site-specific patterns of expression of two genes whose products, leptin and insulin-like growth factor-I (IGF-I), could be involved in mediating differential growth and metabolism of white adipose tissue. We also related these patterns of expression to measures of adipose depot cellularity. RESEARCH METHODS AND PROCEDURES: Male Wistar rats were fed ad libitum and studied from ages 7 weeks to approximately 12 months. Terminal measures of body weights; weights, composition, and cellularity of four white adipose depots; circulating leptin and IGF-I; and adipose depot-specific expression levels of leptin and IGF-I were measured in subsets of rats at 7, 12, 22, 42, and 46 weeks of age. RESULTS: Both leptin and IGF-I mRNAs are quantitatively expressed in a depot-specific manner, in the following order: retroperitoneal approximately equals epididymal > mesenteric > subcutaneous inguinal. Furthermore, there is a marked correlation between the expressions of these hormones in the various regions of adipose tissue of rats during the first year of life. The mechanisms that underlie the parallel expressions of leptin and IGF-I appear to be related to fat-cell volume. DISCUSSION: Because both leptin and IGF-I have been implicated in the regulation of energy homeostasis and are both expressed in adipose tissue, the depot-specific linkage between the two genes suggests interaction at the autocrine level. This interaction may have an important role in determining functional properties particular to individual adipose depots.

Weight control and risk factor reduction in obese subjects treated for 2 years with orlistat: a randomized controlled trial.

CONTEXT: Orlistat, a gastrointestinal lipase inhibitor that reduces dietary fat absorption by approximately 30%, may promote weight loss and reduce cardiovascular risk factors. OBJECTIVE: To test the hypothesis that orlistat combined with dietary intervention is more effective than placebo plus diet for weight loss and maintenance over 2 years. DESIGN: Randomized, double-blind, placebo-controlled study conducted from October 1992 to October 1995. SETTING AND PARTICIPANTS: Obese adults (body mass index [weight in kilograms divided by the square of height in meters], 30-43 kg/m2) evaluated at 18 US research centers. INTERVENTION: Subjects received placebo plus a controlled-energy diet during a 4-week lead-in. On study day 1, the diet was continued and subjects were randomized to receive placebo 3 times a day or orlistat, 120 mg 3 times a day, for 52 weeks. After 52 weeks, subjects began a weight-maintenance diet, and the placebo group (n = 133) continued to receive placebo and orlistat-treated subjects were rerandomized to receive placebo 3 times a day (n = 138), orlistat, 60 mg (n = 152) or 120 mg (n = 153) 3 times a day, for an additional 52 weeks. MAIN OUTCOME MEASURES: Body weight change and changes in blood pressure and serum lipid, glucose, and insulin levels. RESULTS: A total of 1187 subjects entered the protocol, and 892 were randomly assigned on day 1 to double-blind treatment. For intent-to-treat analysis, 223 placebo-treated subjects and 657 orlistat-treated subjects were evaluated. During the first year orlistat-treated subjects lost more weight (mean +/- SEM, 8.76+/-0.37 kg) than placebo-treated subjects (5.81+/-0.67 kg) (P<.001). Subjects treated with orlistat, 120 mg 3 times a day, during year 1 and year 2 regained less weight during year 2 (3.2+/-0.45 kg; 35.2% regain) than those who received orlistat, 60 mg (4.26+/-0.57 kg; 51.3% regain), or placebo (5.63+/-0.42 kg; 63.4% regain) in year 2 (P<.001). Treatment with orlistat, 120 mg 3 times a day, was associated with improvements in fasting low-density lipoprotein cholesterol and insulin levels. CONCLUSIONS: Two-year treatment with orlistat plus diet significantly promotes weight loss, lessens weight regain, and improves some obesity-related disease risk factors.

Qualitative regional differences in adipose tissue growth and cellularity in male Wistar rats fed ad libitum.

Adipose tissue grows primarily by a combination of increases in fat cell volume (hypertrophy) and in fat cell number (hyperplasia), but the regional growth pattern of white adipose tissue depots in animal species and in the human is still unclear. In this study we characterized fully the age-related changes in adipose tissue growth, composition, and cellularity of four fat depots of male Wistar rats that varied in age from 7 wk to 15 mo and in body weight from 178 to 808 g. Body weight and the weight of each of the four adipose depots studied (epididymal, mesenteric, subcutaneous inguinal, and retroperitoneal) increased progressively with age and ad libitum feeding. Comparison of the cellularity of the four adipose depots, however, showed remarkable and significant differences in the pattern of growth within the same animals. The cumulative growth of the two intraabdominal fat depots (mesenteric and epididymal) was due mostly to hypertrophy (increases in cell volume of 83 and 64%, respectively), whereas the growth of the other two depots (retroperitoneal and inguinal) was due predominantly to hyperplasia (increases in cell number of 58- and 65%, respectively). These findings uncover major and unexpected regional differences in the modulation of adipose tissue growth within aging animals fed ad libitum and suggest local, region-specific regulatory controls of this growth.

Lactate production from glucose and response to insulin in perifused adipocytes from mesenteric and epididymal regions of lean and obese rats.

Lactate, an important metabolic substrate for peripheral tissues and the liver, is released in significant amounts from adipose tissue. Using a perifusion system, we measured lactate production from glucose and response to insulin in isolated mesenteric and epididymal adipocytes removed from fed or fasted male Wistar rats at two stages of growth and development: (a) lean rats (7 weeks to 9 weeks old, weighing approximately 250 g), and (b) fatter rats (6 months to 8 months old, weighing approximately 550 g). The results show that lactate production in perifused adipocytes is regulated by the prior nutritional state of the animals, by the adipose tissue region, and by the presence of insulin in the perifusate. In fat cells from lean rats, basal lactate production was significantly higher (p<0.05) in mesenteric cells when compared with epididymal cells, both in the fed state (7.8 nmo/10(7) fat cells per minute vs. 2.9 nmol/10(7) fat cells per minute) and after 2 days of fasting (13.6 nmol vs. 3.5 nmol). When the response to 1 mU/mL insulin was studied, however, the relative increase in lactate production produced by insulin was greater in the epididymal cells than in the mesenteric cells, in both the fed (194% vs. 91% over basal, respectively) and fasted (360% vs. 55% over basal, p<0.05) state. When larger epididymal adipocytes from fatter rats were compared with an equal number of smaller epididymal cells from leaner rats, the larger cells produced 4.99 nmol of lactate/10(7) fat cells per minute, whereas the smaller cells produced 2.93 nmol (p=0.08). Large fat cells showed a small and nonsignificant response to insulin in either type of cell (epididymal vs. mesenteric) or nutritional state (fed vs. fasted). This study indicates that distinct regional differences exist in lactate production and response to insulin. Mesenteric adipose tissue, which drains directly into the portal vein and provides substrates to the liver, may be an important source of lactate for the hepatic processes of gluconeogenesis and glycogenesis.

A simple method to predict cellular density in adipocyte metabolic incubations.

OBJECTIVES: The density of isolated adipocyte suspensions, namely the cellular concentration, influences metabolic results when lipolysis and the pattern of glucose metabolism are studied. It is often difficult to obtain reproducible adipocyte concentrations from experiment to experiment, and investigators usually measure the cell concentration at the end rather than at the initiation of metabolic incubations. METHOD: A simple and rapid method to obtain reliable and predictable adipocyte concentration prior to metabolic incubations is described and validated. The method is based on determination of lipocrit, mean adipocyte diameter (by optical sizing), and calculation of volume, in aliquots of isolated adipocyte suspensions. MAIN OUTCOME MEASURES: Lipocrit, mean adipocyte volume, predicted and observed adipocyte number in isolated cell suspensions. RESULTS: In 15 experiments, adipocyte concentration was accurately predicted within 12-18% of actual concentration. This is in contrast to the four or five-fold differences usually encountered in a series of experiments. CONCLUSION: One can rapidly predict the number of adipocytes present in a given cell suspension with the proposed method, and then correct it to a desired adipocyte concentration at the beginning of metabolic incubations. This method will help to eliminate the confounding effects of variable cell concentrations in in vitro metabolic experiments with isolated adipocytes.

Morphological and metabolic changes associated with large differences in daily food intake in crossed-intestines rats.

Twenty-two inbred male Lewis rats were made into parabiotic pairs and 7 pairs had a further operation in which the small intestines of the 2 rats were connected so that one rat continually lost food into the upper small intestine and bloodstream of its partner. As a result, these rats showed large and sustained changes in daily food intake with one rat (A) in each pair eating more than twice as much as its partner (B) for the rest of their lives. Measurements of plasma levels of glucose, insulin, and glucagon did not vary directly with daily food intake, but integrated plasma lactate values were lower in rats that ate more (A) and higher in rats that ate less (B). At sacrifice, the rats that ate more were found to have less fat with reduced fat cell size but the same cell number in both retroperitoneal and epididymal fat pads. Measurements of the rate and pattern of glucose metabolism in retroperitoneal fat cells with or without insulin stimulation were similar across groups. Rates of lipolysis with and without epinephrine did not differ among groups. Lipoprotein lipase varied directly with fat cell size and indirectly with daily food intake. These studies show that daily food intake varies directly with fat cell size and inversely with plasma lactate and retroperitoneal lipoprotein lipase levels.

Components of the renin-angiotensin system in adipose tissue: changes with maturation and adipose mass enlargement.

White adipose tissue is a rich source of angiotensinogen protein and mRNA. Studies in clonal cells suggest that angiotensinogen, and its cleavage product, angiotensin II, are involved in preadipocyte differentiation into mature fat cells. No studies have determined whether angiotensinogen is also involved in adipose tissue development in vivo. In this report, we studied male Wistar rats at two stages of development to determine if angiotensinogen protein and mRNA are increased in retroperitoneal fat depots of rapidly growing young, lean, 8-week-old rats compared to 26-week-old rats that are fatter, but are undergoing less rapid adipose tissue growth. We also assessed renin mRNA and angiotensin I-generating activity, since it is less clear whether renin is locally produced in adipose tissue. We found that angiotensin I-generating activity was measurable in adipose tissue and adipocytes, but renin mRNA was undetectable by northern blot analysis. Angiotensinogen mRNA was abundant in adipocytes, but was absent in stromal-vascular cells of adipose tissue. Angiotensinogen content per 10 million fat cells was approximately threefold higher in 8-week-old rats compared to 26-week-old rats (p < .0002). Angiotensinogen mRNA was approximately twofold higher in adipocytes of 8-week-old rats compared to 26-week-old rats. The age-related decline in angiotensinogen protein and mRNA indicates that the local renin-angiotensin system may play an important role in adipose tissue growth, and possibly contribute to the changes in adipose mass and cellularity seen in old senescent rats.

Insulin sensitivity accounts for glucose and lactate kinetics after intravenous glucose injection.

Mathematical modeling was used to explore the interaction between glucose, insulin, and lactate during the frequently sampled intravenous glucose tolerance test (FSIGTT). Insulin-modified FSIGTs were performed in 25 lean volunteers, and an additional 5 volunteers underwent FSIGTs with glucose injection alone to illustrate the effect of insulin on both glucose and lactate kinetics. The model chosen as the best representation of the system extended the minimal model of glucose kinetics (MM) by including a two-compartment model of lactate kinetics. The model accounted for both glucose and lactate kinetics, provided traditional MM parameters of insulin sensitivity and glucose effectiveness, and descriptive parameters of lactate kinetics. Modeling suggested that lactate production was limited by the rate of glucose disappearance, with no indication of direct effects of insulin on lactate. Inclusion of lactate kinetics had no adverse effect on MM parameters (SG: 0.023 +/- 0.009 vs. 0.023 +/- 0.010 min-1, SI: 1.01 +/- 0.70 vs. 1.03 +/- 0.71 x 10(4).min-1.pmol-1.1; P > 0.50, lactate model vs. MM), and indicated that approximately 1.2% min-1 of total glucose disappearance during the FSIGT is converted to lactate. An additional benefit of including lactate kinetics was the significant improvement in precision in MM parameter estimates as reflected by the fractional standard deviations (FSDs). This effect was most prominent for SG, in which a threefold improvement in parameter precision was observed (FSD: 13.5 +/- 3.1 vs. 42.5 +/- 48.5; means +/- SD).(ABSTRACT TRUNCATED AT 250 WORDS)

Biennial survey of physician clinical-nutrition training programs.

This is the fourth survey of physician clinical-nutrition training programs. As in previous reports, current fellowship training programs were identified, descriptive information obtained, and program content surveyed. In addition, a questionnaire developed by the American Board of Nutrition Committee on Fellowship Training Programs was used to determine the degree of emphasis given to content in the areas of basic nutrition science, clinical applications, and research training. Among the 38 programs identified, uniform ratings of importance were found in all major topic areas. There was also uniformity in most subtopics, with minor exceptions. As expected, in the area of nutrition in the life cycle, pediatric training programs emphasized infancy and childhood whereas medical-surgical programs emphasized adulthood and aging. Alcoholism was emphasized in medical-surgical training programs whereas cystic fibrosis and inborn errors of metabolism were emphasized in pediatric programs. Nutrition in burn patients received minor emphasis in all programs. The overall uniformity of curricular content in training programs confirms the contention that clinical nutrition has a defined clinical scope and should be considered for establishment as a recognized subspecialty in American medicine.

Effects of cell density on in vitro glucose metabolism by isolated adipocytes.

We studied the effect of variable isolated fat cell concentrations (from 0.17 to 1.25 x 10(6) cells/ml) on rate and pattern of basal and insulin-stimulated glucose metabolism by rat epididymal fat cells. Cell concentration did not affect total glucose utilization, but high cell concentrations increased the absolute and relative conversion of glucose to CO2 and glyceride-fatty acids by two- to threefold and decreased the conversion to lactate, pyruvate, and glyceride-glycerol when compared with values observed at low cell concentration. When effects of adenosine deaminase (ADA) and N-6(2-phenylisopropyl)adenosine (PIA) were examined, addition of ADA to incubated cells produced no significant changes in the rate or pattern of adipocyte glucose metabolism; PIA had a slight and uniform effect on the conversion of glucose to its metabolic products and minimal effect on insulin-stimulated glucose metabolism. Medium free fatty acid concentration did not change during the incubation at various cell density, but intracellular free fatty acids were found to be inversely related to fat cell density in the medium. Thus a variable fat cell density influences the pattern of adipocyte glucose metabolism in vitro. This effect may be due to variable rates of lipolysis and resulting changes in intracellular fatty acid concentration rather than to adenosine per se. This work has practical implications in the need to define cell density when carrying out in vitro measurements of adipocyte glucose conversion to products.

Simplified enzymatic assay of angiotensin-converting enzyme in serum.

We report a simple, enzymatic method for determining angiotensin-converting enzyme (ACE; EC 3.4.15.1) in serum. The proposed method features coupling an established reaction catalyzed by gamma-glutamyltransferase (GGT; EC 2.3.2.2) to the ACE reaction, which releases glycylglycine from the artificial substrate hippuryl-glycyl-glycine. The glycyl-glycine released by the ACE reaction becomes rate-limiting in the GGT reaction, in which it participates as a receptor substrate for a gamma-glutamyl group transferred from a donor substrate, L-gamma-glutamyl-3-carboxy-4-nitroanilide. The reaction releases 3-carboxy-4-nitroaniline, which is monitored spectrophotometrically at 410 nm. The resulting rate of change in absorbance is linearly related to the glycyl-glycine concentration and therefore to the activity of ACE. The linear range of the method extends from ACE values < 50 to at least 1300 U/L of serum. Good precision is indicated by a low CV for replicate analyses (3.6% and 4.6% for within-run and day-to-day assays, respectively, for normal ACE activity, and 3.1% within-run for high ACE activity). Results also correlate well with those of an established colorimetric method (r = 0.978). The major advantages of the method are its procedural simplicity, limited cost, use of readily available reagents, applicability to isoenzyme studies, and adaptability to automation.

Severe vs moderate energy restriction with and without exercise in the treatment of obesity: efficiency of weight loss.

Thirty obese women were randomly assigned to either 40% [severe energy restriction (SER)] or 70% [moderate energy restriction (MER)] of their maintenance energy requirements and to no exercise, aerobic exercise (walking), or aerobic exercise plus circuit weight training. Body composition by hydrostatic weighing and energy expenditure by indirect calorimetry were measured at 0, 3, and 6 mo. In addition, we developed a deficit-efficiency factor (DEF), calculated as body energy loss/dietary energy deficit, to attempt to quantify the effectiveness of the weight-reduction interventions. Subjects in the SER group lost more weight (mean +/- SE: 15.1 +/- 1.4 vs 10.8 +/- 1.0 kg), fat (11.7 +/- 1.1 vs 8.3 +/- 0.6 kg), and fat-free mass (2.8 +/- 0.3 vs 1.8 +/- 0.3 kg) than the MER group (P < or = 0.05). However, the overall DEF was greatest in the MER group (0.80 +/- 0.07) compared with the SER group (0.52 +/- 0.05; P < or = 0.01). Exercise had no significant effect. This study demonstrates that MER may offer an advantage over SER because it produces a greater energy loss relative to energy deficit.

Reversal of high-fat diet-induced obesity in female rats.

The purpose of the present study was to test whether the degree of obesity or the duration of the obese state affects the reversibility of diet-induced obesity. This was accomplished by initially feeding adult female Wistar rats either a low-fat diet (Chow) or one of two high-fat diets (HFDs; 30 and 60% of total calories as dietary fat; 30% HFD and 60% HFD, respectively). Fifty-four days, reversal 1 (R1), or ninety-seven days, reversal 2 (R2), later the HFDs were substituted with the low-fat control diet in subgroups of rats. Animals from all groups were sampled at three intervals: the start of R1 (R1 start), and the completion of R1 (R1 end) and R2 (R2 end). At the end of each interval the 60% HFD-fed group had increased body weight, carcass lipid content, and retroperitoneal and parametrial white adipose tissue (RWAT and PWAT) pad weight, fat cell diameter, and fat cell volume, but not fat cell number (FCN), compared with the other groups. The 60% HFD-fed rats also exhibited a marked and persistent hyperphagia that continued even as most of the indexes of obesity approached their maximal values (R1 end). The 60% HFD group had a transient increase in RWAT and PWAT lipoprotein lipase activity that followed the development of most obesity indicators. A clear intermediate level of obesity did not develop in the 30% HFD-fed group. Instead, these animals had nonsignificant increases in these measures of adiposity, making it impossible to test whether the severity of the obesity affected its reversibility in age-matched groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Habitual dietary intake and insulin sensitivity in lean and obese adults.

Studies in rodents have shown that short-term increases in dietary fat result in fat cell enlargement and insulin resistance. In humans, although high-fat diets have been associated with obesity, little is known about the specific metabolic effects of these diets. In this study we explored possible associations between habitual dietary composition and insulin sensitivity. Twenty-two lean and 23 obese subjects were characterized by dietary history (food frequency questionnaire), anthropometrics, oral glucose tolerance, and insulin sensitivity (SI, from the minimal model). As shown previously, body mass index was positively correlated with percent of energy intake as fat (r = 0.47, P = 0.001). Increasing fat intake was also associated with diminished SI (r = -0.41, P = 0.01). In contrast, SI was positively correlated with fiber intake (r = 0.43, P = 0.007). Multivariate analysis confirmed the importance of dietary fiber for SI. We conclude that habitually low dietary fiber intake, along with elevated dietary fat, correlates with diminished SI in otherwise healthy lean and obese subjects.

Lactate production in adipose tissue: a regulated function with extra-adipose implications.

Estimates of the quantitative contribution of adipose tissue to whole-body glucose metabolism, previously reported as 1-3%, have been revised to be on the order of 10-30%. These revised estimates come, in part, from a recognition that adipose tissue uses glucose to produce lactate and pyruvate, in addition to CO2 and triglycerides. Lactate production by adipose tissue is modulated in vitro by changes in glucose, insulin, and epinephrine concentrations. In vivo, lactate production is regulated acutely by the animal's nutritional state (fed or fasted) and chronically by the degree of obesity. A strong positive correlation exists between rat fat cell size and relative conversion of glucose to lactate (r = 0.89, P less than 0.001). Diabetes is also associated with markedly increased lactate production in adipocytes. Fat cells from obese or diabetic rats (or humans) can metabolize to lactate as much as 50-70% of the glucose taken up. From these recent studies, a picture is emerging in which the adipose organ may provide lactate for hepatic gluconeogenesis during fasting, and also lactate for hepatic glycogen synthesis after food ingestion. Modulation of adipocyte lactate production and contribution of adipose tissue lactate to the body's fuel economy in physiological and pathological states are the focus of this review.