Tumor Necrosis Factor‐α Stimulates Cell Proliferation in Adipose Tissue‐Derived Stromal‐Vascular Cell Culture: Promotion of Adipose Tissue Expansion by Paracrine Growth Factors

Objective: Elevated levels of tumor necrosis factor‐α (TNF‐α) protein and mRNA have been reported in adipose tissue from obese humans and rodents. However, TNF‐α has catabolic and antiadipogenic effects on adipocytes. Addressing this paradox, we tested the hypothesis that paracrine levels of TNF‐α, alone or together with insulin‐like growth factor‐I (IGF‐I), support preadipocyte development. Research Methods and Procedures: Cultured stromal‐vascular cells from rat inguinal fat depots were exposed to serum‐free media containing insulin and 0.2 nM TNF‐α, 2.0 nM TNF‐α, or 0.2 nM TNF‐α + 1.0 nM IGF‐I at different times during 7 days of culture. Results: TNF‐α inhibited adipocyte differentiation as indicated by a reduction in both immunocytochemical reactivity for the preadipocyte‐specific antigen (AD3; early differentiation marker) and glycerol‐3‐phosphate dehydrogenase activity (late differentiation marker). Early exposure (Days 1 through 3 of culture) to 0.2 nM TNF‐α did not have a long term effect on inhibiting differentiation. Continuous exposure to 0.2 nM TNF‐α from Days 1 through 7 of culture resulted in a 75% increase in cell number from control. There was a synergistic effect of 0.2 nM TNF‐α + 1 nM IGF‐I on increasing cell number by Day 7 of culture to levels greater than those observed with either treatment applied alone. Discussion: These data suggest that paracrine levels (0.2 nM) of TNF‐α alone or in combination with IGF‐I may support adipose tissue development by increasing the total number of stromal‐vascular and/or uncommitted cells within the tissue. These cells may then be recruited to become preadipocytes or may alternatively serve as infrastructure to support adipose tissue growth.     

Regional Fat Pad Growth and Cellularity in Obese Zucker Rats: Modulation by Caloric Restriction

Objective : To investigate, in young obese male Zucker rats, the effects of chronic food restriction and subsequent refeeding on: 1) parameters of nonadipose and adipose growth, 2) regional adipose depot cellularity [fat cell volume (FCV) and number], and 3) circulating leptin levels. Research Methods and Procedures : Obese (fa/fa) and lean (Fa/?) male Zucker rats were studied from age 5 to 19 weeks. After baseline food intake monitoring, 10 obese rats were subjected to 58 days of marked caloric restriction from ad libitum levels [obese‐restricted (OR)], followed by a return to ad libitum feeding for 22 days. Ten lean control rats and 10 obese control rats were fed ad libitum for the entire experiment. All rats were fed using a computer‐driven automated feeding system designed to mimic natural eating patterns. Results : After food restriction, OR rats weighed significantly less than did lean and obese rats and showed a significant diminution in body and adipose growth as compared with obese rats. Relative adiposity was not different between obese and OR rats and was significantly higher than that of lean rats. The limitation in growth of the adipose tissue mass in OR rats was due mostly to suppression of fat cell proliferation because the mean FCV in each of the four depots was not affected. Serum leptin levels of OR and obese rats were not different from each other but were significantly higher than those of lean rats. Discussion : Marked caloric restriction affects obese male Zucker rats in a manner different from that of nongenetic rodent models (i.e., Wistar rats). In comparison with the response to caloric deprivation of Wistar rats, these calorically restricted obese male Zucker rats appeared to defend their relative adiposity and mean FCV at the expense of fat cell number. These findings indicate that genetic and/or tissue‐specific controls override the general consequences of food restriction in this genetic model of obesity.     

Estrogen Reduces 11β‐Hydroxysteroid Dehydrogenase Type 1 in Liver and Visceral,but Not Subcutaneous,Adipose Tissue in Rats

Following menopause, body fat is redistributed from peripheral to central depots. This may be linked to the age related decrease in estrogen levels. We hypothesized that estrogen supplementation could counteract this fat redistribution through tissue‐specific modulation of glucocorticoid exposure. We measured fat depot masses and the expression and activity of the glucocorticoid‐activating enzyme 11β‐hydroxysteroid dehydrogenase type 1 (11βHSD1) in fat and liver of ovariectomized female rats treated with or without 17β‐estradiol. 11βHSD1 converts inert cortisone, or 11‐dehydrocorticosterone in rats into active cortisol and corticosterone. Estradiol‐treated rats gained less weight and had significantly lower visceral adipose tissue weight than nontreated rats (P < 0.01); subcutaneous adipose weight was unaltered. In addition, 11βHSD1 activity/expression was downregulated in liver and visceral, but not subcutaneous, fat of estradiol‐treated rats (P < 0.001 for both). This downregulation altered the balance of 11βHSD1 expression and activity between adipose tissue depots, with higher levels in subcutaneous than visceral adipose tissue of estradiol‐treated animals (P < 0.05 for both), opposite the pattern in ovariectomized rats not treated with estradiol (P < 0.001 for mRNA expression). Thus, estrogen modulates fat distribution, at least in part, through effects on tissue‐specific glucocorticoid metabolism, suggesting that estrogen replacement therapy could influence obesity related morbidity in postmenopausal women.     

Normal Distribution of Body Weight Gain in Male Sprague‐Dawley Rats Fed a High‐Energy Diet

Objective: To investigate the effect of a high‐energy (HE) diet on caloric intake, body weight, and related parameters in outbred male Sprague‐Dawley (SD) rats. Research Methods and Procedures: Twenty‐eight SD rats were fed either chow (C) for 19 weeks or HE diet for 14 weeks and then C for 5 weeks. Blood hormones and metabolites were assayed, and expression of uncoupling protein‐1 and hypothalamic energy‐balance‐related genes were determined by Northern blotting and in situ hybridization, respectively. Results: HE rats gained body weight more rapidly than C animals with a range of weight gains, but there was no evidence that weight gain was bimodally distributed. Caloric intake was transiently elevated after introduction of the HE diet. Transfer of HE rats back to C resulted in a drop in caloric intake, but a stable body weight. In terminal analysis, two of four dissected adipose tissue depots were heavier in rats that had previously been fed HE diet. Blood leptin, insulin, glucose, and nonesterified fatty acids were not different between the groups. Uncoupling protein‐1 mRNA was elevated in interscapular brown adipose tissue from HE rats. There was a trend for agouti‐related peptide mRNA in the hypothalamic arcuate nucleus to be higher in HE rats. Discussion: Contrary to other studies of the SD rat on HE diet, body weight and other measured parameters were normally distributed. There was no segregation into two distinct populations on the basis of susceptibility to diet‐induced obesity. This characteristic may be dependent on the breeding colony from which animals were sourced.     

Testes‐specific transgene expression in insulin‐like growth factor‐I transgenic mice

Insulin‐like growth factor‐I (IGF‐I) is a low molecular weight peptide that mediates the cell proliferating actions of growth hormone. Evidence exists indicating that IGF‐I is produced by various cell types and this growth factor has been implicated in a variety of reproductive processes. To investigate the effect of IGF‐I over‐expression on reproductive systems, we generated three independent lines of transgenic mice harbouring a human IGF‐I cDNA (hIGF‐I) under the control of a Cytomegalovirus immediate early (CMV) promoter. The CMV promoter was used in an attempt to direct expression of IGF‐I into a variety of tissues both reproductive and non‐reproductive. Yet expression of the foreign hIGF‐I gene, determined by Northern blot, was found to occur only in the testicular tissues of the male mice, apparently due to methylation of the transgene in all the tissues tested except the testes, which demonstrate transgene hypomethylation. Evaluation of the transgene expression during testicular development revealed that expression begins between 10 and 15 days of development, coinciding with the appearance of the zygotene and pachytene primary spermatocytes during early spermatogenesis, therefore indicating germ line expression of the transgene. Extensive study of the CMV‐hIGF‐I transgenic lines of mice has revealed that the effects of the transgene expression do not extend beyond the testicular tissues. No significant differences (P > 0.05) in the IGF‐I serum levels, growth rates, or testicular histology have been observed between transgenic and non‐transgenic male siblings. The ability of transgenic males to produce offspring also appears unaffected. Evaluation of the IGF binding protein (IGFBP) levels in the testicular tissues of CMV‐hIGF‐I transgenic mice by Western ligand blot revealed an increase in the concentration of testicular proteins with molecular weights corresponding to IGFBP‐2 and IGFBP‐3. These results suggest that the testicular over‐expression of IGF‐I induces increased IGFBP localization in this tissue. Inhibition of IGF activity by the IGFBPs would explain the lack of a dramatic physiological effect in the CMV‐hIGF‐I transgenic mice, despite the presence of elevated testicular IGF‐I. The observation that testis specific IGF‐I overexpression induces localization of IGFBPs in this tissue confirms the existence of a well regulated testicular IGF system and supports the convention that this growth factor plays an important role in testicular function. Mol. Reprod. Dev. 54:32–42, 1999. © 1999 Wiley‐Liss, Inc.     

Obesity and Elevated Plasma Leptin Concentration in oMT1A‐o Growth Hormone Transgenic Mice

OBERBAUER, A. M., JONATHAN A. RUNSTADLER, JAMES D. MURRAY, AND PETER J. HAVEL. Obesity and elevated plasma leptin concentration in oMT1A‐o growth hormone transgenic mice. Objective: This study was undertaken to evaluate plasma leptin concentration in the regulatable ovine metallothionein‐ovine growth hormone (oMT1a‐oGH) transgenic (TG) mouse model of obesity. Research Methods and Procedures: Transgene stimulus (zinc) was provided at 21 days of age to male and female wild‐type (WT) and TG mice. Plasma leptin concentrations were measured by radioimmunoassay at 42, 63, 84, and 105 days of age and from inactivated TG mice at 84 and 105 days. Results: WT and TG mice did not differ significantly in plasma leptin concentration at any of the ages examined (42, 63, 84, and 105 days), although females showed consistently higher plasma leptin concentrations than males regardless of genotype throughout the duration of the study. Male and female TG mice in which the transgene was inactivated at 63 days had a 1.5‐fold to 3.5‐fold increase in plasma leptin concentration over WT mice and continuously activated TG mice at 84 and 105 days of age. The elevated plasma leptin concentration seen in the inactivated TG mice at 84 and 105 days of age reflects the >300% increase in white adipose tissue seen in this model and correlated with all adipose depot weights and overall body lipid at these later ages. When plasma leptin was expressed per gram of total body fat, the leptin adjusted for body lipid was significantly higher in WT mice than either continuously activated TG or activated and then inactivated TG groups. Discussion: The inactivated TG mice in this study had higher plasma leptin levels with increasing total body adiposity, but the relative proportion of circulating leptin, on a total body lipid basis, was reduced when compared with the WT mice. This reduction was also observed in activated TG mice at the older ages. Although the absolute levels of circulating leptin were elevated in the inactivated TG animals, the amount of leptin produced per gram of fat was lowered. With the inactivation of the transgene, the leptin remained depressed after the removal of the elevated growth hormone. This represents a potential explanation for the ensuing hypertrophy of the fat depots and the abnormal phenotypic response of inactivated TG mice to elevated plasma leptin concentrations resulting in the development of obesity.     

Sex‐Dependent Dietary Obesity,Induction of UCPs,and Leptin Expression in Rat Adipose Tissues

Objective: The aim of this study was to determine the sex‐dependent differences in the response of key parameters involved in thermogenesis and control of body weight in brown adipose tissue (BAT) and white adipose tissue (WAT) in postcafeteria‐fed rats, a model of dietary obesity. Research Methods and Procedures: BAT and WAT were obtained from male and female control and postcafeteria‐fed Wistar rats. Postcafeteria‐fed rats were initially fed with cafeteria diet from day 10 of life until day 110 (cafeteria period) and with standard chow diet from then until day 180 of life (postcafeteria period). Body mass and energy intake were evaluated. Biometric parameters were analyzed in interscapular BAT (IBAT). Levels of uncoupling protein 1 (UCP1), α₂‐adrenergic receptor (AR), and β₃‐AR proteins and UCP1, UCP2, UCP3, β₃‐AR, and leptin mRNAs, in IBAT or WAT, were studied by Western blot and Northern blot analyses, respectively. Results: Rats attained 59% (females) and 39% (males) increase in body weight at the end of the cafeteria period. During the postcafeteria period, the rats showed a loss of body weight, which was higher in females. Postcafeteria‐fed female rats also presented higher activation of thermogenic parameters in IBAT, including UCP1, UCP2, and UCP3 mRNAs. Female control rats showed lower levels of both α2 and β₃‐ARs in BAT compared with male rats, but these levels in postcafeteria‐fed female and male rats were the same, because males tended to down‐regulate them. Levels of leptin mRNA in response to the postcafeteria state depended on gender and the specific WAT depot studied. Discussion: It is suggested that in postcafeteria‐fed female rats, BAT thermogenic capacity becomes more efficiently activated than in males. Female rats also showed a bigger weight loss. The parallel regulation of the levels of UCP2 and UCP3 mRNAs, with respect to UCP1 mRNA, with higher activation in female postcafeteria‐fed rats, suggests a possible role of both UCP2 and UCP3 in the regulation of energy expenditure and in the control of body weight. The distinct responses to overweight of α2 and β₃‐ARs—which were sex dependent—and leptin mRNA—which depended on both sex and WAT depot—also support the different response of thermogenesis‐related parameters between overweight males and females.     

The IGF‐I regulatory system and its impact on skeletal and energy homeostasis

Insulin‐like growth factor (IGF)‐I is important in the acquisition and maintenance of both soft and hard tissues. Skeletal remodeling requires energy and recent work has demonstrated that bone can influence insulin sensitivity and thereby regulate metabolic processes. New insights from mouse models into the role of IGF‐binding proteins (IGFBPs) as more than mere depots for the IGFs has reignited investigations into the metabolic targets influenced by the IGF regulatory system and the pathways that link bone to adipose tissue. Although there remains continued uncertainty about the relative balance between the effects of circulating versus tissue IGF‐I actions, the role of the IGFBPs has been redefined both as modulators of IGF‐I action and as independent signaling factors. This review highlights several recent findings that shed new light on the physiologic role of the IGF regulatory system and its influence on skeletal and fat metabolism. J. Cell. Biochem. 111: 14–19, 2010. © 2010 Wiley‐Liss, Inc.     

Sensitivity of ob/ob Mice to Leptin‐Induced Adipose Tissue Apoptosis

Objective: ob/ob mice have increased sensitivity to many of leptin's effects. The primary objective of this experiment was to determine whether ob/ob mice demonstrated increased sensitivity to leptin‐induced adipose tissue apoptosis. Research Methods and Procedures: Fifteen‐week‐old female ob/ob and Ob/? mice received 0 (saline), 2.5, or 10 μg/d leptin for 14 days through subcutaneous (sc) osmotic minipumps. Food intake (FI), body temperature, physical activity, and body weight were measured daily. Body composition and weights and adipose tissue apoptosis (percentage DNA fragmentation) of inguinal, parametrial, and retroperitoneal fat pads were determined at the end of the study. Results: FI decreases were more pronounced in ob/ob. Leptin (10 μg/d) decreased total FI 71% in ob/ob and 34% in Ob/? (p < 0.05). Body weight was decreased by both doses of leptin in ob/ob (p < 0.01) but was unchanged in Ob/?. Leptin increased body temperature in ob/ob but not in Ob/?. Physical activity was increased 400% by 10 μg/d leptin in ob/ob (p < 0.01) but decreased 13% in Ob/? (p < 0.01). Body fat content of ob/ob was reduced by both leptin doses, whereas only 10 μg/d leptin decreased body fat in Ob/?. Fat pad weights were decreased similarly by leptin in both genotypes. However, apoptosis was increased by leptin in all three fat pads in ob/ob, whereas Ob/? showed significant increases only in retroperitoneal. Discussion: ob/ob mice had greater overall sensitivity to leptin. Although ob/ob mice appeared to be more sensitive than Ob/? mice to leptin‐induced adipose tissue apoptosis, there were differences among adipose depots in responsiveness to leptin‐induced apoptosis.     

Medium‐Chain Oil Reduces Fat Mass and Down‐regulates Expression of Adipogenic Genes in Rats

Objective: To test the hypothesis that adipose tissue could be one of the primary targets through which medium‐chain fatty acids (MCFAs) exert their metabolic influence. Research Methods and Procedures: Sprague‐Dawley rats were fed a control high‐fat diet compared with an isocaloric diet rich in medium‐chain triglycerides (MCTs). We determined the effects of MCTs on body fat mass, plasma leptin and lipid levels, acyl chain composition of adipose triglycerides and phospholipids, adipose tissue lipoprotein lipase activity, and the expression of key adipogenic genes. Tissue triglyceride content was measured in heart and gastrocnemius muscle, and whole body insulin sensitivity and glucose tolerance were also measured. The effects of MCFAs on lipoprotein lipase activity and adipogenic gene expression were also assessed in vitro using cultured adipose tissue explants or 3T3‐L1 adipocytes. Results: MCT‐fed animals had smaller fat pads, and they contained a considerable amount of MCFAs in both triglycerides and phospholipids. A number of key adipogenic genes were down‐regulated, including peroxisome proliferator activated receptor γ and CCAAT/enhancer binding protein α and their downstream metabolic target genes. We also found reduced adipose tissue lipoprotein lipase activity and improved insulin sensitivity and glucose tolerance in MCT‐fed animals. Analogous effects of MCFAs on adipogenic genes were found in cultured rat adipose tissue explants and 3T3‐L1 adipocytes. Discussion: These results suggest that direct inhibitory effects of MCFAs on adiposity may play an important role in the regulation of body fat development.     

Upper and Lower Body Adipose Tissue Function: A Direct Comparison of Fat Mobilization in Humans

Objectives: Fat in the lower body is not associated with the same risk of cardiovascular disease as fat in the upper body. Is this explained by differences in the physiological functioning of the two depots? This study had two objectives: 1) to determine whether fat mobilization and blood flow differ between gluteal and abdominal adipose tissues in humans, and 2) to develop a new technique to assess gluteal adipose tissue function directly. Research Methods and Procedures: We performed detailed in vivo studies of adipose tissue function involving the assessment of fat mobilization by measurement of adipose tissue blood flows, arterio‐venous differences of metabolites across each depot, and gene expression in tissue biopsies in a small‐scale physiological study. Results: Gluteal adipose tissue has a lower blood flow (67% lower, p < 0.05) and lower hormone‐sensitive lipase rate of action (87% lower, p < 0.05) than abdominal adipose tissue. Lipoprotein lipase rate of action and mRNA expression are not different between the depots. This is the first demonstration of a novel technique to directly investigate gluteal adipose tissue metabolism. Discussion: Direct assessment of fasting adipose tissue metabolism in defined depots show that the buttock is metabolically “silent” in terms of fatty acid release compared with the abdomen.     

The Yellow Agouti Mutation Alters Some But Not All Responses to Diet and Exercise

Objective: Effects of ectopic expression of the agouti signaling protein were studied on responses to diet restriction and exercise in C57BL/6J (B6) mice and obese B6 mice congenic for the yellow agouti mutation [B6.Cg‐A^y (A^y)]. Research Methods and Procedures: Adult male A^y mice were either kept sedentary or exercised on a running wheel and fed ad libitum or diet restricted until weight matched to ad libitum‐fed B6 control mice. Body composition, plasma lipids, leptin, and adiponectin were measured. mRNA levels for leptin, adiponectin, lipoprotein lipase, and pyruvate dehydrogenase kinase 4 were measured in a visceral (epididymal) and a subcutaneous (femoral) fat depot by real‐time polymerase chain reaction. Results: Correlations among traits exhibited one of three patterns: similar lines for B6 and A^y mice, different slopes for B6 and A^y mice, and/or different intercepts for B6 and A^y mice. Correlations involving plasma leptin, mesenteric and epididymal adipose weights, or low‐density lipoprotein‐cholesterol were most likely to have different slopes and/or intercepts in B6 and A^y mice. mRNA levels for leptin, Acrp30, pyruvate dehydrogenase kinase 4, and lipoprotein lipase in epididymal adipose tissue were not correlated with corresponding levels in femoral adipose tissue. Discussion: The agouti protein interferes with leptin signaling at melanocortin receptors in the hypothalamus of A^y mice. Our results are consistent with the hypothesis that the melanocortin portion of the leptin‐signaling pathway mediates effects primarily on certain fat depots and on some, but not all, components of cholesterol homeostasis.     

Regulation of Acetyl CoA Carboxylase and Carnitine Palmitoyl Transferase‐1 in Rat Adipocytes

Objective: Acetyl CoA carboxylase (ACC) is a key enzyme in energy balance. It controls the synthesis of malonyl‐CoA, an allosteric inhibitor of carnitine palmitoyltransferase‐1 (CPT‐I). CPT‐I is the gatekeeper of free fatty acid (FFA) oxidation. To test the hypothesis that both enzymes play critical roles in regulation of FFA partitioning in adipocytes, we compared enzyme mRNA expression and specific activity from fed, fasted, and diabetic rats. Research Methods and Procedures: Direct effects of nutritional state, insulin, and FFAs on CPT‐I and ACC mRNA expression were assessed in adipocytes, liver, and cultured adipose tissue explants. We also determined FFA partitioning in adipocytes from donors exposed to different nutritional conditions. Results: CPT‐I mRNA and activity decreased in adipocytes but increased in liver in response to fasting. ACC mRNA and activity decreased in both adipocytes and liver during fasting. These changes were not caused directly by fasting‐associated changes in plasma insulin and FFA concentrations because insulin suppressed CPT‐I mRNA and did not affect ACC mRNA in vitro, whereas exogenous oleate had no effect on either. Despite the decrease in adipocyte CPT‐I mRNA and specific activity, CO₂ production from endogenous FFAs increased, suggesting increased FFA transport through CPT‐I for β‐oxidation. Discussion: Stimulation of FFA transport through CPT‐I occurs in both tissues, but CPT‐I mRNA and specific activity correlate with FFA transport in liver and not in adipocytes. We conclude that the mechanism responsible for increasing FFA oxidation in adipose tissue during fasting involves mainly allosteric regulation, whereas altered gene expression may play a central role in the liver.