Barbara A. Horwitz

Hyperprolactinemia stimulates food intake in the female rat

Lactation in the rat is marked by extreme hyperphagia. The present study examined the possibility that elevated prolactin levels contribute to this increase. It also evaluated the effects of hyperprolactinemia on brown adipose tissue and carcass composition. Virgin Osborne-Mendel rats were made hyperprolactinemic via ectopic pituitary transplants (PIT, n = 9) or were sham-operated (SHAM, n = 8). Eight lactating rats (LACT) served as additional controls. Food intake, body weight and rectal temperature were recorded daily. Eleven days postsurgery (or 11-12 days postpartum), the rats were sacrificed, and brown fat (scapular, axillary, cervical and thoracic) was excised, weighed, and assayed for GDP binding, one indicator of thermogenic capacity. Carcasses were subjected to body composition analysis. Although prior to surgery, PIT and SHAM rats weighed the same, PIT rats gained significantly more weight during the experiment than did SHAMs. Percent body fat and food intake (both total intake and intake relative to metabolic body size) were significantly elevated in the PIT rats. GDP binding in both PIT and LACT rats was significantly less than in SHAMs. This was true whether GDP binding was expressed per mg mitochondrial protein or per total amount of mitochondrial protein recovered. These data confirm that brown fat thermogenic capacity is suppressed during lactation. They also demonstrate that elevations of serum prolactin, to levels that are well within physiological limits, are capable of stimulating food intake and white fat deposition in the female rat. It is presently unclear whether these results are a direct or an indirect effect of hyperprolactinemia.

Uncoupling proteins-2 and 3 influence obesity and inflammation in transgenic mice.

OBJECTIVE: To test the hypothesis that either uncoupling protein-2 UCP2 or UCP3 or both together influence obesity and inflammation in transgenic mice.DESIGN: We generated 12 lines of transgenic mice for both human UCP2 and 3 using native promoters from a human bacterial artificial chromosome (BAC) clone. The BAC expresses no genes other than UCP2 and 3. Mice used for experiments are N4 or higher of backcross to C57BL/6J (B6). Each experiment used transgenic mice and their nontransgenic littermates.RESULTS: Northern blots confirmed expression on human UCP2 in adipose and spleen, while human UCP3 expression was detectable in gastrocnemius muscle. Western blots demonstrated a four-fold increase of UCP2 protein in spleens of Line 32 transgenic animals. Heterozygous mice of four lines showing expression of human UCP2 in spleen were examined for obesity phenotypes. There were no significant differences between Lines 1 and 32, but female transgenics of both lines had significantly smaller femoral fat depots than the control (littermate) mice (P=0.015 and 0.005, respectively). In addition, total fat of transgenic females was significantly less in Line 1 (P=0.05) and almost significantly different in Line 32 (P=0.06). Male Line 1 mice were leaner (P=0.04) while male Line 32 mice were almost significantly leaner (P=0.06). Heterozygous mice of Lines 35 and 44 showed no significant differences from the nontransgenic littermate controls. Effects of the UCP2/UCP3 transgene on obesity in Line 32 mice were confirmed by crossing transgenic mice with the B6.Cg-Ay agouti obese mice. B6.Cg-Ay carrying the UCP2/UCP3 transgene from Line 32 were significantly leaner than nontransgenic B6.Cg-Ay mice.Line 32 UCP2/UCP3 transgenics showed increased hypothalamic Neuropeptide (NPY) levels and food intake, with reduced spontaneous physical activity. Transgenic baseline interleukin4 (IL-4) and interleukin6 (IL-6) levels were low with lower or later increases after endotoxin injection compared to wild-type littermates. Endotoxin-induced fever was also diminished in transgenic male animals. Low-density lipoprotein (LDL) cholesterol levels were significantly higher in both Line 1 and 32 transgenics (P=0.05 and 0.001, respectively) after they had been placed on a moderate fat-defined diet containing 32% of calories from fat for 5 weeks.CONCLUSION: Moderate overexpression of UCP2 and 3 reduced fat mass and increased LDL cholesterol in two independent lines of transgenic mice. Thus, the reduced fat mass cannot be due to insertional mutagenesis since virtually identical fat pad weights and masses were observed with the two independent lines. Line 32 mice also have altered inflammation and mitochondrial function. We conclude that UCP2 and/or 3 have small but significant effects on obesity in mice, and that their mechanism of action may include alterations of metabolic rate.

T3 stimulates resting metabolism and UCP-2 and UCP-3 mRNA but not nonphosphorylating mitochondrial respiration in mice

The molecular basis for variations in resting metabolic rate (RMR) within a species is unknown. One possibility is that variations in RMR occur because of variations in uncoupling protein 2 (UCP-2) and uncoupling protein 3 (UCP-3) expression, resulting in mitochondrial proton leak differences. We tested the hypothesis that UCP-2 and -3 mRNAs positively correlate with RMR and proton leak. We treated thyroidectomized and sham-operated mice with triiodothyronine (T(3)) or vehicle and measured RMR, liver, and skeletal muscle mitochondrial nonphosphorylating respiration and UCP-2 and -3 mRNAs. T(3) stimulated RMR and liver UCP-2 and gastrocnemius UCP-2 and -3 expression. Mitochondrial respiration was not affected by T(3) and did not correlate with UCP-2 and -3 mRNAs. Gastrocnemius UCP-2 and -3 expression did correlate with RMR. We conclude 1) T(3) did not influence intrinsic mitochondrial properties such as membrane structure and composition, and 2) variations in UCP-2 and -3 expression may partly explain variations in RMR. One possible explanation for these data is that T(3) stimulates the leak in vivo but not in vitro because a posttranslational regulator of UCP-2 and -3 is not retained in the mitochondrial fraction.The molecular basis for variations in resting metabolic rate (RMR) within a species is unknown. One possibility is that variations in RMR occur because of variations in uncoupling protein 2 (UCP-2) and uncoupling protein 3 (UCP-3) expression, resulting in mitochondrial proton leak differences. We tested the hypothesis that UCP-2 and -3 mRNAs positively correlate with RMR and proton leak. We treated thyroidectomized and sham-operated mice with triiodothyronine (T3) or vehicle and measured RMR, liver, and skeletal muscle mitochondrial nonphosphorylating respiration and UCP-2 and -3 mRNAs. T3 stimulated RMR and liver UCP-2 and gastrocnemius UCP-2 and -3 expression. Mitochondrial respiration was not affected by T3 and did not correlate with UCP-2 and -3 mRNAs. Gastrocnemius UCP-2 and -3 expression did correlate with RMR. We conclude 1) T3 did not influence intrinsic mitochondrial properties such as membrane structure and composition, and 2) variations in UCP-2 and -3 expression may partly explain variations in RMR. One possible explanation for these data is that T3 stimulates the leak in vivo but not in vitro because a posttranslational regulator of UCP-2 and -3 is not retained in the mitochondrial fraction.

Brown Adipose Tissue Thermogenesis During Aging and Senescence

We have found that cold- and norepinephrine-induced brown adipose tissue (BAT) nonshiveringthermogenesis (NST) is significantly lower in old male Fischer 344 rats and is associatedwith the decreased ability of these animals to maintain homeothermy. This decline in BATthermogenesis is not as great in females. Although the mechanism(s) underlying this genderdifference in the age-related decrease in brown fat NST are not completely elucidated, theydo not appear to reflect decreased sympathetic neural activity of BAT in the older males vs.females. Rather, our investigations, strongly suggest that the blunted cold-induced heatproduction of BAT reflects less functional BAT. The fact that the older animals have less functionalBAT than do their younger counterparts may predispose them to the accumulation of excessbody fat. Our studies have also found that near the end of the natural life of these rats, theyenter a state of senescence that can be identified by spontaneous rapid body weight loss,resulting from decreased food intake. In this state, the rats are considerably more susceptibleto cold than are comparably aged presenescent (body weight stable) rats of the samechronological age. The greater hypothermia exhibited by the senescent vs. presenescent rats during coldexposure is associated with a significant reduction in the amount of functional brown fat andin the amount of heat each brown fat cell can generate. It is the intent of this review to discussthe findings of these investigations.

The effect of adrenergic agonists and cyclic AMP on the Na+/K+ ATPase activity of brown adipose tissue.

The Na+/K+ ATPase activity of membrane fractions prepared from brown adipose tissue of cold-acclimated rats could be stimulated by addition of any one of the following: norepinephrine, isoproterenol, cyclic AMP or phenylephrine. These results are consistent with the proposal that in the intact brown adipocyte, the norepinephrine-induced stimulation of the Na+/K+ membrane pump is associated with alpha- as well as beta-adrenergic pathways and is, in part, mediated via cyclic AMP.

Functional and anatomical characteristics of the nerve-brown adipose interaction in the rat

SummaryTwo aspects of the coupling of neural information to brown fat thermogenesis were examined—namely, the thermal responses to increasing neural stimulation and the anatomical nature of the brown fat innervation. Upon stimulation of the nerves to the interscapular brown fat pad, there ensued a biphasic response. This response was manifested by an initial, but transient temperature decrease, followed by a rise in brown fat temperature. The magnitude of both components of this response increased with increasing stimulus strength, thereby demonstrating the ability of the tissue to respond in a graded manner—a feature which may underlie the controlled thermogenic response of brown fat observed in the cold-exposed intact animal.No anatomically unique fiber types appeared to be specifically associated with innervation to the brown adipocytes or to the vessels within the fat pad. On the other hand, the nerves entering the interscapular fat pad were morphologically dissimilar, a finding consonent with their functional dissimilarity (i.e., innervation of adipocytes, innervation of blood vessels in the fat pad, and innervation of areas in the overlying skin).

Alterations in endogenous circadian rhythm of core temperature in senescent Fischer 344 rats

We assessed whether alterations in endogenous circadian rhythm of core temperature (CRT) in aging rats are associated with chronological time or with a biological marker of senescence, i.e., spontaneous rapid body weight loss. CRT was measured in male Fischer 344 (F344) rats beginning at age 689 days and then continuously until death. Young rats were also monitored. The rats were housed under constant dim red light at 24-26°C, and core temperature was recorded every 10 min via biotelemetry. The CRT amplitude of the body weight-stable (presenescent) old rats was significantly less than that of young rats at all analysis periods. At the onset of spontaneous rapid weight loss (senescence), all measures of endogenous CRT differed significantly from those in the presenescent period. The suprachiasmatic nucleus (a circadian pacemaker) of the senescent rats maintained its light responsiveness as determined by an increase in c- fos expression after a brief light exposure. These data demonstrate that some characteristics of the CRT are altered slowly with chronological aging, whereas others occur rapidly with the onset of senescence.

Effect of aging, caloric restriction, and uncoupling protein 3 (UCP3) on mitochondrial proton leak in mice

Mitochondrial proton leak may modulate reactive oxygen species (ROS) production and play a role in aging. The purpose of this study was to determine proton leak across the life span in skeletal mitochondria from calorie-restricted and UCP2/3 overexpressing mice. Proton leak in isolated mitochondria and markers of oxidative stress in whole tissue were measured in female C57BL/6J mice fed ad-libitum (WT-Control) or a 30% calorie-restricted (WT-CR) diet, and in mice overexpressing UCP2 and UCP3 (Positive-TG), their non-overexpressing littermates (Negative-TG) and UCP3 knockout mice (UCP3KO). Proton leak in WT-CR mice was lower than that of control mice at 8 and 26 months of age. The Positive-TG mice had greater proton leak than the Negative-TG and UCP3KO mice at 8 months of age, but this difference disappeared by 19 and 26 months. Lipid peroxidation was generally lower in WT-CR vs. WT-Control mice and UCP3KO mice had greater concentrations of T-BARS (thiobarbituric acid reactive substances, a measure of lipid peroxidation) than did Positive-TG and Negative-TG. The results of this study indicate that sustained increases in muscle mitochondrial proton leak are not responsible for alterations in life span with calorie restriction or UCP3 overexpression in mice. However, UCP3 may contribute to the actions of CR through mechanisms distinct from increasing basal proton leak.

Meal-induced changes in extracellular 5-HT in medial hypothalamus of lean (Fa/Fa) and obese (fa/fa) Zucker rats.

Hypothalamic serotonin (5-HT) is involved in appetite regulation and sympathetic stimulation of thermogenesis. This study tested the hypothesis that the enhanced energetic efficiency of obese Zucker rats involves blunted serotonergic release within the medial hypothalamus (MH). We used microdialysis and HPLC-EC to measure dynamic changes in extracellular 5-HT levels in the MH of 10-13-week-old male lean (Fa/Fa) and obese (fa/fa) Zucker rats before and after a meal. No differences were noted in basal levels of 5-HT between lean and obese rats. Consistent with the suggestion that hypothalamic 5-HT plays a physiological role in feeding, extracellular 5-HT levels increased significantly in both lean and obese rats given a meal. This increase was observed in the 20 min interval in which they ate the 8.1 kcal meal and remained for an additional 60 min. The net release of 5-HT during the meal interval was comparable in the lean (1.46+/-0.38 fmol/microl) and obese (1.21+/-0.82 fmol/microl) rats. However, the 5-HT levels of the leans (1.80+/-0.29 fmol/microl) plateaued in the next 20 min interval, whereas they continued rising (2.74+/-0.53 fmol/microl) in obese rats and were significantly higher than those in the leans during the 40 and 60 min intervals after the meal was presented. This resulted in a total net release during the meal plus the next three 20 min intervals that was significantly higher in obese (9.83+/-1.16 fmol/microl) than in lean (5.59+/-0.85 fmol/microl) rats. Thus, the enhanced energetic efficiency of the obese Zucker rats may not be associated with attenuated serotonin release in response to a meal. Rather their enhanced release of 5-HT in the MH may reflect compensatory mechanisms for the elevated orexigen NPY, the reduction in meal-induced CCK release, and/or a functional resistance to 5-HT.

Thermogenic capacity and brown fat in rats exercise-trained by running

Brown adipose tissue, a major effector of nonshivering thermogenesis (NST) in mammals, is activated by the sympathetic neurotransmitter norepinephrine. Prolonged increases in norepinephrine levels, whether elicited by cold exposure or exogenous application of catecholamines, lead to increased NST and increased thermogenic capacity of brown fat. Exercise training is also accompanied by enhanced sympathetic activity. The possibility exists that this enhancement may alter brown fat function. The present study was designed to assess the effect of a running exercise regimen on whole animal NST and the in vivo response of brown fat. Rats were trained by running on a treadmill (an average of 17 m/min, 0 degrees incline, for 90 min/d) for a period of at least 6 weeks. Whole animal NST capacity was assessed by monitoring oxygen consumption in response to infusion of norepinephrine. As a measure of the contribution of brown fat to whole body NST, the mass and norepinephrine-stimulated blood flow (microsphere technique) to the tissue were measured. None of these variables differed between the exercised (n = 10) and sedentary (n = 10) groups. That is, there were no significant differences between the two groups with respect to resting oxygen consumption, norepinephrine-induced oxygen consumption, brown fat mass, and brown fat blood flow--whether expressed per gram of tissue or as total tissue blood flow (ie, tissue mass X blood flow per gram). Further study is needed to explain the differential responses of brown fat to the increased sympathetic activity occurring during exercise v that occurring during cold exposure.