Acetylated and nonacetylated forms of beta-endorphin in rat brain and pituitary

Extracts of rat posterior intermediate pituitary and extracts of brains from normal and hypophysectomized rats were separated by gel filtration chromatography and fractions were analyzed by both a classical β-endorphin radioimmunoassay and by a radioimmunoassay specific for α-N-acetyl β-endorphin. In posterior intermediate pituitary extracts, more than 90 percent of the β-endorphin-sized immunoreactive material was α-N-acetylated. In extracts of brains from normal rats, less than 2 percent of the β-endorphin-sized immunoreactive material corresponded to α-N-acetylβ-endorphin, whereas in brains from hypophysectomized animals, no α-N-acetylβ-endorphin-like material could be detected. Immunofluorescence on normal brain sections, using either affinity purified antibodies to α-N-acetylβ-endorphin or conventional β-endorphin antibodies, showed no α-N-acetylβ-endorphin immunoreactivity in β-endorphin neurons. Only in brain sections which had been acetylated $\text{in}\text{vitro}$ prior to immunostaining could α-N-acetylβ-endorphin-like material be detected in the β-endorphin neurons. These results suggest that—in contrast to the cells in the intermediate lobe of the pituitary—the β-endorphin in brain neurons is not α-N-acetylated and that the small amount of α-N-acetyl β-endorphin which can be found in extracts of brains from normal animals is probably of pituitary origin.     

Development and validation of a sensitive radioimmunoassay for naturally occurring beta-endorphin-like peptides in human plasma

β-Endorphin-like peptides in blood plasma of normal human subjects were studied by means of a radioimmunoassay (RIA) and gel filtration. Plasma was extracted with silica gel, which was washed with water and 1 n HCl, and eluted with 50% acetone. Plasma extracts thus obtained and standard synthetic human β-endorphin yielded parallel RIA curves. Total immunoreactivity in normal donors ranged from 1.2 to 10.4 fmol/ml (21 subjects). The immunoreactivity was completely destroyed by treatment with papain. Gel filtration indicated the presence of three components-one of unknown nature at the void volume and the others at elution positions characteristic of β-lipotropin and β-endorphin. Recoveries of human β-endorphin and β-lipotropin added to plasma were 53 and 58%, respectively. Addition of N-ethylmaleimide to plasma or of aprotinin to blood immmediately following collection had no effect on the amount of total immunoreactivity. Furthermore, a large amount of β-endorphin-like immunoreactivity. The above results lead us to conclude that a β-endorphin-like immunoreactive peptide occurs naturally in plasma of normal human subjects.     

Naltrexone reduces weight gain, alters “β-endorphin”, and reduces insulin output from pancreatic islets of genetically obese mice

Naltrexone, an opiate antagonist, was administered to young obese (ob/ob) and lean mice for five weeks. Animals had continuous access to food and received 10 mg/kg SC twice daily with equivalent volumes of saline given to controls. The effects on body weight, and pituitary and plasma levels of β-endorphin-like material were measured. Naltrexone-injected obese animals gained weight more slowly over the first three weeks while the weight gain of lean animals was not affected by naltrexone. Plasma levels of β-endorphin were shown to be significantly higher in untreated ob/ob mice and this difference increased with age (4–20 weeks). With naltrexone treatment, plasma levels in +/? mice rose and exceeded those in ob/ob. Saline treatment appeared to be a stress, and pituitary β-endorphins rose 4–6 fold in ob/ob compared with +/?. While naltrexone reduced the levels in ob/ob pituitary towards normal, no effect on β-endorphin levels in pituitary of lean mice was obtained. In vitro studies of effects of the opiate antagonists, naloxone, on insulin secretion by isolated islets provided additional evidence of resistance of lean mice to naloxone relative to ob/ob. (IRI secretion fell only in naloxone treated ob/ob islets.) These observations support the contention that this form of genetic obesity is characterized by elevated endogenous opiate levels and an increased sensitivity to opiate antagonists such as naltrexone or naloxone.     

Localization of multiple forms of ACTH- and β-endorphin-related substances in the pituitary of the reptile, Anolis carolinensis

Immunohistochemical studies on the pituitary of Anolis carolinensis detected ACTH-like, β-endorphin-like, and 16K fragment-like immunoreactivity in distinct clusters of cells in the anterior lobe; ACTH-like, αMSH-like, β-endorphin-like, and 16K fragment-like immunoreactivity was detected in all the cells of the intermediate lobe. Crude acid extracts of both lobes, when alayzed by radioimmunoassay, gave displacement curves in ACTH and β-endorphin assays which were parallel to the appropriate synthetic standard. Only extracts of the intermediate lobe gave parallel displacement curves in an αMSH radioimmunoassay. Extracts of both lobes crossreacted with antiserum to 16K fragment, but the displacement curves were not parallel to that of mouse 16K fragment standard. The levels of immunoreactive ACTH and β-endorphin in the intermediate lobe were approximately 8-fold higher than in the anterior lobe. Fractionation of anterior lobe and intermediate lobe extracts by either gel filtration on Sephadex G-75 in 10% formic acid or sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed multiple forms of ACTH-related and β-endorphin-related substances in both lobes. In the anterior lobe the major forms of immunoreactivity were, respectively, ACTH-sized and β-endorphin-sized. In the intermediate lobe the major forms of immunoreactivity were αMSH-sized, CLIP-sized, and β-endorphin-sized. In both lobes, antisera directed against ACTH and β-endorphin detected high molecular weight material with an apparent molecular weight slightly less than that of mouse pro-ACTH/endorphin; this material probably represents the putative common precursor for ACTH and β-endorphin in this species.     

Isolation and characterization of α-N-acetylβ-endorphin(1–26) from the rat posterior/intermediate pituitary lobe

An extract from 50 rat posterior intermediate pituitaries was fractionated by gel filtration followed by cation exchange chromatography. α-N-Acetylated derivatives of β-endorphin-like molecules were detected with a specific radioimmunoassay for α-N-acetylβ-endorphins. Six peaks of α-N-acetylβ-endorphin-like immunoreactivity were observed in the cation exchange chromatography fractions. One of these peaks was purified to homogeneity using reverse phase high performance liquid chromatography (RP-HPLC). The isolated peptide was characterized by tryptic digestion followed by RP-HPLC and by amino acid analysis. The results showed that the isolated peptide was α-N-acetylβ-endorphin(1–26) with an oxidized methionine residue at position 5. Two previously unrecognized α-N-acetylβ-endorphin derivatives were also observed during the isolation procedure.     

Naltrexone reduces weight gain, alters “β-endorphin”, and reduces insulin output from pancreatic islets of genetically obese mice

Naltrexone, an opiate antagonist, was administered to young obese (ob/ob) and lean mice for five weeks. Animals had continuous access to food and received 10 mg/kg SC twice daily with equivalent volumes of saline given to controls. The effects on body weight, and pituitary and plasma levels of β-endorphin-like material were measured. Naltrexone-injected obese animals gained weight more slowly over the first three weeks while the weight gain of lean animals was not affected by naltrexone. Plasma levels of β-endorphin were shown to be significantly higher in untreated ob/ob mice and this difference increased with age (4–20 weeks). With naltrexone treatment, plasma levels in +/? mice rose and exceeded those in ob/ob. Saline treatment appeared to be a stress, and pituitary β-endorphins rose 4–6 fold in ob/ob compared with +/?. While naltrexone reduced the levels in ob/ob pituitary towards normal, no effect on β-endorphin levels in pituitary of lean mice was obtained. In vitro studies of effects of the opiate antagonists, naloxone, on insulin secretion by isolated islets provided additional evidence of resistance of lean mice to naloxone relative to ob/ob. (IRI secretion fell only in naloxone treated ob/ob islets.) These observations support the contention that this form of genetic obesity is characterized by elevated endogenous opiate levels and an increased sensitivity to opiate antagonists such as naltrexone or naloxone.     

Evidence for a common precursor for αMSH and β-endorphin in the intermediate lobe of the pituitary of the reptile Anolis carolinensis

Immunohistochemical studies on the pituitary of Anolis carolinensis detected ACTH-like, β-endorphin-like, and 16K fragment-like immunoreactivity in distinct clusters of cells in the anterior lobe; ACTH-like, αMSH-like, β-endorphin-like, and 16K fragment-like immunoreactivity was detected in all the cells of the intermediate lobe. Crude acid extracts of both lobes, when alayzed by radioimmunoassay, gave displacement curves in ACTH and β-endorphin assays which were parallel to the appropriate synthetic standard. Only extracts of the intermediate lobe gave parallel displacement curves in an αMSH radioimmunoassay. Extracts of both lobes crossreacted with antiserum to 16K fragment, but the displacement curves were not parallel to that of mouse 16K fragment standard. The levels of immunoreactive ACTH and β-endorphin in the intermediate lobe were approximately 8-fold higher than in the anterior lobe. Fractionation of anterior lobe and intermediate lobe extracts by either gel filtration on Sephadex G-75 in 10% formic acid or sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed multiple forms of ACTH-related and β-endorphin-related substances in both lobes. In the anterior lobe the major forms of immunoreactivity were, respectively, ACTH-sized and β-endorphin-sized. In the intermediate lobe the major forms of immunoreactivity were αMSH-sized, CLIP-sized, and β-endorphin-sized. In both lobes, antisera directed against ACTH and β-endorphin detected high molecular weight material with an apparent molecular weight slightly less than that of mouse pro-ACTH/endorphin; this material probably represents the putative common precursor for ACTH and β-endorphin in this species.     

Elevated plasma beta-endorphin levels in pregnant women and their neonates.

Using radioimmunoassay technique β-endorphin levels were measured in the plasma of women undergoing labour and partirition and in the plasma of their neonates. The level of immunoreactive β-endorphin in the plasma of women undergoing labour was found to be significantly elevated (mean values: 38–135 fmoles/ml) above the levels found in non-pregnant women (mean values: 5–10 fmoles/ml). After birth, the level of β-endorphin-like immunoreactivity in maternal venous plasma was significantly higher than that in the umbilical vein and artery plasma of the new-borns, but there was no arterio-venous difference in the neonatal plasma. Since the antiserum used displayed the same avidity for human β-endorphin and β-lipotropin chromatographic separation of the immunoreactive components was performed by gelfiltration. Both peptides were found in the plasma of non-pregnant women, in maternal plasma and in the plasma of the neonates. In addition, high amounts of both peptides were found in the fetal pituitary gland showing that the fetus can probably produce its own peptides.     

Wide distribution of β-endorphin-like immunoreactivity in extrapituitary tissues of rat

Immunoreactive β-endorphin (IR-β-EP) was detected by radioimmunoassay in boiled acetic acid extracts of rat pituitary, eye, pineal, kidney, pancreas, gut and adrenal gland. Fractionation of the immunoreactivity by gel filtration and high pressure liquid chromatography revealed that in pituitary 5 % was due to material behaving like synthetic β-endorphin (β-EP), while in extrapituitary tissues 60–100 % of the immunoreactive material eluted identically with β-EP. Hence, the comparison between the quantity of β-EP in pituitary and extrapituitary tissues showed that in rat more than half of the total β-EP-like material is situated outside pituitary. The whole IR-β-EP (β-EP, β-lipotropin and pro-opiocortin) of pituitary, however, far exceeds that of extrapituitary tissues.     

Dysfunction of endothelium-dependent relaxation to insulin via PKC-mediated GRK2/Akt activation in aortas of ob/ob mice

In diabetic states, hyperinsulinemia may negatively regulate Akt/endothelial nitric oxide synthase (eNOS) activation. Our main aim was to investigate whether and how insulin might negatively regulate Akt/eNOS activities via G protein-coupled receptor kinase 2 (GRK2) in aortas from ob/ob mice. Endothelium-dependent relaxation was measured in aortic rings from ob/ob mice (a type 2 diabetes model). GRK2, β-arrestin2, and Akt/eNOS signaling-pathway protein levels and activities were mainly assayed by Western blotting. Plasma insulin was significantly elevated in ob/ob mice. Insulin-induced relaxation was significantly decreased in the ob/ob aortas [vs. age-matched control (lean) ones]. The response in ob/ob aortas was enhanced by PKC inhibitor or GRK2 inhibitor. Akt (at Thr(308)) phosphorylation and eNOS (at Ser(1177)) phosphorylation, and also the β-arrestin2 protein level, were markedly decreased in the membrane fraction of insulin-stimulated ob/ob aortas (vs. insulin-stimulated lean ones). These membrane-fraction expressions were enhanced by GRK2 inhibitor and by PKC inhibitor in the ob/ob group but not in the lean group. PKC activity was much greater in ob/ob than in lean aortas. GRK2 protein and activity levels were increased in ob/ob and were greatly reduced by GRK2 inhibitor or PKC inhibitor pretreatment. These results suggest that in the aorta in diabetic mice with hyperinsulinemia an upregulation of GRK2 and a decrease in β-arrestin2 inhibit insulin-induced stimulation of the Akt/eNOS pathway and that GRK2 overactivation may result from an increase in PKC activity.     

Growth hormone in normal female rat plasma appears on gel filtration as a large molecular weight form

Gel filtration of female rat plasma with normal growth hormone (GH) concentrations (less than 100 ng/m1) showed that nearly all the immuno-reactivity was centred on a peak with an apparent molecular weight in the region of 82,000. In contrast, pituitary GH was almost entirely monomeric. The majority of plasma prolactin (PRL) in the same samples had a molecular weight of 23,000 (i.e. monomeric), and was similar in profile to pituitary PRL. Samples from male rats showed some GH immunoreactivity at the 82,000 molecular weight position but more than 65% coeluted with monomeric PRL. In female plasma with GH concentration between 300 and 1,000 ng/ml, immuno-reactivity resolved into peaks at the void volume, the monomeric position, and a peak at 82,000 that decreased, as a percentage of the total, with increasing GH concentration. These results indicate the possible presence of a GH binding factor, with greater activity in female than male rat plasma.     

Existence of a common precursor to ACTH and endorphin in the anterior and intermediate lobes of the rat pituaitary

Extracts of rat anterior and intermediate-posterior pituitary were fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and assayed for immunoactive ACTH and endorphin. In both lobes the major forms of immunoactive ACTH have apparent molecular weights of 31,000 (31K), 20–21K, 14K, and 4.5K, and the major forms of immunoactive endorphin have apparent molecular weights of 31K (coincident with the peak of immunoactive ACTH), 13K (a βLPH-like peptide), and 3.5K (a β-endorphin-like peptide). However, the quantitative distribution of immunoactivity among the various forms differs greatly between the lobes. Assays using an extreme COOH-terminal ACTH antiserum indicate that the 31K ACTH/endorphin molecule in rat antierior and intermediate pituitary is similar to the pro-ACTH/endorphin molecule from mouse pituitary tumor cells. A radioimmunoassay that is specific for the NH₂-terminal non-ACTH, nonendorphin segment (referred to as 16K fragment) of the mouse pro-ACTH/endorphin molecule was used to assay extracts of rat pituitary. In addition to detecting material at 31K and 20–21K, the 16K fragment radioimmunoassay detects significant amounts of cross-reactive material with an apparent molecular weight of 16K in extracts of both lobes. This result also suggests that the structure and processing of the rat 31K ACTH/endorphin molecule is similar to that of mouse tumor cell pro-ACTH/endorphin. Cell suspensions were prepared from the anterior and intermediate lobes of the rat pituitary and maintained in culture for a 24-h period. The isolated cells from both lobes incorporate [³H] phenylalanine into immunoprecipitable ACTH- and endorphin-containing molecules. By sequential immunoprecipitation with ACTH and endorphin antisera, it is possible to demonstrate directly that a single molecule (31K ACTH/endorphin) has antigenic determinants for both ACTH and endorphin. Significant amounts of 31K ACTH/endorphin are released into the culture medium by isolated anterior lobe and intermediate lobe cells. The isolated intermediate lobe cells synthesize and secrete relatively large amounts of a β-endorphin-like molecule; the isolated anterior lobe cells secrete significant amounts of both a βLPH-like molecule and a β-endorphin like molecule. These same quantitative differences between anterior and intermediate lobe tissue were observed in immunoassays of extracts of the separated lobes and probably reflect differences in the processing of the common precursor. The isolated anterior lobe cells can be stimulated to release increased amounts of immunoprecipitable ACTH and endorphin by incubation with a cyclic AMP analog and a phosphodiesterase inhibitor.     

Affinity purification of β-endorphin-like material from NG108CC15 hybrid cells by means of the monoclonal β-endorphin antibody 3-E7

Neuroblastoma × glioma hybrid cells (NG108CC15) were examined for the presence of β-endorphin-like material. In order to differentiate this β-endorphin-like material from crude cell extract, a procedure for immunoaffinity chromatography was developed. The monoclonal antibody 3-E7 employed possesses the unique property of recognizing the N-terminal sequence of virtually all endogenous opioid peptides, but not their precursors. By means of this immunoaffinity procedure about 90% of exogenous β-endorphin was recovered from 10 ml phosphate buffered saline samples. Affinity chromatography served as first-step purification of crude NG108CC15 cell extract for the separation and concentration of β-endorphin-like material. The eluate of the immunoaffinity gel was subjected either to Sephadex gel filtration or to high pressure liquid chromatography. Under either condition, immunoreactive β-endorphin which eluted with synthetic β-endorphin was detected. The concentration in six different batches varied from 4 to 17 fmol/10⁸ cells. This would be 10–200-fold lower than that observed for the enkephalins or dynorphin A/α-neo-endorphin. It is concluded that the utilization of the monoclonal antibody 3-E7 for a first-step purification of cell extracts was an essential pre-requisite for the separation of β-endorphin-like material from the hybrid cells. The presence of enkephalin-like material, of dynorphin A/α-neo-endorphin-like material and of β-endorphin immunoreactive material suggests that NG108CC15 cells are able to generate opioid peptides related to the precursors pre-proenkephalin A, pre-proenkephalin B and pro-opiomelanocortin.     

beta-Endorphin in human plasma: basal and pathologically elevated levels.

β-Endorphin-like immunoreactivity was measured in plasma of normal human subjects and in plasma of patients with pathologically elevated ACTH levels. The antiserum used displayed the same avidity for human β-endorphin and human β-lipotropin (detection limit for both peptides 1–2 fmoles/tube). Gel chromatography of the immunoreactive components in plasma of normal subjects indicated the presence of both β-lipotropin (2.1 -10.1 fmoles/ml) and β-endorphin (3.5–6.4 fmoles/ml). A close correlation between immunoreactive β-endorphin and ACTH was found in plasma of patients suffering from Addison's disease, Cushing's disease and exhibiting Nelson's syndrome. Elevated levels of β-endorphin-like immunoreactivity in plasma of these patients were due to both β-lipotropin and β-endorphin.     

Desaturation of stearic acid by liver and adipose tissue from obese-hyperglycaemic mice (ob/ob).

Stearic acid desaturase activity was assayed in preparations from perigenital adipose tissue and liver from lean and genetically obese female mice (ob/ob). The total activity in the perigenital adipose tissue from obese mice was threefold greater than in the tissue from lean mice, but per g of adipose tissue the activity was twofold greater in tissue from lean mice. In liver, the activity in obese mice was elevated at 8 weeks of age, remained elevated up to 24 weeks and then decreased by half at 48 weeks, but at all ages was higher than that in lean mice. The decrease in desaturase activity of liver from obese mice at 48 weeks corresponded to a change in the fatty acid composition of liver lipids toward that found in lean mice. Whereas in adipose tissue much of the increased enzyme activity may be due to tissue hyperplasia, in liver it is mainly an increased activity per cell.     

Liver monoamine oxidase in the obese-hyperglycaemic (ob/ob) mouse.

1. The specific activity of monoamine oxidase was found to be greater in liver mitochondria from ob/ob mice than from lean mice. The activities of marker enzymes were similar in both tissues. 2. Experiments with various substrates (5-hydroxytryptamine, benzylamine and tyramine) and inhibitors (clorgyline and deprenyl) indicated that, unlike rat liver mitochondria, mouse liver mitochondria contain a predominance of the B-form of monoamine oxidase. 3. The Km values for lean and ob/ob mice were the same for any given substrate and were in the increasing order 5-hydroxytryptamine less than tyramine less than benzylamine. Vmax. was approximately 50% greater in obese than in lean mice. 4. Extraction of liver mitochondria with acetone/water or acetone/water/NH3 to remove lipids decreased the enzyme activity relatively more in obese- than in lean-mice preparations, but residual activity was the same in both preparations.     

Glucoregulatory effects of bombesin in lean and genetically obese hyperglycaemic (ob/ob) mice

1. Plasma glucose and insulin responses to bombesin were examined in 12-15-week-old 12 hr fasted lean and genetically obese hyperglycaemic (ob/ob) mice. 2. Bombesin (1 mg/kg ip) produced a prompt but transient increase of plasma insulin in lean mice (maximum increase of 50% at 5 min), and a more slowly generated but protracted insulin response in ob/ob mice (maximum increase of 80% at 30 min). Plasma glucose concentrations of both groups of mice were increased by bombesin (maximum increases of 40 and 48% respectively in lean and ob/ob mice at 15 min). 3. When administered with glucose (2 g/kg ip), bombesin (1 mg/kg ip) rapidly increased insulin concentrations of lean and ob/ob mice (maximum increases of 39 and 63% respectively at 5 min). Bombesin did not significantly alter the rise of plasma glucose after exogenous glucose administration to these mice. 4. The results indicate that bombesin exerts an insulin-releasing effect in lean and ob/ob mice. The greater insulin-releasing effect in ob/ob mice renders bombesin a possible component of the overactive entero-insular axis in the ob/ob mutant, especially if it acts within the islets as a neurotransmitter or paracrine agent.     

Studies on the role of opiate peptides in two forms of genetic obesity: ob/ob mouse and fa/fa rat

Recent reports have indicated that genetically obese hyperinsulinemic mice (ob/ob) and Zucker rats (fa/fa) compared with their lean controls have elevated levels of pituitary and plasma B-endorphins, opiates that can stimulate insulin secretion. In this study we have measured opiate levels by a radio-receptor assay in gastro-intestinal tissues and pancreas in ob/ob and fa/fa animals and their controls. Ob/ob mice showed significantly higher levels than control mice (+/+) in most gastro-intestinal tissues and pancreas. Levels in fa/fa rats did not differ from their controls. Radioimmunoassay of pancreas for B-endorphins, revealed higher levels in ob/ob vs +/+ mice, while there was no difference in the obese and lean rats. Fasting tended to decrease gastro-intestinal opioids in mice, while B-endorphin levels rose. It is concluded that opiates may play a significant role in the obesity of the ob/ob mouse and that this genetic obesity differs from that in Zucker rats.     

Zinc attenuation of GDP binding to brown adipocytes mitochondria in genetically obese (ob/ob) mice

In this study, we investigate the in vitro effect of zinc addition on guanosine diphosphate (GDP) binding to mitochondria in brown adipocytes of genetically obese (ob/ob) mice. Interscapular brown adipocytes of male mice (obese; lean) at 4 and 12 wk of age were incubated with 0, 50, 100, or 200 μM zinc sulfate. Mitochondria were then isolated and their GDP binding capacities were measured. The GDP-binding capacities of ob/ob mice were lower than lean mice, with or without zinc addition, in both age groups (p<0.05). Zinc addition did not have any significant effect on GDP binding in lean mice. GDP binding decreased with increasing zinc addition in ob/ob mice, and this attenuation was more predominant in 12-wk old ob/ob mice. Moreover, we found that high magnesium addition (5 mM) increased GDP binding in lean mice, but this effect was not significant in ob/ob mice. This study reveals that brown adipose tissue thermogenesis in ob/ob mice could be greatly attenuated by zinc addition, suggesting that zinc may play a regulatory role in obesity.     

Adrenalectomy increases norepinephrine turnover in brown adipose tissue of obese (ob/ob) mice

The hyperphagia and rapid body weight gain normally observed in young obese (ob/ob) mice were abolished by removal of their adrenal glands, whereas food intake and weight gain of lean mice were not significantly affected by adrenalectomy. Adrenalectomy lowered body energy density (kcal/g carcass) in obese mice more than could be attributed to reduced food intake per se, suggesting that their energy expenditure was also increased. In control obese mice, low stimulation of brown adipose tissue by the sympathetic nervous system, as indicated by the low fractional rates of norepinephrine (NE) turnover in their brown adipose tissue may have contributed to the reduced energy expenditure in these animals. Adrenalectomy increased the rates of NE turnover in brown adipose tissue of obese mice to rates nearly equal to those observed in lean mice without affecting NE turnover in this tissue of lean mice. Likewise, removal of the adrenals normalized the low rates of NE turnover in hearts of obese mice without affecting lean mice. Rates of NE turnover in two other organs, white adipose tissue and pancreas, of control and adrenalectomized obese mice were similar to rates observed in lean counterparts. The adrenal may thus contribute to both the hyperphagia and the low energy expenditure by brown adipose tissue that together cause gross obesity in ob/ob mice.