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Leptin, the gene product of the obese gene, may play an important role in regulating body weight by signalling the size of the adipose tissue mass. Plasma leptin was found to be highly correlated with body mass index (BMI) in rodents and in 87 lean and obese humans. In humans, there was variability in plasma leptin at each BMI suggesting that there are differences in its secretion rate from fat. Weight loss due to food restriction was associated with a decrease in plasma leptin in samples from mice and obese humans.

Previous studies have demonstrated that in vitro treatment of adipocytes with catecholamines results in a decrease in the activity of the enzyme lipoprotein lipase (LPL). To examine the mechanism of this effect, primary cultures of rat adipocytes were cultured in the presence of various concentrations of epinephrine (10(-9)-10(-5) M). Epinephrine yielded a dose-dependent decrease in LPL activity; heparin-releasable LPL activity was reduced to 66% of control values after exposure to 10(-5) M epinephrine for 2 h. However, there was no effect of epinephrine on LPL immunoreactive mass, as measured by enzyme-linked immunosorbent assay. When cells were pulse labeled with [35S]methionine, there was a rapid and dose-dependent decrease in immunoprecipitable LPL. In spite of the decrease in LPL translation, neither epinephrine nor other catecholamines altered the level of LPL mRNA or the rate of LPL transcription. To further examine LPL posttranslational processing, cells were pulse labeled with [35S]methionine in the absence of epinephrine and then chased with unlabeled methionine in the presence of epinephrine. Cells exposed to epinephrine during the chase demonstrated a decrease in LPL secretion into the medium as well as a decrease in LPL degradation. The addition of epinephrine during LPL posttranslational processing did not alter the sensitivity of the newly synthesized LPL protein to endo-beta-N-acetylglucosaminidase-H. Thus, epinephrine had multiple effects on adipocyte LPL. Although there was a rapid decrease in LPL synthesis that was not due to changes in LPL mRNA, the level of LPL protein was unchanged under these conditions due to a decrease in LPL degradation and secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

Patients with diabetes commonly manifest hypertriglyceridemia along with decreased adipose tissue lipoprotein lipase (LPL) activity, and improved diabetes control tends to reverse these abnormalities. To better understand the mechanism of regulation of LPL in diabetes, 11 diabetic patients (3 type I, 8 type II) were brought under improved glycemic control, and adipose tissue LPL gene expression was assessed by performing paired fat biopsies. Six of the 11 patients attained improved control with insulin, with a decrease in glycohemoglobin (glyc Hgb) from 13.8 +/- 0.9 to 10.4 +/- 0.6%; 5 patients attained improved control with glyburide (glyc Hgb fell from 14.2 +/- 2.4 to 8.8 +/- 0.6%), and together they demonstrated a lowering of serum triglycerides and total cholesterol. No changes were observed in HDL cholesterol. Improved diabetes control resulted in a significant increase in LPL activity in both the heparin-releasable (HR) and extractable (EXT) fractions of adipose tissue, as well as in LPL immunoreactive mass. The change in LPL activity with improved control was variable, and showed a positive correlation with the HDL levels prior to treatment (r = 0.74, P less than 0.02). When adipose tissue was pulse-labeled with [35S]methionine, there was an increase in isotope incorporation into LPL after treatment, indicating an increase in LPL synthetic rate. However, improved diabetes control resulted in no significant change in LPL mRNA levels. Thus, improved glycemic control resulted in an increase in LPL activity which correlated with each patient's basal high density lipoprotein. This increase in LPL activity was accompanied by an increase in LPL immunoreactive mass, and an increase in LPL synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

purpose: The effects of hydroxychloroquine (HCQ) on serum levels of cholesterol, triglycerides, and high- (HDL) and low-density lipoprotein (LDL) were studied in patients with rheumatoid arthritis or systemic lupus erythematosus. patients and methods: A total of 155 women were divided into the following treatment groups: Group A: patients taking HCQ and no steroids (n = 58); Group B: patients taking steroids and no HCQ (n = 35); Group C: patients receiving both HCQ and steroids (n = 18); and Group D: patients receiving neither HCQ nor steroids (n = 44). results: HCQ therapy had a high statistical association with low serum levels of cholesterol (181 mg/dL; p=0.0006), triglycerides (106 mg/dL; p=0.0459), and LDL (101 mg/dL; p=0.0004), irrespective of concomitant steroid administration. The HCQ-treated group (A) had lower cholesterol (181 mg/dL; p=0.0039) and LDL (101 mg/dL; p=0.007) levels than those receiving neither HCQ nor steroids (205 mg/dL) and 128 mg/dL) (Group D). No HDL differences were observed. conclusion: The effects of HCQ do not appear to be due to changes in diet or weight, and the drug was well tolerated. Although the mechanism of cholesterol lowering by HCQ is not known, this drug deserves further investigation for its lipid-lowering properties.

Lipoprotein lipase is an enzyme in adipose tissue that hydrolyzes circulating triglycerides and thereby generates the fatty acids used in the synthesis of triglyceride in fat cells. To determine whether the activity and expression of lipoprotein lipase are affected by weight loss, we studied lipoprotein lipase in the adipose tissue of nine very obese subjects before and after a program of weight reduction. The subjects' mean (+/- SEM) initial weight was 136 +/- 7.3 kg, and the body-mass index (weight in kilograms divided by the square of the height in meters) ranged from 33.3 to 52.8 (mean, 43.0 +/- 2.5). Biopsies of adipose tissue were performed before weight loss and after it, when weight had been stable for three months. The weight reduction was achieved by a very-low-calorie diet (mean weight loss, 42.5 +/- 6.8 kg). After weight loss, the level of heparin-releasable lipoprotein lipase activity increased in all patients, from 3.8 +/- 1.1 to 7.1 +/- 1.6 neq of free fatty acid released per minute per 10(6) cells (P less than 0.05). In addition, the amount of lipoprotein lipase immunoreactive protein increased from 6.3 +/- 1.7 to 24.4 +/- 6.9 ng per 10(6) cells (P less than 0.05), and there was also an increase in the level of lipoprotein lipase messenger RNA as measured by Northern blotting. There was a strongly positive correlation between the initial body-mass index and the magnitude of the increase in lipoprotein lipase activity (r = 0.80, P less than 0.01) and immunoreactive protein (r = 0.92, P less than 0.01). We conclude that weight loss in very obese subjects leads to the increased activity and expression of lipoprotein lipase, thereby potentially enhancing lipid storage and making further weight loss more difficult.

Postheparin plasma is a convenient source for the measurement of lipoprotein lipase (LPL) in humans. Previous studies have focused on the measurement of LPL catalytic activity, and have been unable to conveniently measure the LPL protein or identify possibly different plasma forms of the enzyme. Pre- and postheparin plasma was treated with a highly specific antibody raised against bovine milk LPL and the immunoprecipitate was analyzed by Western blotting. In normal subjects there were several species of LPL in plasma. A 56 kD protein increased after heparin injection, and likely represented active LPL. The anti-LPL antibody reacted specifically with this 56 kD protein, and also reacted specifically with proteins at 52 kD, 69 kD, as well as a 20 kD breakdown product. In addition, using peptide mapping, the 56 kD protein was structurally similar to the 52 and 69 kD LPL proteins. The antibodies were affinity purified, biotinylated, and used to quantitate LPL immunoreactive mass using an enzyme-linked immunosorbent assay (ELISA). LPL immunoreactive mass was present in all subjects in preheparin plasma. In postheparin plasma, five patients with type I hyperlipoproteinemia displayed decreased LPL immunoreactive mass when compared to normal subjects, although there was a wide range of specific activity of the small amount of enzyme present. When the LPL from the plasma of the patients was immunoprecipitated and Western blotted, there was considerable heterogeneity in the appearance of the LPL forms, and an overall decrease in LPL protein. Thus, several different immunoreactive LPL proteins were present in pre- and postheparin plasma. In preheparin plasma, as well as in patients with type I hyperlipoproteinemia, there was decreased immunoreactive LPL protein, and the LPL protein that was present was of low specific activity.

Previous studies of human adipose tissue lipoprotein lipase (LPL) have focused on enzyme catalytic activity, and have not measured the LPL protein directly. To study the regulation of the LPL protein, an antibody against purified bovine LPL was used. To demonstrate the specificity of the antiserum, adipose homogenates were Western blotted, and adipocytes were radiolabeled and the cell homogenates immunoprecipitated, yielding a single specific band at 53 kD. Breakdown products of LPL were demonstrated at 35 and 20 kD by Western blotting. An ELISA for human adipose LPL was established, in which LPL was sandwiched between affinity-purified antibody and biotinylated affinity-purified antibody. The standard curves for bovine LPL and human adipose LPL were parallel, and LPL activity correlated strongly with LPL immunoreactive mass. Thus, the bovine LPL standard curve was used to estimate LPL immunoreactive mass from human adipose tissue. The regulation of LPL activity and immunoreactive mass were compared in cultured adipocytes in the presence an absence of insulinlike growth factor-I/somatomedin C (IGF-I), insulin, and fetal bovine serum. IGF-I and a high insulin concentration (70 nM) stimulated only the heparin-releasable (HR) component of LPL activity and immunoreactive mass, and neither IGF-I nor insulin affected LPL specific activity. In contrast, 10% fetal bovine serum stimulated HR activity, HR mass, and cellular extractable (EXT) immunoreactive mass, with no effect on EXT activity. This resulted in a decrease in EXT specific activity in response to serum. The effects of the locally produced nucleosides adenosine and inosine were studied in a similar manner. As with serum, adenosine stimulated HR activity, HR mass, and EXT immunoreactive mass, resulting in a decrease in EXT specific activity. Inosine stimulated an increase in HR activity and HR mass, but had no effect on EXT, and thus did not change LPL specific activity. Thus, a sensitive ELISA for adipose tissue LPL has been developed using a specific, well-characterized antibody. Regulation of human LPL immunoreactive mass was demonstrated in vitro by IGF-I, serum, high concentrations of insulin, adenosine, and inosine. This method will permit further investigations into the regulation of the LPL protein.

To develop a system for studying the regulation of adipose tissue lipoprotein lipase (LPL) in human cells, we isolated cells from the stromalvascular fraction of human adipose tissue and propagated them in tissue culture. These cells were cultured in medium containing fetal bovine, horse, or human sera in the absence or presence of aminoglycoside antibiotics. After the cells had been at confluence for at least 1 week, they were exposed to medium containing insulin, 3-isobutyl-l-methylxanthine (IBMX), and dexamethasone, in an attempt to induce differentiation into cells which produce LPL. Stromal cells in 62 cultures from 20 subjects were monitored for any LPL activity secreted into the culture medium. In addition, cultures were assayed for activity releasable with heparin, and extractable from cell digests in nonionic detergent. Upon reaching confluence, the human adipose tissue stromal cells began to accumulate lipid droplets. However, no consistent LPL activity was measured in the culture medium or after exposure to heparin. Five cultures contained a minimal amount of detergent extractable hydrolytic activity. In contrast, cultures of rat stromal cells not exposed to insulin, IBMX, and dexamethasone, contained assayable LPL in the culture medium (3.3 +/- 1.6 nEq/min/ml) and in the heparin-releasable fraction (1.7 +/- 0.5 nEq/min/10(6)cells). Thus, human adipose tissue stromal cells do not represent a useful system for studying the regulation of LPL. Although these cells accumulate some lipid, the absence of measurable LPL suggests that complete differentiation has not yet occurred.

Previous studies have demonstrated that cachectin/tumor necrosis factor (TNF) inhibits lipoprotein lipase (LPL) activity in cultures of 3T3-L1 cells. To determine whether TNF also inhibits LPL in human adipocytes, primary cultures of isolated human adipocytes were exposed to a spectrum of concentrations of recombinant human TNF. TNF concentrations up to 1000 pM had no effect on either LPL activity or LPL immunoreactive mass in the human adipocytes. Specific binding of 125I-labeled TNF was demonstrated in human adipocytes, and a TNF concentration of 100 pM competed for approximately 50% of the 125I-labeled TNF binding sites. In contrast, the same TNF in the same concentrations progressively inhibited LPL activity and immunoreactive mass in 3T3-L1 cells. Thus, human adipocytes respond to TNF in a different manner than 3T3-L1 cells. TNF may not cause the cachexia of cancer or chronic infection by directly inhibiting LPL in adipose tissue.

Polyclonal antibodies against bovine milk lipoprotein lipase (LPL) were used to generate an enzyme-linked immunosorbent assay (ELISA) for rat LPL. The antibodies to LPL were affinity purified on bovine LPL columns and were shown to be specific for LPL by immunoprecipitation and enzyme inhibition. The solid-phase ELISA was sensitive from 1.0 to 20 ng/ml of LPL and paralleled enzyme activity. Denatured rat LPL showed the same LPL mass as undenatured samples, allowing LPL mass to be quantitated effectively in a variety of rat tissue extracts.

The syndrome of inappropriate antidiuresis (SIAD) is usually associated with inappropriately elevated plasma arginine vasopressin (AVP) concentrations. We describe herein a patient with a macroprolactinoma who had symptomatic hyponatremia due to SIAD. Although the patient had excessive thirst, severe plasma hypoosmolality, and hyperosmolar urine, no immunoassayable AVP could be detected. During long term treatment with bromocriptine, there was gradual shrinkage of the prolactinoma coincident with improvement in the ability to excrete a water load and normalization of the thirst threshold. At this point, plasma immunoactive AVP was measurable during a hypertonic saline infusion for the first time. By high pressure liquid chromatographic analysis, this immunoactive substance coeluted with AVP. These studies suggest that the SIAD in this patient was due to the production of an antidiuretic substance distinct from AVP in association with his prolactinoma.

Several studies have suggested that insulin and glucose increase adipose tissue lipoprotein lipase (LPL). To study the mechanism of the glucose-induced stimulation of LPL, the effects of glucose and glycosylation were examined in primary rat adipocyte cultures. In cells cultured in the presence of 1 mg/ml glucose, a 55-kDa LPL protein was synthesized and secreted into the medium, whereas cells cultured in glucose-free medium synthesized a 49-kDa form of LPL which was not secreted. The treatment of the mature 55-kDa form of LPL with peptide:N-glycosidase-F resulted in the formation of a 49-kDa form of LPL. When cells were cultured in the presence of tunicamycin, a 49-kDa form of LPL was synthesized by the cells but was not secreted. In addition, LPL activity was reduced by 90% when glycosylation was blocked by either tunicamycin or glucose deprivation. LPL synthetic rate was examined in cells cultured in a spectrum of glucose concentrations. LPL synthetic rate increased directly with medium glucose concentration and was decreased 80% in the absence of glucose compared to the synthetic rate in the presence of 1 mg/ml glucose. In addition, LPL synthetic rate in the presence of insulin was approximately 200% of the synthetic rate in untreated control cells at all glucose concentrations and even in the absence of glucose. In spite of the effect of glucose on LPL synthetic rate, glucose had no effect on the level of LPL mRNA. In contrast, the mRNA for the 78-kDa glucose-regulated protein (GRP78) was increased in adipocytes cultured in glucose-free medium. In summary, glucose was essential for glycosylation of LPL, and glycosylation was essential for LPL catalytic activity and secretion. In addition, glucose stimulated LPL synthetic rate and potentiated the stimulatory effects of insulin, but had no specific effect on LPL mRNA. Whereas insulin stimulates LPL by increasing the level of LPL mRNA, glucose stimulates LPL translation and post-translational processing.

Previous studies have demonstrated higher levels of adipose tissue lipoprotein lipase (LPL) catalytic activity in obese subjects, and in response to a meal. To examine the cellular mechanism of this increase in activity, LPL activity, immunoreactive mass, and mRNA level were measured in lean and obese subjects both before and 4 h after a carbohydrate-rich meal. Heparin-releasable (HR) LPL activity was approximately 2.5-fold higher in the 15 obese subjects, when compared with six lean subjects. However, there was no difference in LPL immunoreactive mass between the lean and obese subjects. In response to the meal, there was a 2.2-fold increase in total adipose tissue LPL activity in the lean subjects due to an increase in both the HR fraction, as well as the adipose fraction extracted with detergents. However, no increase in LPL immunoreactive mass was observed in any adipose tissue LPL fraction, resulting in an increase in LPL specific activity in response to the meal. In the obese subjects, there was no significant increase in LPL activity in response to feeding, and also no increase in immunoreactive mass or specific activity. After extraction of RNA, there was no difference in either the relative proportion of the 3.6- and 3.4-kb human LPL mRNA transcripts, nor in the quantity of LPL mRNA in response to feeding. Thus, these data suggest that the increase in LPL activity under these conditions occurs through a posttranslational activation of a previously inactive LPL precursor.

Human adipose tissue lipoprotein lipase (LPL) is stimulated in vivo by an insulin-glucose infusion. However, previous work by us showed no effect of physiologic insulin concentrations on LPL in isolated human adipocytes. To pursue further the regulation of LPL in vitro, primary cultures of isolated human adipocytes were prepared and exposed to glucose concentrations of 0–4.5 mg/ml. LPL activity was measured as activity secreted into the culture medium (CM), released from cells by heparin (HR), and extracted from cell digests (EXT). After 5 h in culture, a stimulatory effect of glucose on HR was observed. After 24 h there was a gradual increase in CM, HR, and EXT in parallel with increasing glucose concentrations of 0—1.0 mg/ml. At glucose concentrations >1.0 mg/ml, however, there was a decrease in CM. At a glucose concentration of 4.5 mg/ml, CM was only 51 ± 14% (P < .02) of its value at glucose concentrations of 1.0 mg/ml. Cellular LPL (HR and EXT) was not affected by high glucose concentrations. Response of cellular LPL to the hormonal regulator insulin-like growth factor I (IGF-I) was modulated by medium glucose. HR in cultures treated with 50 ng/ml IGF-I was 166 ± 40 and 147 ± 23% of HR in control cultures at glucose concentrations of 1.0 and 2.5 mg/ml, respectively (P ≤.05). However, IGF-I failed to stimulate HR at glucose concentrations >2.5 mg/ml or

Primary cultures of microvascular endothelial cells and isolated adipocytes were prepared from human omental adipose tissue to study the potentially overlapping roles of insulin and insulinlike growth factors (IGFs) in human adipose tissue. To determine whether adipocytes contain type I IGF receptors, binding experiments were carried out with 125 I-labeled IGF-I. At 16°C, saturation of specific binding to adipocytes was reached after 30 min and was 0.7% per 10 6 cells. At 37°C, chloroquine produced an increase in cell-associated 125 I-IGF-I, suggesting that IGF-I is internalized and degraded in a manner analogous to insulin. In competition experiments, IGF-I competed for binding more effectively than rat IGF-II or insulin. The concentrations of IGF-I, rat IGF-II, and insulin necessary to displace 50% of 125 I-IGF-I binding were 2.5,15, and 90 nM, respectively. In addition, a monoclonal antibody (α-IR 3 ) that has been shown to block the type I IGF receptor was used in competition binding experiments. The antibody also inhibited binding of 125 I-IGF-I to adipocytes. The biological effects of insulin and IGF-I were examined by studying adipocyte lipoprotein lipase (LPL). Insulin stimulated [ 14 C]glucose incorporation into cellular lipid in a dose-dependent manner, with 50% effective concentration (EC 50 ) of ∼.3 nM. However, an increase in LPL activity was observed only at a high insulin concentration, with an EC S0 of ∼30 nM. In contrast, IGF-I stimulated a progressive increase in LPL, with an EC 50 of 3.2 nM. In addition, α-IR 3 blocked the stimulatory effect of IGF-I on adipocyte LPL. To study the possible local production of IGFs in adipose tissue, adipose-derived human microvascular endothelial cells were cultured. The conditioned medium from these cells contained significant quantities of immunoreactive IGF. After passing the conditioned medium through a Deltapak C 4 100-A column and eluting with an acetonitrile gradient, IGF was separated from binding protein, and most of the immunoreactive IGF in endothelial cell-conditioned medium consisted of IGF binding protein, with smaller contributions from IGF-II and IGF-I. Thus, in human adipose tissue, IGFs and IGF binding protein are produced by microvascular endothelial cells. In addition, type I IGF receptors are present on adipocytes, and LPL is stimulated through type I receptors.

Reported herein is the possible interaction between two drugs used to treat a man with a large prolactin-secreting pituitary adenoma. The patient had a long history of schizophrenia that was treated with many different medications, including phenothlazines. Evaluation of progressive lethargy led to the discovery of a large parasellar tumor and a prolactin level of 7,981 ng/ml. His serum prolactln level fell to the 400 ng/ml range during bromocriptine therapy but rose whenever the antipsychotic thioridazine was added to his regimen. A marked deterioration of his visual fields was noted after 3 months' therapy with both drugs, and this abnormality resolved five days after the thioridazine was stopped. The use of dopamlne antagonists such as thioridazine in patients with prolactinoma may interfere with bromocriptine's action, resulting in potentially serious complications.

The cellular regulation of adipose tissue lipoprotein lipase by insulin was investigated using cultured isolated rat adipocytes. Evidence for sustained cell viability over 3 days included stability of specific [125I]insulin binding and adipocyte number. Lipoprotein lipase was measured in three functional compartments: 1) enzyme activity secreted into the culture medium, 2) activity releasable from cell suspensions by heparin, and 3) activity extractable from cells (after maximal heparin release) in deoxycholate and detergent. One day after preparation, these activities stabilized and were 1.3 +/- 0.2, 1.4 +/- 0.2, and 7.7 +/- 0.9 neq/10(6) cells X min, respectively (n = 24, mean +/- SEM). Insulin, added the day after preparation, produced a dose-dependent (1-400 ng/ml) increase in lipoprotein lipase releasable from cells by heparin at 2, 4, and 24 h. Insulin also increased intracellular enzyme measured as deoxycholate-detergent-solubilized activity extracted from previously heparin-released cells. However, insulin-mediated increases in culture medium enzyme only occurred subsequent to cellular effects. All insulin-mediated effects were prevented by cycloheximide (1 microgram/ml). Thus, insulin increased two cellular pools of adipocyte lipoprotein lipase in a dose-dependent manner, but had no direct effect on enzyme secretion. Overall, cultured isolated rat adipocytes appear to be a valuable system for the study of lipoprotein lipase regulation at the level of the adipocyte.

In Reply. —Dr Daniels is correct to point out that a low dose of levothyroxine can produce thyrotoxicosis in the presence of autonomous thyroid tissue. As stated in the figure legend in our article, the patient had not been receiving levothyroxine therapy three weeks before the preoperative sodium iodide I 123 thyroid scan. Several weeks later, the patient became clinically mildly hyperthyroid, with a serum thyroxine uptake of 16.0 μg/dL. At that time, she had not received any levothyroxine therapy for six weeks. Nevertheless, the indication for surgery was not the mild hyperthyroidism, but the mass effect of the enlarged left thyroid gland, which was causing a pressure sensation in the patient's neck. Potassium iodide in pharmacologic doses has been used for many years to prepare thyrotoxic patients for surgery. In addition to producing a prompt block to thyroid hormone secretion, iodide decreases vascularity within 14 days, 1 making the