Effect of feeding and obesity on lipoprotein lipase activity, immunoreactive protein, and messenger RNA levels in human adipose tissue.

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.

The regulation of adipose tissue and muscle lipoprotein lipase in runners by detraining.

To study the mechanism of lipoprotein lipase (LPL) regulation by exercise, we recruited 16 healthy athletes to undergo a 2-wk period of detraining. Fasting fat and muscle biopsies were performed both before and after the detraining period. In muscle, detraining resulted in a decrease in LPL activity in both the heparin-releasable (HR) (-45%, P < 0.05) and cellular (extractable [EXT]) (-75%, P < 0.005) fractions, with no significant changes in LPL immunoreactive mass and mRNA levels. However, several subjects demonstrated parallel decreases in LPL mass and mRNA levels with detraining, suggesting that there is some degree of heterogeneity in response. In adipose tissue, detraining had the opposite effects on LPL activity. In the HR fraction, detraining resulted in an 86% increase (P < 0.005) in LPL activity, which was paralleled by a 100% (P = 0.02) increase in HR mass. However, there was no significant change in EXT LPL activity or EXT LPL mass. There were no changes in adipose LPL synthetic rate or LPL mRNA levels with detraining. The ratio of adipose tissue/muscle LPL, which may be an important indicator of the tendency for storage of circulating lipids in adipose tissue, increased significantly after detraining. The adipose/muscle LPL ratio was 0.51 +/- 0.17 in the exercising runners, and 4.45 +/- 2.46 in the same runners after detraining (P < 0.05). Thus, detraining of athletes resulted in a decrease in muscle LPL that occurred through post-translational mechanisms, whereas adipose tissue LPL increased, also due to posttranslational changes. This decrease in muscle LPL, coupled with an increase in adipose LPL, yielded a condition favoring adipose tissue storage.

Isolation and culture of microvascular endothelium from human adipose tissue.

The study of human endothelial cells in tissue culture has been previously limited to umbilical vein, a large vessel source, and microvascular endothelium from human foreskin, spleen, and adrenal. Microvascular endothelium cultured from these sources have required matrix-coated culture flasks, tumor-conditioned medium, or 50% human serum for growth and subcultivation. To obtain cultures of microvascular endothelium with less stringent growth requirements, human adipose tissue was digested with collagenase and endothelial cells were separated from other stromal elements by sequential filtration and layering cells onto 5% albumin. Using standard medium containing 10% fetal calf serum, these cells grew readily to confluence and survived serial passages. When the cultures were subconfluent, cytoplasmic extensions and a capillary-like morphology were observed. Confluent cultures displayed the "cobblestone" appearance characteristic of other endothelial preparations. Electron microscopy demonstrated the presence of characteristic tight junctions and pinocytotic vesicles. Immunofluorescent staining for Factor VIII was positive, and cultures contained angiotensin-converting enzyme activity. Thus, cultures of human microvascular endothelium were readily obtained from adipose tissue and required only standard medium with 10% serum for growth and subcultivation. This system can be used to study human endothelial cell biology and may prove useful in the study of pathologic states such as diabetic microvasculopathy and tumor angiogenesis.

Regulation of lipoprotein lipase in primary cultures of isolated human adipocytes.

To study the regulation of adipose tissue lipoprotein lipase (LPL) in human adipocytes, omental adipose tissue was obtained from healthy subjects and digested in collagenase. The isolated adipocytes thus obtained were suspended in Medium 199 and cultured at 37 degrees C. Cell viability was demonstrated in adipocytes cultured for up to 72 h by constancy of cell number, cell size, trypan-blue exclusion, and specific 125I-insulin binding. In addition, chloroquine induced an increase in cell-associated 125I-insulin at 24, 48, and 72 h after preparation. Thus, isolated adipocytes retained their ability to bind, internalize, and degrade insulin. LPL was measured as activity secreted into the culture medium (CM), released from cells by heparin (HR), and extracted from cell digests. A broad range of heparin concentrations produced a prompt release of LPL from a rapidly replenishable pool of cellular activity. When cells were cultured in medium containing 10% fetal bovine serum, there was a marked stimulation of CM and HR. The secretory response to serum (CM) correlated strongly with HR 24 h after preparation (rs = 0.731, P less than 0.001). In addition, HR was found to correlate logarithmically and inversely with body mass index (r = -0.731, P less than 0.001). Insulin, at 400 ng/ml only, increased HR by 36 +/- 10%, an effect simulated by lower concentrations of insulin-like growth factor-1 (IGF1). Thus, LPL is produced and regulated in isolated human adipocytes. The degree of adiposity and serum are important regulators of HR activity, whereas insulin is stimulatory only at a pharmacologic concentration. This effect of insulin may be mediated through the IGF1 receptor. Isolated human adipocytes represent a novel and useful system for the study of LPL and lipid metabolism as well as for other aspects of adipocyte biology.

Regulation of lipoprotein lipase immunoreactive mass in isolated human adipocytes.

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.

Regulation of lipoprotein lipase translation by epinephrine in 3T3-L1 cells. Importance of the 3' untranslated region.

Lipoprotein lipase (LPL) is a central enzyme in lipoprotein metabolism and is in part responsible for adipocyte lipid accumulation. Catecholamines are known to decrease the activity of LPL in adipocytes, and we have previously demonstrated that this inhibition occurs posttranscriptionally, with a prominent inhibition of LPL translation. To better characterize the inhibition of LPL translation, 3T3-L1 cells were differentiated into adipocytes, and exposed to epinephrine. Epinephrine induced a dose-dependent decrease in LPL synthesis using [35S]methionine incorporation, with no change in LPL mRNA levels, demonstrating translational regulation of LPL in this cell line. The poly A-enriched RNA from epinephrine-treated cells was translated well in vitro, and there was no difference in the polysome profiles from control and epinephrine-treated cells, suggesting that epinephrine did not affect mRNA editing, and did not induce an inhibition of translation initiation. To obtain evidence for the presence of an inhibitory factor, a cytoplasmic extract from control, and epinephrine-treated adipocytes was human. When compared to the control cell extract, the epinephrine-treated cell extract sharply inhibited LPL translation in vitro, yet had no effect on the translation of other mRNAs. Epinephrine-treated cells had fourfold more of this inhibitor activity than control cells, and this translation inhibition was partially reversed by heat treatment. To determine what region of the LPL mRNA was involved in the translation inhibition, different LPL constructs were synthesized. The inhibitory effect of the epinephrine-treated cell extract was dependent on the presence of the first 40 nucleotides of the 3' (untranslated region UTR) (nucleotides 1599-1638), whereas deletion of the 5' UTR and other areas of the 3' UTR had no effect on translation inhibition. When a sense RNA strand corresponding to this region was added to the in vitro translation reaction, it restored translation towards normal, suggesting that the sense strand was competing for a transacting binding protein. Thus, epinephrine-treated adipocytes produced a transacting factor, probably a protein, that interacted with a region on the LPL mRNA between nucleotides 1599 and 1638, resulting in an inhibition of translation. These studies add new insight into the hormonal regulation of LPL.

The expression of tumor necrosis factor in human adipose tissue. Regulation by obesity, weight loss, and relationship to lipoprotein lipase.

A previous study reported the increased expression of the cytokine TNF in the adipose tissue of genetically obese rodents. To examine this paradigm in humans, we studied TNF expression in lean, obese, and reduced-obese human subjects. TNF mRNA was demonstrated in human adipocytes and adipose tissue by Northern blotting and PCR. TNF protein was quantitated by Western blotting and ELISA in both adipose tissue and the medium surrounding adipose tissue. Using quantitative reverse transcriptase PCR (RT-PCR), TNF mRNA levels were examined in the adipose tissue of 39 nondiabetic subjects, spanning a broad range of body mass index (BMI). There was a significant increase in adipose TNF mRNA levels with increasing adiposity. There was a significant correlation between TNF mRNA and percent body fat (r = 0.46, P < 0.05, n = 23). TNF mRNA tended to decrease in very obese subjects, but when subjects with a BMI > 45 kg/m2 were excluded, there was a significant correlation between TNF mRNA and BMI (r = 0.37, P < 0.05, n = 32). In addition, there was a significant decrease in adipose TNF with weight loss. In 11 obese subjects who lost between 14 and 66 kg (mean 34.7 kg, or 26.6% of initial weight), TNF mRNA levels decreased to 58% of initial levels after weight loss (P < 0.005), and TNF protein decreased to 46% of initial levels (P < 0.02). TNF is known to inhibit LPL activity. When fasting adipose LPL activity was measured in these subjects, there was a significant inverse relationship between TNF expression and LPL activity (r = -0.39, P < 0.02, n = 39). With weight loss, LPL activity increased to 411% of initial levels. However, the magnitude of the increase in LPL did not correlate with the decrease in TNF. Thus, TNF is expressed in human adipocytes. TNF is elevated in most obese subjects and is decreased by weight loss. In addition, there is an inverse relationship between TNF and LPL expression. These data suggest that endogenous TNF expression in adipose tissue may help limit obesity in some subjects, perhaps by increasing insulin resistance and decreasing LPL.

The expression of TNF alpha by human muscle. Relationship to insulin resistance.

TNFalpha is orverexpressed in the adipose tissue of obese rodents and humans, and is associated with insulin resistance. To more closely link TNF expression with whole body insulin action, we examined the expression of TNF by muscle, which is responsible for the majority of glucose uptake in vivo. Using RT-PCR, TNF was detected in human heart, in skeletal muscle from humans and rats, and in cultured human myocytes. Using competitive RT-PCR, TNF was quantitated in the muscle biopsy specimens from 15 subjects whose insulin sensitivity had been characterized using the glucose clamp. technique. TNF expression in the insulin resistant subjects and the diabetic patients was fourfold higher than in the insulin sensitive subjects, and there was a significant inverse linear relationship between maximal glucose disposal rate and muscle TNF (r = -0.60, P < 0.02). In nine subjects, muscle cells from vastus lateralis muscle biopsies were placed into tissue culture for 4 wk, and induced to differentiate into myotubes. TNF was secreted into the medium from these cells, and cells from diabetic patients expressed threefold more TNF than cells from nondiabetic subjects. Thus, TNF is expressed in human muscle, and is expressed at a higher level in the muscle tissue and in the cultured muscle cells from insulin resistant and diabetic subjects. These data suggest another mechanism by which TNF may play an important role in human insulin resistance.

Regulation of lipoprotein lipase by glucose in primary cultures of isolated human adipocytes. Relevance to hypertriglyceridemia of diabetes.

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 greater than 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 less than .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 less than or equal to .05). However, IGF-I failed to stimulate HR at glucose concentrations greater than 2.5 mg/ml or less than 1.0 mg/ml.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulinlike growth factor action and production in adipocytes and endothelial cells from human adipose tissue.

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 125I-labeled IGF-I. At 16 degrees C, saturation of specific binding to adipocytes was reached after 30 min and was 0.7% per 10(6) cells. At 37 degrees C, chloroquine produced an increase in cell-associated 125I-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 125I-IGF-I binding were 2.5, 15, and 90 nM, respectively. In addition, a monoclonal antibody (alpha-IR3) that has been shown to block the type I IGF receptor was used in competition binding experiments. The antibody also inhibited binding of 125I-IGF-I to adipocytes. The biological effects of insulin and IGF-I were examined by studying adipocyte lipoprotein lipase (LPL). Insulin stimulated [14C]glucose incorporation into cellular lipid in a dose-dependent manner, with 50% effective concentration (EC50) of 0.3 nM. However, an increase in LPL activity was observed only at a high insulin concentration, with an EC50 of approximately 30 nM. In contrast, IGF-I stimulated a progressive increase in LPL, with an EC50 of 3.2 nM. In addition, alpha-IR3 blocked the stimulatory effect of IGF-I on adipocyte LPL.(ABSTRACT TRUNCATED AT 250 WORDS)

Epinephrine inhibits lipoprotein lipase gene expression in rat adipocytes through multiple steps in posttranscriptional processing.

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)

Unilateral toxic multicystic goiter.

We treated a 43-year-old woman who had multiple thyroid cysts and coexistent toxic nodules localized to the left lobe. After surgical excision of this lobe, the patient's right lobe proceeded to function normally, displaying no evidence of nodules, cysts, or hyperfunction. Since toxic multinodular goiter is usually a diffuse process involving the whole gland, this case is unique and underlines the wide variability in the manifestation of this disorder.

The regulation of lipoprotein lipase gene expression by dexamethasone in isolated rat adipocytes.

Lipoprotein lipase (LPL) is an enzyme found in adipose tissue that is important in the hydrolysis of triglyceride rich lipoproteins, and in the uptake of FFA lipid into the adipocyte. To examine the effects of glucocorticoids on adipose tissue LPL, male Sprague-Dawley rats were injected with dexamethasone (1 mg/kg) every other day for 10 days, followed by measurement of LPL in epididymal adipose tissue. Compared to sham-injected controls, heparin-releasable LPL activity and LPL mass in the dexamethasone-treated rats were 44% and 62% of those in control rats, respectively. Adipocytes were prepared from the fat pads and pulse labeled with [35S]methionine, demonstrating a decrease in the LPL synthetic rate in the treated rats to 57% of the rate in control rats. In addition, LPL mRNA was quantitated by Northern blotting, demonstrating a decrease in LPL mRNA in the dexamethasone-treated rats. A simultaneous decrease in the message for gamma-actin was also noted. To examine the effects of dexamethasone on LPL in vitro, adipocytes were prepared from normal rats and treated with dexamethasone for 24 h in vitro. Dexamethasone decreased heparin-releasable LPL activity in cultured adipocytes to 40 +/- 6% of the control value (P less than 0.01). This decrease in LPL activity was accompanied by a decrease in the LPL synthetic rate using [35S]methionine labeling, to 33% of the control value, and no specific change in LPL turnover or secretion. In addition, dexamethasone added to adipocytes decreased LPL mRNA levels. Because the combination of insulin plus dexamethasone has been shown to yield synergistic increases in LPL in adipose tissue pieces, insulin was added to isolated adipocytes in combination with dexamethasone. Whereas insulin and dexamethasone individually had opposite effects on LPL, the combination of insulin plus dexamethasone resulted in no change in any aspect of LPL gene expression. Thus, dexamethasone resulted in a decrease in adipocyte LPL mRNA levels both when added to cultured adipocytes in vitro as well as when injected into rats. This decreased LPL mRNA level yielded corresponding changes in the LPL synthetic rate and LPL activity.

Insulin regulation of lipoprotein lipase in cultured isolated rat adipocytes.

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.

1,25-Dihydroxyvitamin D induces lipoprotein lipase expression in 3T3-L1 cells in association with adipocyte differentiation.

1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] is known to modulate the development of bone and other mesenchymal cell types. Since osteoblasts and adipocytes are thought to arise in bone marrow from a common progenitor, this work examined the effects of 1,25-(OH)2D3 on adipocyte development, and in particular on the expression of lipoprotein lipase (LPL), which is an early marker for the differentiated adipocyte. 3T3-L1 preadipocytes were cultured in the presence of 1,25-(OH)2D3 (10(-9) to 10(-7) M) for up to 7 days. LPL activity was measured in the medium and cell extracts, and LPL messenger RNA levels were measured by Northern blotting. When compared to control cells, 10(-7) M 1,25-(OH)2D3 increased medium LPL activity by 2- to 3-fold and cellular LPL by 1.5-fold. Significant increases in medium and cellular LPL were observed at 10(-9) M and were maximal at 10(-7) M. Along with the increase in LPL activity, there was an increase in LPL messenger RNA by 2-fold at 5 days, and by 5-fold at 7 days. In addition to an increase in LPL, 1,25-(OH)2D3 increased expression of aP2, an adipocyte-specific marker associated with differentiation. After the addition of 1,25-(OH)2D3, there was a decrease in 3T3-L1 cell number, which is consistent with differentiation, and a decrease in vitamin D receptors. Finally, these cells developed a different morphology. 1,25-(OH)2D3-treated cells assumed a rounded appearance, although without detachment from the dish and without the degree of lipid accumulation usually associated with the addition of insulin, isbutylmethylxanthine, and dexamethasone. It is concluded that 1,25-(OH)2D3 induced LPL expression in 3T3-L1 cells through an induction of differentiation-dependent mechanism(s). These findings suggest an important role for 1,25-(OH)2D3 in normal adipocyte differentiation.

Effects of tumor necrosis factor-alpha on glucose metabolism in cultured human muscle cells from nondiabetic and type 2 diabetic subjects.

The effects of tumor necrosis factor-alpha (TNF alpha) on glucose uptake and glycogen synthase (GS) activity were studied in human skeletal muscle cell cultures from nondiabetic and type 2 diabetic subjects. In nondiabetic muscle cells, acute (90-min) exposure to TNF alpha (5 ng/ml) stimulated glucose uptake (73 +/- 14% increase) to a greater extent than insulin (37 +/- 4%; P < 0.02). The acute uptake response to TNF alpha in diabetic cells (51 +/- 6% increase) was also greater than that to insulin (31 +/- 3%; P < 0.05). Prolonged (24-h) exposure of nondiabetic muscle cells to TNF alpha resulted in a further stimulation of uptake (152 +/- 31%; P < 0.05), whereas the increase in cells from type 2 diabetics was not significant compared with that in cells receiving acute treatment. After TNF alpha treatment, the level of glucose transporter-1 protein was elevated in nondiabetic (4.6-fold increase) and type 2 (1.7-fold) cells. Acute TNF alpha treatment had no effect on the fractional velocity of GS in either nondiabetic or type 2 cells. Prolonged exposure reduced the GS fractional velocity in both nondiabetic and diabetic cells. In summary, both acute and prolonged treatment with TNF alpha up-regulate glucose uptake activity in cultured human muscle cells, but reduce GS activity. Increased skeletal muscle glucose uptake in conditions of TNF alpha excess may serve as a compensatory mechanism in the insulin resistance of type 2 diabetes.

Lack of effect of leptin on glucose transport, lipoprotein lipase, and insulin action in adipose and muscle cells.

The effect of leptin on glucose transport, lipogenesis, and lipoprotein lipase activity was studied in cultured rat adipocytes and 3T3-L1 adipocytes. Leptin had no effect on basal and insulin stimulated glucose transport in isolated adipocytes from the rat and the genetically obese mouse. The incorporation of glucose into lipids was also unaffected. Lipoprotein lipase (LPL) activity remained unchanged in response to leptin in these cells, as well as in minced adipose tissue. Leptin also had no effect on both basal and insulin-stimulated glucose transport in cultured rat and human skeletal muscle cells. These studies showed that leptin had no effect on glucose transport, lipoprotein lipase activity, and insulin action in fat and muscle cells in vitro.

Effect of weight loss on muscle fiber type, fiber size, capillarity, and succinate dehydrogenase activity in humans.

To examine the effects of weight loss on muscle oxidative properties, nine obese subjects (body mass index, 34 +/- 1.5) had muscle biopsies before and after weight loss and weight stabilization. Weight loss ranged from 13-32 kg and represented 20.8 +/- 2.1% of initial weight. After weight loss, there was no change in the proportions of oxidative (type I and type IIa) fibers and also no change in mean fiber cross-sectional area, whereas there was a small, but significant, decrease in the relative interstitial space (P < 0.05). However, weight loss resulted in a 32 +/- 6% (mean +/- SEM) increase in capillary/fiber ratio and a 54% increase in capillary density (P < 0.05). In addition, there was a 41 +/- 13% increase in succinate dehydrogenase (SDH) activity (P < 0.05). This increase in muscle capillarization and SDH activity was seen in all fiber types, even the relatively lower oxidative type IIx fibers. There was a strong correlation between the change in capillary/fiber ratio and the change in SDH activity (r = 0.82; P < 0.02). Thus, weight loss resulted in no change in muscle fiber type or cross-sectional area, but produced increases in capillary/fiber ratio, capillary density, and SDH activity, suggesting an increase in muscle oxidative capacity.

Syndrome of inappropriate antidiuresis in the absence of arginine vasopressin.

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.

Effects of acromegaly treatment and growth hormone on adipose tissue lipoprotein lipase.

Lipoprotein lipase (LPL) hydrolyzes lipoprotein triglyceride into nonesterified fatty acids, which are then reesterified and stored in adipose tissue. Previous studies have demonstrated increases in LPL in response to insulin-like growth factor I and GH when added in vitro. This study examined the effects of acromegaly treatment on adipose tissue LPL. Ten patients with clinically active acromegaly were recruited. A fasting adipose tissue biopsy was performed both before and 3 months after treatment with octreotide (8 patients) or surgery plus octreotide (2 patients). With treatment, mean baseline insulin-like growth factor I levels fell from 6.41 to 3.98 U/mL (normal, < 2.2 U/mL; P < 0.05), and serum glycohemoglobin fell from 8.6 to 7.2 (normal, < 6.8). Adipose LPL was measured in the heparin-released fraction as well as the cellular fraction extracted with nonionic detergent (EXT). After treatment of acromegaly, there was no change in heparin-released fraction LPL activity or immunoreactive mass. However, there was an increase in EXT activity from 0.73 +/- 0.33 to 1.83 +/- 0.58 nEq/min.10(6) cells (mean +/- SEM; P < 0.05) and an increase in EXT mass from 4.1 +/- 0.89 to 11.4 +/- 2.0 ng/10(6) cells (P < 0.05). There was no change in LPL messenger ribonucleic acid levels with treatment, determined using both quantitative polymerase chain reaction and Northern blotting. Thus, treatment of acromegaly resulted in an increase in the intracellular level of the LPL protein, with no change in messenger ribonucleic acid levels, suggesting posttranscriptional regulation of LPL. These changes in LPL may be due to improved insulin sensitivity, or to other changes associated with acromegaly treatment.