Leptin does not affect adipocyte glucose metabolism: Studies in fresh and cultured adipocytes

Leptin Does Not Affect Adipocyte Glucose Metabolism: Studies in Fresh and Cultured Adipocytes Gall Mick, Tracy Vanderbloomer, Chang Ling Fu, and Kenneth McCormick Leptin, the 16-kd hormone produced by white fat cells, regulates energy homeostasis, satiety, and multiple sites in the neuroendocrine system. Leptin receptors have been identified in the central nervous system (CNS) and are widespread in peripheral tissues, including fat. Given the association between insulin resistance and obesity, it is important to establish whether leptin has additional effects on peripheral insulin action and glucose metabolism. This study examined whether leptin has a direct autocrine/paracrine action on glucose metabolism in both freshly isolated and 24-hour cultured rat fat cells. Freshly isolated rat adipocytes were incubated for 30 minutes with 200 ng/mL recombinant murine leptin. Thereafter, basal and insulin-stimulated (10 s mol/L) glucose transport, glycolysis-Krebs oxidation and lipogenesis ([6-14C]glucose conversion to [14C]O2 and to [14C]triglyceride), and lipolysis were measured. Upon leptin exposure, no statistical differences were detected in glucose transport or metabolism. Increasing the leptin concentration to 1 to 2 iLg/mL or prolonging the duration of preincubation with the fat cells to 60 minutes before the metabolic assays did not alter the results. Finally, using two disparate fat cell culture methods with differing substrate additions (pyruvate and high or low glucose concentrations), there was no effect of 24-hour exposure to leptin (200 ng/mL) on basal and insulin-stimulated glucose transport or lipogenesis. We conclude that leptin does not modulate basal or insulin-stimulated glucose metabolism in isolated and cultured fat cells in vitro. However, in vivo, higher pericellular leptin concentrations, as well as other cellular or soluble serum factors, may exist that might lead to a physiologically relevant autocrine action of leptin.

Copyright© 1998by W.B. Saunders Company ONVINCING EVIDENCE can be adduced for the central nervous system (CNS) as the primary locus of action for leptin, the 16-kd hormone specifically coded in white fat cells. 1,2 This peptide is thought to govern energy homeostasis and satiety, but may also mediate several disparate neuroendocrine systems) Initial in vivo studies in rodents, either normal or genetically predisposed to obesity, confirmed that leptin could reduce caloric intake and body weight. Moreover, leptin was proven to be effective either when given parenterally or via injection into the brain ventricles. 2 In humans and all normal animals examined until now, there is an affirming positive correlation between circulating leptin levels and total body fat. 4"7 Hence, an attractive hypothesis which further supports a CNS site for this hormone is that it acts as a peripheral indicator of body nutritional status. Maiden studies in the diabetes-prone ob/ob mouse demonstrated that leptin (or perhaps concomitant weight loss) ameliorated the hyperglycemia and/or hyperinsulinemia in these animals. 8,9 Subsequent studies using pair-fed controls supported a independent, albeit modest, antidiabetic role of leptin. ~° In contrast to its well-described central neurogenic action, a peripheral physiologic role for leptin, either paracrine, autocrine, or as a traditional circulating hormone, is less well characterized. In the pancreas, the reported effects of leptin are inconsistent. In perfused normal rat pancreas, either no effect of leptin H or a potent inhibition of insulin secretion ~2 was re-

C

ported. In isolated islets obtained from the pancreas of ob/ob mice, leptin caused a dose-dependent inhibition of insulin release. 13 In hepatocarcinoma cells, leptin (3 to 60 nmol/L) produced insulin resistance? 4 but this was not affirmed in a later report. 15And, insofar as the protein is a member of the cytokine family, leptin was demonstrated to enhance cytokine production and induce proliferation and differentiation in cultured murine and human hematopoietic cells.16 The available data regarding the potential metabolic action of leptin on fat tissue have also been conflicting. Using rat fat cells cultured in hyperglycemic (21 mmol/L glucose) media, several hours of exposure to physiologic concentrations of recombinant leptin were required to elicit effects on insulin-stimulated glucose metabolism. 17 However, another laboratory, in subsequent abstracts, did not observe an acute autocrine metabolic effect of leptin (1 lag/mL) on lipolysis, glucose uptake, or tyrosine phosphorylation of proteins.18,19 This report explores the potential actions of leptin on insulin-stimulated glucose metabolism in both freshly isolated rat fat cells and adipocytes that were cultured for 24 hours under various conditions with and without recombinant murine leptin. Given the known pathophysiologic linkage between obesity and insulin resistance, it is of major concern clinically whether leptin can modify the cellular actions of insulin.

MATERIALS AND METHODS Materials From the Department of Pediatrics, Medical College of Wisconsin, Milwaukee, W1. Submitted October 29, 1997; accepted May 17, 1998. Supported by grants from the American Diabetes Association, the American Heart Association, and the Max McGee Fund for Juvenile Diabetes at Children's Hospital of Wisconsin. Address reprint requests to Gail Mick, MD, The University of Illinois College of Medicine at Peoria, Pediatric Endocrinology, 530 NE Glen Oak Ave, Peoria, IL 61637. Copyright © 1998 by W.B. Saunders Company 0026-0495/98/4711-0011503.00/0

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General chemicals were obtained from Sigma (St Louis, MO). Murine leptin (recombinant) was from Biomol Research Laboratories (Plymouth Meeting, PA). Animals

Male Sprague-Dawley rats (150 to 200 g) were fed standard chow ad libitum. Rats were killed by cervical dislocation between 8 and 9 AM. They were maintained in accordance with the National Institutes of Health guide for the care and use of laboratory animals. Metabolism, Vo147,No 11 (November), 1998:pp 1360-1365

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LEPTIN DOES NOT AFFECT FAT CELL METABOLISM

Table 1. Effect of Leptin on Adipocyte Glucose Metabolism

Preparation of lsolated Adipocytes Epididymal fat pads were excised and collagenase-treated as described previously,z° Cells were washed in the following (Krebs-Ringer phosphate [KRP]) buffer: 10 mmol/L HEPES (pH 7.4), 130 mrnol/L NaCI, 5 mmol/L KC1, 1.25 mmol/L MgSO4, 2 mmol/L CaC12, and 10 mmol/L NaI-I2PO4 with 2% bovine serum albumin prior to metabolic studies. Cells were diluted to a final concentration of 0.5 to 1 × 106/mL in all metabolic assays. Cell viability was determined by propidium iodide flttorescence staining. 2°

Cultured Fat Cells Method L Fat cells were cotlagenase-isolated as already described and prepared for primary culture per previous methods,zl In this system, cells were incubated with and without 200 ng/mL leptin for 24 hours in sterile media containing 20 mmol/L HEPES, 120 mmol/L NaCI, 1.2 mmot/L MgSO4, 2 mmol/L CaClz, 2.5 lrmaol/L KC1, 1.0 mmoUL NaHzPO4, 1% bovine serum albumin, and 1 mmol/L sodium pyruvate. The antibiotic concentration was decreased to 12.5 U/mL penicillin and 2.5 mg/mL streptomycin. After culture, the cells were washed three times in fresh, leptin-free culture buffer (minus antibiotics) mad glucose transport measurements were made. Celt viability was routinely determined just prior to glucose transport studies and was grea~er than 95% by propidium iodide fluorescence staining, z0 Method II. In separate experiments, freshly isolated adipocytes were prepared for culture by an alternative method. I7 Briefly, fat cells were isolated from epididymal fat pads by collagenase and washed twice in Krebs-Ringer (KRH; 25 mmol/L HEPES free acid, 25 mmol/L HEPES sodium salt, 80 mmol/L NaC1, 1 rnmol/L MgSO4, 2 mrnol/L CaClz, 6 m.rnol/L KCI, 1 mmol/L sodium pyruvate, and 0.5% bovine sermn albumin, pH 7.4) and then once in a 5-mmol/L glucose Dulbecco's modified Eagle's medium (DMEM) culture media (Life Technologies, Grand Island NY) containing 20 mmol/L HEPES, pH 7.4, 2% fetal calf serum, l% bovine serum albumin, 12.5 U/mL penicillin, and 2.5 mg/mL streptomycin. After the cell content was adjusted to 2.5 × 105/mL in this same buffer, the cells were further diluted by adding 1 mL of cells to 4 mL of either 25 mmogL glucose DMEM (with the above-mentioned additions plus 100 nmol/L N6phenylisopropyl adenosine), 5-mmol/L glucose DMEM (with additions as already noted), or 5-mmoI/L glucose DMEM plus 20 mmol/L mannitol (with additions as already noted). Incubations (37°C with 5% COt) and subsequent washing procedures were as previously detailed.17 For glucose transport studies, the washing buffer was KRH, and for lipogenesis it was KRH with 0.1 mmol/L glucose.

Parameter

Control

Glucose transport Glycolysis-Krebs oxidation Lipogenesis Totallipids Free fatty acids Glyceride-glycerol

100 100

452 ± 33 95 -+ 7 453 ± 78 104 ± 9

432 _+ 25 508 _+ 165

100 100 100

196 + 50 102 ± 6 338±36 79-+7 238 ± 13 111 _+ 6

194 +_ 49 381±57 186 ± 80

Leptin

Leptin + insulin

NOTE. Results are presented as a percent of the control value (no additions). Isolated adipocytes were incubated according to the methods. Control rates (n = 3, all groups) were as follows: glucose transport, 1.42 ± 0.35 nmol/10 s cells/3 rain; glycolysis-Krebs ([6~4Clglucoset oxidation, 6.8 _+ 0.4 nmol/h/10 ~ ce/~s; lipogenesis-totaf lipids, 19 + 3.4 nmol/h/105 cells. There were no statistical differences between groups with and without leptin by paired ttest.

Statistical Analysis All metabolic assays were performed in triplicate to quadruplicate except for the data in Table 2 (n = 2). Statistical analysis was performed by the paired Student's t test for cells incubated with and without leptin. ANOVA was used for the data in Table 2 and there were no statistical differences by this method. RESULTS

Adipocyte Metabolism in Freshly Isolated Rat Fat Cells Neither glycolysis-Krebs oxidation ([6JgC]glucose oxidation) nor concomitantly measured lipogenesis were altered by the acute addition o f 200 n g / m L leptin ( m a x i m u m total time o f exposure to leptin, 2 hours, Table 1). Lipolysis was similarly unaffected (Fig 1).

Glucose Transport in Freshly Isolated Rat Fat Cells Exposure to 200 n g / m L leptin for 30 minutes did not modify basal or maximally insulin-stimulated (10 -8 tool/L) glucose transport (Table 1). In separate experiments (not included), the insulin concentration was further adjusted between 10 -12 and 10 -8 mol/L to determine whether leptin might act within a wide 600 500

Metabolic Assays Glucose transport was determined by the uptake of [UA4C]2 deoxyglucose.22 Briefly, 500 gL ceils were preincubated fur 30 minutes at 37°C with and without leptin (200 ng/mL) and subsequently for another 30 minutes with and without insulin (10 -s tool/L) prior to addition of [UJ4C]2-deoxyglucose. Uptake was calculated from the linear slope of the cell-associated radioactivity versus time. Given the rapid nature of these uptake experiments, care was taken to assay each paired sample (with and without leptin) in sequential chronological order. Glycolysis-Krebs oxidation and lipogenesis were determined as previously reported,z3 except that the cells were in KRP and the substrate was 1 mmol/L glucose. Preincubations with leptin and insulin were as before for glucose transport. Lipolysis was measured over 90 minutes. Cells were preincubated with and without the following (in sequential order): leptin 200 ng/mL for 30 minutes, insulin iO-~molB~for 30 minutes, and ~nalty, isoproterenol 1 ~tolfL for 30 minutes. The assay was terminated by the addition of 4N perchloric acid, neutralized with 4N KOH, and then analyzed for glyercol,z4

insulin

g

400

~o 300 o~ 200 100 0

basal

insulin (104M)

isoproterenol (1 gM)

isoproterenol and insulin

Fig 1. Effect of leptin 200 ng/mL on lipolysis in freshly isolated fat cells. Fat cells were incubated with and without leptin. The basal rate of lipolysis was 25 nmol/h/10 s cells. There were no statistical differences between groups with and without leptin by paired t test (n = 3).

MICK ET AL

1362

range encompassing physiologic insulin concentrations. Leptin did not shift the dose-response curve.

Conditions for Acute Metabolic Assays in Freshly Isolated Rat Fat Cells In five separate experiments not shown (two measuring glucose transport, one lipogenesis, and two lipolysis), leptin was increased to a maximum concentration of 1 to 2 gg/mL and the duration of leptin preincubation was increased to 60 minutes--no metabolic effects were noted despite these high concentrations of leptin and longer exposure times. The addition of leptin did not alter cell viability (as determined by cell count and propidium iodine fluorescent staining).2°

Cultured Fat Cell Experiments Culture method 1. When fat cells were exposed to 200 ng/mL leptin for 24 hours (per culture method 1), there was no effect on basal or insulin-stimulatedglucose transport (Fig 2). Culture method 2. When fat cells were exposed to 200 ng/mL leptin for 24 hours (per culture method 2), there was no effect of leptin on basal and insulin-stimulatedglucose transport or lipogenesis (Table 2). DISCUSSION

We examined the effects of leptin on intermediary metabolism and glucose transport in freshly isolated rat adipocytes

_ _

leptin

l

~7-

=6Im=~

¢~5E4-

eF=~

.= 3 =2

0

--//i 0

, I I 0.001 0.01 0.1 1 Insulin (nM)

I 10

I 100

Fig 2. Effect of leptin 200 ng/mL on cultured fat cells. Fat cells were incubated with and without leptin (culture method 1). The insulin stimulation factor is the ratio of the insulin-stimulated to basal glucose transport rate at each of the indicated insulin concentrations. Data are expressed as the means -+ SD of 4 experiments. Basal (no insulin) rates of glucose transport were 1.55 -+ 0,31 and 1.55 + 0.65 nmol/10 s cells/3 rain in control and leptin-treated fat cell groups, respectively. There were no statistical differences with and without leptin by paired ttest.

exposed acutely to this hormone and in cultured fat cells after 24 hours. Despite incubating the fat cells with leptin concentrations far exceeding those found in the sera of obese humans or normal ratsy no differences in glucose transport or its intracellular metabolism were uncovered. To date, various tissues have been examined following either in vivo or in vitro leptin treatment to determine whether the hormone has meaningful extraneural actions. For example, in human renal carcinoma cells, leptin caused tyrosine phosphorylation of STAT-1 and other cellular proteins. 26 Additional reported or proposed systemic hormonal actions include the following: dose-dependent differentiation and proliferation of hematopoietic cells,~6inhibitionof acetyl coenzyme A carboxylase activity and lipogenesis in a preadipocyte (30A5) cell line,27 and inhibition of glucose-induced insulin release in perfused rat pancreas, found in some studies 12 but not others, 11 as well as prevention of triglyceride formation from fatty acids in isolated islets. 28 Of major interest, inasmuch as the hyperglycemia and hyperinsulinemia of ob/ob mice are improved following exogenous leptin administration,9,1°is whether leptin might alter the cellular action of insulin. Numerous in vivo studies have addressed this important question. In an examination of the effects of short-term (5-hour) intravenous versus intracerebroventricular leptin infusion on in vivo measurements of glucose turnover and uptake in normal C57BL/6J mice,29 leptin was shown to increase glucose turnover and uptake while decreasing liver glycogen content. Since serum glucose, insulin, and glucagon levels were unchanged and intracerebroventricular infusion was as effective as intravenous infusion, the investigators proposed that leptin stimulated efferent signals from the CNS which in turn altered glucose metabolism. However, this was not the case in studies of lean Zucker Fa/fa rats, 3° wherein a 4-day intravenous, but not intracerebroventricular, leptin infusion increased glucose utilization. Similarly, using normal rats, treatment with subcutaneous leptin for 48 hours31 increased insulin sensitivity without changing adipose tissue GLUT 4 levels. Others showed that a brief intravenous infusion of leptin (6 hours) enhanced certain aspects of hepatic (decreased glycogenolysis) but not peripheral insulin action) 2 However, long-term (8-day) leptin infusion caused a striking reduction in visceral adiposity, augmented peripheral glucose uptake (which, in vivo, is largely into muscle), and altered hepatic glucose production (again, by enhanced sensitivity to the inhibitory action of insulin on glycogen breakdown).33 In contradistinction to these rodent studies, there was no significant relationship in normal women, between blood leptin and in vivo measures of insulin sensitivity and secretionY Turning to the in vitro data regarding the potential direct actions of leptin on extraneural cellular metabolism, particularly insulin action, one study using a human hepatocellular carcinoma line proports that leptin alters the intracellular signaling mechanism of insulin.14 Exposure to leptin for 10 minutes resulted in reduced phosphorylation of insulin receptor substrate-1, and upon incubation for a maximal 2 hours, leptin upregulated the insulin inhibition of phosphoenolpyruvate carboxylase mRNA. However, in these cell studies, some of the

LEPTIN DOES NOT AFFECT FAT CELL METABOLISM

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Table 2. Basal and Insulin-Stimulated Lipogenesis and 2-Deoxyglucose Transport in Rat Adipocytes Following 24-Hour Culture in Various Media With and Without 200 ng/mL Leptin Control Media Lipogenesis 5 mmol/L glucose 21 mmol/L glucose 5 mmol/L glucose + 16 mmol/L mannitol 2-Deoxyglucose transport 5 mmol/L glucose 21 mmol/L glucose 5 mmel/L glucose + 16 mmol/L mannitol

Leptin

Basal

Insulin

Fold Increase by insulin

Basal

Insulin

Fold Increase by Insulin

0.58 _+ 0.26 0.56 -+ 0.28 0.62 _+ 0.22

1.92 + 0.35 2.19 -+ 0.01 1.85 + 0.31

3.3 3.9 3.0

0.57 ~ 0.35 0.59 ± 0.21 0,61 -+ 0.15

1.82 + 0.25 2.28 -+ 0.62 1.68 ± 0.68

3.2 3,8 2.8

0.33+0.04 0,7 _+ 0.21 1.1_+0

2.0-+0.7 2.2 4- 0.5 2.4_+1.0

6.0 3.0 2.1

0.8-+0.4 1.1 -+ 0.1 1.1-+0.1

3.3-+0.5 3.5 -+ 0.4 2.6-+0.5

4,1 3.1 2.3

NOTE. Rat adipocytes were cultured for 24 hours according to Mfiller et ai, ~7 but in various final media concentrations of glucose and mannitol and with and without added leptin (200 ng/mL) and in the presence and absence of 10 nmol/L insulin. Following 24-hour incubation, the cells were washed in either KRH containing 0.1 mmol/L glucose and 1% BSA (for lipogenesis) or KRH containing 1 mmol/L pyruvate and 1% BSA (for transport). Lipogenesis rates are presented as nmol glucose converted to triglyceride/h/lO 5 cells, mean _+ SD (n = 2). There were no statistical differences among the different culture media or with or without leptin by ANOVA. The fold increase by insulin refers to the rate in the presence of 10 nmol/L insulin divided by the rate without added insulin.

reported actions were found at a leptin concentration of 900 ng/mL, a level not yet approached in humans. In contrast, others did not observe leptin effects on cellular insulin action with a hepatoma cell line into which the long form of the leptin receptor was stably expressed. 15 Surprisingly, in C2C~2 myotubules, leptin caused a stimulation of glucose transport and glycogen synthesisY Perhaps most germane to our study are the recent observations regarding the metabolic effects of leptin on cultured rat fat cells. I7 In these studies, supraphysiologic leptin concentrations (50 nmol/L = 800 ng/mL) inhibited insulin-stimulated glucose transport by 50% within 1 hour of exposure. Physiologic leptin concentrations (0.5 to 2 nmol/L) required longer exposure times: for example, incubation for 8 hours at 2 nmol/L leptin caused a 50% inhibition of insulin-stimulated glucose transport. Since we found no effect of leptin on rat fat cells after 24 hours of exposure (culture method), we considered whether our assay conditions and, in particular, the media glucose concentration obscured a potential leptin effect. However, there was no inhibition by 200 ng/mL leptin of either 2-deoxyglucose uptake or lipogenesis after 24 hours regardless of the glucose concentration (Table 2). It therefore does not appear that the ambient glucose level explains the discrepancy. It is also possible that the nutritional state of the animals, fat cell size, or perhaps the adipose tissue depot of origin may alter potential extraneural actions of leptin analogously to the manner by which these same parameters affect insulin sensitivity.36 Since we explored a wide range of leptin concentrations (--<2 pg/mL), it is unlikely that factors which might diminish cellular leptin sensitivity would be missed. In the adipose studies to date, both small rats (<200 g, M~iller et a117 and this study) and large rats 37 have been examined for the potential metabolic response to leptin. Our observations corroborate the later study, which showed that a 2-hour incubation with murine recombinant leptin (300 ng/mL) did not modify basal or insulin-stimulated glucose transport 37 in either adipocytes or epitrochlearis muscle from normal rats. Our experiments extend these studies further by including measurements of intracellular metabolism (glycolysis-Krebs oxidation, lipogenesis, and lipolysis) and examining the effects of leptin on

cultured adipocytes. Finally, concerning the discrepancy between our data and the report by Mtiller etal, 17 other variables such as the diet and the adipose tissue depot could potentially affect the cellular action of leptin on fat tissue, since these factors clearly influence ob mRNA levels. 38-4~Of the potential fat depots (retroperitoneal, omental, subcutaneous, and epididyreal), omental fat would be potentially most interesting given the association between visceral obesity and diabetes, dyslipidemia, and cardiovascular disease. 39,42,43 However, thus far, the above-mentioned studies and others 44,45 do not appear to link leptin with central obesity. However, one in vivo rodent study did find a selective reduction in visceral adiposity with infused leptin. 33 Since gut perfusion and the locally increased lactate formation and lipolysis are unique to visceral fat, it is also unclear whether freshly isolated or cultured fat cell preparations (regardless of the in vivo site of origin) would address potential regional differences in leptin action. In contrast to the lipolysis data presented herein (Fig 1), other investigators,3° using fat pads from normal rats, demonstrated a large increase in lipolysis following 2-hour exposure to 10 nmol/L leptin. This discrepancy is unexplained, but may relate to important intrinsic differences between isolated cells versus fat pads (paracrine or membrane-related factors) or possibly the nutritional state of the animals. Our lipolysis data in isolated rat fat cells are closer to the results observed in freshly isolated mouse adipocytes, 46 wherein leptin (10 -I2 mol/L or 0.02 ng/mL) caused only a minor (1.2-fold) non-dose-dependent increase in lipolysis. In these same studies, there was a greater (2.6-fold maximal) effect of leptin in fat cells from ob/ob mice, but no effect of the adipocyte hormone in fat cells from db/db (mutant receptor) mice. In summary, using isolated rat adipocytes, brief (-<2 hours) or prolonged (24-hour culture) leptin exposure did not disturb the stimulatory action of insulin on 2-deoxyglucose transport or glucose metabolism using high to supraphysiologic leptin concentrations and different cell substrate conditions. Hence, while emerging evidence supports a direct extraneural action of leptin on hepatic glucose metabolism and peripheral glucose uptake (muscle), white adipocyte glucose metabolism and

1364

MICK ET AL

uptake is not controlled by leptin in an autocrine fashion. Regional differences in adipose tissue leptin sensitivity and factors related to the [33-adrenergic system, or other yet undescribed neuronally mediated signal cascades or tissue paracrine factors, may be important qualifiers to this conclusion in vivo. It is germane to the pathophysiology accompanying human obesity whether prolonged leptin exposure promotes

autocrine insulin resistance, which may contribute, in part, to the peripheral defect in diabetes. NOTE ADDED IN PROOF

While this report was in press, the lack of an effect of leptin on glucose transport, lipoprotein lipase, and insulin action in epididymal adipocytes from small fasted rats was reported. 47

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