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Molecular Endocrinology 22 (10): 2260-2267
Copyright © 2008 by The Endocrine Society

The Mammalian Target of Rapamycin Complex 1 Regulates Leptin Biosynthesis in Adipocytes at the Level of Translation: The Role of the 5'-Untranslated Region in the Expression of Leptin Messenger Ribonucleic Acid

Partha Chakrabarti, Takatoshi Anno, Brendan D. Manning, Zhijun Luo and Konstantin V. Kandror

Boston University School of Medicine (P.C., T.A., Z.L., K.V.K.), Boston, Massachusetts 02118; and Harvard School of Public Health (B.D.M.), Boston, Massachusetts 02115

Address all correspondence and requests for reprints to: K. V. Kandror, Boston University School of Medicine, Department of Biochemistry, K124D, 715 Albany Street, Boston, Massachusetts 02118. E-mail: kandror{at}biochem.bumc.bu.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Leptin production by adipose cells in vivo is increased after feeding and decreased by food deprivation. However, molecular mechanisms that control leptin expression in response to food intake remain unknown. Here, we test the hypothesis that leptin expression in adipose cells is regulated by nutrient- and insulin-sensitive mammalian target of rapamycin complex 1 (mTORC1)-mediated pathway. The activity of mTORC1 in 3T3-L1 adipocytes was up-regulated by stable expression of either constitutively active Rheb or dominant-negative AMP-activated protein kinase. In both cases, expression of endogenous leptin was significantly elevated at the level of translation. To investigate the role of leptin 5'-untranslated region (UTR) in the regulation of protein expression, we created bicistronic reporter constructs with and without the 5'-UTR. We found that the presence of leptin 5'-UTR renders mRNA resistant to regulation by mTORC1. It appears, therefore, that mTORC1 controls translation of leptin mRNA via a novel mechanism that does not require the presence of either the 5'-terminal oligopyrimidine tract or the 5'-UTR.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
LEPTIN IS AN adipocyte-made hormone that plays a central role in the regulation of food intake and body weight (1). Circulating leptin levels increase after feeding (2) and decrease after food deprivation (3), suggesting that nutritional status of the body has a profound effect on leptin production by adipocytes. However, the mechanistic connection between nutrient intake and leptin levels is not yet completely understood.

Because insulin levels also rise after feeding and diminish upon starvation, the predominant hypothesis for the last 10–12 yr has been that leptin production is controlled by insulin (1). In particular, it was proposed that insulin activated leptin expression at the level of transcription (4, 5, 6). This idea, however, has not been confirmed by experiments in vitro. Indeed, regulation of leptin production by insulin and nutrients is sustained in isolated adipocytes and samples of fat tissue (7, 8, 9, 10, 11, 12, 13) where neither insulin (12, 13, 14) nor nutrient status (7, 8) affects the levels of leptin mRNA.

Alternatively, it has been suggested that insulin exerts its effect on leptin expression via the mammalian target of rapamycin complex 1 (mTORC1)-mediated pathway. This is an attractive hypothesis, because the activity of mTORC1 depends not only on insulin levels but also on nutrient availability (15, 16, 17), providing an additional physiological dimension to the regulation of leptin production. To this end, it has been shown that the specific mTORC1 inhibitor, rapamycin, efficiently blocks leptin production in isolated adipocytes (8, 12, 13) and that mTORC1 activator, leucine, stimulates leptin production both in vitro (13) and in vivo (18). However, a pharmacological approach alone may not provide sufficient evidence for this model. In addition, it remains unclear whether mTORC1 regulates leptin production at the level of transcription or translation.

We decided to determine, in direct experiments, whether or not leptin expression in adipocytes is regulated by the mTORC1-mediated pathway. For that, we activated mTORC1 in 3T3-L1 adipocytes by stable transfection of the dominant active form of Rheb (19), a low-molecular-weight GTPase, which represents a physiological activator of mTORC1 in cells (20). In parallel, we have activated mTORC1 by overexpression of the dominant-negative form of AMP-activated protein kinase (AMPK). We found that in both cases, leptin translation is significantly stimulated. The effect of mTORC1 on translation often requires the presence of structured 5'-untranslated region (UTR) in mRNA (21). Moreover, the 5'-UTR of leptin mRNA was previously suggested (13) and even shown (8) to regulate the efficiency of leptin translation. Therefore, we decided to explore the significance of the 5'-UTR in the regulation of leptin expression by mTORC1. Unexpectedly, we found that the presence of the 5'-UTR in leptin mRNA does not account for up-regulation of its translation in response to activation of mTORC1. It appears, therefore, that mTORC1 controls translation of leptin mRNA via a novel mechanism that requires neither the presence of the 5'-terminal oligopyrimidine tract nor the 5'-UTR.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Biosynthesis of Leptin in 3T3-L1 Adipocytes Is Activated by Constitutively Active Rheb
Constitutively active hS16H Rheb (hereafter referred to as Rheb) (19), was tagged with the V5 epitope at the carboxy terminus and stably overexpressed in 3T3-L1 adipocytes with the help of the lentiviral expression vector. Figure 1AGo shows that the level of Rheb overexpression is moderate and exceeds the level of the endogenous protein by about 50%. Nonetheless, mTORC1 in Rheb-transfected cells is significantly activated as judged by increased phosphorylation of the ribosomal protein S6 and 4E-BP both in the presence of serum and in serum-starved adipocytes (Fig. 1BGo).


Figure 1
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  		Fig. 1. Stable Overexpression of Rheb in 3T3-L1 Adipocytes Increases Leptin Biosynthesis

A, Total lysates (50 µg per lane) of 3T3-L1 adipocytes stably expressing S16H hRheb (Rheb) or EV-transfected control adipocytes were analyzed by Western blotting. Irrelevant protein cellugyrin was used as loading control. B, Differentiated EV or Rheb-expressing 3T3-L1 cells were incubated in serum-free DMEM for 6 h, and whole-cell lysates were analyzed by Western blotting. C, Levels of leptin mRNA in differentiated (d 7) EV and Rheb-overexpressing 3T3-L1 adipocytes were determined in triplicate by quantitative PCR and normalized by 36B4 mRNA. Data are expressed as mean ± SD of leptin mRNA/36B4 mRNA in Rheb-transfected cells vs. EV cells. D, Differentiated 3T3-L1 adipocytes (d 7) were metabolically labeled with [35S]methionine/cysteine for 45 min as described in Materials and Methods. Cells were homogenized, and leptin was immunoprecipitated from total cell lysates after correction for trichloroacetic acid precipitable counts. Immunoprecipitated samples were separated in 15% SDS-PAGE, and incorporation of 35S into leptin was measured by autoradiography of dried gels. Data are presented in arbitrary units (a.u) as mean ± SD for three independent experiments. The bottom panel shows a representative gel. *, P < 0.05.

 
Activation of the mTORC1-mediated signaling pathway in 3T3-L1 adipocytes by overexpression of Rheb does not lead to a statistically significant increase in leptin mRNA (Fig. 1CGo). Nonetheless, we decided to measure the rate of leptin translation in these cells. Rheb- and empty vector (EV)-transfected adipocytes were cultured in the methionine- and cysteine-free medium for 1 h and then incubated with 750 µCi/ml of [35S]methionine/cysteine for 45 min. After that, 35S-labeled leptin was immunoprecipitated from cell lysates as described in Materials and Methods. As shown in Fig. 1DGo, leptin translation is activated about 2-fold in Rheb-expressing adipocytes in comparison with EV-infected cells. Similar results were obtained with the individual clone and with pooled clones of cells stably transfected with Rheb.

Biosynthesis of Leptin in 3T3-L1 Adipocytes Is Activated by Dominant-Negative AMPK
To confirm these results, we have measured leptin translation in 3T3-L1 adipocytes that stably express FLAG epitope-tagged dominant-negative isoform of AMPK {alpha}1 (DN-AMPK) where aspartate 157 in the conserved ATP-binding motif is substituted for alanine (22). In 3T3-L1 adipocytes, expression of DN-AMPK exceeded levels of the endogenous enzyme at least 5-fold (Fig. 2AGo). It has been previously shown that AMPK inhibits mTORC1 by phosphorylating tuberous sclerosis complex 2 (TSC2) (23) and raptor (24). In agreement with these results, expression of DN-AMPK activates mTORC1 (Fig. 2AGo). The levels of leptin mRNA in DN-AMPK-expressing cells are slightly decreased (Fig. 2BGo). Nevertheless, in agreement with results shown in Fig. 1Go, translation of leptin is increased about 2.5-fold (Fig. 2CGo). Thus, activation of mTORC1 in adipocytes either by constitutively active Rheb or by DN-AMPK stimulates leptin expression at the level of translation.


Figure 2
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  		Fig. 2. Stable Overexpression of DN-AMPK in 3T3-L1 Adipocytes Increases Leptin Biosynthesis

A, Total lysates (50 µg per lane) of 3T3-L1 adipocytes stably expressing FLAG-tagged DN-AMPK or EV-transfected control adipocytes were analyzed by Western blotting. Irrelevant protein cellugyrin was used as loading control. B, Expression of leptin mRNA in EV and DN-AMPK-overexpressing 3T3-L1 adipocytes was measured as indicated in the legend to Fig. 1CGo. C, Rate of leptin biosynthesis was assayed in EV and DN-AMPK-overexpressing 3T3-L1 adipocytes as described in the legend to Fig. 1DGo. *, P < 0.05.

 
The Role of the 5'-UTR in Leptin Translation
It is thought that the structural features that render mRNAs susceptible for the translational regulation via the mTORC1-mediated pathway include the presence of the 5'-terminal oligopyrimidine tract (5'-TOP) or the structured 5'-UTR (21). Leptin mRNA lacks 5'-TOP but has putative hairpin structures in its 57-nucleotide-long 5'-UTR (13). Thus, we decided to determine whether or not the 5'-UTR is responsible for the translational regulation of leptin mRNA.

With this end in view, we have constructed bicistronic constructs consisting of the luciferase open reading frame, internal ribosomal entry site (IRES), and green fluorescence protein (GFP) with and without leptin’s 5'-UTR (Fig. 3Go). In this experimental set-up, leptin’s 5'-UTR should exclusively affect translation of luciferase, whereas expression of GFP driven by the internal ribosomal entry site remains constant. Results of transfection experiments are expressed as luciferase activity normalized by the levels of constitutively expressed GFP. Because both reporter genes are localized in the same mRNA molecules expressed in the same transfected cells, the experimental error should be minimal.


Figure 3
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  		Fig. 3. A Schematic Representation of the Bicistronic Constructs with and without the 5'-UTR of Leptin mRNA.

 
First, we used these bicistronic constructs in HEK293 cells to determine whether leptin’s 5'-UTR affects luciferase translation under nutrient-poor conditions [Krebs-Ringer-Hepes (KRH) plus 2% BSA] and in response to nutrients (DMEM) and insulin (DMEM+Ins). Unexpectedly, we did not detect any inhibitory effect of the 5'-UTR on the efficiency of luciferase expression under basal conditions (Fig. 4AGo). In fact, leptin 5'-UTR has a small positive effect on the expression of luciferase (Fig. 4Go, A and B). Insulin and nutrients activate expression of luciferase vs. GFP regardless of the presence of leptin’s 5'-UTR in mRNA (Fig. 4Go, A and B, top panels). Because both insulin and nutrients activate the mTORC1-mediated pathway (Fig. 4Go, A and B, bottom panels), these results argue against the specific role of the 5'-UTR in the regulation of leptin translation by mTORC1. Analogous results were obtained in NIH-3T3 cells (data not shown).


Figure 4
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  		Fig. 4. Effects of Nutrients and Insulin on the Expression of the Leptin 5'-UTR(–) and 5'-UTR(+) Constructs

A, Top panel, HEK293 cells were transiently transfected with 5'-UTR(–) and 5'-UTR(+) constructs. After 48 h, cells were transferred to KRH with 2% BSA for 6 h. At the end of the incubation, cells were either maintained in KRH with 2% BSA or transferred to DMEM for another 3 h. Cells were washed three times with cold PBS and harvested in reporter lysis buffer. Luciferase activity was assayed in cell lysates and normalized by GFP fluorescence. Data are presented as mean ± SD. *, P < 0.05. NS, Not significant. Bottom panels, cell lysates were analyzed by Western blotting (50 µg per lane). B, HEK293 cells were transiently transfected with 5'-UTR(–) and 5'-UTR(+) constructs. After 48 h, cells were transferred to KRH with 2% BSA for 6 h. At the end of the incubation, cell medium was replaced with either DMEM or DMEM with 100 nM insulin for another 3 h. Luciferase assay (top panel) and Western blotting (bottom panels) were carried out as described above.

 
Although nutrients and insulin activate mTORC1 (Fig. 4Go, A and B, bottom panels), they may also have additional mTORC1-independent effects on translation. Therefore, to test the role of leptin’s 5'-UTR specifically in mTORC1-regulated translation, we carried out double transfection of cells with the bicistronic constructs and constitutively active Rheb. Transfection of Rheb results in a significant stimulation of mTORC1 activity as compared with EV-transfected cells (Fig. 5Go, bottom panels). Under these conditions, expression of luciferase directed by the bicistronic 5'-UTR(–) mRNA is increased. Interestingly, the presence of leptin’s 5'-UTR renders mRNA resistant to up-regulation by mTORC1 (Fig. 5Go, top panel).


Figure 5
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  		Fig. 5. Leptin 5'-UTR Blocks the Effect of Rheb on the Reporter Gene Activity

Top panel, HEK293 cells were transiently transfected with 5'-UTR(–) and 5'-UTR(+) constructs together with pRK5-myc (EV) or pRK5-myc Rheb (Rheb). After 48 h, cells were incubated in serum-free DMEM for another 6 h. Luciferase activity was measured as described in the legend to Fig. 4Go. Data ar presented as mean ± SD; *, P < 0.05. NS, Not significant. Bottom panels, cell lysates were pooled and analyzed by Western blotting (50 µg per lane).

 
To confirm this unexpected result in a different cell type, we transfected bicistronic constructs into wild-type (TSC2+/+) and TSC2-null (TSC2–/–) mouse embryonic fibroblasts (MEFs). The TSC1/2 complex (also referred to as hamartin and tuberin, respectively) inhibits mTORC1 by acting as GTPase activating protein for Rheb (20, 25). In TSC2–/– MEFs, similar to Rheb-transfected HEK293 cells, mTORC1 is significantly activated (Fig. 6AGo). In wild-type MEFs, the reporter plasmids show no difference in the efficiency of luciferase expression (Fig. 6BGo). However, in agreement with data demonstrated in Fig. 5Go, the presence of leptin’s 5'-UTR inhibits translation of luciferase in TSC2–/– MEFs in comparison with the 5'-UTR(–) construct (Fig. 6BGo).


Figure 6
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  		Fig. 6. Leptin 5'-UTR Inhibits the Reporter Gene Activity in TSC2–/– Cells

A, TSC2+/+ and TSC2–/– cells were incubated in serum-free DMEM for 6 h, and whole-cell lysates were analyzed by Western blotting (50 µg per lane). Actin served as a loading control. B, Top panel, TSC2+/+ and TSC2–/– cells were transiently transfected with 5'-UTR(–) and 5'-UTR(+) constructs. After 48 h, cells were transferred to serum-free DMEM for 6 h, and luciferase activity was measured as described in the legends to Figs. 4Go and 5Go. Bottom panel, cell lysates were pooled and analyzed by Western blotting (100 µg per lane) to show equal transfection efficiency.

 
These results suggest that although the expression of leptin in adipose cells is activated by the mTORC1-mediated pathway at the level of translation, this effect is not likely to be mediated by previously described mechanisms that depend on the presence of either 5'-TOP or 5'-UTR in mRNA and should thus be attributed to some novel mechanisms of translational control.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
In this study, we show that the biosynthesis of leptin in cultured adipocytes is controlled at the level of translation via the insulin- and nutrient-sensitive mTORC1-mediated pathway. We believe that this type of regulation plays an important role in the control of postprandial leptin levels and provides a direct mechanistic connection between food intake and leptin output by adipocytes. Indeed, mTORC1 in adipocytes is known to be regulated by insulin (26, 27), a hormone that correlates with circulating leptin in vivo (1) and directly activates its translation in vitro in isolated adipocytes (8). Our results confirm the latter report by Fried and colleagues (8) and further identify mTORC1 as the downstream insulin target that regulates leptin translation. This conclusion is also consistent with the earlier report of Barthel et al. (28) showing that leptin production in adipocytes is increased by constitutively active Akt.

The activity of mTORC1 is coupled to the energy status of the cell via the AMPK-mediated axis (23, 24). Thus, our results about the role of AMPK in regulation of leptin translation (Fig. 2Go) are highly consistent with the earlier data showing that leptin expression in adipocytes depends on energy metabolism (10, 11) and, in particular, correlates with the levels of intracellular ATP (29).

The involvement of mTORC1 in the control of leptin expression may help to explain a long known correlation between adipocyte size and the amount of leptin produced by the cell (1). Indeed, mTORC1 directly regulates cell mass and size (16, 30) and may thus be involved in both adipocyte growth and leptin expression. In fact, this may be a case of feedback regulation in the organism when adipocytes use the same molecular mechanism for the regulation of cell growth and, simultaneously, production of leptin, which limits food intake and uncontrolled weight gain.

In rats, plasma leptin levels rise about 2.5- to 3-fold within 3 h after food intake (2). Activation of mTORC1 in cultured adipocytes by either Rheb or DN-AMPK does not significantly increase leptin mRNA but causes a comparable activation of leptin biosynthesis (Figs. 1Go and 2Go). This suggests that regulation at the level of translation via mTORC1 plays the predominant role in the control of leptin levels in vivo in response to insulin and nutrients irrespective of the effect of insulin on the transcription of the ob gene. This idea is in line with several independent observations showing that neither insulin (12, 13, 14) nor nutrients (7, 8) increase leptin mRNA in cultured adipocytes.

Previously, we and others have demonstrated that leptin production from adipocytes is regulated by insulin not only at the level of translation but also at the level of secretion (7, 9, 12, 31), suggesting the existence of a rapidly released regulatable pool of presynthesized leptin molecules in adipose cells. Insulin- and serum-responsive leptin-containing vesicles identified in our previous report (31) may constitute such a storage pool. We estimate, however, that the capacity of the leptin storage pool in adipocytes is moderate and is roughly equivalent to 1 h of constitutive secretion, although this number may be underestimated because adipocytes tend to lose leptin in the process of cell isolation and, especially, during collagenase treatment (9). We suggest that the acute type of regulation at the level of secretion may be responsible for the rapid pulsatile oscillations in circulating leptin levels that take place in vivo (32, 33, 34), whereas translational control of leptin expression may couple leptin production to food intake.

In the second part of our study, we attempted to characterize the role of the 5'-UTR in the translational control of leptin expression. Along with the recent report by Fried and colleagues (8), we have found that the 5'-UTR does not inhibit but, rather, slightly activates translation of the message in full media and under nutrient-poor conditions (Fig. 4AGo). Although in our hands, the stimulatory effect of the 5'-UTR is more modest than it has been previously reported by Fried’s group (8), we believe that our results regarding the role of the 5'-UTR in translation of leptin mRNA under nonstimulated conditions are conceptually similar. However, in contrast to the previous report (8), we have determined that insulin has a small but statistically significant positive effect on translation of both 5'-UTR(–) and 5'-UTR(+) reporter mRNAs (Fig. 4BGo). We believe that this discrepancy may be explained by differences in the experimental protocols used by our and Fried’s groups. In any case, the presence of the 5'-UTR does not render the reporter construct more sensitive to the regulation by insulin and nutrients (see also Ref. 8) and, therefore, is not likely to account for the stimulatory effect of insulin on leptin translation.

Pharmacological evidence (8, 12) as well as results presented here (Figs. 1Go and 2Go) strongly suggest that insulin increases the efficiency of leptin translation via the mTORC1-mediated pathway. It is thought that mTORC1 controls translation of mRNAs that have either structured 5'-UTR or the 5'-TOP (21). Because leptin mRNA does not have 5'-TOP, we decided to reexamine the role of the 5'-UTR in leptin translation specifically in response to activation of the mTORC1. Our results suggest that the 5'-UTR does not play any major role in the activation of leptin translation by mTORC1 (Figs. 5Go and 6Go), indicating that control of leptin biosynthesis may be executed by a novel mechanism.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Antibodies
Polyclonal serum against mouse leptin as well as recombinant mouse leptin were purchased from Dr. A. F. Parlow (National Hormone and Peptide Program, Torrance, CA). Rabbit polyclonal antibody against cellugyrin was described previously (35). Monoclonal anti-Myc tag, polyclonal anti-Rheb, polyclonal anti-AMPK, polyclonal anti-4E-BP1 and all phospho-specific antibodies were purchased from Cell Signaling Technology, (Beverly, MA). Polyclonal anti-GFP antibody was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); monoclonal anti-actin and anti-FLAG antibodies were from Sigma Chemical Co. (St. Louis, MO); monoclonal anti-HA tag antibody was from Covance (Berkeley, CA); monoclonal anti-V5 antibody was from Invitrogen (Carlsbad, CA).

Plasmids and Constructs
Leptin cDNA was obtained from the total RNA isolated from differentiated 3T3-L1 adipocytes by RT-PCR with primers 5'-GGATCCCTGCTCCAGCAGCTG-3' and 5'-CTCAGATCCAAAATCTACCTCCAAC-3' and cloned into the pGEM-T vector (Promega Corp., Madison, WI). The 57-bp 5'-UTR of leptin mRNA (starting from the 2784 bp of leptin cDNA) was subcloned upstream of the luciferase reporter gene in the pGL3 vector (Promega) via HindIII and NcoI sites. Bicistronic constructs were then generated by subcloning the luciferase cassette with and without leptin 5'-UTR in the recombinant adeno-associated viral vector pTRUF12-GFP (UF Vector Core; http://www.clas.ufl.edu/jur/200206/papers/paper_buethe.html) via HindIII and SpeI sites.

Open reading frame of constitutively active S16H hRheb in the pRK5 vector (19) was amplified by PCR and subcloned into the pLenti-m1 vector via BamHI and SalI sites.

The open reading frame of DN-AMPK 1{alpha} in the pDK6 vector was amplified by PCR and subcloned into the lentiviral vector constructed and provided by Dr. Wu (35).

Cell Culture
3T3-L1 preadipocytes were cultured and differentiated as described previously (36). Human embryonic kidney (HEK293) cells as well as TSC2–/– MEFs and littermate control wild-type MEFs (37) were cultured in DMEM supplemented with 10% FBS and 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. Murine 3T3-L1 preadipocytes were infected with pLenti-m1-V5-S16H hRheb according to the Invitrogen’s protocol. EV cells were stably transfected with pLenti-m1 empty vector and used as controls. Selection of cells was performed with blasticidin (10 µg/ml) for 7–10 d, and pooled as well as individual stable clones were used for further experiments.

Transient Transfections and Reporter Gene Assays
Transient transfections were performed with the help of Lipofectamine 2000 (Invitrogen Life Technologies, Grand Island, NY) according to the manufacturer’s instructions. Briefly, HEK293 cells or MEFs were grown in six-well plates to about 80 and 50% confluency, respectively, and transfected with 250 ng of the bicistronic construct and 500 ng pRK5-myc-S16H hRheb (19). All experiments were performed in triplicate. After 48 h of transfection, cells were incubated either in serum-free DMEM with or without 100 nM insulin or in nutrient-poor media [KRH buffer consisting of 120 mM NaCl, 5 mM KCl, 1.2 mM MgSO4, 0.33 mM CaCl2, 12 mM HEPES (pH 7.4) with 2% fatty acid-free BSA] and harvested in Reporter Lysis Buffer (Promega). Luciferase activity was measured in whole-cell lysates using the Promega luciferase assay kit and expressed as relative light units. Expression of GFP was measured fluorometrically with the help of TD-700 Fluorometer (Turner Designs, Sunnyvale, CA), and luciferase activity was normalized by GFP fluorescence. Pooled cell lysates from triplicate experiments were assessed for protein expression by Western blotting.

Gel Electrophoresis and Western Blotting
Proteins were separated in SDS-polyacrylamide gels and transferred to Immobilon-P membranes (Millipore Corp., Bedford, MA) in 25 mM Tris, 192 mM glycine. After transfer, the membrane was blocked with 10% nonfat milk in PBS with 0.5% Tween 20 for 2 h. The blots were probed overnight with specific primary antibodies at 4 C followed by 1 h incubation at room temperature with horseradish peroxidase-conjugated secondary antibodies (Sigma). Protein bands were detected with the enhanced chemiluminescence substrate kit (PerkinElmer Life Sciences, Boston, MA) using a Kodak Image Station 440CF (Eastman Kodak, Rochester, NY).

Measurement of Relative Rates of Leptin Biosynthesis
The rate of leptin biosynthesis was measured as previously described (8) with minor modifications. Briefly, differentiated 3T3-L1 adipocytes (d 7) infected with either constitutively active Rheb in the lentiviral vector or EV were incubated in the absence of serum in MEM (without methionine and cysteine, GIBCO, Carlsbad, CA) for 1 h at 37 C and labeled with [35S]methionine/cysteine (750 µCi/ml, Easy-Tag EXPRE35S35S protein labeling mix; PerkinElmer) for 45 min under the same conditions. After labeling, cells were harvested in lysis buffer containing 1% Triton X-100, and total cell lysates with equal amounts of trichloroacetic acid-precipitable radioactivity were immunoprecipitated with leptin antiserum (5 µl) and protein G-agarose (60 µl of 50% slurry; Sigma). Immunoprecipitates were washed three times with lysis buffer and once with PBS and separated by 15% SDS-PAGE using recombinant mouse leptin as standard. [35S]Leptin bands were detected in dried gels with the help of Instantimager (Packard Instruments, Downers Grove, IL). Analysis of postadsorptive supernatants demonstrated that immunoprecipitation of leptin was virtually complete (not shown).

RNA Extraction and Quantitative PCR
Total RNA was extracted from differentiated 3T3-L1 cells (d 7) using TRIzol reagent (Invitrogen). Reverse transcription of 1 µg total RNA was performed using random decamers (RETROscript kit; Ambion, Austin, TX) and leptin mRNA was determined by quantitative PCR (MX4000 Multiplex qPCR system; Stratagene, La Jolla, CA). Reactions were performed in triplicate in the total volume of 25 µl containing 2.5 µl 1:100-diluted cDNA, 1x SYBR green master mix (Brilliant II SYBR Green qPCR Master Mix; Stratagene), and gene-specific primers (for leptin, 5'-CAGACCAGTG GCCTGCAGAA-3' and 5'-AGAGCCCTGCAGCCTGCTC-3'; for 36B4, 5'-TCATCCAGCAGGTGTTTGACA-3' and 5'-GGCACCGAGGCAACAGTT-3'). Reactions were run at 95 C for 10 min followed by 40 cycles of 95 C for 15 sec and 60 C for 1 min. Leptin expression was normalized by 36B4 expression by {Delta}{Delta}Ct method. Deoxyribonuclease-treated samples and no-template controls were analyzed in parallel experiments to confirm specificity.

Statistics
Student’s paired two-tailed t test was used to evaluate statistical significance of the results.


   ACKNOWLEDGMENTS
 
We thank Drs. G. Navruzbekov, G. Thoidis, and A. Tzatsos as well as Rong Tao for help with some experiments. We are grateful to Drs. D. Carling, R. Lamb, J. Procter, S. Pyronnet, and X. Wu for their gifts of cDNA.


   FOOTNOTES
 
This work was supported by Research Grants DK52057 and DK56736 from the National Institutes of Health and Research Award from the American Diabetes Association (to K.V.K.).

Disclosure Statement: The authors have nothing to disclose.

First Published Online July 24, 2008

Abbreviations: AMPK, AMP-activated protein kinase; DN-AMPK, dominant-negative AMPK; EV, empty vector; GFP, green fluorescence protein; MEF, mouse embryonic fibroblast; mTORC1, mammalian target of rapamycin complex 1; 5'-TOP, 5'-terminal oligopyrimidine tract; TSC2, tuberous sclerosis 2; 5'-UTR, 5'-untranslated region.

Received for publication May 5, 2008. Accepted for publication July 16, 2008.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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M.-J. Lee and S. K. Fried
Integration of hormonal and nutrient signals that regulate leptin synthesis and secretion
Am J Physiol Endocrinol Metab, June 1, 2009; 296(6): E1230 - E1238.
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