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Molecular Endocrinology, doi:10.1210/me.2005-0144
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Molecular Endocrinology 19 (10): 2527-2534
Copyright © 2005 by The Endocrine Society

Role of A Kinase Anchor Proteins in the Tissue-Specific Regulation of Lipoprotein Lipase

Gouri Ranganathan, Irina Pokrovskaya, Subramanian Ranganathan and Philip A. Kern

The Central Arkansas Veterans HealthCare System, and Department of Medicine, Division of Endocrinology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205

Address all correspondence and requests for reprints to: Philip A. Kern, M.D. or Gouri Ranganathan, Ph.D, Research, 151 LR, Central Arkansas Veterans Healthcare System, 4300 West 7th Street, Little Rock, Arkansas 72205. E-mail: kernphilipa{at}uams.edu or Ranganathangouri{at}uams.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Activation of protein kinase A by catecholamines inhibits lipoprotein lipase (LPL) activity through the elaboration of an RNA binding complex, which inhibits LPL translation by binding to the 3'-untranslated region of the LPL mRNA. To better define this process, we reconstituted the inhibitory RNA binding complex in vitro and demonstrated that the K homology (KH) domain of A kinase anchor protein (AKAP) 121/149 plays a vital role in the inhibition of LPL translation. Inhibition of LPL translation occurred in vitro only when the C{alpha} subunit, R subunit, and AKAP 149 were present. Using different glutathione-S-transferase fusion proteins of AKAP 149, sequences containing the KH domain were required for inhibition of LPL translation, and the inhibition of AKAP 121 expression in 3T3-F442A adipocytes with short interfering RNA resulted in loss of epinephrine-mediated translation inhibition. After epinephrine injection into mice, LPL activity was inhibited in white adipose tissue but not in brown adipose tissue (BAT) or muscle. LPL activity and synthetic rate were inhibited in vitro by the addition of epinephrine to 3T3-F442A adipocytes, but there was no effect in L6 muscle cells and cultures of brown adipocytes. Corresponding with these differences in LPL translation, AKAP 121 protein and mRNA were abundantly expressed in mouse white adipose tissue, but was either very low or undetectable in BAT and muscle. Thus, AKAP 121/149 contains a KH region that is essential to the translation inhibition of LPL in response to epinephrine. BAT and muscle do not express significant AKAP 121/149, and this likely explains some of the tissue-specific differences in LPL regulation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
LIPOPROTEIN LIPASE (LPL) is a central enzyme in triglyceride-rich lipoprotein hydrolysis and in adipose tissue lipogenesis. LPL hydrolyzes the triglyceride core of circulating chylomicrons and very low-density lipoprotein into nonesterified fatty acids and monoacylglycerol. This hydrolysis results in the accumulation of chylomicron remnants and intermediate density lipoprotein particles, along with the release of nonesterified fatty acids for tissue uptake and lipogenesis (1). LPL is expressed at low levels in many tissues, but high levels of expression are found in white adipose tissue (WAT), brown adipose tissue (BAT), and muscle.

The regulation of LPL is complex and occurs at multiple levels of gene expression. In response to a hyperinsulinemic or postabsorptive environment, LPL activity in adipose tissue is increased primarily due to posttranslational mechanisms resulting in enzyme activation (2, 3), although the addition of insulin to adipocytes in vitro yielded increased LPL mRNA levels (4). There are several known instances of LPL regulation through alterations of translation. After stimulation of protein kinase A (PKA) with epinephrine, adipose LPL activity is decreased, primarily due to a decrease in LPL translation due to the interaction of an RNA binding complex that interacts with the 3'-untranslated region (UTR) of the LPL mRNA (5). Changes in LPL translation in adipose tissue also occur after depletion of protein kinase C (6, 7), induction of diabetes in rats (8), and after the treatment of diabetes in humans (9).

LPL is also expressed at a high level in muscle. Muscle LPL regulation occurs through different mechanisms and often in an opposite direction to LPL regulation in adipose tissue. For example, after feeding in rats, there is an increase in adipose tissue LPL along with a decrease in muscle LPL, both occurring through posttranslational mechanisms (10). In normal weight humans, studies using insulin glucose infusion resulted in an increase in adipose LPL activity but a decrease in muscle LPL activity (11, 12). In response to detraining, adipose LPL increases whereas muscle LPL decreases, both through posttranscriptional mechanisms (13). The mechanism for this inverse regulation between adipose tissue and muscle is not known.

In previous studies, we characterized the translational regulation of LPL in 3T3-F442A adipocytes in response to epinephrine. After activation of PKA, an RNA binding complex, consisting of the catalytic (C) subunit of PKA and A kinase anchor protein (AKAP) 121, interacts with the 3'-UTR of LPL mRNA to inhibit translation (5, 14). The AKAP family of proteins function to tether PKA to specific cellular sites and play an important role in localizing signals (15). AKAP 121 includes a K homology (KH) domain, which is an RNA-binding motif, and previous studies have demonstrated the PKA-mediated binding of AKAP 121 to the 3'-UTR of transcripts encoding ATPase and MnSOD (16).

In this study, we have reconstituted the inhibitory RNA binding complex in vitro and demonstrated that the KH region of AKAP 121 plays a vital role in the inhibition of LPL translation. Inhibition of AKAP 121 expression using small interfering RNA (siRNA) resulted in the loss of LPL translational regulation in response to epinephrine. Thus AKAP 121 is a key component of the RNA protein interaction. Therefore the levels of AKAP 121 expression in various tissues may provide an explanation for the tissue-specific regulation of LPL.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In Vitro Interactions between AKAP 149 and PKA Subunits with LPL mRNA
To further understand the role of RNA-protein interactions in the inhibition of LPL translation, we examined the combination of PKA C{alpha}, PKA R, and AKAP 149 constructs on the inhibition of LPL translation. AKAP 149 was purified after expression in glutathione-S-transferase (GST) fusion vectors as illustrated in Fig. 1Go, and in vitro translation of LPL mRNA was performed as described in Materials and Methods in the presence of different combinations of the three proteins. As shown in Fig. 2Go, lane1, LPL mRNA [nucleotides (nt) 1–2435] containing the complete coding sequence and 836 nt of 3'-UTR was translated using rabbit reticulocyte in vitro translation system in the presence of [35S]methionine (control). In vitro translation reactions lanes 2–8 contained combinations of the C{alpha} subunit, R subunit, GST 149, or GST 149–84 proteins. LPL translation in each lane was analyzed with reference to the control lane. LPL translation was not inhibited by the addition of C{alpha} subunit, GST 149, or GST 149–84 when these proteins were added alone (lanes 2, 3, and 4). C{alpha} subunit in the presence of GST 149 or GST 149–84 did not inhibit LPL translation (lanes 5 and 6). LPL translation was inhibited by 50–60% only when PKA-R and C{alpha} subunit were present along with either GST 149 or GST 149–84 (lanes 7 and 8). The presence of the PKA R subunit was essential to obtain significant inhibition of LPL translation (lanes 6 and 8).



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  		Fig. 1. Diagrammatic Representation of the GST AKAP 149 Constructs Expressed in E. coli BL 21.

 


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  		Fig. 2. Effects of AKAP 149 Constructs and PKA Subunits on LPL in Vitro Translation

A, The LPL mRNA containing 836 nt of 3'-UTR was translated in vitro in the presence of PKA subunits C{alpha}, R subunit, and GST AKAP 149 or GST AKAP 149–84 expressed proteins from constructs illustrated in Fig. 1Go. B, Densitometric analysis of three experiments.

 
The above experiments indicated that the C-terminal region of AKAP 149, which includes the KH region, is important to LPL translation. To more precisely localize this region and to further examine the interactions with PKA subunits, in vitro translation was performed in the presence of the C{alpha} and R subunits of PKA, along with the GST 84 and GST KH constructs, as well as the GST protein alone. As shown in Fig. 3Go, PKA C{alpha} and R subunits, by themselves or in the presence of GST protein, had no inhibitory effect on LPL translation. LPL mRNA translation was inhibited slightly, but not significantly, in the presence of PKA C{alpha}, R, and GST 84 (lane 4). However, a large and significant (P < 0.05) 70 ± 5% inhibition of LPL translation was observed only when PKA R and C{alpha} subunit were present along with GST KH (lane 5).



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  		Fig. 3. Effect of the KH Region of AKAP 149 on LPL Synthesis

A, Using an LPL transcript containing 836 nt of 3'-UTR, the in vitro translation reaction was performed in the presence of the PKA C{alpha} and R subunits with the addition of the indicated GST-AKAP fusion proteins, which are illustrated in Fig. 1Go. B, Densitometric analysis of three experiments.

 
To study the specificity of LPL translational inhibition, three transcripts were examined. In addition to the LPL mRNA 1–2435, which contains 836 nt of 3'-UTR, we examined LPL 1–1599, which contains all of the LPL coding sequence until the stop codon but lacks the complete 3'-UTR, or an irrelevant mRNA encoding the luciferase coding sequence. As shown in Fig. 4Go, LPL 1599 translation was not inhibited in the presence of PKA-R, C{alpha} and GST 149, or GST 149–84, which contained all three components of the inhibitory complex, as described above. Luciferase mRNA translation was also not inhibited by the addition of PKA-R, C{alpha}, and GST 149 full-length or GST 149–84 proteins. However, translation of LPL mRNA 2435, which contains 835 bases of 3'-UTR was inhibited by 60 ± 15%, by the addition of PKA-R, C{alpha}, and GST-149 proteins, as shown in lanes 7 and 8, indicating that inhibition of translation mediated by PKA-R, C{alpha}, and GST 149 or GST 149–84 proteins is specific to LPL transcript containing the 3'-UTR. Figure 4BGo represents densitometric analysis of the data from at least two similar experiments.



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  		Fig. 4. Specificity of mRNA in the Translational Inhibition

A, Three different transcripts were studied. LPL mRNA lacking the 3'-UTR, an irrelevant mRNA and LPL transcript containing 836 nt of 3'-UTR. In vitro translation reaction was performed in the presence of the PKA C{alpha} and R subunits with the addition of the indicated GST 149–84 or GST 149 fusion proteins. B, Densitometric analysis of three experiments. LUC, Luciferase.

 
To determine whether the inhibition of LPL translation by this complex of PKA R and C{alpha} subunits and AKAP 149 was dependent on the ratio of R/C subunit, we studied translation of LPL mRNA in the presence of a fixed concentration of C{alpha} and AKAP 149, but with varying concentrations of R subunit. As shown in Fig. 5AGo, increasing concentrations of R subunit was added to an in vitro translation reaction containing 25 U of C{alpha} and 50 ng of GST AKAP construct. At an R subunit concentration of 25 U, translation was inhibited about 55–65%, and the addition of more R subunit did not further inhibit translation (data not shown). Decreasing the concentration of R subunit to 12.5 or 6.25 U was not sufficient to inhibit translation. Similar results were obtained with two different GST AKAP proteins, both of which contain the KH region of AKAP 149. These data indicate that the appropriate ratio of R to C{alpha} subunits is 1:1 because 1 U of R subunit can inhibit 1 U of C subunit phosphorylation activity in the absence of cAMP. Figure 5BGo represents densitometric analysis of data from two similar experiments.



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  		Fig. 5. Role of R Subunit in the Regulation of LPL Translation

In vitro translation of LPL transcript containing 836 nt of 3'-UTR was studied in the presence of 25 U of PKA C{alpha} subunit, and GST 149 or 149–84, with increasing concentration of R subunit. Lanes 1 and 5 received 0 U of R subunits, lanes 2 and 6 received 6.25 U of R subunit, lanes 3 and 7 received 12.5 U of R subunit, and lanes 4 and 8 received 25 U of R subunit. Lane 9 (controls) represents in vitro translation of LPL mRNA in the absence of components of the RNA binding complex. B, Densitometric analysis of two similar experiments.

 
AKAP 149 anchors PKA to cellular sites, and these data indicate that AKAP 149 is important in the translational regulation of LPL. To demonstrate the importance of this in a physiological system, we used siRNA to inhibit AKAP expression in adipocytes and then measured the inhibition of LPL translation in response to epinephrine. AKAP 121 is the mouse homolog of human AKAP 149. An AKAP 121 siRNA transfected into 3T3-F442A adipocytes inhibited AKAP 121 expression significantly as shown in Fig. 6AGo (lanes 1 and 3); however, a scrambled siRNA with the same GC content did not inhibit AKAP expression (lanes 1 and 2). LPL synthetic rate was measured in cells treated with AKAP siRNA followed by epinephrine treatment (Fig. 6BGo). Epinephrine inhibited LPL synthesis by 55 ± 10% in mock-transfected cells (lanes 1 and 2) and cells transfected with scrambled siRNA oligonucleotides (lanes 3 and 4); this inhibition was lost in cells transfected with AKAP 121 siRNA (lanes 5 and 6).



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  		Fig. 6. Effect of Inhibition of AKAP 121 on LPL Translation

Adipocytes were treated with siRNA to AKAP 121, followed by epinephrine treatment. A, Representative Western blot of 3T3-F442A adipocytes treated with transfecting agent lipid alone, a scrambled siRNA, or AKAP 121 siRNA. B, LPL synthetic rate after treatment with transfecting agent lipid alone, a scrambled siRNA, or AKAP 121 siRNA. LPL synthetic rate was compared in control and epinephrine-treated cells in three independent experiments. KD, Kilodalton.

 
LPL is expressed in many tissues, with high level expression in WAT, BAT, and muscle. However, the hormonal regulation of LPL is different in different tissues. We examined the effects of epinephrine on LPL in WAT, BAT, and muscle both in vivo and in vitro. As described in Materials and Methods, mice were injected with epinephrine, and WAT, BAT, and muscle were removed 2 h later. As shown in Fig. 7AGo, epinephrine treatment resulted in a 30% reduction in LPL activity in WAT, with no inhibitory effect in BAT or muscle. To assess the effects of epinephrine in vitro, we used the 3T3-F442A adipocyte cell line as a cell representative of WAT, and for muscle we used the L6 cell line. For BAT cells, we prepared primary cultures of BAT cells from mice and induced these cells to differentiate, as described in Materials and Methods. As shown in Fig. 7BGo, LPL activity was inhibited by 50% after epinephrine treatment in 3T3-F442A adipocytes but not in cultures of BAT cells or L6 myotubes. To determine whether epinephrine inhibited LPL translation, we compared LPL synthesis after epinephrine treatment in all three cell systems. As shown in Fig. 7CGo, LPL synthetic rate was inhibited only in 3T3-F442A adipocytes after epinephrine treatment, and there was no change after epinephrine treatment in BAT cultures or L6 muscle cells. This inhibition of LPL synthesis in 3T3-F442A adipocytes after epinephrine treatment is not accompanied by a decrease on LPL mRNA (17).



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  		Fig. 7. LPL Activity in WAT, BAT, and Muscle after Epinephrine Treatment

A, As described in Materials and Methods, mice were injected with epinephrine, and LPL activity was measured in WAT, BAT, and muscle 2 h post treatment. B, LPL activity was measured in 3T3-F442A adipocytes, L6 rat muscle cells, and cultured brown adipocytes after epinephrine treatment. C, LPL synthetic rate was compared in 3T3-F442A adipocytes, L6 rat muscle cells, and cultured brown adipocytes after 2 h of epinephrine (10–5 M) treatment. Lane 1 represents in vitro translated LPL mRNA standard for LPL protein. Con, Control; Epi, epinephrine.

 
Because LPL translation is inhibited by epinephrine in WAT cells, but not in BAT cells or muscle, we wondered whether AKAP expression could explain the tissue-specific response of LPL. Therefore, we compared the expression of AKAP 121 in mouse WAT, BAT, and skeletal muscle. As shown in Fig. 8AGo, Western blot analysis indicated the predominant presence of AKAP 121 in WAT, much lower levels of AKAP 121 were detected in BAT, and no AKAP was detected in skeletal muscle. In addition, no AKAP 121 was detected in the cultured L6 muscle cells described in Fig. 7Go, and previous studies have demonstrated expression of AKAP 121 in 3T3-F442A adipocytes (5). Analysis of several Western blots indicated that the levels of expression of AKAP 121 in WAT/BAT were about 20:1 (Fig. 8CGo). These data were confirmed by RT PCR of mRNA isolated from WAT, BAT, and skeletal muscle tissues (Fig. 8BGo).



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  		Fig. 8. Tissue-Specific Expression of AKAP 121

A, Western blot analysis comparing the expression of AKAP121 in WAT, BAT, and muscle of mouse total tissue lysates. B, RT-PCR of AKAP 121 from mouse WAT, BAT, and skeletal muscle. C, Densitometric analysis three different Western blots analyzed for the expression of AKAP 121 comparing total tissue lysates of WAT, BAT, and muscle from mouse.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The regulation of adipose tissue lipid accumulation involves several key enzymes, which respond in an opposite fashion to the hormonal milieu of the feeding-fasting environment (18). In response to the hyperinsulinemic environment of the postprandial state, LPL activity is increased, and hormone-sensitive lipase activity is inhibited, resulting in a net increase in adipose tissue lipid accumulation. In contrast, during fasting, insulin levels are low and the influence of catecholamines and glucagon on PKA and cAMP generation predominate. Hormone-sensitive lipase is stimulated, LPL is inhibited, and the net lipolysis of adipose tissue lipid occurs.

Although it has long been known that these inverse changes in lipase regulation occur, the mechanisms by which they occur are complex. The regulation of LPL is complicated, such that this enzyme is regulated at the level of LPL mRNA in some circumstances, and posttranscriptionally in others (19). For example, LPL activity is increased in the hyperinsulinemic postprandial state due to increased LPL posttranslational processing (3, 10). However, changes in LPL activity are accompanied by increases in LPL mRNA levels in other rodents (20), and in vitro studies have demonstrated increases in LPL mRNA levels in rat adipocyte primary cultures (4) and increased posttranscriptional processing in 3T3-L1 adipocytes (21).

The decrease in LPL activity in adipocytes after cAMP stimulation by catecholamines is well documented; however, the cellular mechanisms controlling this regulation are very complex. In response to catecholamines, we have demonstrated decreased LPL synthesis with no change in LPL mRNA levels in rat adipocytes and in 3T3-adipocytes (14, 17, 22). The inhibition of LPL translation by epinephrine involved RNA binding proteins that interact with the proximal 3'-UTR of the LPL mRNA (17), and a 30-kDa RNA binding protein was identified using UV cross-linking experiments (14), later identified as the catalytic (C) subunit of PKA. AKAPs are a family of proteins that tether PKA to specific locations in the cell and help to target specific intracellular changes (23). Phosphorylated AKAP 121 and PKA C{alpha} subunit were identified as proteins that interacted with the 3'-UTR of LPL mRNA (5). AKAP 149 and the mouse homolog AKAP 121 both have a unique KH domain (24, 25), suggesting that they may participate in RNA/protein binding interactions (26).

In the present study, we have further characterized the proteins participating in the LPL translation-inhibitory RNA binding complex. Using in vitro translation experiments, we have reconstituted the inhibitory complex. This complex consisted of PKA R subunits, C{alpha} subunits, and AKAP 149, and all three components were required for translational inhibition. With regard to the AKAP protein, our data suggest that the KH domain of AKAP 149 was essential. LPL mRNA translation was not inhibited in the presence of PKA R and C subunits alone, but the addition of GST AKAP 149 full length or a fusion protein containing the KH domain of AKAP 149 resulted in the inhibition of translation in vitro. The importance of the KH domain in the regulation of LPL translational was further confirmed because GST AKAP 149–84 inhibited LPL translation in the presence of PKA subunits; however, GST AKAP 84 (which does not include the KH domain) had no inhibitory effect on LPL translation.

To further demonstrate the importance of AKAP 149/121 in the regulation of LPL expression, we inhibited AKAP 121 expression in vitro with siRNA. We compared LPL synthetic rate in 3T3-F442A adipocytes transfected with a scrambled siRNA sequence or a specific AKAP-inhibitory siRNA. After epinephrine treatment, LPL synthetic rate was inhibited in cells that expressed AKAP 121, and this effect was lost in cells that were depleted of AKAP 121, indicating further that AKAP 121 is an essential part of the inhibitory RNA binding complex. It is not known whether AKAP 121 is regulated by physiological conditions, such as feeding/fasting.

One of the mysteries of LPL regulation is centered on the tissue specificity. The reciprocal regulation of LPL by insulin and catecholamines applies largely to adipose tissue, and LPL is regulated quite differently in BAT and skeletal muscle (27, 28, 29). Our experiments measured LPL activity in mice after ip epinephrine treatment and indicated that LPL activity was inhibited in WAT; however BAT and skeletal muscle LPL were not inhibited. Similar results were obtained when epinephrine was added to cultured cells; LPL activity was inhibited after epinephrine treatment in 3T3 adipocytes, but LPL activity in L6 muscle cells or primary cultures of BAT cells were not inhibited. LPL synthetic rate was examined in all three cultured cell lines after epinephrine treatment, and LPL synthesis was decreased corresponding to the decrease in LPL activity only in the 3T3 adipocytes, with no change in LPL synthesis in the BAT or muscle cells. The changes in LPL synthesis in 3T3-F442A adipocytes have previously been shown to occur without a corresponding change in LPL mRNA, indicating translational regulation (5, 14). Because the PKA signaling system is ubiquitous, we hypothesized that a possible reason for the lack of LPL response to epinephrine in BAT and muscle could be a lack of AKAP 149/121. As shown in Fig. 8Go, BAT and muscle express little or no AKAP 121 in comparison with WAT, suggesting that the absence of expression of this PKA-anchoring protein with RNA binding properties accounts for the tissue-specific regulation of LPL by epinephrine. Nevertheless, the regulation of LPL by complex metabolic conditions, such as fasting, is complex. Although fasting is associated with elevated epinephrine, which may result in inhibition of LPL translation, there are likely multiple causes for the decrease in LPL activity during fasting. Indeed, feeding/fasting studies in both rodents and humans have suggested that fasting results in a decrease in LPL posttranslational processing (3, 10, 30, 31).

Therefore, PKA activation by epinephrine resulted in LPL inhibition by a mechanism involving inhibition of translation. This inhibition of LPL translation was dependent on the simultaneous presence of the C{alpha} and R subunits of PKA, along with AKAP constructs that contain a KH RNA binding region. This phenomena occurred only in WAT, which is the only major LPL-expressing tissue that also expresses AKAP 121/149. Thus, the expression levels of AKAP 121 protein may be responsible for the tissue-specific regulation of LPL that is characteristic of WAT.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cell Culture and Differentiation
3T3-F442A cells were obtained from Dr. Howard Green (Harvard Medical School, Boston, MA). Cells were maintained in DMEM (Life Technologies, Inc., Gaithersburg, MD), supplemented to 10% with calf serum. For experiments, cells were grown to confluence and stimulated to differentiate in DMEM containing 10% fetal calf serum and 100 nM insulin for 5–7 d. L6 muscle cells were obtained from American Type Culture Collection (Manassas, VA) and grown in MEM supplemented with 10% fetal bovine serum; to induce differentiation, confluent cultures were maintained in MEM with 2% fetal bovine serum for 5–7 d. Primary cultures of brown adipocytes were isolated from interscapular BAT of 8- to 9-wk-old mice, as previously described (32). In brief, cultures were digested using collagenase, and precursor cells isolated by filtration followed by centrifugation were plated in DMEM containing 10% fetal calf serum and penicillin (10 µg/ml) and streptomycin (10 µg/ml) (Life Technologies). Confluent cultures were differentiated with medium supplemented with isobutyl methyl xanthine (0.5 mM), dexamethasone (1 µM), and insulin (100 nM) for 48 h and maintained in plating medium supplemented with insulin for 7–8 d.

Preparation and Expression of GST Fusion Constructs of AKAP
Human AKAP 149 is the human homolog of mouse AKAP 121. AKAP 149 cDNA was a generous gift from Dr. Hiroyoshi Ariga (Hokkaido University, Sapporo, Japan) (33), and it was amplified using Vent polymerase, and cloned into the Escherichia coli GST expression vector pGEX-4T-2 (Amersham Pharmacia Biotech, Piscataway, NJ). Four different GST AKAP constructs were used to examine the role of AKAP 149 in LPL translation (Fig. 1Go). In addition to the full-length GST 149 (construct 1 in Fig. 1Go), the fusion construct GST 84 was used, which spanned the region between nt 134 and 1828 and did not contain the KH region. The construct GST 149–84 (construct 3 in Fig. 1Go) included nt 1828–2838, which included the KH region, and the fusion construct GST-KH spanned the KH domain nt 1932 and 2164. All fusion constructs were sequenced and verified as correct.

Expression and Purification of GST-AKAP Fusion Proteins
All GST-AKAP plasmids were expressed in E. coli BL 21. Cultures at the log phase of growth were induced with 1.0 mM isopropyl ß-D-thiogalactoside for 3 h. GST fusion proteins were purified using glutathione sepharose affinity chromatography. The recombinant plasmids express the predicted size of GST-AKAP fusion proteins as verified using Western blot (data not shown). Proteins were quantitated by separation on acryl amide gels followed by staining using colloidal Coomassie.

In Vitro Translation
In vitro translation of RNA transcripts was performed as described previously (17). RNA transcripts were made from a LPL cDNA construct (nt 1–2435) (34). Equal quantities of RNA transcripts (0.1 µg) were translated in a rabbit reticulocyte lysate system (Promega Corp., Madison, WI) in the presence of [35S]methionine. Before the addition of rabbit reticulocyte lysates, 25 U of PKA C{alpha} subunit (Calbiochem, La Jolla, CA), 25 U of R subunit (Sigma Chemical Co., St. Louis, MO) and purified GST fusion protein of AKAP 149 approximately 50 ng protein prepared as described above were added. Reactions were incubated at 30 C for 1 h and terminated by transferring to 4 C. The products of reactions were analyzed on 10% SDS-PAGE followed by autoradiography. Images are quantitated using ImageQuant software (Amersham Biosciences, Sunnyvale, CA).

Transfection of siRNA
siRNA to AKAP 121 was synthesized by Ambion, Inc (Austin, TX) and annealed siRNA was made to the sense strand sequence: 5'-GGUUCGACGAAGAUCAGAGtt-3' and antisense sequence: 5'-CUCUGAUCUUCGUCGAACCtg, corresponding to nt 485–503 of mouse AKAP 121 mRNA with overhangs. Cells were transfected 5–7 d after initiation of differentiation using serum free OPTIMEM (Life Technologies) containing 3 µl of lipid reagent (Ambion, Inc.) and 100 nM annealed double-stranded RNA for 4 h (35). After transfection, differentiation medium DMEM with serum and insulin was added to the cultures for 48–72 h.

LPL Synthetic Rate
The synthetic rate of LPL was measured in adipocytes using a 40-min pulse with [35S] methionine (100 µCi/ml), as described previously (4). The medium containing unincorporated label was aspirated, and the total cellular proteins were extracted in cell lysis buffer containing 50 mM phosphate buffer, pH 7.4, 2% deoxycholate, 1% sodium dodecyl sulfate, 20 mM phenylmethylsulfonylfluoride, 2 mM leupeptin, and 2 mM EDTA. The extracts were immunoprecipitated using specific polyclonal antibodies as described previously (36). Immunoprecipitated samples were analyzed on 10% SDS-PAGE, followed by autoradiography.

Western Blot Analysis
The detection of AKAP 121/149 was performed essentially as described earlier (6). The tissue was minced and rinsed in cold PBS, and total protein was extracted using the cell lysis buffer containing 50 mM phosphate buffer (pH 7.4), 1.0% Nonidet P-40, 0.1% sodium dodecyl sulfate, 20 mM phenylmethylsulfonylfluoride, and protease inhibitor cocktail (Sigma). Proteins (15 µg) were fractionated by 10% SDS-PAGE and transferred onto nitrocellulose membranes using 200 mA current for 2–3 h. Membranes were treated with PBS (pH 7.6), 0.2% Tween 20, and 5% nonfat dry milk overnight at 4 C. To identify AKAP 121 protein, polyclonal antibody (Transduction Laboratories, Inc., Lexington, KY) was applied at 1:200, followed by antirabbit horseradish peroxidase conjugate at 1:5000 (Sigma). The reaction product was visualized with chemiluminescence reagents (Amersham Pharmacia Biotech).

Intraperitoneal Injections of Mice with Catecholamine
Normal C57/BL6 male mice (20 ± 2 g) were fasted for 4 h and injected ip with 2.5 µg/kg of epinephrine; tissues were collected 2 h after epinephrine treatment, and LPL activity was measured as described.

Measurement of LPL Activity
Heparin-releasable and extractable LPL activities were determined as described previously (37). Heparin-releasable LPL was measured by incubating adipocytes in 1 ml of DMEM containing 10 U/ml heparin for 60 min at 37 C. LPL catalytic activity was measured as previously described using a substrate containing [3H]triolein and fetal bovine serum as a source of apolipoprotein C-II (38). LPL activity was expressed as nanomoles free fatty acid released/h·mg protein.


   ACKNOWLEDGMENTS
 
We thank Dr. Tong Lu for his expert technical assistance and suggestions during the course of the experiments. We also acknowledge the valuable secretarial assistance of Ms. Sarah Dunn.


   FOOTNOTES
 
This work was supported by a grant from the American Diabetes Association (to G.R.), DK 39176 from the National Institute of Health, and a Merit Review Grant from the Veterans Administration.

First Published Online June 16, 2005

Abbreviations: AKAP, A kinase anchor protein; BAT, brown adipose tissue; GST, glutathione-S-transferase; KH, K homology; LPL, lipoprotein lipase; PKA, protein kinase A; nt, nucleotides; siRNA, small interfering RNA; UTR, untranslated region; WAT, white adipose tissue.

Received for publication April 5, 2005. Accepted for publication June 6, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Goldberg IJ, Merkel M 2001 Lipoprotein lipase: physiology, biochemistry, and molecular biology. Front Biosci. 6:D388–D405
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