Differential function of Akt1 and Akt2 in human adipocytes

Molecular and Cellular Endocrinology 358 (2012) 135–143

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Molecular and Cellular Endocrinology journal homepage: www.elsevier.com/locate/mce

Differential function of Akt1 and Akt2 in human adipocytes Pamela Fischer-Posovszky a,1, Daniel Tews a,1, Sina Horenburg a, Klaus-Michael Debatin b, Martin Wabitsch a,⇑ a b

Division of Pediatric Endocrinology and Diabetes, Department of Pediatrics and Adolescent Medicine, Ulm University Medical Center, Eythstr. 24, 89075 Ulm, Germany Department of Pediatrics and Adolescent Medicine, Ulm University Medical Center, Eythstr. 24, 89075 Ulm, Germany

a r t i c l e

i n f o

Article history: Received 28 November 2011 Received in revised form 22 March 2012 Accepted 22 March 2012 Available online 29 March 2012 Keywords: Obesity Akt1 Akt2 SGBS Adipocyte

a b s t r a c t Adipose tissue mass is determined by both cell size and cell number. Mouse models suggest that Akt isoforms are involved in the determination of fat mass by interfering with preadipocyte-to-adipocyte transition and regulating lipid storage. Here, we took advantage of a lentiviral mediated shRNA approach to study the role of Akt1 and Akt2 in differentiation and metabolism of human SGBS adipocytes. Adipogenic differentiation as measured by lipid accumulation was robustly inhibited in Akt2 deficient cells, whereas it was not affected by knockdown of Akt1. The knockdown of Akt2 caused an almost complete inhibition of preadipocyte proliferation. Furthermore, Akt2 deficient preadipocytes were significantly more sensitive to apoptosis induction by death receptor stimulation compared to Akt1 deficient cells. Both the knockdown of Akt1 or Akt2 equally affected insulin-stimulated lipogenesis as well as the anti-lipolytic effect of insulin. We conclude that Akt2 is indispensable for the regulation of preadipocyte and adipocyte number, whereas Akt1 and Akt2 are equally important for the regulation of insulin-stimulated metabolic pathways in human adipocytes. Recently proposed as an attractive target for the treatment of cancer, modulating Akt2 activity might also be a new molecular strategy to control adipose tissue mass. Ó 2012 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Obesity is characterized by increased white adipose tissue mass and is a risk factor for developing secondary disorders like noninsulin dependent diabetes mellitus (NIDDM), cardiovascular diseases and certain types of cancer (Guh et al., 2009). The size of the adipose organ is governed by cell volume and number and is therefore regulated by the interplay of cell renewal and cell death on one hand and triglyceride storage and release on the other hand (Arner and Spalding, 2010). A key contributor of adipose tissue mass regulation is the serine/threonine kinase Akt, which is part of the intracellular signaling cascade activated by insulin and IGF-1 (Schultze et al., 2011). There is evidence that, despite a close homology of the three isoforms of Akt, the regulation of adipose tissue mass is isoform specific (Dummler and Hemmings, 2007). The three Akt isoforms are expressed in a tissue-dependent manner. While Akt1 is expressed ubiquitously at high levels with exception of the kidney, spleen and liver (Coffer and Woodgett, 1992; Jones et al., 1991; Bellacosa et al., 1993), expression of Akt2 is elevated in insulin-sensitive tissues such as fat, skeletal ⇑ Corresponding author. Tel.: +49 (0)731 500 57401; fax: +49 (0)731 500 57407. 1

E-mail address: [email protected] (M. Wabitsch). These authors contributed equally to this work.

0303-7207/$ - see front matter Ó 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.mce.2012.03.018

muscle and liver (Calera et al., 1998; Hill et al., 1999; Summers et al., 1999). Akt3 is predominantly expressed in the brain, with low levels in skeletal muscle and liver (Nakatani et al., 1999). Several groups have generated Akt1 knockout mice by deleting different exons of the Akt1 gene (exon1, exon 3–4, exon 4–7, exon 8–13) (Chen et al., 2001; Cho et al., 2001a; Yang et al., 2003; Wan et al., 2012). All Akt1 transgenes were not lethal, but were characterized by decreased fetal and postnatal growth, persisting into adulthood (Cho et al., 2001a). The life span of Akt1/ mice, upon exposure to genotoxic stress, was decreased (Chen et al., 2001). Deficiency of Akt1 has also been implicated in angiogenesis and tumor development (Cho et al., 2001a). Interestingly, none of the Akt1/ mutants displayed a diabetic phenotype; the mice were either normal with regard to glucose tolerance and insulin-stimulated disposal of blood glucose (Cho et al., 2001a), or displayed an improved insulin sensitivity and enhanced energy expenditure compared to wt animals (Buzzi et al., 2010; Wan et al., 2012). An ex vivo approach revealed that Akt1 is essential for adipocyte differentiation in mouse embryonic fibroblasts (MEFs), while differentiation capacity was restored by re-expression of Akt1 (Yun et al., 2008; Baudry et al., 2006). Experimental as well as clinical studies suggest that Akt2 is a key regulator of adipose tissue mass. Two different strains of Akt2 knockout mice both displayed an impediment of glucose

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metabolism. Cho et al. showed that Akt2/ animals had impaired insulin sensitivity in liver and skeletal muscle (Cho et al., 2001b). Garofalo et al. reported that Akt2-null mice exhibit mild growth deficiency, an age-dependent loss of adipose tissue (lipodystrophy) and severe insulin resistance (Garofalo et al., 2003). In accordance with this phenotype, insulin-stimulated glucose uptake was inhibited in MEFs generated from Akt2/ mice (Bae et al., 2003). Surprisingly, adipocyte differentiation was not altered in these cells (Bae et al., 2003). In 2004, George et al. described a family with severe insulin resistance and diabetes due to a mutation in Akt2 (George et al., 2004). Interestingly, these patients had also a reduced body fat content. Overexpression of the mutant kinase in human liver cells and rodent adipocytes disrupted insulin signaling and inhibited the function of co-expressed, wild-type Akt. These findings demonstrated for the first time that Akt signaling plays a central role for insulin sensitivity in humans (George et al., 2004). The function of Akt1 and Akt2 in human adipocyte biology has not been intensively studied. Therefore, we developed a lentiviral mediated shRNA approach to specifically inhibit the expression of Akt1 and Akt2. We studied preadipocyte proliferation and adipogenic differentiation to elucidate the impact of both isoforms on adipocyte number. Insulin-regulated metabolic pathways were analyzed to uncover the potential isoform-specific impact on the adipocyte de novo lipogenesis and lipolysis.

visualize and quantitate cellular lipid content. In brief, cells were washed with PBS, fixed for 2 h in 10% PFA and stained with Oil Red O for 1 h at room temperature. Unbound dye was removed by washing with water. After documentation with a digital camera, incorporated dye was further solubilized in isopropanol and OD550 was determined as a measure of relative lipid content. 2.3. Cell proliferation assay Preadipocytes were seeded at low density (1300 cells/cm2) in medium containing 10% FCS. After 24 h, cells were washed twice with PBS and were cultivated in medium in presence or absence of 10% FCS for another 48 h. Cell numbers were counted microscopically using a net micrometer and were expressed as ratio (cell count 10% FCS)/(cell count 0% FCS). 2.4. Apoptosis induction and quantitation Preadipocytes were treated with the CD95 agonistic antibody anti-APO-1 IgG3 (1 lg/ml) for 72 h in serum-free medium. Quantitation of specific apoptosis was carried out as described elsewhere (Nicoletti et al., 1991; Fischer-Posovszky et al., 2004). In the experiments the percentage of specific apoptosis was calculated as follows to normalize data: 100  [experimental apoptosis (%)  spontaneous apoptosis in vehicle (%)]/[100%  spontaneous apoptosis in vehicle (%)].

2. Materials and methods 2.5. Western blot and phospho-MAPK analysis 2.1. Cell culture and lentiviral transduction Cell culture of SGBS preadipocytes and differentiation into adipocytes was carried out as described before (Fischer-Posovszky et al., 2008, 2006; Wabitsch et al., 2001). Knockdown of Akt1 and Akt2 was performed by using the BLOCK-iT™ Inducible H1 Lentiviral RNAi System (Invitrogen, Darmstadt, Germany) according to the manufacturer’s protocol. Specific shRNA constructs against Akt1 (50 -ggacaaggacgggcacattaa-30 ), Akt2 (50 -ggttcttcctcagcatcaact-30 ) and a sequence with no corresponding counterpart in the human genome (hyper random sequence, HRS) (50 -gatcatgtagatacgctca-30 ) were cloned into the pENTR™/H1/TO vector using the BLOCK-iT™ Inducible H1 RNAi Entry vector kit. After propagation of the vector, the shRNA expression cassettes were transferred into the pLenti4/BLOCK-iT™-DEST vector by targeted recombination. Viral particles were produced after transfection of 293FT cells in the presence of Lipofectamine2000 using the ViraPower™ Lentiviral Expression Systems (Invitrogen, Darmstadt, Germany). Three days after transfection, viral supernatants were harvested, filtered through a 0.45 lm PVDF filter and stored at 80 °C upon usage. SGBS cells were transduced with an MOI = 1 in presence of 8 lg/ml polybrene. Stable bulk cultures were generated by selection with 0.2 mg/ml Zeocin for 14 days. For transient knockdown, SGBS adipocytes were transduced at day 10 of differentiation. The knockdown of Akt1 and Akt2 was controlled by qRT-PCR and Western Blot analysis. For all experiments, cells from at least three independent lentiviral transductions were used. 2.2. Adipogenic differentiation assay Akt1 and Akt2 deficient cell strains as well as HRS control cells were differentiated until day 14 as outlined before (Fischer-Posovszky et al., 2006; Wabitsch et al., 2001). The number of morphologically differentiated adipocytes was determined by direct microscopic counting using a net micrometer. Furthermore, cells were stained with Oil Red O (Grigem et al., 2005), in order to

For determination of insulin signaling, SGBS adipocytes were pre-incubated in serum-free medium for 24 h. After stimulation with 100 nM insulin for 15 min, cells were washed with ice–cold PBS and lysed as described before (Keuper et al., 2011). Lysates were either subjected to phospho-MAPK or Western blot analysis. Phosphorylation of different MAP kinases was performed using the Proteome Profiler MAPK Array (R&D Systems, Wiesbaden, Germany) according to the manufacturer’s protocol. Western blot analysis was carried out as described elsewhere (Fischer-Posovszky et al., 2006). For detection, enhanced chemiluminescence was used. Following antibodies were used: rabbit anti-phospho(Ser473) Akt, mouse anti-p38, rabbit anti-phospho(Ser 21/9) GSK3a/b (all from Cell Signaling Technology, Beverly, USA), mouse anti-Akt1, mouse anti-GSK3b, (both from BD Biosciences, Heidelberg, Germany), rabbit anti-Akt2 (Millipore, Schwalbach, Germany), rabbit anti-phospho(Thr202/185, Tyr204/187) Erk1/2, rabbit anti-phospho(Thr180,Tyr182)-p38 (all from New England Biolabs, Frankfurt, Germany), anti phospho-(Ser 380) RSK1, anti RSK1 (both from Abcam, Cambridge, UK), rabbit anti-Erk1/2 (Sigma–Aldrich, Munich, Germany), mouse anti-alpha-tubulin (Merck, Darmstadt, Germany). 2.6. Insulin-stimulated glucose uptake and de novo lipogenesis After washing twice with PBS, SGBS adipocytes on day 14 of adipogenic differentiation were incubated in serum-free medium for 24 h. For glucose uptake, cells were stimulated without or indicated doses of insulin for 15 min and 2-deoxy-D-[14C]-glucose (0.2 lCi/well) was added for another 15 min. Cells were washed twice in ice–cold PBS, harvested with 100 mM NaOH and incorporation of 2-deoxy-D-[14C]-glucose was measured on a ß-counter. For lipogenesis, indicated doses of insulin and D-[14C]-glucose (0.1 lCi/well) were added for 24 h. Thereafter, cells were washed twice in ice–cold PBS, harvested in 100 mM NaOH, lysed in a lipophilic scintillation fluid and incorporation of 14C was determined on a ß-counter.

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50 -GGACAGGGTGTGCTTCTTCCAGTGCTC-30 50 -AAAAGCTTCTCATGGTCCTG-30 50 -ACTTCCATCTCCTCAGTCGT-30 50 -GAGGGAGTTGGAAGGCTCTTC-30 50 -CATCCCACTGGTACACCTTCCCACTCAC-30 50 -ACACTGATGATCATGTTAGGTTTGG-30 50 -AAGCACCGCAGAGAACACAG-30

5 -CATGCTGCCGTGTTCCGTGTGGG-3 50 -GCCCTGAAGTACTCTTTCCA-30 50 -GGTACTTCCTGCTGAAGAGC-30 50 -GATCCAGTGGTTGCAGATTACAA-30 50 -CTACCTGAGCATAGTGTGGAAGACGCTG-30 50 -GCTTTTGTAGGTACCTGGAAACTT-30 50 -TTCCAACAGATAGGCTCCGAAG-30

2.7. Lipolysis

A

2.9. Statistics If not otherwise stated, data from three independent experiments were expressed as mean ± standard error of means (SEM). For statistical comparison, one-way analysis of variants (ANOVA) test was used, considering p < 0.05 statistically significant. For the analysis, we used GraphPad Prism version 5.0 (GraphPad Software Inc., San Diego, USA). 3. Results 3.1. Expression of Akt1 and Akt2 in SGBS adipocytes It is evident that Akt activity is necessary for a wide range of insulin’s actions in the cell. Akt1 is the most ubiquitously expressed isoform in mammalian cells and tissues. Akt2 is also expressed at low levels in most tissues and organs, but it is highly abundant in insulin-responsive tissues (Altomare et al., 1995, 1998), indicating a specific role of this isoform in insulin-stimulated metabolism. We used SGBS preadipocytes as a model system for human adipocyte biology. These cells have been intensively characterized and behave functionally and biochemically like human primary adipocytes (Fischer-Posovszky et al., 2008) and examined the expression of Akt1 and Akt2 during adipogenic differentiation. Both isoforms were present at the mRNA (Fig. 1A) and the protein level (Fig. 1B) confirming previous data (Altomare et al., 1995, 1998). The expression of Akt1 was clearly down-regulated during adipogenesis, while Akt2 expression was slightly upregulated at the protein level.

relative expression

*

*

0.5

* *

d1 4

d1 1

d7

d0

0.0

B 60 kDa

Akt1

60 kDa

Akt2 -actin d14

d10

42 kDa d7

Total RNA was prepared using the RNeasy Lipid Tissue Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. cDNA was synthesized as described elsewhere (Fischer-Posovszky et al., 2006). qPCR was performed on a LightCycler™ 2.0 (Roche Diagnostics, Mannheim, Germany). Relative mRNA levels were determined by comparison to a reference gene (succinate dehydrogenase subunit alpha, SDHA) using the DDCT method. The sequences of the gene-specific primers are presented in Table 1.

1.0

d0

2.8. RNA preparation and real-time expression analysis

Akt1 Akt2

1.5

Basal and isoproterenol-stimulated lipolysis was determined as described before (Wabitsch et al., 1996) with some modifications. After starvation of in vitro differentiated SGBS adipocytes in insulin-free medium for 24 h, cells were washed in PBS and were pre-treated with 1010 M insulin for 15 min in Krebs–Ringer-bicarbonate buffer supplemented with 1% fatty acid-free BSA. Subsequently, cells were stimulated with 1 lM isoproterenol for 4 h. Cell-free supernatants were harvested by centrifugation and glycerol content was determined using the Free Glycerol Assay Kit (Sigma–Aldrich, Munich, Germany) following the manufacturer’s protocol.

d4

0

d4

SDHA AKT1 AKT2 PPARg FASN FABP4 GLUT4

Forward primer

d1

Gene name

Fig. 1. Expression of Akt isoforms during adipogenic differentiation. At the indicated time points during adipogenic differentiation, SGBS cells were collected and RNA and protein was isolated. Relative mRNA expression was determined by qRT-PCR using gene-specific primers (A). Protein expression was determined by Western blot (B, one representative blot shown) and ECL signals were quantified densitometrically (C). Data are expressed as means ± SEM and are from three independent experiments, ⁄p < 0.05.

3.2. Generation of Akt1 and Akt2 deficient SGBS preadipocytes Transgenic mouse models pointed to an important role of Akt in maintaining white adipose tissue mass (Garofalo et al., 2003; Peng et al., 2003). However, the role of Akt isoforms in preadipocytes and adipocytes from human origin is still not completely understood. To this end, we established SGBS preadipocytes stably overexpressing shRNA constructs specifically targeting Akt1 or Akt2. The mRNA expression of both Akt1 and Akt2 was successfully inhibited by 85 ± 4% and 76 ± 3%, respectively (Fig. 2A). Also, protein expression of both isoforms was blunted to near knockout levels. (Fig. 2B). 3.3. Akt2 deficient preadipocytes display reduced proliferative and differentiation capacity and enhanced sensitivity towards apoptosis induction The number of fat cells within adipose tissue is balanced through generation of new cells by adipogenic differentiation and by elimination of old cells at the same time. First, we aimed at identifying the role of Akt isoforms in adipogenesis. The rate of differentiation was comparable in HRS express-

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Akt1

Akt2

1.5

1.5

relative expression (Akt2/SDHA)

relative expression (Akt1/SDHA)

A

1.0

0.5

** 0.0

ing controls and parental SGBS preadipocytes (85%) (Fig. 3A). The number of lipid-laden adipocytes was robustly diminished in Akt2 deficient cells (differentiation rate 25%), while Akt1 deficient cells were not statistically different from HRS control cells (Fig. 3 A and B). Likewise, the triglyceride content was significantly decreased in cultures with Akt2 knockdown (Fig. 3C), paralleled by decreased mRNA expression of PPARc and FABP4 (Fig. 3D). Again, Akt1 deficient cells were comparable to control cells in terms of lipid accumulation and adipocyte marker gene expression. The number of precursor cells present in adipose tissue might be an important regulator of adipose mass. We therefore studied the proliferation rate of Akt1 and Akt2 deficient preadipocytes. We detected a 2-fold increase in cell numbers of HRS control cells as well as Akt1 knockdown cells after 48 h (Fig. 4A). In contrast, proliferation of Akt2 deficient cells was almost completely blunted (1.26 ± 0.21-fold increase after 48 h). The naturally occurring cell death, apoptosis, provides a mechanism for the elimination of preadipocytes and adipocytes. We therefore studied the impact of Akt1 and Akt2 in death-ligand induced apoptosis. Upon treatment with agonistic CD95 antibodies, Akt1 deficient cells showed a trend towards specific apoptosis induction compared to control cells, however without statistical significance (Fig. 4B). In comparison, specific apoptosis of Akt2 deficient cells was enhanced by twofold compared to control cells. This set of data clearly shows that Akt2 is an important regulator of adipocyte number. The presence of Akt2 is required for the process of adipogenic differentiation as well as for proliferation of preadipocytes.

1.0

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H

sh A

kt

R S sh A kt 1 sh A kt 2

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1 kt sh A

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Akt1 Akt2

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panAkt

42 kDa

-actin shAkt2

wt

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shAkt1

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3.4. Both Akt1 and Akt2 are required for insulin-dependent metabolism in adipocytes

Fig. 2. Isoform-specific knockdown of Akt in SGBS cells. Stable bulk cultures of Akt1 and Akt2 deficient SGBS cells were generated by lentiviral transduction as outlined in section 2. Relative mRNA expression was determined by qRT-PCR using genespecific primers. Data are expressed as means ± SEM and are from three independent experiments, ⁄p < 0.05. Protein expression was determined by Western blot. One representative experiment of three performed is shown.

A

The volume of adipocytes is regulated by the competing processes of lipid storage and lipid breakdown (Large et al., 2004). Insulin is essential for key anabolic pathways in adipocytes and

wt

HRS

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shAkt2

differentiation rate (%)

B 100 80 60 40

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20

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sh A

kt

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HRS shAkt1 shAkt2

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P4 FA B

R

g

2 kt

1 sh A

kt sh A

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0.0 PP A

OD550 (fold basal)

C

2

0

Fig. 3. Akt2 is involved in adipocyte differentiation. Different SGBS cell strains were differentiated in vitro into adipocytes. On day 14, cells were stained with Oil Red (A) and differentiation rate was determined by counting (B). Relative lipid contents were determined by measuring uptake of the dye (C). mRNA levels of PPARg and FABP4 were determined by qRT-PCR (D). Representative pictures are shown. Data are from three independent experiments and are shown as means ± SEM, ⁄p < 0.05.

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cells (fold of basal)

A

Deficiency of both isoforms decreased the uptake of radioactive labeled deoxy-glucose (approx. 20% at 107 M insulin) (Fig. 5C) and the incorporation of radioactive labeled glucose into cellular lipids to a comparable extent (approx. 35% at 107 M insulin) (Fig. 5D). Insulin at 1010 M inhibited isoproterenol-stimulated lipolysis in control cells and to a comparable extent also in Akt1 and Akt2 deficient cells (Fig. 5E). There was no significant inhibition of basal lipolysis by insulin and also no difference between control cells and Akt1 and Akt2 deficient cells (Fig. 5E). Taken together, human Akt1 and Akt2 deficient adipocytes showed a phenotype of reduced insulin sensitivity as seen by decreased insulin-stimulated lipid synthesis, and diminished insulin-stimulated anti-lipolysis. In contrast to published data in murine model systems (Cho et al., 2001a,b; Bae et al., 2003), there was no significant difference between the isoforms. The single isoform knockdown was not sufficient to completely block these insulin-stimulated processes to unstimulated levels. This suggests that either the respective other isoform of Akt or any other kinase partially compensates the deficiency in one single signaling molecule. To this end, we studied insulin-related signal transduction in our knockdown cells.

3

2

*** 1

specific apoptosis (%)

kt 2 sh A

kt 1 sh A

H

R S

0

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60

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20

2 sh A

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0 H

139

Fig. 4. Proliferation and apoptosis in Akt deficient SGBS preadipocytes. (A) Preadipocytes were seeded at low density and were allowed to recover for 24 h. Cells were then cultivated for 48 h in medium containing 10% FCS or not, and cell numbers were determined by direct counting using a net micrometer. Cell growth is expressed as ratio treated:untreated cells. Data (mean + SEM) from three experiments are shown, ⁄⁄⁄p < 0.001. (B) SGBS preadipocytes were treated with 1 lg/ml of the CD95 agonist anti-APO-1 IgG3 in the absence of serum. After 72 h, the content of hypodiploid DNA was determined by flow cytometry. Specific apoptosis was calculated as follows to normalize data: 100  [experimental apoptosis (%)  spontaneous apoptosis in vehicle (%)]/[100%  spontaneous apoptosis in vehicle (%)]. Data are expressed as means ± SEM and are from three independent experiments, ⁄⁄p < 0.05.

3.5. Insulin signaling in Akt1 and Akt2 deficient adipocytes We stimulated Akt1 and Akt2 deficient adipocytes with 107 M insulin for 15 min and subjected protein samples to a protein array to study the phosphorylation state of a large panel of down-stream kinases (summary of all data given in Supplementary Fig. S1). As expected, phosphorylation of Akt1 was decreased in adipocytes with Akt1 knockdown; phosphorylation of Akt2 was decreased in adipocytes with Akt2 knockdown. Surprisingly, out of all downstream targets of Akt, only phosphorylation of RSK1 seemed inhibited while GSK3b was not affected by the knockdown. Also the phosphorylation of several other kinases acting at the same level as Akt including Erk1/2, p38 kinase and JNK was not changed as compared to control cells. To confirm these findings, we performed Western blot analysis using phospho-specific antibodies for several key kinases. The insulin-stimulated phosphorylation of RSK1, GSK3ab, Erk1/2 and p38 was not altered upon Akt1 or Akt2 knockdown, although expression levels reached near knockout levels (Fig. 6A). We next analyzed the mRNA expression of lipogenic genes including Glut4, fatty acid synthase (FASN) and acetyl-CoA carboxylase. None of these genes was significantly changed upon knockdown of Akt1 or Akt2 (Fig. 6B). We conclude from these results that reduction in insulin sensitivity by loss of Akt1 or Akt2 is not mediated by changes in gene expression, but rather by modulating Glut4 translocation to the membrane as described previously by Bae and colleagues (Bae et al., 2003). 4. Discussion

also other insulin-sensitive cells by triggering glucose uptake via glucose transporter 4 and by exerting anti-lipolytic effects at the same time (Kanzaki, 2006; Bézaire and Langin, 2009). Despite their close homology, Akt isoforms serve very distinct metabolic functions (Bae et al., 2003), at least in mice. Here, we sought to identify the metabolic function of Akt isoforms in human adipocytes. In order to avoid an interference of Akt knockdown with adipogenic differentiation we transiently transduced SGBS adipocytes at day 10 of differentiation. Four days post transduction, expression levels of Akt1 and Akt2 were markedly reduced as shown by qRT-PCR and Western blot analysis (Fig. 5A and B). The number of adipocytes was comparable in parental cells, HRS controls and Akt knockdown cells (data not shown).

The adipose organ represents a tissue with a continuous turnover of cells (Arner and Spalding, 2010; Spalding et al., 2008). Adipose tissue homeostasis involves the new generation of fat cells from precursors on the one side, and the elimination of old cells on the other side. Adipose tissue mass is predominantly determined by the volume of adipocytes, which in turn is regulated by competing processes of lipolysis and lipid storage (Large et al., 2004). Insulin as well as IGF-1 are important extracellular regulators of both adipocyte number and function. Inside of the cell, the serine/threonine kinase Akt is a key molecular player in these processes. The three existing isoforms of Akt, Akt1, Akt2, and Akt3, hold specific physiological functions in different cell types including proliferation, growth, survival and differentiation as well

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1.5

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C glucose incorporation into cellular lipids (cpm)

D 2000

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C deoxy glucose uptake (cpm)

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glycerol release (fold basal)

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insulin (log M)

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HRS Akt1 Akt2

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ve h

0

Fig. 5. Metabolic characterization of Akt deficient SGBS adipocytes. In vitro differentiated adipocytes were transduced with lentivirus on day 10 of adipogenic differentiation. On day 14, knockdown efficiency was controlled by qRT-PCR (A) and Western Blot (B). (C) Insulin-stimulated glucose uptake was assessed by incorporation of [14C]-deoxyglucose in the presence of increasing insulin concentrations (0–1000 nM). p values for shAkt1 vs. HRS were ⁄⁄p < 0.01, ⁄⁄⁄p < 0.001. p values for shAkt2 vs. HRS were ##p < 0.01, ### p < 0.001. (D) De novo lipogenesis was assessed by incorporation of [14C]-D-glucose into lipids in the presence of increasing insulin concentrations (0–1000 nM). p values for shAkt1 vs. HRS were ⁄p < 0.05, ⁄⁄p < 0.01. p values for shAkt2 vs. HRS were ##p < 0.01. (E) As a measure for lipolysis the accumulation of glycerol in medium supernatants was determined. Cells were pre-treated with 0.1 nM insulin for 15 min and then stimulated with or without 1 lM isoproterenol for 4 h. Data (mean + SEM) from three to four independent experiments are shown.

as several metabolic functions (Brazil et al., 2004; Schultze et al., 2011). In this study, we explored the role of the Akt isoforms, Akt1 and Akt2 in the regulation of human preadipocyte and adipocyte biology. Our results clearly demonstrate that Akt2 is indispensable for human preadipocyte proliferation, apoptosis sensitivity, and adipogenesis, whereas Akt1 played a minor role here. However, both Akt1 and Akt2 were equally important for mediating the effects of insulin on cellular metabolism. The expression profile of Akt1, Akt2, and Akt3 may in part reflect their specific physiological functions in different tissues. Akt1 is relatively ubiquitously expressed (Coffer and Woodgett, 1992; Jones et al., 1991; Bellacosa et al., 1993), while Akt2 is elevated in insulin-sensitive tissues such as fat, skeletal muscle and liver (Calera et al., 1998; Hill et al., 1999; Summers et al., 1999).

In line with this, both Akt1 and Akt2 were expressed during adipogenic differentiation of SGBS cells. Several recent studies showed that the PI3K/Akt pathway is required for preadipocyte proliferation, survival and adipogenic differentiation. Akt1 and not Akt2 is required for cell cycle progression from G1 to S-phase in murine cells (Yun et al., 2009). As concerns cellular survival pathways, all three isoforms of Akt seem to possess a redundant anti-apoptotic function (Liu et al., 2006). The histological analysis of brown adipose tissue from Akt1 and Akt2 double knockout neonates revealed no visible lipid droplets and MEFs derived from these animals were unable to differentiate in vitro (Peng et al., 2003). Both MEFs derived from Akt1 knockout mice and Akt1 deficient 3T3L1 preadipocytes showed an attenuated capacity of adipogenic differentiation, whereas Akt2 knockout MEFs differentiated normally (Yun et al., 2008; Baudry

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A

83 kDa

p-RSK1

83 kDa

RSK1

60 kDa

p-Akt

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Akt1

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Akt2

51 kDa

p-GSK3

46 kDa 51 kDa

GSK3

44 kDa 42 kDa

p-ERK1/2

44 kDa 42 kDa

ERK1/2

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43 kDa

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60 kDa

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Fig. 6. Expression and signaling of key targets in Akt deficient adipocytes. In vitro differentiated adipocytes were transduced with lentivirus on day 10 of adipogenic differentiation. On day 14, cells were cultured in basal medium without differentiation factors for 24 h. Cells were stimulated with 100 nM Insulin for 15 min and protein was harvested subsequently. Protein expression and phosphorylation was then analyzed by Western blot (A). RNA was of transduced cells was also extracted and expression analyses were performed by qPCR (B). Data (mean + SEM) from three independent experiments are shown.

et al., 2006; Peng et al., 2003). These data suggest that murine adipogenesis is mainly controlled by Akt1. In contrast, in our human model system, adipogenesis was only affected by knockdown of Akt2 whereas Akt1 deficient cells retained their differentiation capacity. Key adipocyte markers such as PPAR (peroxisome proliferator activated receptor)-c or FABP (fatty acid binding protein) 4 were only down-regulated in Akt2 deficient cells while being unchanged in Akt1 deficient cells compared to control cells. Proliferative capacity was diminished in Akt2, and not Akt1 knockdown cells. Furthermore, sensitivity for apoptosis induced by CD95 stimulation was only significantly increased in Akt2 deficient cells. Our set of data points to species-specific function of Akt1 and Akt2.

Akt2 is of essential importance for insulin sensitivity. Different Akt2 knockout mice strains all displayed a disturbed glucose metabolism and impaired insulin responsiveness in skeletal muscle as well as in liver (Cho et al., 2001b; Garofalo et al., 2003). Moreover, one strain displayed mild growth deficiency and an age-dependent loss of adipose tissue or lipodystrophy (Garofalo et al., 2003). In 3T3L1 cells, Akt2 turned out to be predominantly responsible for the regulation of cellular glucose transport (Katome et al., 2003). Most importantly, George et al. described a point mutation in the gene encoding the protein kinase Akt2 in a family that shows autosomal dominant inheritance of severe insulin resistance and diabetes mellitus (George et al., 2004). This study

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demonstrated for the first time that Akt2 signaling is essential for maintaining insulin sensitivity in humans. Disruption of Akt1 however did not cause a diabetic phenotype and knockout animals had either normal or even improved glucose tolerance and insulinstimulated blood glucose disposal (Cho et al., 2001a; Buzzi et al., 2010; Wan et al., 2012). In fact, Akt1 deficiency resulted in decreased fetal and postnatal growth and in a decreased life span (Chen et al., 2001; Yang et al., 2003). In addition, an attenuation of spermatogenesis and increased spontaneous apoptosis in testes and thymus were observed (Chen et al., 2001). In order to study the impact of Akt1 and Akt2 concerning glucose metabolism in adipocytes we performed glucose uptake and de novo lipogenesis assays in transiently transduced adipocytes. In this experiment, Akt1 and Akt2 deficiency was reducing the insulin effect to the same extent. Also, inhibition of adipocyte lipolysis by insulin was equal in both cell lines. Surprisingly, although we achieved a down-regulation of both isoforms to near knockout levels, the effect of Akt1 or Akt2 deficiency on insulin-stimulated metabolic processes was relatively small. This suggests that other signaling molecules of the highly abundant system of insulin-stimulated kinases or the remaining other Akt isoform might take over the function of the knocked out isoform of Akt. However, neither the respective other isoform of Akt, nor Erk or p38 kinase showed changes in phosphorylation upon insulin stimulation. Taken together, our data suggest, in contrast to mouse experiments, that both Akt isoforms are involved in insulin-stimulated glucose metabolism without any apparent specificity for one isoform. Interestingly, classical downstream targets such as GSK3 and p70S6 K were not affected in terms of phosphorylation after transient knockdown of either Akt1 or Akt2. Moreover, expression of the glucose transporter 4 (GLUT4) and key regulators of lipogenesis (ACACA, FASN) as well as lipolysis (HSL, ATGL, PLIN) was unchanged. Lacking any regulation on the level of expression, insulin-stimulated de novo lipogenesis are most likely regulated by the interplay with glucose transporter translocation events at the plasma membrane. The regulation of GLUT4 translocation to the plasma membrane might therefore serve as a rate-limiting step, affecting downstream glucose uptake and lipogenesis. In accordance, it has been shown that Akt is involved in GLUT4 translocation in adipocytes (Bae et al., 2003). Hoffstedt et al. recently reported that the regional morphology of adipose tissue is associated with the metabolic profile in obesity (Hoffstedt et al., 2010). In general, a phenotype of many, but small adipocytes might be protective against obesity-related metabolic disturbances. Hypoplasia (few, but large fat cells) in the subcutaneous fat depot seems to predispose to glucose intolerance, while it is associated with dyslipidemia when found in the visceral depot. It will be of great importance to identify factors regulating the local adipose tissue morphology. It is well conceivable that Akt2 plays an important role in these processes. 5. Conclusion In conclusion, we demonstrate that both proliferation and adipogenic differentiation of human adipocytes is dependent on Akt2 in human preadipocytes, whereas Akt1 and Akt2 are equally important for the regulation of insulin-stimulated metabolic pathways. Acknowledgements This study was supported by the German Research Association (WA1096/3-3), the German Ministry of Education and Research (NGFNplus)(BMBF01GS0824), and the Centre of Excellence Baden-Württemberg ‘‘Metabolic Disorders’’ to MW. PFP is funded

by a Margarete von Wrangell scholarship financed by the BadenWuerttemberg Ministry of Science, Research and Arts, the European Social Fund, and Ulm University.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.mce.2012.03.018.

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