Photoactivation of GLUT4 translocation promotes glucose uptake via PI3-K/Akt2 signaling in 3T3-L1 adipocytes

Insulin resistance is a hallmark of the metabolic syndrome and type 2 diabetes. Dysfunction of PI-3K/Akt signaling was involved in insulin resistance. Glucose transporter 4 (GLUT4) is a key factor for glucose uptake in muscle and adipose tissues, which is closely regulated by PI-3K/Akt signaling in response to insulin treatment. Low-power laser irradiation (LPLI) has been shown to regulate various physiological processes and induce the synthesis or release of multiple molecules such as growth factors, which (especially red and near infrared light) is mainly through the activation of mitochondrial respiratory chain and the initiation of intracellular signaling pathways. Nevertheless, it is unclear whether LPLI could promote glucose uptake through activation of PI-3K/Akt/GLUT4 signaling in 3T3L-1 adipocytes. In this study, we investigated how LPLI promoted glucose uptake through activation of PI-3K/Akt/GLUT4 signaling pathway. Here, we showed that GLUT4 was localized to the Golgi apparatus and translocated from cytoplasm to cytomembrane upon LPLI treatment in 3T3L-1 adipocytes, which enhanced glucose uptake. Moreover, we found that glucose uptake was mediated by the PI3-K/Akt2 signaling, but not Akt1 upon LPLI treatment with Akt isoforms gene silence and PI3-K/Akt inhibitors. Collectively, our results indicate that PI3-K/Akt2/GLUT4 signaling act as the key regulators for improvement of glucose uptake under LPLI treatment in 3T3L-1 adipocytes. More importantly, our ̄ndings suggest that activation of PI3-K/Akt2/GLUT4 signaling by LPLI may provide guidance in practical applications for promotion of glucose uptake in insulin-resistant adipose tissue.


Introduction
Insulin resistance is a hallmark in which peripheral tissues fail to properly respond to insulin stimulation, leading to abnormal glucose and lipid metabolism, which contributes to the risk of developing cardiovascular disease and type 2 diabetes. The e®ects of insulin are largely mediated by the activation of phosphatidylinositol3-kinase (PI3)kinase/protein kinase B (PI3-K/Akt) signaling pathway. PI3-K/Akt signaling pathway regulates many physiological processes, including cell proliferation, survival, anti-apoptosis, development and metabolism. 1 In mammals, three highly conserved isoforms of Akt (PKB/Akt1, PKB/Akt2 and PKB/Akt3) encoded by three separate genes have been identi¯ed. All three genes products share a high degree of amino acid identity and seem to be regulated by similar mechanisms. 2 Recent studies have reported that PI3-kinase/Akt signaling pathway regulated glucose metabolism upon insulin exposure. Glucose transporter 4 (GLUT4) is a predominant downstream factor of PI3-k/Akt signaling pathway and responsible for glucose uptake in muscle and adipose tissues. 3 Once GLUT4 was activated by Akt, GLUT4 translocated from cytosol to cytomembrane and promoted glucose absorption. Nevertheless, if this process is disordered, glucose metabolism will be dysregulated and lead to hyperglycemia and insulin resistance.
Low-power laser irradiation (LPLI) has been found to regulate various biological processes in cell and animal models. It has been widely applied in neck pain care, 4 treatment of skeletal muscle regeneration, 5 wound healing 6 and diverse neurological diseases. 7,8 A large body of evidence has shown that LPLI can promote gene transcription, 9 movement 10 and di®erentiation 11 in di®erent cell types, which are determined by activation of diverse signaling pathways, such as mitogen-activated protein kinase/extracellular regulated protein kinase (MAPK/ERK), 5 Src, 12 protein kinase C (PKC) 13 and PI3-K/Akt. 14 Moreover, it has been reported that LPLI can signi¯cantly prevent cell apoptosis induced by A in PC12 cells through activation of Akt/GSK3 signaling. 15 LPLI can e±ciently penetrate into biological tissues including the central nervous system, producing noninvasive bene¯cial photobiomodulation e®ects such as promoting nerve regeneration and increasing ATP synthesis. 16 All the evidence suggests that LPLI regulates di®erent biological processes through diverse signaling pathways in many cell types and ultimately a®ects cell physiological processes.
Although the e®ects of LPLI have been illustrated in vitro and in vivo, the roles of LPLI on GLUT4 activation and glucose uptake in 3T3-L1 adipocytes are poorly understood and the underlying molecular mechanisms are currently unknown. In the present study, we attempted to clarify how LPLI induced GLUT4 translocation and promoted glucose uptake. Based on various techniques and approaches, we found that GLUT4 was localized to the Golgi apparatus and translocated from cytoplasm to cytomembrane upon LPLI treatment in 3T3L-1 adipocytes. We also showed that glucose uptake induced by LPLI was almost completely prevented by PI3-K/Akt inhibitors. In addition, the results of shRNA-medicated knock down of Akt isoforms revealed that glucose uptake was mediated by the PI3-K/Akt2 signaling, but not PI3-K/Akt1 upon LPLI treatment. Collectively, we demonstrate that LPLI promotes glucose uptake through PI3-K/Akt2/GLUT4 signaling pathway. Our¯ndings suggest that LPLI may have the potential therapeutic value in insulin-resistant adipose tissue.

Cell culture and di®erentiation
3T3-L1¯broblast were grown in DMEM (GBICO, Co. Ltd., Grand Island, NY) containing 10% FCS, 50 units/mL penicillin and 50 g/mL streptomycin. The cells were maintained in a humidi¯ed, 37 C incubator with 5% CO 2 and 95% air. For di®erentiation experiments, 3T3-L1 cells were induced 2 days post con°uence and then were incubated in DMEM supplemented with 10% FCS, 500 M 3-isobutyl 1-methylxanthine (IBMX), 0.25 M dexamethasone and 10 g/mL insulin (sigma) for 60 h. Finally, the cells were maintained in the same medium without IBMX and dexamethasone. All experiments were performed on day 8 after di®erentiation.

Cell transfection and LPLI treatment
Transient transfection was carried out using X-tremeGENE HP DNA transfection reagent (Roche Applied Science, Mannheim, Germany) according to the manufacturer's instructions. 3T3-L1 adipocytes were seeded on 22-mm culture glasses or 60-mm plates one day prior to transfection. Cells were maintained in culture medium (Invitrogen) during transfection, and replaced with fresh culture medium 6 h later. About 48 h after transfection, cells were subjected to di®erent treatments. 3T3-L1 adipocytes were serum-starved for at least 6 h in serum-free medium. For irradiation of these cells, the cells were irradiated with a He-Ne laser (632.8 nm, HN-1000; Guangzhou, China) in the dark with the°uence of 5 J/cm 2 . The internal wells were¯lled with ink in order to minimize the scattered or re°ected light. The power intensity was maintained at 10 mW/cm 2 .

GLUT4 translocation assay
3T3-L1 adipocytes were co-transfected with GFP-GLUT4, YFP-TGN38 and RFP-F-actin expression plasmids. About 48 h after transfection, the cells were serum-starved for 6 h and then treated with LPLI (5 J/cm 2 Þ. The dynamic changes of GLUT4 in single living cell were observed by confocal microscopy (LSM510 META; Carl Zeiss Co., Ltd. Jena, Germany). Cells were observed with a 40 x oil objective lens (NA ¼ 1:3). The stage of LSM was equipped with a temperature-controlled and CO 2controlled small incubator (CTI-controller 3700 digital and Tempcontrol 37-2 digital; Zeiss, Jena, Germany), which maintained the cells at 37 C, 5% CO 2 during the whole experiment. GFP or YFP°u orescence was excited with a 458-or 514-nm Ar-Ion laser, and the°uorescence emission was detected through a 465-510 nm or a 520-555 nm band pass¯lter, respectively. RFP°uorescence was excited with a 543-nm laser and the°uorescence emission was detected through a 650-nm long pass¯lter. During the experiments, the exciting power of 458-, 514-, or 543-nm laser was reduced to the minimal level (1-3%) to reduce the possible e®ects of exciting light. Data were analyzed with Zeiss Rel 3.2 image processing software (Zeiss, Germany).

Glucose uptake assay
The glucose consumption in vitro was measured by glucose oxidase method (GOD) using glucose assay kit (Applygen Company, China) according to manufacturer's instructions. 3T3-L1 adipocytes were serum-starved for 6 h and then treated with LPLI (5 J/cm 2 Þ in the presence or absence of PI3-K/Akt inhibitors. After 2 h, glucose uptake in cells was detected with an In¯nite 200 plate reader (TECAN, M€ onnedorf, Switzerland).

Subcellular fractionation
E±cient extraction of transmembrane proteins was extracted with ProteoExtract r Transmembrane Protein Extraction Kit (Novagen) according to manufacturer's protocols. The extraction of transmembrane proteins was used in western blot analysis.

Cell lysates collection
For Western blot analysis, 3T3-L1 adipocytes were washed three times with PBS and then lysed with 200 mL of lysis bu®er (20 mM Tris, pH 7.4, 150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 1 mM sodium orthovanadate, 50 mM sodium°uoride, 1% TritonX-100, 0.1% SDS and 100 mM phenylmethylsulfonyl°u oride). The lysates were collected in microcentrifuge tubes and centrifuged. Protein concentrations were determined using the Bradford method. The lysates were stored at À80 C for Western blot analysis.

Western blot analysis
Western blot analysis was performed as described previously. 22 Brie°y, the extracted proteins were separated in SDS-polyacrylamide gels and transferred to polyvinylidene di°uoride (PVDF) membranes. The membranes were washed three times for 10 min each time with tris bu®ered saline with tween 20 (TBST) and incubated with indicated primary antibodies at 4 C overnight. After incubation, the membranes were labeled with goat antimouse conjugated to IRDyeTM 700 or goat antirabbit conjugated to IRDyeTM 800 secondary antibodies (Rockland Immunochemicals, Gilbertsville, PA). Detection was performed using LI-COR Odyssey Scanning Infrared Fluorescence Imaging System (LI-COR, Inc., Lincoln, NE). Data were analyzed using LI-COR Image Studio Software (LI-COR, Biosciences, Lincoln, NE).

Statistical analysis
We performed each study at least three times under identical conditions. Data were represented as means AE SEM. Statistical analysis was applied using Student's paired t-test. Di®erences were considered statistically signi¯cant at p < 0:05.

Subcellular localization of GLUT4
In order to explore the subcellular localization of GLUT4, GFP-GLUT4, YFP-TGN38 and RFP-F-actin, expression plasmids were co-transfected into 3T3-L1 adipocytes. The subcellular localization of GLUT4 in single living cell was observed with confocal microscopy. As shown in Fig. 1(a), GLUT4 (green emission) was co-localized with trans-Golgi network (TGN38, yellow emission). This result indicates that GLUT4 is localized in the Golgi apparatus in 3T3-L1 adipocytes. Subsequently, the dynamic changes of GLUT4 in single living cell upon insulin (1 nM) exposure were observed, because insulin is a well-known factor which induces GLUT4 translocation. As shown in Fig. 1(b), GLUT4 was predominant in the cytosol under the normal condition, whereas it was translocated to the cytomembrane in response to insulin stimulation.

LPLI induces GLUT4 translocation
Next we investigate whether GLUT4 could be translocated from cytosol to cytomembrane upon LPLI treatment. To this end, GFP-GLUT4, YFP-TGN38 and RFP-F-actin expression plasmids were co-transfected into 3T3-L1 adipocytes and then treated with LPLI. The dynamic changes of GFP-GLUT4 in single living cell were monitored in real time by confocal microscopy. As shown in Fig. 2(a), GFP-GLUT4 was predominantly localized in the cytosol under normal conditions. When cells were stimulated with LPLI, GFP-GLUT4 was translocated from cytosol to cytomembrane within 90 min [see Fig. 2(b)]. This result indicates that LPLI can induce GLUT4 translocation in 3T3-L1 adipocytes.
To further con¯rm the result that GLUT4 translocation was induced by LPLI, we isolated the cell membrane and cytoplasm of 3T3-L1 adipocytes and measured the subcellular localization of GLUT4  When cells were treated with LPLI, GLUT4 mainly existed in the cytomembrane. Taken together, these results demonstrate that GLUT4 can be activated by LPLI.

PI3-K/Akt signaling mediates glucose uptake upon LPLI treatment
We have demonstrated that LPLI can induce GLUT4 translocation in 3T3-L1 adipocytes. However, it is unclear whether LPLI could promote glucose uptake, which is mediated by the PI3-K/Akt/GLUT4 signaling axis. To further investigate whether LPLI could promote glucose uptake through activation of PI3-K/Akt signaling, 3T3-L1 adipocytes were treated with LPLI in the presence or absence of PI3-K or Akt speci¯c inhibitors, respectively. As shown in Fig. 3(a), the phosphorylation levels of Akt were signi¯cantly increased by LPLI, whereas PI3-K/Akt inhibitors completely prevented the activation of Akt induced by LPLI. Subsequently, we measured the glucose uptake under LPLI treatment in the presence or absence of PI3-K or Akt speci¯c inhibitors, respectively. As shown in Fig. 3(b), we found that glucose uptake was dramatically increased by LPLI. In contrast, PI3-K inhibitors (wortmannin and LY294002) almost completely prevented LPLIinduced glucose uptake compared to LPLI treatment only, revealing that PI3-K was indeed involved in LPLI-induced glucose uptake. Consistent with the above results, two Akt speci¯c inhibitors (Akt inhibitor IV and Akt inhibitor V) signi¯cantly reduced LPLI-induced glucose uptake compared to LPLI treatment only [see Fig. 3(c)]. Taken together, these results demonstrate that LPLI induces glucose uptake through activation of PI3-K/Akt signaling axis.

Akt2, but not Akt1 regulates glucose uptake upon LPLI treatment
We next asked if glucose uptake could be regulated by Akt isoform-speci¯c signaling upon LPLI treatment. RNA interference technology was used to knock down the expression levels of Akt1, Akt2 and Akt1/2 in 3T3-L1 adipocytes. As shown in Fig. 4(a), down-regulation of Akt2 speci¯cally reduced LPLI-stimulated glucose uptake, whereas Akt1 knockdown had no e®ect on glucose uptake induced by LPLI. When Akt1 and Akt2 were simultaneously knocked down, glucose uptake induced by LPLI was dramatically reduced, but simultaneous knockdown of both Akt1 and Akt2 did not have a greater inhibitory e®ect than Akt2 knockdown. The expression levels of Akt1 and Akt2 in 3T3-L1 adipocytes were detected by western blot analysis [see Fig. 4(b)]. These results demonstrate that Akt2 regulates glucose uptake in response to LPLI stimulation, but not Akt1.

Conclusion
In this study, we showed a key regulatory mechanism of LPLI for GLUT4 translocation and glucose uptake in 3T3-L1adipocytes. Our major¯ndings found that LPLI induced GLUT4 translocation from cytosol to cytomembrane and resulted in glucose uptake by activating the PI3-K/Akt signaling axis ( Figs. 1 and 2). Further study revealed that promotion of glucose uptake caused by LPLI was controlled by Akt2 isoform-speci¯c signaling axis, but not Akt1 (see Fig. 4). Understanding the molecular mechanisms and functional signi¯cance of LPLIinduced glucose uptake may lead to development of a new approach for diabetes therapy. Akt is considered to be an important downstream target of insulin receptor substrate (IRS)/ PI3-K signaling in regulation of glucose metabolism. 23 However, it remains completely unknown whether LPLI could promote glucose uptake through activation of PI-3K/Akt/GLUT4 signaling axis in 3T3L-1 adipocytes. Our study is designed to evaluate the relevance of Akt isoforms in control of GLUT4 translocation and glucose uptake upon LPLI treatment. In the present study, we found that LPLI not only induced activation of Akt, but also promoted GLUT4 translocation in 3T3L-1 adipocytes, which increased glucose uptake. Knockdown of Akt isoforms by shRNAs revealed that Akt2 mediated LPLI-induced glucose uptake, but not Akt1, which suggested that the functions of Akt isoforms in 3T3L-1 adipocytes were di®erent, though their structures were very similar in mammalian cells. Recent studies suggested that Akt2 was required for glucose metabolism 24 and de¯ciency of Akt2 led to insulin resistance. 25 All the evidence suggests that Akt isoforms (Akt1 and Akt2) exert di®erent functions in mammalian cells, although their regulation mechanisms are similar.
In summary, our data demonstrate that LPLI induced GLUT4 translocation and glucose uptake through PI3-K/Akt2 isoform-speci¯c signaling axis, but not the PI3-K/Akt1. Activated Akt2 promoted downstream signaling pathways activaton that were closely related to regulation of glucose transport. These observations may provide a potentially therapeutic strategy for type 2 diabetes through increasing glucose absorption in the peripheral tissues.