AICAR was purchased from Toronto Chemicals. Antibodies against phosphospecific as well as nonphospho-AMPKα and ACC were from Cell Signaling (Beverly, MA). Antibodies against AMPK α1 and α2 were from Abcam (Cambridge, MA). Antibodies against C/EBPα, C/EBPβ, SREBP1, PPARγ, β actin and proteinA/G plus agarose were from Santa Cruz Biotechnology (Santa Cruz, CA). [³H]Thymidine was from PerkinElmer Life Sciences (Boston, MA). 5-iodotubercidin was purchased from Calbiochem (San Diego, CA).

3T3-L1 preadipocytes were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10 % (v/v) NBS. AICAR was dissolved in DMSO for cell culture studies (0.5 M, stock). On day 0 (2 day after 3T3-L1 preadipocytes reached confluence), cells were induced to differentiate by insulin (1.7 μmol/L), IBMX (0.5 mmol/L), and DEX (1 μmol/L). On day 3 and every other 3^rd day thereafter, fresh regular medium was substituted until day 9 19. Differentiation was monitored by morphological assessment and Oil red O staining 20. For Oil red O staining, cells were washed twice with PBS, fixed in 3.7 % formaldehyde for 1 h, and stained for 30 min with 0.2 % (w/v) oil red O solution in 60 % (v/v) isopropanol. Cells were then washed several times with water, and excess water was evaporated by placing the stained cultures at approximately 32°C. In order to determine the extent of adipose conversion, 0.2 ml of isopropanol was added to the stained culture dish. The extracted dye was immediately removed by gentle pipetting and its optical density was monitored spectrophotometrically at 510 nm. For cell proliferation, 3T3L1 cell were kept in differentiating media in the presence of AICAR/iodotubercidin (0.5 mM/0.1 μM) for three days. [³H] thymidine (1 μCi/well) was added at the end of the treatment for 6 h and mean incorporation of thymidine in DNA was measured by a 1450 Microbeta Wallac Trilux Liquid Scintillation Counter (Perkin-Elmer Life Sciences) as described before 26.

Male C57BL/6 mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Mice were fed a high-fat diet (60% kcal% fat) (Research Diets, New Brunswick, NJ) ad libitum for 6 weeks. At 1, 2, 4 and 6 weeks, animals were sacrificed and various tissues harvested, weighed, and processed for subsequent analysis. All procedures were performed in accordance with the experimental guidelines for animal care and use at the Medical University of South Carolina (Charleston, SC, USA).

To study the effect of AICAR, five groups of mice were allocated into group 1: mice fed with standard diet (STD); group 2: STD mice treated with AICAR (0.5 mg/g bw); group 3: mice fed high fat diet (DIO); group 4: DIO mice treated with AICAR (0.1 mg/g bw); group 5: DIO mice treated with AICAR (0.5 mg/g bw). The mice were treated with AICAR (0.1 or 0.5 mg/g body weight, dissolved in PBS, i.p. injection volume of 200 μl/mouse) three times a week for 6 weeks (Monday, Wednesday and Friday). Mice in the control group received 200 μl of PBS.

Freshly isolated epididymal white adipose tissue from treated and untreated mice was fixed in 10% formalin and embedded in paraffin. Sections (8 μm) were stained with hematoxylin and eosin, and cell morphology and size were analyzed.

After extraction of total RNA from white adipose tissue using of TRIzol (Invitrogen) as per the manufacturer's protocol, total RNA (5 μg) was used to prepare cDNA using iScript cDNA Synthesis Kit (BioRad Laboratories). Real-time PCR was performed as described previously utilizing Bio-Rad iCycler (iCycler iQ Multi-Color Real-Time PCR Detection System) using iQ SYBR Green Supermix reagent as per the instructions from BioRad Laboratories. The primer sets for use were designed (Oligoperfect Designer, Invitrogen) and synthesized from Integrated DNA Technologies. The primer sequences for PPARγ, SREBP1, C/EBPα, aP2, Adipsin and 18S taken from Le Lay et al 27. Primer sequences are follows: DGAT1 for, 5'-TTCCGCCTCTGGGCATT-3'; rev, 5'-AGAATCGGCCCACAATCCA-3'; GPAT for, 5'-ATTGACACCTGCTGCTTTTGA-3'; rev, 5'-CCTACCCACTACAACAGAG-3'. Thermal cycling conditions were as follows: activation of DNA polymerase at 95°C for 10 min, followed by 40 cycles of amplification at 95°C for 30 sec and at 58.3°C for 30 sec. The normalized expression of target gene with respect to 18S was computed for all samples by using the Microsoft Excel data spreadsheet.

AMPKα1 activity was assayed in isolated epididymal white adipose tissue from different groups using anti-AMPKα1 antibody for immunoprecipitation. For this, isolated epididymal white adipose tissues were homogenized in lysis buffer (50 mM Tris-HCl, pH 7.4, containing 50 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 10% glycerol, 1% triton X-100 and protease inhibitor mixture). Approximately 200 μg of tissue homogenate was incubated with anti-AMPKα1 antibody for 2 hr, then 30 μl of protein A/G plus agarose was added and incubated for an additional 1 hr at 4°C. The immune complexes were washed twice in lysis buffer and twice in kinase buffer (62.5 mM HEPES, pH 7.0, 62.5 mM NaCl, 62.5 mM NaF, 6.25 mM sodium pyrophosphate, 1.25 mM EDTA, 1.25 mM EGTA, and 1 mM dithiothreitol), incubated at 30°C in 30 μl of kinase assay buffer containing 200 μM AMP, ATP mixture (200 μM ATP and 1.5 μCi of [γ-³²P]ATP), with or without 250 μM SAMS peptide (HMR

SAMS

Lipids were extracted by the Folch method in a mixture of 2:1 chloroform/methanol (vol/vol). The extract was washed with 0.2 volumes of saline (NaCl 0.9%) and centrifuged at 2,000 rpm for 10 min. The organic phase was recovered and serum triglycerides, cholesterol and free fatty acids (FFAs) were analyzed using HPTLC as described earlier 29. Fatty acid composition of adipose tissue was determined using GC17A and capillary column as described 30.

Blood glucose was measured with a glucometer (AccuChek; Roche Diagnostics, Indianapolis, IN). Serum adiponectin and leptin concentrations were determined using a mouse adiponectin and mouse leptin enzyme-linked immunosorbent assay kits (BioVendor Laboratory Medicine, Inc.), respectively.

A glucose tolerance test was performed on mice fasted for 12 h. Blood glucose levels were determined at 0, 15, 30, 60, and 120 min after an intraperitoneal injection of glucose (2 g/kg body wt). For the insulin tolerance test, animals fasted for 5 h were injected intraperitoneally with 0.5 units insulin/kg body wt (sigma) and glucose levels were measured at 0, 15, 30, 60, and 120 min postinjection.

Results shown represent means ± SD. Statistical analysis was performed by ANOVA by the Student-Neumann-Keuls test using GraphPad InStat software (San Diego, CA).

We investigated the effect of AICAR on the induction of terminal differentiation markers at end of the differentiation period (day 9). Oil Red O staining showed that untreated differentiated cells had many lipid droplets indicating lipid accumulation. However, lipid accumulation was inhibited by AICAR treatment in a dose dependent manner (Fig. 1a). This observation was further supported with the quantitative analysis of neutral lipid content by measuring the absorbance at 510 nM (Fig. 1b). Untreated and AICAR treated (0.1 mM) differentiated 3T3L1 cells showed higher levels of lipid staining with the Oil Red O dye, which was significantly reduced by higher doses of AICAR (0.25–1.0 mM) treatment (Fig. 1b).

To examine further the effect of AICAR on cell proliferation, undifferentiated 3T3L1 cells were treated with increasing concentrations of AICAR (0.1–1.0 mM) in proliferative media for 3 days and cell proliferation was examined using [H3] thymidine uptake. AICAR at low concentrations (0.1 and 0.25 mM) did not have any effect on 3T3L1 cell proliferation but at higher concentrations (0.5–1 mM) it completely blocked cell proliferation (Fig. 2).

Adipogenesis is a highly regulated process requiring coordinated expression and activation of key transcription factors which include CCAAT/enhancer binding proteins (C/EBPs), peroxisome proliferators activated receptor gamma (PPARγ), and sterol regulatory element-binding proteins (SREBPs). To identify the molecular mechanism for the inhibition of adipocyte differentiation by AICAR treatment, the protein expression of key transcriptional factors for adipocyte differentiation was examined (Fig. 3a). The levels of C/EBPα protein expression in 3T3L1 during differentiation increased at day 6 and 9, which was inhibited by AICAR treatment in a dose dependent manner (Fig. 3a). However, the expression of C/EBPβ levels was unaffected by AICAR treatment. The expression of PPARγ and SREBP-1 was also significantly inhibited by AICAR treatment at 0.5 to 1 mM concentration (Fig. 3a). To support further these observations, the mRNA levels of these transcription factors were examined by quantitative analysis using real time PCR. As depicted in figure 3b, the mRNA expression of C/EBPα, PPARγ and SREBP-1 was significantly increased with the progression of adipocyte differentiation. AICAR treatment at the concentration of 0.5 mM significantly blocked the expression of these key adipogenic transcription factors (Fig. 3b), further confirming that AICAR inhibits adipocyte differentiation by inhibiting expression of C/EBPα, PPARγ and SREBP-1. Modulation of the expression of other adipocyte-specific markers including Pref-1, aP2 and adipsin, further supported these observations (Fig. 3b). Pref-1 is a marker for preadipocytes and inhibits adipocyte differentiation 31, whereas, aP2 and adipsin are markers for differentiated adipocytes 3233. The expression of Pref-1 was significantly reduced with increase in adipocyte differentiation; however, its expression was restored with AICAR treatment (Fig. 3b). AICAR treatment also reduced the expression of aP2 and adipsin mRNA as compared to untreated differentiated cells (Fig. 3). Taken together, AICAR inhibited the expression of key transcription factors (C/EBPα, PPARγ and SREBP-1), adipocyte specific markers, thereby blocking the adipocyte differentiation.

We were further interested in examining the role of ZMP (a phosphorylated form of AICAR, which mimics AMP to activate AMPK) in the regulation of AICAR mediated inhibition of adipocyte differentiation. 3T3L1 cells were treated with AICAR (0.5 mM) on different days (from 0, 3 and 6^th day) in the presence or absence of iodotubercidin. Iodotubercidin is an inhibitor of adenosine kinase, and inhibits the conversion of AICAR to its activated form ZMP inside the cell, and thus inhibits activation of AMPK by AICAR 2834. Treatment with AICAR at any stage on either day 0 (proliferation phase), day 3 (differentiation phase) or day 6 (late differentiation phase), significantly blocked the accumulation of neutral lipid content as evident from Oil Red staining (Fig. 4a). Addition of iodotubercidin together with AICAR at different stages (day 0, 3^rd or 6th) completely abolished the AICAR mediated inhibition of neutral lipid content accumulation (Fig. 4a), while iodotubercidin treatment alone had no effect on adipocyte differentiation. These results were further confirmed by examining for total lipid staining where differentiated 3T3L1 cells have high levels of lipids, which was completely abolished by AICAR treatment (Fig. 4b). Addition of iodotubercidin significantly reversed AICAR mediated inhibition in total lipid content (Fig. 4b). Moreover, iodotubercidin also reversed the AICAR induced inhibition of cell proliferation in 3T3L1 cells as well as the expression of SREBP1 and PPARg (Fig. 4c, 4d), further suggesting the involvement of ZMP in AICAR mediated inhibition of adipocyte differentiation of 3T3L1.

Since ZMP is known to mimic AMP and activates AMPK, the effect of AICAR on the phosphorylation status of AMPK and ACC (acetyl CoA carboxylase) at various time points was examined. As shown in figure 5a, the adipocyte differentiation progressed with time; the phosphorylation status of ACC and AMPK also increased, suggesting that activation of AMPK occurs during adipocyte differentiation. Treatment with AICAR further significantly increased the phospho-status of ACC and AMPK in a dose dependent manner, suggesting that AICAR induce the activation of AMPK (Fig. 5).

To investigate whether the activation of AMPK is important or not for adipocyte differentiation, cells were treated with iodotubercidin together with AICAR for 9 days. Chronic treatment with AICAR reduced the levels of phospho-status of ACC, which was restored by iodotubercidin treatment (Fig. 5b).

To assess whether AICAR can affect adipocyte formation in vivo, we used the

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Since AICAR administration restricted body weight as a consequence of reduction in fat content of DIO mice, it was of interest to examine whether AICAR treatment controlled the adipose size or restricted the number of cells. The size of adipocytes were ascertained by microscopic analysis and it was observed that DIO vehicle treated mice had a significant increase in adipocyte size, which was significantly restricted in AICAR treated mice at the dose 0.5 mg/g bw while there was no effect at 0.1 mg/g bw (Fig. 7a). On the other hand, no change in the total DNA content in the adipose tissue of the AICAR treated and vehicle treated DIO mice was observed, indicating that cell number was maintained in both cases (Fig. 7b). This suggested that the decrease in epididymal adipose tissue in AICAR treated DIO mice as compared to DIO vehicle treated mice was not due to a decrease in cell number.

To examine further whether the decrease in epididymal fat content was due to an increase in the adipocyte differentiation process, the expression levels for several adipocyte markers were examined by immunoblot analysis. As shown in Fig. 8a, the decrease in epididymal fat content observed in AICAR treated mice (0.5 mg/g bw) was not reflected in the change in the expression of adipocyte marker genes such as C/EBPα, C/EBPβ, PPARγ, and SREBP1. However, we observed a significant increase in the expression of DGAT and mitochondrial GPAT, two enzymes involved in the synthesis of triglycerides, by AICAR treatment in STD mice as well as in DIO treated mice (data not shown).

Biologically active molecules secreted from adipose tissue called adipokines, have been implicated in the development of obesity. The levels of leptin are significantly increased with the increase in adipose tissue content in DIO vehicle treated mice. However, AICAR (0.5 mg/g bw) treatment for 6 weeks brought these back to the normal levels on par with STD mice (Fig. 9a). In contrast to leptin levels, adiponectin levels were drastically reduced in DIO vehicle mice, which also normalized with AICAR treatment (Fig. 9b). Moreover, DIO mice exhibited pronounced dyslipidemia compared to STD mice, as evident from the elevated levels of triglyceride (TAG), cholesterol and free fatty acids (FFA), which were significantly reduced by chronic treatment of AICAR (0.5 mg/g bw) (Fig. 9c). Furthermore, diet-induced increase in saturated fatty acids (16:0, 18:0) and decrease in major polyunsaturated fatty acid (18:2) in adipose tissue were also normalized by chronic AICAR treatment (Table 1).

Lipid accumulation and alterations in fatty acid composition in various tissues as a result of obesity is considered to be a cause of development of insulin resistance. Therefore, we examined the glucose tolerance test and insulin tolerance test in DIO-vehicle and AICAR treated mice after 6 weeks of treatment. As depicted from figure 10a &10b, DIO-vehicle and AICAR treatment at a dose of 0.1 mg/g bw showed hyperglycemia and hyperisulinemia, however, AICAR treated DIO mice at the dose of 0.5 mg/g bw completely ameliorated hyperglycemia and hyperisulinemia induced by the high fat diet. We also determined the TGA and FFA content in liver of DIO vehicle and AICAR treated DIO (0.5 mg/g bw) mice and observed that DIO vehicle treated mice exhibited a significant increase in TGA and FFA levels, which were significantly inhibited by AICAR treatment (data not shown). This indicates that the differences in glucose tolerance and insulin sensitivity observed between DIO and AICAR treated DIO (0.5 mg/g bw) mice is due to a decrease in lipid storage in adipose tissue and liver in AICAR treated DIO mice (0.5 mg/g bw), which probably prevented the further impairment of glucose homeostasis. Since lipid accumulation in tissues other than adipose tissue, such as liver, is considered to be a cause of the development of insulin resistance associated with obesity.

We examined further the possibility of activation of AMPK in adipose tissue by chronic treatment with AICAR. For this, we examined the phospho-status of AMPKα and ACC as well as the total level of AMPKα1 in adipose tissue after 6 weeks of AICAR treatment in STD and DIO mice. AICAR treatment at both doses induced the phosphorylation of AMPKα as compared to STD and DIO vehicle treated mice (Fig. 11a). However, the phospho-status of ACC was only increased in the AICAR treated STD mice, and did not show any change in DIO-vehicle treated, as well as in AICAR treated DIO mice. No change was observed in the total level of AMPKα1 in any treatment in STD and DIO mice. However, a significant increase in the enzymatic activity of AMPKα1 was observed in AICAR treated STD, in DIO-vehicle treated and AICAR treated (0.1 mg/g bw) adipose tissue (Figure 11b). AICAR treated adipose tissue at a dose of 0.5 mg/g bw also showed a significant increase in AMPKα1 activity but the increase in its activity was not significantly higher than that observed in other group.

Proliferator-activated receptor γ coactivator 1α (PGC-1α) is a coactivator of nuclear receptors and other transcription factors 35. PGC-1α controls mitochondrial biogenesis and oxidative metabolism in many tissues, including brown adipose tissue, skeletal muscle, heart, and liver 36373839. We examined the effect of AICAR on DIO vehicle treated mice and observed that AICAR treatment of STD mice significantly induced protein expression as compared to that in untreated STD mice (Fig. 12a). The expression of PGC1α was found to be significantly down in adipose tissue in DIO vehicle and AICAR (0.1 mg/g bw) treated mice, however AICAR at the dose of 0.5 mg/g bw significantly reversed the protein expression of PGC1α induced in DIO as documented by immunoblot and its densitometry analysis (Figure 12ai &12aii). These observations were further supported by quantitative PCR for PGC1α in adipose tissue from AICAR treated STD and DIO mice (Fig. 12b). To examine the effect of AICAR on the expression of PGC1α, 3T3L1 cells were treated with increasing concentrations of AICAR (0.1–1.0 mM) under differentiating condition and the expression of PGC1 α was examined at 3, 6 and 9 days using immunoblot analysis. As depicted from figure 12c, treatment of AICAR induced the protein expression of PGC1α at day 3 in dose dependent manner, however, higher concentration of AICAR (0.5 and 1 mM) induced the highest expression at day 3 after that there was a decrease in its expression.