3T3L1 (Mouse embryonic fibroblast), C2C12 (Mouse myoblasts cell line) and HepG2 (human liver hepatocytes) cell lines are from ATCC. Antibodies against phospho-AMPKα, AMPKα, phospho-AKT, AKT, phospho-HSL, phospho-mTOR, phospho-JNK, p-eIF2a and β-Actin are from Cell Signaling. Anti-HNF4α antibody is from BioVision. Glucose estimation kit and free glycerol estimation kit from Sigma.
Accu-check glucometer from Roche Diagnostics (Germany) and Ultra-sensitive insulin ELISA kit from Crystal Chem Inc (USA). Triglyceride and Cholesterol estimation reagents are from Diagnostic Systems (Germany). FFA estimation kit from Randox (UK) and Glycerol estimation kits from Sigma. Triton-X and Fluromount were procured from Sigma-Aldrich.
C2C12 cells were seeded in 24-well plates (3 × 104 cells/well) at 37°C in DMEM containing 25 mM Glucose and 10% FBS. When the cells were confluent, the media was supplemented with 25 mM glucose and 2% FBS for myotubes formation. After 4 days of differentiation and myotubes formation, cells were used for the different treatments.
3 T3-L1 cells were cultured in DMEM supplemented with 10% bovine calf serum (Hyclone, USA) and 25 mM glucose in 96 or 24-well tissue culture plates. To induce differentiation, the culture media was supplemented with 100 nM insulin, 1 μM dexamethasone, and 500 μM isobutylmethylxanthine. Media was changed every 2 days with fresh media containing 100 nM insulin until day 5. The differentiation continued for further 3 days in media without insulin.
HepG2 cells were maintained in MEM media with 10% FBS. Cells were seeded in appropriate culture plates two days prior to the treatment.
Tissue distribution and AMPK activation studies
Swiss albino mice (10 wk, 30 ± 3 g) were orally dosed with CNX-012-570 (10 mg/kg body wt,) once-daily for 3 days (n = 5). After 3 h of final dose, animals were killed by cervical dislocation. 100 mg of different tissues like liver, adipose and skeletal muscle were collected and homogenized for the analysis of AMPK activation and its downstream target engagement studies using specific antibodies by Western blotting.
Efficacy studies in disease models
DIO mice study
Sixteen week old male C57BL/6 J mice were fed on either high fat diet (HFD) (D12492; 60% kcal from fat; Research Diets, Inc., New Jersey, USA) or chow diet (10% kcal from fat) for 11 weeks. After acclimatization period, animals were selected for the study. Animals were housed in polypropylene cages, maintained at 23 ± 1°C, 60 ± 10% humidity, exposed to 12 h cycles of light and dark and provided ad libitum access to either chow or HFD and water throughout the acclimatization and experimental period. Animal experiment protocols and experimental procedures were approved by the Connexios Institutional Animal Ethics Committee which are in accordance with the ARRIVE guidelines .
DIO animals were assigned to specific treatment groups based on body weight, glucose AUC during OGTT, fasting blood glucose and fasting serum TG levels. DIO animals were randomized into control and CNX-012-570 treatment groups (3 mg/kg, orally once a day). Animals (n = 8) fed on normal chow diet were served as lean control. DIO animals in the treatment groups (n = 8) and HFD control group (n = 8) were fed with HFD throughout the experimental period. Animals in the treatment group received CNX-012-570, orally once a day as solution in 10% dimethyl acetamide and 8% cremophor as vehicle for 8 weeks. Lean control and HFD control animals were received vehicle orally once a day. Body weight (weekly) and feed consumption (daily) were recorded. Blood was collected from tail vein for glucose and triglyceride estimation. OGTT was performed on the 8th week of treatment after 6 h fasting with 2 g/kg of oral glucose challenge. After 8 weeks of treatment blood was collected from retro-orbital bleeding for glycerol, free fatty acid, cholesterol, LDL-C estimation. Animals were then euthanized and necropsied; liver was excised immediately, weighed and taken for estimation of triglyceride. Different adipose depots were separated and weighed.
db/db mice study
Six week old male db/db mice from Jackson Lab were acclimatized for one week and randomized to either vehicle control or CNX-012-570 (2.5 mg/kg, orally once a day) treatment groups (n = 8) based on the body weight, fed glucose and fasting glucose. Age matched db/+ animals served as lean control. Treatment group animals were administered CNX-012-570, orally once a day as solution in 10% dimethyl acetamide and 8% cremophor as vehicle for 6 weeks. Lean control and db/db controls animals received vehicle orally once a day. Body weight, fed glucose, fasting glucose (tail snip) were monitored weekly. At the end of the treatment HbA1c (Siemens DCA Vantage system kit) and insulin levels (Downers Grove, USA) were measured. Animals were sacrificed and liver, adipose depots, muscle and serum were collected.
Fasting and fed glucose
Blood samples were collected by the tail nipping method after 6 h fasting and glucose levels were measured using Accu-check glucometer (Roche diagnostics) once a week during the entire duration of study. Fed state blood samples were collected as mentioned above and glucose levels were measured at every 2 h time intervals upto 16 h and then followed by 4 h intervals upto 24 h.
Oral glucose tolerance test
OGTT was determined after oral glucose load at a dose of 2 g/kg body weight by oral gavage. Blood glucose was measured by tail clip method at intervals of 0, 15, 30, 60, 90 and 120 min using Accu-check glucometer (Roche diagnostics).
Fed and fasting insulin
Fed state and 6 h fasted blood samples were collected by the retro-orbital puncture and insulin levels were estimated using Crystal chem kit (ELISA). Homeostasis model assessment of insulin resistance (HOMA-IR) was employed to assess the status of insulin action.
Blood samples collected at end of the study termination and total high molecular weight adiponectin levels were measured using adiponectin ELISA kit from ALPCO diagnostics according to the manufacturer’s instruction.
HepG2 cells, C2C12 myotubes and 3T3L1 adipocytes were treated with serum free media containing 0.3 μM of CNX-012-570 and monitored AMPK activation time course for 24 h (0.25, 2,4,6,8,12,16 and 24 h). At each time point, cells were harvested and lysed in lysis buffer. 50 μg of the lysate were separated on 12% SDS-PAGE and transferred to nitrocellulose membrane and probed with primary antibody against phospho-AMPKα and total AMPKα. Signals were developed by enhanced chemiluminescence (West Pico, Thermo Scientific, USA).
For ex vivo analysis of protein markers, after the CNX-012-570 treatment period (Swiss albino mice, DIO mice and db/db mice), mice were killed by cervical dislocation. 100 mg of tissue from different organs were collected (gastronomes muscle, adipose and liver) in lysis buffer. Lysates were prepared by homogenization and 50 μg of the lysate from each treatment was used for the Western blot analysis for different protein markers as mentioned above.
Measurement of thermogenesis
Mice were housed individually and transferred to a cold environment with an ambient temperature of 4°C. Rectal temperature was measured for every 15 min for a total of 75 min and animals were then brought to room temperature. The temperature was measured further for 20 min at 10 min intervals.
Estimation of total cholesterol, LDL-C, glycerol and FFA in serum
Blood was collected from retro orbital under isoflurane anesthesia was allowed to clot for 30 min at room temperature followed by centrifugation for 10 minutes at 4°C and serum was collected for analysis. Serum total cholesterol was measured using fully automated clinical chemistry analyzer EM360, (Transasia Bio-medicals Ltd) with ERBA Kits. LDL-C and glycerol were estimated by colorimetric analysis as per manufacturer’s instruction.
Estimation of liver TG and cholesterol
Tissue TG was extracted according to Folch’s method. Briefly, lipids were extracted with chloroform: methanol (2:1) mixture, the organic layer was separated and dried in a speed vac. The residue was re-suspended in isopropyl alcohol and assayed for TG and cholesterol levels by using TG and cholesterol quantification kit respectively (Diagnostic systems, Germany).
Quantitative PCR analysis
After the study termination, 100 mg of liver tissue was collected from each animal across treatment groups in TRIZOL (Sigma, USA). RNA was isolated and later converted to cDNA as per the standard protocol. Relative mRNA levels of SREBP1c (fwd: 5′-AGCAGCCCCTAGAACAAACAC-3′; rev: 5′-CAGCAGTGAGTCTGCCTTGAT-3′), MCP-1 (fwd: 5′- AGCACCAGCCAACTCTCACT-3′; rev: 5′-TCATTGGGATCATCTTGCTG-3′) and APOB100 (fwd: 5′- AAGCACCCCAAGTGTCACAA-3′; rev: 5′- ATTTGTACTGCAGGGCGTCA-3′) were analyzed using SYBR green chemistry.
Formalin-fixed, paraffin-embedded tissue sections from liver and inguinal adipose depot were sectioned at 4 μm and stained with hematoxylin and eosin (H&E) stain. Histopathological examination was carried out in a blinded fashion using Carl Zeiss Axio Scope A1 microscope. The images were captured at magnification of ×400 using Prog Res C3 camera attached to the microscope.
Formalin-fixed, paraffin-embedded tissue sections from inguinal adipose depot were sectioned at 4 μm and stained with H&E for adipose morphometry. From each section 10 different randomly selected microscopic fields at ×400 magnification were used for evaluation. Adipose morphometry was carried out using ProgRes Pro, v.2.8.8 image analysis suite. The mean area of adipocytes from all the groups were statistically analyzed using Graphpad Prism, v.5.0.
Immunofluorescence and image analysis
For UCP1 immunostaining, subcutaneous adipose sections were de-paraffinized and blocked in 1% BSA in PBS for 30 min at RT and incubated with anti-UCP1 (Abcam, USA) primary antibody for 90 min at RT, followed by washing with PBS. Sections were then incubated with secondary antibody (goat anti-rabbit IgG Alexa Fluor 555, Molecular Probes, Invitrogen) for 30 min at RT and mounted using fluromount. Localization of UCP1 was assessed and the images were captured at ×400 magnification. All adipose sections were viewed at ×400 magnification, and images were captured using Zeiss microscope connected via camera to a computer (progres® capture pro 2.1 camera). Adipocytes size measurement was performed using a computer-assisted image analysis progres® capture pro software. From each animal 10 images were captured.
All the values are expressed as mean ± SEM; Students unpaired t test was used for comparing cell based assay results. One way analysis of variance was performed followed by Dunnets test for establishing level of significance between treatment and control animals. p < 0.05 was considered as significant.