Involvement of Glucagon in Preventive Effect of Menthol Against High Fat Diet Induced Obesity in Mice

Abstract

Glucagon mediated mechanisms have been shown to play clinically significant role in energy expenditure. The present study was designed to understand whether pharmacological mimicking of cold using menthol (TRPM8 modulator) can induce glucagon-mediated energy expenditure to prevent weight gain and related complications. Acute oral and topical administration of TRPM8 agonists (menthol and icilin) increased serum glucagon concentration which was prevented by pre-treatment with AMTB, a TRPM8 blocker. Chronic administration of menthol (50 and 100 mg/kg/day for 12 weeks) to HFD fed animals prevented weight gain, insulin resistance, adipose tissue hypertrophy and triacylglycerol deposition in liver. These effects were not restricted to oral administration, but also observed upon the topical application of menthol (10% w/v). The metabolic alterations caused by menthol in liver and adipose tissue mirrored the known effects of glucagon, such as increased glycogenolysis and gluconeogenesis in the liver, and enhanced thermogenic activity of white and brown adipose tissue. Correlation analysis suggests a strong correlation between glucagon dependent changes and energy expenditure markers. Interestingly, in-vitro treatment of the serum of menthol treated mice increased energy expenditure markers in mature 3T3L1 adipocytes, which was prevented in the presence of non-competitive glucagon receptor antagonist, L-168,049, indicating that menthol-induced increase in serum glucagon is responsible for increase in energy expenditure phenotype. In conclusion, the present work provides evidence that glucagon plays an important role in the preventive effect of menthol against HFD-induced weight gain and related complications.

Bioavailable Menthol (Transient Receptor Potential Melastatin-8 Agonist) Induces Energy Expending Phenotype in Differentiating Adipocytes

“…Oral administration of menthol was given at the dose of 200 mg/Kg in 0.1% tween-80, while topical administration of menthol was given on abdominal area by 1 mL topical application of 10% menthol in 0.1% tween 80. Animals were euthanized after 30, 60 and 120 min of menthol administration by cervical dislocation, blood was collected by cardiac puncture for serum isolation and subcutaneous white adipose tissues were collected for pharmacokinetic profiling of menthol [14].…”

“…Cells from control and test samples (1, 50 and 200 µM administration of menthol during differentiation) were pellet down and digested with nitric acid using Microwave Reaction System (Mars 6, CEM Corporation, Matthews, NC, USA). The concentration of different elements was estimated in the digested samples using standard protocol for inductively coupled plasma mass spectrometry (ICP-MS:77006, Agilent Technologies, Santa Clara, CA, USA) [14].…”

“…Quantitative real time-polymerase chain reaction (qRT-PCR) was performed with the cDNA as templates in 40 cycles using Custom RT 2 PCR Array Kit (CLAM 30615C, Qiagen Lifesciences, Waltham, MA, USA) and RT 2 SYBR Green ROX qPCR Mastermix (Qiagen Lifesciences) keeping GAPDH as internal control and 7 gene specific primers for adipogenesis including TNFRSF9, PPARGC1A, PRDM16, CIDEA, TLE3, HOXC10 and NRIP1 . The qRT-PCR process was performed by programming thermocycler (CFX96 Touch Real-Time PCR Detection System (Bio-Rad Laboratories Inc., Hercules, CA, USA)) to apply initial cycle for denaturation at 95 °C for 2 min, followed by 40 cycles of annealing and elongation at 60 °C for 30 s and denaturation at 95 °C for 5 s. The complete experiment was performed using three biological replicates [14]. Analysis of relative gene expression was done using 2 −ΔΔct method [24] and values were expressed in the terms of fold change with reference to control [14].…”

“…The qRT-PCR process was performed by programming thermocycler (CFX96 Touch Real-Time PCR Detection System (Bio-Rad Laboratories Inc., Hercules, CA, USA)) to apply initial cycle for denaturation at 95 °C for 2 min, followed by 40 cycles of annealing and elongation at 60 °C for 30 s and denaturation at 95 °C for 5 s. The complete experiment was performed using three biological replicates [14]. Analysis of relative gene expression was done using 2 −ΔΔct method [24] and values were expressed in the terms of fold change with reference to control [14].…”

“…Involvement of TRPM8 dependent and independent pathways in mediating cold-induced energy expenditure is well reported. Recently we have published that anti-obesity effect of menthol, a TRPM8 agonist is mediated through glucagon dependent mechanisms particularly in adipose tissues [14]. Activation of TRPM8 receptors in the gut and skin by oral and topical administration of menthol leads to increase in serum glucagon levels, thus activating several downstream catabolic processes like glycogenolysis, gluconeogenesis, browning of white adipose tissue (WAT) and activation of energy expenditure markers in WAT and brown adipose tissue (BAT) [14].…”