Mechanisms Of Inhibited Lipolytic Response To Isoproterenol And High Glucose By Metformin In Primary Rat Adipocytes

Objective: To investigate the inhibitory effects of metformin on isoproterenol or high-concentration glucose induced lipolysis in primary rat adipocytes and further elucidate the underlying mechanisms, for better understanding of the improvement of insulin sensitivity by metformin.Methods: Adipocytes were isolated from epididymal fat pads of Sprague-Dawley (SD) rats. After isolation and digestion, packed adipocytes were incubated in the presence or absence of excess glucose or/and metformin at the concentrations as planed, washed and then treated for 30 min after the addition of isoproterenol or not. Glycerol released into the media was determined by use of a colorimetric assay and served as an index of lipolysis. The expressions of perilipin and its phosphorylation state, phosphorylated-extracellular signal-related kinase (p-ERK1/2) and total ERK1, hormone-sensitive lipase (HSL) and adipose triglyceride lipase (ATGL) were examined by Western blot. Adipose lipases activity was assayed by using an enzymatic assay. Cyclic adenosine monophosphate (cAMP) level and protein kinase A (PKA) activity were examined by immunoradioassay using 125I and 32P respectively. HSL translocation was detected by Western blot and immunofluorescence. Data were statistically analyzed by GraphPad Prism version 4.0. Results: 1. Metformin inhibits isoproterenol-stimulated lipolysis in primary adipocytes. Isoproterenol at 1μmol/L induced a 3.8-fold (P < 0.001) increase in glycerol production over the 30 min control. The antilipolytic action of metformin at 500μmol/L was more effective than that at 250μmol/L. They inhibited the isoproterenol-stimulated lipolysis by 56.7%(P <0.001)and 43.6%(P <0.01)respectively. The time course study depicted that the inhibition began as early as 4 h after the addition of 500μmol/L metformin and lasted to 24 h incubation. The addition of isoproterenol elevated intracellular cAMP level and PKA activity by 1.5-fold (P<0.05)and 3.0-fold(P <0.01)approximately, while metformin caused a 50.3%(P <0.001)and 70.5% (P <0.001)inhibition respectively. Metformin greatly inhibited the double augment of isoproterenol-mediated ERK1/2 phosphorylation by 54.7%(P <0.001), and also reduced the increment of perilipin phosphorylation caused by isoproterenol. Isoproterenol enhanced the total lipolytic lipases activity to round 200% (P <0.01) over that in control, while treatment with metformin reduced the lipases activity by 44.5% (P <0.01). In addition, metformin had no effect on the expression of HSL and ATGL as well as HSL translocation. 2. Metformin restricts the chronic lipolytic action upon high-concentration glucose. High-concentration glucose stimulated lipolysis; glucose at 25 mmol/L elevated glycerol release by 1.72-fold(P <0.001), and such lipolytic response occurred apparently at 16 h and remained high after 24 h incubation, whereas glucose at 10 mmol/L did not produce stimulative effect on basal lipolysis. Additionally, in the presence of insulin, the incubation of adipocytes with high-concentration glucose further enhanced the lipolytic response upon adrenergic stimulation of isoproterenol, while insulin did not affect the intrinsic lipolytic action of high-concentration glucose. Metformin at 500μmol/L showed more potent antilipolytic effect than that at 250μmol/L. They could reduce high glucose-induced lipolysis by 69.1%(P <0.001)and 31.8%(P <0.001)separately, meanwhile, metformin at 500μmol/L markedly inhibited basal and promoted lipolysis induced by isoproterenol in the combination of high-concentration glucose and insulin. High-concentration glucose increased the phosphorylation of perilipin, however metformin significantly attenuated it. Neither high glucose nor metformin altered the expression of perilipin. Exposure of the adipocytes to a medium with high glucose led to an increase by a factor of 1.59-fold (P <0.05) in adipose lipases activity, whereas pretreatment with metformin caused a reduction of 35.8% (P <0.05). Both high glucose and metformin upregulated the expression of HSL with cooperative effect in combination, though neither of them affected the expression of ATGL. Conclusion: Metformin reduces lipolysis in primary rat adipocytes stimulated by isoproterenol or high-concentration glucose. It also restrains the lipolytic stimulation of isoproterenol enhanced by the combination of high-concentration glucose and insulin. Metformin attenuates isoproterenol-mediated lipolysis by inhibiting the elevation of cellular cAMP level and PKA activity, suppressing phosphorylation of ERK1/2 and perilipin and also abrogating the increased activity of adipose lipases. Metformin restricts high glucose-induced lipolysis through eliminating perilipin phosphorylation and decreasing the augment of lipolytic lipases activity. This study provides novel evidences that metformin reduces plasma free fatty acid (FFA) through antagonizing isoproterenol- and high glucose-induced lipolysis in adipocytes that leads to the improvement of insulin sensitivity.