Abstract
The current literature on lipolysis in murine primary adipocytes is rife with experiments performed under conditions not optimized for reproducible and reliable results. Here, we present conditions for optimizing the measurement of lipolysis in murine adipocytes. We demonstrate that adenosine management is of paramount importance in evaluating the lipolytic response under basal and stimulated conditions. Also, adipocyte concentrations in the 10,000-15,000 cells per milliliter range produce a greater increase in stimulated lipolysis than higher concentrations, and the response is further enhanced by agitating the cells.
Highlights
The current literature on lipolysis in murine primary adipocytes is rife with experiments performed under conditions not optimized for reproducible and reliable results
An increasing number of papers have appeared on the behavior of isolated murine adipocytes, and once again, the literature reveals a high level of variability in the magnitude of stimulation achieved with badrenergic receptor agonists or with other stimulants that increase cAMP concentrations and PKA activity
The optimal concentration of adenosine used for adipocyte isolation and washes was based on the finding that mouse adipocytes were z10-fold less sensitive compared with rat adipocytes
Summary
All reagents were obtained from Sigma-Aldrich (St. Louis, MO) and were prepared and manipulated in an Adipocyte Incubation Solution (AIS) unless indicated otherwise. Manuscript received 7 February 2006 and in revised form 23 March 2006 and in re-revised form 18 April 2006 The cells were isolated in AIS fortified with 500 nM adenosine and 3 mg/ml type 1 collagenase (Worthington Biomedical Corp., Lakewood, NJ). The optimal concentration of adenosine used for adipocyte isolation and washes was based on the finding that mouse adipocytes were z10-fold less sensitive compared with rat adipocytes Because of lot-to-lot variation in collagenase activity, the optimal concentration of collagenase may range from 1 to 3 mg/ml; we determined that 3 mg/ml collagenase stock was optimal for the digestion of adipose tissue samples in this study
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