Specific Desensitization of Glycogen Synthase Activation by Insulin in 3T3-L1 Adipocytes

Abstract

A protocol was developed in 3T3-L1 adipocytes that resulted in the specific desensitization of glycogen synthase activation by insulin. Cells were pretreated for 15 min with 100 nM insulin, and then recovered for 1.5 h in the absence of hormone. Subsequent basal and insulininduced phosphorylation of the insulin receptor, IRS-1, MAPK, Akt kinase, and GSK-3 were similar in control and pretreated cells. Additionally, enhanced glucose transport and incorporation into lipid in response to insulin were unaffected. However, pretreatment reduced insulin-stimulated glycogen synthesis by over 50%, due to a nearly complete inhibition of glycogen synthase activation. Removal of extracellular glucose during the recovery period blocked the increase in glycogen levels, and restored insulin-induced glycogen synthase activation. Furthermore, incubation of pretreated 3T3-L1 adipocytes with glycogenolytic agents reversed the desensitization event. Separation of cellular lysates on sucrose gradients revealed that glycogen synthase was primarily located in the dense pellet fraction, with lesser amounts in the lighter fractions. Insulin induced glycogen synthase translocation from the lighter to the denser glycogen-containing fractions. Interestingly, insulin preferentially activated translocated enzyme while having little effect on the majority of glycogen synthase activity in the pellet fraction. In insulin-pretreated cells, glycogen synthase did not return to the lighter fractions during recovery, and thus did not move in response to the second insulin exposure. These results suggest that, in 3T3-L1 adipocytes, the translocation of glycogen synthase may be an important step in the regulation of glycogen synthesis by insulin. Furthermore, intracellular glycogen levels can regulate glycogen synthase activation, potentially through modulation of enzymatic localization.

Inhibition of glycogen synthesis by increased lipid availability is associated with subcellular redistribution of glycogen synthase

“…The insulin-dependent translocation of glycogen synthase which we have observed in muscle of chow-fed rats and in control cells agrees with previous studies (Brady et al 1999, Jensen et al 2000, and in this case the enzyme is probably associated either with cytoskeletal elements (Garcia-Rocha et al 2001) or with glycogen itself (Brady et al 1999, Nielsen et al 2001). An insulin-dependent association with glycogen is supported by the fact that the TIF contained the largest amount of total glycogen, and that glycogen synthesized in response to the hormone was mostly recovered in this fraction.…”

“…Our observations are thus similar to those made with adipocytes (Jensen et al 2000), in which insulin reduced a minor pool of glycogen synthase present in lighter fractions derived from sucrose gradients, and promoted its recovery in a denser fraction. Insulin pretreatment, which was associated with diminished glycogen synthesis upon subsequent insulin stimulation, caused prior partitioning of the enzyme to the denser fraction.…”

“…Insulin pretreatment, which was associated with diminished glycogen synthesis upon subsequent insulin stimulation, caused prior partitioning of the enzyme to the denser fraction. It was concluded that a minor component of glycogen synthase was responsible for most of the newly synthesized glycogen in adipocytes, and that correct basal localization was required for activation of the enzyme by insulin (Jensen et al 2000). Our data support a similar situation in muscle, in that the cytosolic component may contribute most to glycogen synthesis, and furthermore suggest that lipid oversupply, like insulin pretreatment, can lead to a sequestration of the enzyme that contributes to a reduction in subsequent, insulin-stimulated glycogen synthesis.…”

“…However, there have been no studies of the possible link between lipid-induced insulin resistance and a change in the subcellular localization of glycogen synthase, although the inhibition of glycogen synthesis by insulin pretreatment has been associated with prior translocation of a minor pool of glycogen synthase (Jensen et al 2000). The aim of the present study was therefore to examine the potential role of glycogen synthase compartmentalization in the adverse effects of lipids on glycogen synthesis.…”

Structural Origins of the Insulin-mimetic Activity of Bis(acetylacetonato)oxovanadium(IV)

“…Prior to insulin stimulation or treatment with VO 2ϩ compounds, cells were washed twice with phosphate-buffered isotonic saline at 37°C and serum-starved for 2.5 h at 37°C in 1 ml/well KRBH with 5 mM glucose and 25 mM HEPES, pH 7.4. The basal and insulin-stimulated rates of glucose transport in adipocytes treated in this manner were identical to cells serum-starved in KRBH/glucose plus 0.5% BSA and were similar to previous results when Dulbecco's modified Eagle's medium plus 0.5% fetal bovine serum was used (22). The serum starvation medium was then removed, the cells were washed two times with phosphate-buffered isotonic saline (37°C), and the adipocytes were placed in 0.5 ml/well KRBH containing insulin, the VO 2ϩ compound desired (accordingly in the absence or presence of BSA), or no insulin-mimetic compound.…”