Reduction of O-GlcNAc protein modification does not prevent insulin resistance in 3T3-L1 adipocytes

Abstract

3T3-L1 adipocytes develop insulin-resistant glucose transport upon preincubation with high (25 mM) glucose, provided that insulin (0.6 nM) is included, Akt activation is impaired, and high glucose and insulin act synergistically. Considerable evidence suggests that increased glucose flux via the hexosamine biosynthesis pathway enhances the O-GlcNAc modification (O-GlcNAcylation) of some critical protein(s) that may contribute to insulin resistance. However, whether enhanced protein O-GlcNAcylation is necessary for the development of insulin resistance is unknown. We used two strategies to test this hypothesis. The first strategy was the overexpression of O-GlcNAcase, which removes O-GlcNAc from Ser/Thr of proteins. Cells were infected with O-GlcNAcase-expressing adenovirus (or empty virus) 5 days before they were submitted to protocols that elicit (or not) insulin resistance. O-GlcNAcase was highly expressed and functional as assessed by Western blot, O-GlcNAcase assay, and marked reduction of O-GlcNAcylated proteins. The activity was mainly cytosolic. The second strategy was the expression of O-GlcNAc transferase (OGT) being markedly reduced by transfection of OGT siRNA, resulting in an approximately 90% decrease of nuclear and cytosolic OGT protein expression and similar reduction in O-GlcNAcylated proteins. Nontargeting siRNA had no effect. Preincubation in high glucose with low-dose insulin decreased the acute insulin response of glucose transport by at least 50% and impaired Akt activation. None of these parameters were affected by overexpression of O-GlcNAcase or by OGT knockout. Excess O-GlcNAcylation is one of many factors that can cause insulin resistance. It does not seem to be required for the development of glucose/insulin-induced insulin resistance of glucose transport and Akt activation in 3T3-L1 adipocytes.

Fig. 1

O-GlcNAcase amount and activity are markedly increased in cells infected with O-GlcNAcase virus. A: O-GlcNAcase activity was measured in postnuclear supernatants of cell lysates infected with the adenovirus expressing the enzyme or cells infected with empty virus using a method modified from Gao et al. (9), as described in MATERIALS AND METHODS. Means + SE are shown; n = 16 observations from 4 separate experiments. B: Western blots prepared from postnuclear supernatants of cell lysates developed with an antibody (110.6, Pierce) that specifically recognizes O-GlcNAc-modified proteins. 3T3-L1 adipocytes, which had been incubated for 18 h with 25 mM glucose plus 0.6 nM insulin, show clearly increased O-GlcNAcylation of several protein bands if infected with empty virus. However, total O-linked N-acetylglucosamine (O-GlcNAc) modification is diminished in cells overexpressing OGlcNAcase. C: the same cells developed with another O-GlcNAc-specific antibody, RL-2 (Affinity Bioreagents). O-GlcNAcylation is reduced in cells overex-pressing O-GlcNAcase. D: Western blot developed with an anti-O-GlcNAcase antibody of postnuclear supernatants prepared from cells preincubated in high glucose plus 0.6 nM insulin and infected with empty virus (lane 1) or with O-GlcNAcase virus (lane 2).

Fig. 2

Overexpression of O-GlcNAcase does not affect the development of insulin resistance in cells preexposed to high glucose plus low-dose insulin. 3T3-L1 adipocytes were infected with adenovirus expressing O-GlcNacase or with empty virus 5 days before experiments. More than 90% of cells expressed adenovirus, and 95% were fully differentiated at the time of the experiment. Cells were preincubated for 18 h with either 5 mM glucose or 25 mM glucose plus low-dose insulin (6 × 10⁻¹¹ to 6 × 10⁻¹⁰ M), serum and insulin deprived for 2 h, and then either not stimulated (basal) or stimulated for 15 min with insulin at a half-maximally or maximally stimulating dose. Glucose transport was then measured for 3 min [as 2-deoxyglucose (2-DOG) transport]. Means + SE are shown; n = 4–8. *Significantly decreased from corresponding observation in cells pretreated with 5 mM glucose, P < 0.03–0.001. There are no differences between cells treated with O-GlcNAcase or empty virus.

Fig. 3

O-GlcNAcase overexpression fails to restore the impaired activation of Akt by acute insulin stimulation in cells preincubated in high glucose plus low-dose insulin. Cells were treated as described in Fig. 1. Postnuclear supernatants were prepared from detergent-treated cell lysates in the presence of phosphatase and protease inhibitors. Proteins were separated by SDS-PAGE and immunoblotted with a p-Akt antibody that recognizes pSer⁴⁷³. A: representative Western blot. Basal Akt phosphorylation was too low to quantify. The bands shown are from cells that were acutely stimulated with insulin (10⁻⁷M). B: results from 4 experiments. Data are normalized to Akt phosphorylation after acute insulin stimulation in cells preincubated in 5 mM glucose and infected with empty virus. Means + SE are shown; n = 4. *Significantly different from corresponding cells preincubated in 5 mM glucose. The expression of O-GlcNAcase had no significant effect.

Fig. 4

Nuclear extracts show little evidence of increased O-GlcNAcase activity in cells infected with virus expressing the enzyme. Nuclear extracts were prepared as described in MATERIALS AND METHODS. The infranatant below the lipid layer overlying the nuclear pellet was designated cytosol. A: O-GlcNAcase activity measurements (as in Fig. 1A) in nuclear extracts and cytosol. Although the cytosol shows marked increases in O-GlcNAcase activity in cells infected with virus expressing the enzyme, there is no evidence of this in the nuclear extracts; n = 3 experiments. *Different from cells expressing empty virus, P < 0.01–0.001. B: Western blots of nuclear extracts or cytosol developed with an antibody (110.6 from Pierce) that specifically recognizes O-GlcNAc modified proteins. O-G, O-GlcNAcase-expressing virus. In cells infected with empty virus, there is a trend supporting increased glycosylation of proteins in cells preexposed to high glucose plus insulin. Although in the cytosol there is a marked decline in protein glycosylation in cells infected with O-GlcNAc-expressing virus, this trend is barely discernible in the nucleus (compare lanes 2 and 4). The gel is representative of 3 similar experiments.

Fig. 5

Knockdown of O-GlcNAc transferase (OGT) does not prevent glucose/insulin-induced insulin resistance. In the experiments shown here, OGT knockdown was carried out using the “Smartpool Reagent”(Dharmacon), which consists of 4 unique siRNAs against mouse OGT; as nontargeting controls, we used Dharmacon’s nontargeting siRNA pool of 4. Cells were transfected using a transfection reagent obtained from Genospectra, as described in MATERIALS AND METHODS, 48 h prior to initiating experiments and then cells were placed into conditions for 18 h, which will (or will not) cause insulin resistance. Thus cells were processed ~72 h after transfection. A: expression of OGT protein assessed by Western blots in nuclei and cytosol of cells that were incubated for 18 h in 25 mM glucose plus 0.6 nM insulin. NA, no RNA addition, although the cells underwent all the manipulations of the transfection protocol; NT, transfected with nontargeting siRNA; OGT, transfected with siRNA against OGT. Top of the graph shows a representative Western blot, whereas the bar graph (bottom) illustrates means + SE of 4 observations. *OGT < NA or NT, P < 0.001. B: representative Western blot of nuclear extracts and cytosol developed with the anti-O-GlcNAc antibody. The cells were transfected and preincubated under exactly the same conditions as in A, and the gel is representative of 4 similar experiments. Clearly, OGT knockdown markedly reduced protein O-GlcNAcylation both in the nucleus and in the cytosol. C: comparison of basal and maximally insulin-stimulated (0.1 uM) glucose transport between cells preincubated in 5 mM glucose, with cells preincubated in 25 mM glucose plus 0.6 nM insulin. High glucose/insulin caused insulin resistance, P < 0.01 vs. corresponding 5 mM glucose-treated cells. OGT knockdown had no effect; n = 4. D: same experimental design as C, except that insulin-stimulated Akt activation is studied, which is measured as the phosphorylation of Akt on Ser⁴⁷³. Basal phosphorylation of Akt in this system is barely detectable and is not shown. Cells preincubated in high glucose plus low insulin demonstrated impaired insulin-stimulated Akt activation, P < 0.01. OGT knockdown had no effect; n = 4.