Glycogen synthase kinase 3␤ (GSK3␤) phosphorylates substrates, including the microtubule-associated protein tau, at both primed and unprimed epitopes. GSK3␤ phosphorylation of tau negatively regulates tau-microtubule interactions; however the differential effects of phosphorylation at primed and unprimed epitopes on tau is unknown. To examine the phosphorylation of tau at primed and unprimed epitopes and how this impacts tau function, the R96A mutant of GSK3␤ was used, a mutation that prevents phosphorylation of substrates at primed sites. Both GSK3␤ and GSK3␤-R96A phosphorylated tau efficiently in situ. However, expression of GSK3␤-R96A resulted in significantly less phosphorylation of tau at primed sites compared with GSK3␤. Conversely, GSK3␤-R96A phosphorylated unprimed tau sites to a significantly greater extent than GSK3␤. Prephosphorylating tau with cdk5/p25 impaired the ability of GSK3␤-R96A to phosphorylate tau, whereas GSK3␤-R96A phosphorylated recombinant tau to a significantly greater extent than GSK3␤. Moreover, the amount of tau associated with microtubules was reduced by overexpression of GSK3␤ but only when tau was phosphorylated at primed sites, as phosphorylation of tau by GSK3␤-R96A did not negatively regulate the association of tau with microtubules. These results demonstrate that GSK3␤-mediated phosphorylation of tau at primed sites plays a more significant role in regulating the interaction of tau with microtubules than phosphorylation at unprimed epitopes.

Site‐specific phosphorylation of tau negatively regulates its ability to bind and stabilize microtubule structure. Although tau is a substrate of glycogen synthase kinase 3β (GSK3β), the exact sites on tau that are phosphorylated by this kinase in situ have not yet been established, and the effect of these phosphorylation events on tau–microtubule interactions have not been fully elucidated. GSK3β phosphorylates both primed and unprimed sites on tau, but only primed phosphorylation events significantly decrease the ability of tau to bind microtubules. The focus of the present study is on determining the importance of the GSK3β‐mediated phosphorylation of a specific primed site, Thr231, in regulating tau's function. Pre‐phosphorylation of Ser235 primes tau for phosphorylation by GSK3β at Thr231. Phosphorylation by GSK3β of wild‐type tau or tau with Ser235 mutated to Ala decreases tau–microtubule interactions. However, when Thr231 alone or Thr231 and Ser235 in tau were mutated to Ala, phosphorylation by GSK3β did not decrease the association of tau with the cytoskeleton. Further, T231A tau was still able to efficiently bind microtubules after phosphorylation by GSK3β. Expression of each tau construct alone increased tubulin acetylation, a marker of microtubule stability. However, when cells were cotransfected with wild‐type tau and GSK3β, the level of tubulin acetylation was decreased to vector‐transfected levels. In contrast, coexpression of GSK3β with mutated tau (T231A/S235A) did not significantly decrease the levels of acetylated tubulin. These results strongly indicate that phosphorylation of Thr231 in tau by GSK3β plays a critical role in regulating tau's ability to bind and stabilize microtubules.

Tau is a substrate of caspases, and caspase-cleaved tau has been detected in Alzheimer's disease brain but not in control brain. Furthermore, in vitro studies have revealed that caspase-cleaved tau is more fibrillogenic than full-length tau. Considering these previous findings, the purpose of this study was to determine how the caspase cleavage of tau affected tau function and aggregation in a cell model system. The effects of glycogen synthase kinase 3␤ (GSK3␤), a well established tau kinase, on these processes also were examined. Tau or tau that had been truncated at Asp-421 to mimic caspase cleavage (Tau-D421) was transfected into cells with or without GSK3␤, and phosphorylation, microtubule binding, and tau aggregation were examined. Tau-D421 was not as efficiently phosphorylated by GSK3␤ as fulllength tau. Tau-D421 efficiently bound microtubules, and in contrast to the full-length tau, co-expression with GSK3␤ did not result in a reduction in the ability of Tau-D421 to bind microtubules. In the absence of GSK3␤, neither Tau-D421 nor full-length tau formed Sarkosyl-insoluble inclusions. However, in the presence of GSK3␤, Tau-D421, but not full-length tau, was present in the Sarkosyl-insoluble fraction and formed thioflavin-Spositive inclusions in the cell. Nonetheless, co-expression of GSK3␤ and Tau-D421 did not result in an enhancement of cell death. These data suggest that a combination of phosphorylation events and caspase activation contribute to the tau oligomerization process in Alzheimer's disease, with GSK3␤-mediated tau phosphorylation preceding caspase cleavage.

BackgroundObesity is a health hazard that is associated with a number of diseases and metabolic abnormalities, such as type-2 diabetes, hypertension, dyslipidemia, and coronary heart disease. In the current study, we investigated the effects of Citrus aurantium flavonoids (CAF) on the inhibition of adipogenesis and adipocyte differentiation in 3T3-L1 cells.MethodsDuring adipocyte differentiation, 3T3-L1 cells were treated with 0, 10, and 50 μg/ml CAF, and then the mRNA and protein expression of adipogenesis-related genes was assayed. We examined the effect of CAF on level of phosphorylated Akt in 3T3-L1 cells treated with CAF at various concentrations during adipocyte differentiation.ResultsThe insulin-induced expression of C/EBPβ and PPARγ mRNA and protein were significantly down-regulated in a dose-dependent manner following CAF treatment. CAF also dramatically decreased the expression of C/EBPα, which is essential for the acquisition of insulin sensitivity by adipocytes. Moreover, the expression of the aP2 and FAS genes, which are involved in lipid metabolism, decreased dramatically upon treatment with CAF. Interestingly, CAF diminished the insulin-stimulated serine phosphorylation of Akt (Ser473) and GSK3β (Ser9), which may reduce glucose uptake in response to insulin and lipid accumulation. Furthermore, CAF not only inhibited triglyceride accumulation during adipogenesis but also contributed to the lipolysis of adipocytes.ConclusionsIn the present study, we demonstrate that CAF suppressed adipogenesis in 3T3-L1 adipocytes. Our results indicated that CAF down-regulates the expression of C/EBPβ and subsequently inhibits the activation of PPARγ and C/EBPα. The anti-adipogenic activity of CAF was mediated by the inhibition of Akt activation and GSK3β phosphorylation, which induced the down-regulation of lipid accumulation and lipid metabolizing genes, ultimately inhibiting adipocyte differentiation.

This study examined the anti-obesity effect and mechanism of action of blueberry peel extracts (BPE) in 3T3-L1 cells and high-fat diet (HFD)-induced obese rats. The levels of lipid accumulation were measured, along with the changes in the expression of genes and proteins associated with adipocyte differentiation in 3T3-L1 cells. Evidenced by Oil-red O staining and triglyceride assay, BPE dose-dependently inhibited lipid accumulation at concentrations of 0, 50, and 200 µg/ml. BPE decreased the expression of the key adipocyte differentiation regulator C/EBPβ, as well as the C/EBPα and PPARγ genes, during the differentiation of preadipocytes into adipocytes. Moreover, BPE down-regulated adipocyte-specific genes such as aP2 and FAS compared with control adipocytes. The specific mechanism mediating the effects of BP revealed that insulin-stimulated phosphorylation of Akt was strongly decreased, and its downstream substrate, phospho-GSK3β, was downregulated by BPE treatment in 3T3-L1 cells. Together, these data indicated that BP exerted anti-adipogenic activity by inhibiting the expression of PPARγ and C/EBPβ and the Akt signaling pathway in 3T3-L1 adipocytes. Next, we investigated whether BP extracts attenuated HFD-induced obesity in rats. Oral administration of BPE reduced HFD-induced body weight gain significantly without affecting food intake. The epididymal or perirenal adipose tissue weights were lower in rats on an HFD plus BPE compared with the tissue weights of HFD-induced obese rats. Total cholesterol and triglyceride levels in the rats fed BPE were modestly reduced, and the HDL-cholesterol level was significantly increased in HFD plus BP-fed rats compared with those of HFD-fed rats. Taken together, these results demonstrated an inhibitory effect of BP on adipogenesis through the down-regulation of C/EBPβ, C/EBPα, and PPARγ and the reduction of the phospho-Akt adipogenic factor in 3T3-L1 cells. Moreover, BPE reduced body weight gain and inhibited fat accumulation in an HFD-induced animal model of obesity.