Insulin-Mediated Phosphorylation of the Proline-Rich Akt Substrate PRAS40 Is Impaired in Insulin Target Tissues of High-Fat Diet–Fed Rats

Abstract

Clinical insulin resistance is associated with decreased activation of phosphatidylinositol 3-kinase (PI3K) and its downstream substrate protein kinase B (PKB)/Akt. However, its physiological protein substrates remain poorly characterized. In the present study, the effect of in vivo insulin action on phosphorylation of the PKB/Akt substrate 40 (PRAS40) was examined. In rat and mice, insulin stimulated PRAS40-Thr246 phosphorylation in skeletal and cardiac muscle, the liver, and adipose tissue in vivo. Physiological hyperinsulinemia increased PRAS40-Thr246 phosphorylation in human skeletal muscle biopsies. In cultured cell lines, insulin-mediated PRAS40 phosphorylation was prevented by the PI3K inhibitors wortmannin and LY294002. Immunohistochemical and immunofluorescence studies showed that phosphorylated PRAS40 is predominantly localized to the nucleus. Finally, in rats fed a high-fat diet (HFD), phosphorylation of PRAS40 was markedly reduced compared with low-fat diet-fed animals in all tissues examined. In conclusion, the current study identifies PRAS40 as a physiological target of in vivo insulin action. Phosphorylation of PRAS40 is increased by insulin in human, rat, and mouse insulin target tissues. In rats, this response is reduced under conditions of HFD-induced insulin resistance. Diabetes 55:3221-3228, 2006 I nsulin resistance and type 2 diabetes are associated with impaired insulin action in peripheral tissues like skeletal muscle, adipose tissue, liver, and the heart (1). Insulin action is initiated by binding of insulin to its receptor, leading to activation and phosphorylation of the receptor tyrosine kinase, which in turn phosphorylates several endogenous substrates, including the insulin receptor substrate proteins (1). Tyrosine phosphorylation of the insulin receptor substrate proteins facilitates the binding and activation of phosphatidylinositol 3Ј-kinase (PI3K), thus catalyzing the formation of phosphatidylinositol 3,4,5-trisphosphate and providing a platform for the binding and activation of protein kinase B (PKB)/Akt (1). Numerous studies have linked PKB/Akt to the regulation of glucose metabolism, cell growth, and antiapoptosis (2,3). However, the endogenous substrates regulating these responses are only starting to become characterized (4).The PKB/Akt protein kinase phosphorylates proteins on serine (S/Ser) or threonine (T/Thr) residues within a RxRxxpS/pT motif (5). The use of phospho-specific antibodies recognizing this PKB/Akt consensus sequence led to the identification of multiple novel proteins, including proline-rich Akt substrate 40 (PRAS40; also known as Akt1 substrate 1 [Akt1S1]) (6,7). PRAS40 is ubiquitously expressed and appears to be localized to the nucleus (7,8). In response to growth factors, PRAS40 is phosphorylated on Thr246 via PI3K-and PKB/Akt-dependent signaling pathways (6,8). In vitro, phosphorylation of PRAS40 facilitates the binding of 14-3-3 proteins, and this protein complex has been implicated in nerve growth factor-mediated neuroprotection from ischemia (8). Al...

Denervation and High-Fat Diet Reduce Insulin Signaling in T-Tubules in Skeletal Muscle of Living Mice

“…Based on biochemical measurements, some have argued that defects in insulin signaling in muscle are late events seen after 30 but not after 8 weeks of high-fat diet (7). In contrast, other studies have reported reduced insulin-stimulated PI3-K and Akt/ protein kinase B activities after only 4 weeks of high-fat diet (5,37) and reduced Akt/protein kinase B signaling after 7 weeks of high-fat diet (38). Our study, for the first time, shows that 12 weeks of high-fat diet reduce PI3-K activity in the entire length of the T-tubules and, to a lesser extent, in sarcolemma (Figs.…”

“…Previous biochemical studies have found either normal (4,5) or reduced (6,37,39) overall GLUT4 content in muscle from high fat-fed animals, while GLUT4 translocation was reduced (4,5,7). One study using subcellular fractionation found completely abrogated insulin-stimulated GLUT4 translocation to both sarcolemma and T-tubules (38).…”

Inhibition of c-jun N terminal kinase (JNK) improves functional beta cell mass in human islets and leads to AKT and glycogen synthase kinase-3 (GSK-3) phosphorylation

“…Since the integrity of the insulin signalling cascade in beta cells is responsible for preservation of beta cell mass and for insulin secretion [43], and since JNK inhibition affects IRS-1/AKT in peripheral tissues [13][14][15], we tested the effect of L-JNKI in human islet insulin pathway with our main focus on AKT and its substrates. Among those substrates, GSK-3B and p70S6K participate in the regeneration of beta cell mass via increased mitogenesis [44,45], while PRAS40 is also activated by insulin in peripheral tissues and is implicated in protection of neurons against ischaemic injury [46,47]. We found that JNK inhibition in human islets treated with either L-JNKI or SP600125 specifically stimulated the phosphorylation of AKT and GSK-3B (Fig.…”

Knockdown of PRAS40 inhibits insulin action via proteasome-mediated degradation of IRS1 in primary human skeletal muscle cells

“…Disturbances in the insulin-mediated activation of the IRS1/PI3K/Akt pathway, which facilitates GLUT4-dependent glucose disposal in skeletal muscle, characterise insulin resistance [12]. The impaired activity of the IRS1/PI3K/Akt pathway also results in reduced insulinmediated phosphorylation of one of the most prominent Akt substrates, the proline-rich Akt substrate of 40 kDa (PRAS40), in skeletal muscle of humans with type 2 diabetes and rodents fed a high-fat diet [13][14][15]. PRAS40 is a component of mTORC1 [16,17].…”