Bandyopadhyay Gautam, Standaert Mary L, Sajan Mini P, Kanoh Yoshinori, Miura Atsushi, Braun Uschi, Kruse Friederike, Leitges Michael, Farese Robert V
James A Haley Veterans Hospital and University of South Florida College of Medicine, Tampa, 33612, USA.
Mol Endocrinol. 2004 Feb;18(2):373-83. doi: 10.1210/me.2003-0087. Epub 2003 Nov 13.
Atypical protein kinase C (aPKC) isoforms have been suggested to mediate insulin effects on glucose transport in adipocytes and other cells. To more rigorously test this hypothesis, we generated mouse embryonic stem (ES) cells and ES-derived adipocytes in which both aPKC-lambda alleles were knocked out by recombinant methods. Insulin activated PKC-lambda and stimulated glucose transport in wild-type (WT) PKC-lambda(+/+), but not in knockout PKC-lambda(-/-), ES cells. However, insulin-stimulated glucose transport was rescued by expression of WT PKC-lambda in PKC-lambda(-/-) ES cells. Surprisingly, insulin-induced increases in both PKC-lambda activity and glucose transport were dependent on activation of proline-rich tyrosine protein kinase 2, the ERK pathway, and phospholipase D (PLD) but were independent of phosphatidylinositol 3-kinase (PI3K) in PKC-lambda(+/+) ES cells. Interestingly, this dependency was completely reversed after differentiation of ES cells to adipocytes, i.e. insulin effects on PKC-lambda and glucose transport were dependent on PI3K, rather than proline-rich tyrosine protein kinase 2/ERK/PLD. As in ES cells, insulin effects on glucose transport were absent in PKC-lambda(-/-) adipocytes but were rescued by expression of WT PKC-lambda in these adipocytes. Our findings suggest that insulin activates aPKCs and glucose transport in ES cells by a newly recognized PI3K-independent ERK/PLD-dependent pathway and provide a compelling line of evidence suggesting that aPKCs are required for insulin-stimulated glucose transport, regardless of whether aPKCs are activated by PI3K-dependent or PI3K-independent mechanisms.
非典型蛋白激酶C（aPKC）亚型被认为可介导胰岛素对脂肪细胞和其他细胞中葡萄糖转运的作用。为了更严格地验证这一假说，我们通过重组方法构建了敲除aPKC-λ两个等位基因的小鼠胚胎干细胞（ES细胞）和ES细胞来源的脂肪细胞。胰岛素可激活野生型（WT）PKC-λ（+/+）ES细胞中的PKC-λ并刺激葡萄糖转运，但在敲除型PKC-λ（-/-）ES细胞中则无此作用。然而，在PKC-λ（-/-）ES细胞中表达WT PKC-λ可挽救胰岛素刺激的葡萄糖转运。令人惊讶的是在PKC-λ（+/+）ES细胞中，胰岛素诱导的PKC-λ活性和葡萄糖转运增加依赖于富含脯氨酸的酪氨酸蛋白激酶2、ERK途径和磷脂酶D（PLD）的激活，但不依赖于磷脂酰肌醇3激酶（PI3K）。有趣的是，ES细胞分化为脂肪细胞后这种依赖性完全逆转，即胰岛素对PKC-λ和葡萄糖转运的作用依赖于PI3K，而非富含脯氨酸的酪氨酸蛋白激酶2/ERK/PLD。与ES细胞一样，胰岛素对PKC-λ（-/-）脂肪细胞的葡萄糖转运无作用，但在这些脂肪细胞中表达WT PKC-λ可挽救此作用。我们的研究结果表明，胰岛素通过一种新发现的不依赖PI3K、依赖ERK/PLD的途径激活ES细胞中的aPKC和葡萄糖转运，并提供了令人信服的证据表明，无论aPKC是通过依赖PI3K还是不依赖PI3K的机制被激活，aPKC都是胰岛素刺激葡萄糖转运所必需的。
