Effect of activating and inactivating mutations of Gs- and Gi2-alpha protein subunits on growth and differentiation of 3T3-L1 preadipocytes

J Cell Biochem. 1997 Feb;64(2):242-57. doi: 10.1002/(sici)1097-4644(199702)64:2<242::aid-jcb8>3.0.co;2-x.

Abstract

Previous investigations have demonstrated that both Gs- and the Gi-family of GTP-binding proteins are implicated in differentiation of the 3T3-L1 preadipocyte. In order to further analyze the role of Gs alpha vs. Gi2 alpha, which are both involved in adenylate cyclase modulation, we transfected undifferentiated 3T3-L1 cells with two sets of G-protein cDNA: the pZEM vector with either wild type, the activating (i.e., GTP-ase inhibiting) R201C-Gs alpha or the inactivating G226A(H21a)-Gs alpha point mutations, or the pZIPNeoSV(X) retroviral vector constructs containing the Gi2 alpha wild type or the missense mutations R179E-Gi2 alpha, Q205L-Gi2 alpha, and G204A(H21a)-Gi2 alpha. The activating [R201C]Gs alpha-mutant did not significantly affect the differentiation process, i.e., increase in the steady-state levels of G-protein subunits, gross appearance, or insulin-elicited deoxy-glucose uptake into 3T3-L1 adipocytes, despite a marked initial increase in hormone-elicited adenylate cyclase activity. The [H21a]Gs alpha-mutant, on the other hand, enhanced the degree of differentiation slightly, as evidenced by an augmented production of lipid vesicles and insulin-stimulated deoxy-glucose uptake. However, an expected increase in mRNA for hormone-sensitive lipase was not seen. Secondly, it appeared that both activating [R179E]Gi2 alpha or [Q205L]Gi2 alpha mutants reduced cell doubling time in non-confluent 3T3-L1 cell cultures, while [H21a]Gi2 alpha slowed proliferation rate. Furthermore, it seemed that cell proliferation, as evidenced by thymidine incorporation, ceased at a much earlier stage prior to cell confluency when cultures were transfected with the [R179E]Gi2 alpha or [Q205L]Gi2 alpha mutants. Upon differentiation with insulin, dexamethasone, and iBuMeXan, the following cell characteristics emerged: the [R179E]Gi2 alpha and [Q205L]Gi2 alpha mutants consistently enhanced adenylate cyclase activation and cAMP accumulation stimulated by isoproterenol and corticotropin over controls. Deoxy-glucose uptake was also super-activated by the [R179E]Gi2 alpha and [Q205L]Gi2 alpha mutants. Finally, steady-state levels of hormone sensitive lipase mRNA were dramatically increased by [R179E]Gi2 alpha and [Q205L]Gi2 alpha over differentiated controls. The inactivating [H21a]Gi2 alpha-mutant obliterated all signs of preadipocyte differentiation. It is concluded that Gi2 plays a positive and much more important role than Gs in 3T3-L1 preadipocyte differentiation. Cyclic AMP appears to play no role in this process.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 3T3 Cells
  • Adenylyl Cyclases / metabolism
  • Adipocytes / cytology*
  • Adipocytes / enzymology
  • Animals
  • Cell Differentiation / genetics*
  • Cell Division / genetics*
  • Cyclic AMP / metabolism
  • Deoxyglucose / metabolism
  • Enzyme Activation
  • GTP-Binding Proteins / genetics
  • GTP-Binding Proteins / metabolism*
  • Mice
  • Mutagenesis, Site-Directed
  • Rats
  • Sterol Esterase / genetics
  • Thymidine / metabolism
  • Transcription, Genetic
  • Transfection

Substances

  • Deoxyglucose
  • Cyclic AMP
  • Sterol Esterase
  • GTP-Binding Proteins
  • Adenylyl Cyclases
  • Thymidine