mRNA of the Pro-Apoptotic Gene BBC3 Serves as a Molecular Marker for TNF-α-induced Islet Damage in Humans

Diabetologia. 2011 Aug;54(8):2056-66. doi: 10.1007/s00125-011-2183-8. Epub 2011 May 13.

Abstract

Aims/hypothesis: TNF-α plays important roles in the pathogenesis of type 1 and type 2 diabetes mellitus. In light of this, we examined the involvement of a pro-apoptotic gene, BBC3 (also known as PUMA), in TNF-α-mediated beta cell dysfunction and destruction in human islets.

Methods: Human islets were exposed in vitro to TNF-α alone or in combination with IFN-γ. Gene expression was assessed by RT-PCR using a set of single islets. Protein abundance and cellular localisation of BBC3 were assessed by immunoblot and immunohistochemistry. A marginal number of islets were transplanted into diabetic NODscid mice to correlate in vivo islet function with BBC3 expression.

Results: BBC3 and IL8 mRNA were upregulated in TNF-α-stimulated islets in a dose-dependent manner and enhanced through addition of IFN-γ, but not upregulated by IFN-γ alone. Immunohistochemistry revealed that TNF-α in combination with IFN-γ upregulated basal BBC3 abundance in the cytoplasm of beta cells along with the perinuclear clustering of mitochondria partially co-localised with BBC3. TNF-α alone did not induce beta cell death, but did abrogate preproinsulin precursor mRNA synthesis in response to high glucose stimulation, which was inversely associated with upregulation of BBC3 mRNA expression by TNF-α. Higher BBC3 mRNA expression in islets correlated with decreased graft function in vivo.

Conclusions/interpretation: These results suggest that BBC3 mRNA can serve as a molecular marker to detect early TNF-α-induced beta cell stress and may help identify islet-protective compounds for the treatment of diabetes.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult
  • Aged
  • Animals
  • Apoptosis Regulatory Proteins / genetics
  • Apoptosis Regulatory Proteins / metabolism*
  • Blotting, Western
  • Cell Death / drug effects*
  • Cells, Cultured
  • Female
  • Flow Cytometry
  • Humans
  • Immunohistochemistry
  • In Vitro Techniques
  • Insulin-Secreting Cells / cytology
  • Insulin-Secreting Cells / drug effects
  • Insulin-Secreting Cells / metabolism
  • Interferon-gamma / pharmacology
  • Interleukin-8 / metabolism
  • Islets of Langerhans / drug effects*
  • Islets of Langerhans / metabolism*
  • Male
  • Mice
  • Mice, Inbred NOD
  • Mice, SCID
  • Middle Aged
  • Proto-Oncogene Proteins / genetics
  • Proto-Oncogene Proteins / metabolism*
  • Tumor Necrosis Factor-alpha / pharmacology*
  • Young Adult

Substances

  • Apoptosis Regulatory Proteins
  • BBC3 protein, human
  • Interleukin-8
  • Proto-Oncogene Proteins
  • Tumor Necrosis Factor-alpha
  • Interferon-gamma