PDX-1/VP16 Fusion Protein, Together With NeuroD or
Ngn3, Markedly Induces Insulin Gene Transcription and
Ameliorates Glucose Tolerance Hideaki Kaneto, Yoshihisa Nakatani, Takeshi Miyatsuka, Taka-aki Matsuoka, Munehide Matsuhisa,
Masatsugu Hori, and Yoshimitsu Yamasaki Diabetes is the most prevalent and serious metabolic
disease, and the number of diabetic patients worldwide
is increasing. The reduction of insulin biosynthesis in
pancreatic -cells is closely associated with the onset and progression of diabetes, and thus it is important to
search for ways to induce insulin-producing cells in
non -cells. In this study, we showed that a modied form of the pancreatic and duodenal homeobox factor 1
(PDX-1) carrying the VP16 transcriptional activation
domain (PDX-1/VP16) markedly increases insulin bio-
synthesis and induces various pancreas-related factors
in the liver, especially in the presence of NeuroD or
neurogenin 3 (Ngn3). Furthermore, in streptozotocin-
induced diabetic mice, PDX-1/VP16 overexpression, to-
gether with NeuroD or Ngn3, drastically ameliorated
glucose tolerance. Thus PDX-1/VP16 expression, to-
gether with NeuroD or Ngn3, markedly induces insulin
gene transcription and ameliorates glucose tolerance.
This approach warrants further investigation and may
have utility in the treatment of diabetes. Diabetes 54:
1009 1022, 2005 T he pancreatic and duodenal homeobox factor 1
(PDX-1), a member of the homeodomain-con-
taining transcription factor family (also known
as IDX-1/STF-1/IPF1) (13), is expressed in the pancreas and duodenum and plays a crucial role in pan-
creas development (4 14), -cell differentiation (1526), and maintenance of normal -cell function by regulating several -cell related genes (14,2737). At early stages of embryonic development, PDX-1 is initially expressed in
the gut region when the foregut endoderm becomes com- mitted to common pancreatic precursor cells (1,4
7,11,13). During pancreas development, PDX-1 expression
is maintained in precursor cells that coexpress several
hormones; later, its expression is restricted to -cells (13,5,6,11,13,28). Mice homozygous for a targeted muta-
tion in the PDX-1 gene are apancreatic and develop fatal
perinatal hyperglycemia (4), and heterozygous PDX-1 de-
cient mice have impaired glucose tolerance (12); both of
these ndings suggest the crucial role PDX-1 plays in
pancreas development. In clinical manifestations, muta-
tions in PDX-1 are known to cause maturity-onset diabetes
of the young (MODY) (38). NeuroD, a member of the basic helix-loop-helix (bHLH) transcription factor family, also known as BETA2, is
expressed in pancreatic and intestinal endocrine cells and
neural tissue. NeuroD also plays an important role in
pancreas development and the regulation of insulin gene
transcription (39 41). It has been reported that the insu-
lin-enhancer elements E-box and A-box play an important
role in regulating cell-specic expression of the insulin
gene (42,43), and that NeuroDs binding to the E-box as
well as PDX-1s binding to the A-box are very important for
insulin gene transcription. Mice homozygous for the null
mutation in NeuroD have a striking reduction in the
number of -cells, develop severe diabetes, and die peri- natally (40). In clinical manifestations, mutations in Neu-
roD cause MODY (44). Neurogenin 3 (Ngn3) is also a bHLH
transcription factor that binds to the E-box and is involved
in pancreas development (4550). Transgenic mice over-
expressing Ngn3 early in their development show a
marked increase in endocrine cell formation, indicating
that Ngn3 induces differentiation of islet cell precursors
(46,47). In contrast, mice with targeted disruption of Ngn3
have no endocrine cells (48). Insulin plays a crucial role in maintaining blood glucose levels; insulin facilitates glucose uptake into muscle and
adipose tissue and suppresses gluoconeogenesis in the
liver. In addition, insulin signaling has a functional role in -cells themselves; it has been reported that insulin sig- naling is crucial for insulin gene expression and glucose-
stimulated insulin secretion (49 52). In the diabetic state,
however, hyperglycemia (53 60) and subsequent produc-
tion of reactive oxygen species (61 65) decrease insulin
gene expression and secretion, accompanied by reduced
PDX-1 expression and DNA binding activity. Although
pancreas and islet transplantation efciently restore nor-
moglycemia, such treatment requires lifelong immunosup- From the Department of Internal Medicine and Therapeutics, Osaka Univer-
sity Graduate School of Medicine, Osaka, Japan. Address correspondence and reprint requests to Hideaki Kaneto, MD, PhD, Department of Internal Medicine and Therapeutics, Osaka University Gradu-
ate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan. E-mail:
kaneto@medone.med.osaka-u.ac.jp. Received for publication 28 July 2004 and accepted in revised form 7 January 2005. H.K. and Y.N. contributed equally to this work.
ABC, avidin-biotin complex; Ad, adenovirus; ALT, alanine aminotransferase; AST, aspartic acid aminotransferase; bHLH, basic helix-loop-helix; DAB, 3,
3 -diaminobenzidine tetrahydrochloride; GFP, green uorescent protein;
MODY, maturity-onset diabetes of the young; Ngn3, neurogenin 3; PDX-1,
pancreatic and duodenal homeobox factor 1; PFU, plaque forming unit; STZ,
streptozotocin; TBS, Tris-buffered saline.