Download 384 The non-canonical Wnt ligand, Wnt4, is highly expressed in

384
The non-canonical Wnt ligand, Wnt4, is highly expressed in pancreatic beta cells and its
expression is negatively correlated with cell growth
H.J. Welters, A. Henderson, A. Bowen;
Institute of Biomedical and Clinical Science, University of Exeter Medical School, UK.
Background and aims: We have previously published data showing that in beta-cells most Wnt
ligands are either not present or expressed at very low levels. The exception to this is Wnt4 which is
expressed at levels 10 fold higher than any other Wnt ligand. Wnt4 has been suggested to have a
negative effect on beta-cell biology, with the levels in islets increased in rodent models of insulin
resistance. The aim of this study is to investigate the regulation of Wnt4 expression in beta-cells and
the impact of Wnt4 on beta-cell growth.
Materials and methods: mRNA and protein expression levels were measured by qRT-PCR and
western blotting.
Results: We investigated Wnt4 mRNA expression in mouse organs and found that islets expressed
Wnt4 at 10 fold higher levels than kidney, liver, muscle or brain (Wnt4 mRNA expression relative to
housekeeping genes; kidney: 0.01±0.004, liver: 0.01±0.003, brain: 0.02±0.002, muscle: 0.02±0.009
and islets: 0.25±0.018, (mean±sem)), suggesting an important role for Wnt4 in islet cells. We used the
rodent beta-cell line, INS-1 to investigate regulation of Wnt4 expression. In agreement with islet data,
we found that INS-1 cells expressed high levels of Wnt4 mRNA and protein. To determine whether a
diabetogenic environment altered Wnt4 expression, we treated INS-1 cells with high glucose (Wnt4
mRNA expression normalised to housekeeping genes and expressed relative to 5.5mM glucose;
5.5mM glucose: 1±0.07, 16.7mM glucose: 1.07±0.13, (mean±sem) n=3) or 0.25 and 0.5mM palmitate
(Wnt4 mRNA expression normalised to housekeeping genes and expressed relative to control; control:
1.0±0.06, 0.25mM palmitate: 1.1±0.1, 0.5mM palmitate: 0.94±0.06, (mean±sem) n=3) for 24hrs.
However none of these treatments altered Wnt4 mRNA expression. We did though find that as cell
confluence increased (with an associated decrease in cell proliferation) levels of Wnt4 mRNA
increased (Fig 1). In contrast the levels of the proposed Wnt4 receptor, Fzd6, remained unchanged
(Fig 1). In addition levels of Wnt4 mRNA decreased upon 6hr treatment with the growth stimulating
protein, HGF (Wnt4 mRNA expression normalised to housekeeping genes and expressed relative to
control; control: 1±0.08, 10ng/ml HGF: 0.47±0.07, (mean±sem) n=3, p<0.01), suggesting a negative
correlation between Wnt4 expression and beta-cell proliferation. In agreement with this we find that
treatment of INS-1 cells with Wnt4 is able to inhibit cell growth stimulated by the canonical Wnt
ligand, Wnt3a (% change in cell growth over 72hrs compared to control; 10ng/ml Wnt3a:
114.8±1.03% (p<0.0001), 10ng/ml Wnt4: 97.1±1.04%, 10ng/ml Wnt3a+10ng/ml Wnt4: 97.3±0.97%
(mean±sem), n=4).
Conclusion: Our data suggests that Wnt4 may act as a negative regulator of canonical Wnt signalling
in beta-cells, leading to inhibition of beta-cell proliferation.