Study on the role of beta cell serotonin during pregnancy in the mouse.

Pregnancy is period in which the maternal body has to undergo several adaptations to meet the

demands of the fetus, for example in pancreatic beta cells. Pregnancy hormones stimulate prolactin

receptors (PRLRs) on beta cells to increase their capacity to produce insulin. In the last years, several

studies have been conducted on these changes in beta cells. Pancreatic islets of non-pregnant and

pregnant mice (at different time points) were compared for their mRNA expression profile. Two

genes were found to be highly induced during pregnancy in islets, Tph1 and Tph2, encoding the

enzymes tryptophan hydroxylase 1 and 2. These enzymes are responsible for the rate-limiting step

of serotonin biosynthesis, leading to the production and secretion of beta cell serotonin.

Remarkable about this serotonin is that not all beta cells produce serotonin to the same extent, but

that it displays a heterogeneous production pattern. Questions raised about the function of

serotonin in beta cells. A first study showed that serotonin acts locally to increase beta cell

proliferation during pregnancy via the 5-HTR2B receptor. Another study claimed that serotonin is

responsible for the stimulation of glucose-induced insulin secretion via the 5-HTR3A receptor.

In this thesis, pregnancy-related beta cell adaptations were investigated with a focus on the role of

serotonin in the process. In a first part, it was shown that both female and male islets are capable

of undergoing pregnancy-induced changes. Using an in vitro and an in vivo approach, it was

demonstrated that islets of both genders upregulate the same set of genes, produce serotonin and

have an increased beta cell proliferation rate. Furthermore, it was shown that the upregulation of

the set of genes was caused by stimulation of prolactin receptors by placental lactogens.

The main finding of this thesis was that neither beta cell serotonin nor 5-HTR2B are needed for the

increase in beta cell proliferation during pregnancy, which is in contrast with the results of another

research group. The percentage of proliferating beta cells was assessed in three different mouse

models in non-pregnant and pregnant condition. A first model was the total Tph1KO mouse, the

second model a newly discovered spontaneous “beta cell-specific” Tph1betaKO model. Both models

underwent a normal increase in proliferation compared to control mice. The third studied model

was the Htr2b deficient mouse strain, in which, again, no difference was found between Htr2b+/+

and Htr2b-/- mice regarding beta cell proliferation. In the future, more experiments are needed to

unravel the function of serotonin. In the Tph1betaKO mice, the influence of local serotonin on insulin

secretion and on islet blood flow will be investigated.

The discovery of the spontaneous “beta cell-specific” Tph1betaKO mouse is an important step

forward in the search to the role of beta cell serotonin. However, the path towards a useful model

was highly convoluted and full of hurdles and pitfalls. At first, the Cre/LoxP technology was used to

generate a mouse model. In total, four different models were tested (Pdx1-CreLate, Ngn3-Cre, Rip-

CreERTR26LacZ and Tph1fl/fl) and in three out of four models, problems were found. The Pdx1-CreLate

mouse contained the full sequence of the human growth hormone gene (hGH) and this led to a

pregnancy phenotype in non-pregnant mice. The Ngn3-Cre father mice were demonstrated to have

aspecific recombination in testes, giving rise to total Tph1KO mice among the offspring. Finally, the

Tph1fl/fl mouse turned out to be no good due to poor design of the floxed allele, as a fully active

truncated TPH1 enzyme was expressed in the Tph1betaKO mice. Therefore, Cre/LoxP technology

could not be used for the study. The discovery of all these artefacts in just one study suggests that

these artefacts sneak into the design of the conditional knock-out mice in other scientific fields,

beside the beta cell field.

The role of 5-HTR2B was already studied in islets, however, its highest mRNA signal was found in

placenta. Moreover, the signal for Htr2b mRNA was the second highest of all serotonin receptors in

the mouse body. The possibility of an endocrine communication axis between islets and placenta

was studied. In this model, beta cell serotonin would stimulate 5-HTR2B on placental cells providing

the sufficient production of pregnancy hormones. On their turn, pregnancy hormones stimulate

PRLRs on beta cells. Htr2b deficient placentas were compared with wild-type placentas for their

gene expression levels. None of the expressed genes, including Prl genes, in placenta were altered

in Htr2b-/- mice. Furthermore, placentas looked normal on macroscopic level. The function of 5-

HTR2B in placenta remains an open question and requires further research.