By studying the effects of prenatal metformin exposure in mice, we showed that exposure to metformin at fetal phase programs the metabolic phenotype of the offspring. In our study, we first showed that metformin treatment led to a slight but significant decrease in fetal weight at E18.5 without affecting maternal body weight development or food intake. In addition to this, we showed that prenatal metformin exposure caused long-term effects that became particularly evident during the HFD-phase at later development in both sexes: prenatal metformin exposure led to an increased body weight gain, a significant reduction in total body water content, a significant increase in mesenteric fat and liver weight at the end of the HFD-phase. Furthermore, metformin exposed male offspring showed impaired glucose tolerance and elevated fasting glucose during the high fat diet.
The expression of Insig-1, one of the hepatic genes up-regulated by metformin, which was observed in the microarray data, was detected to be up-regulated by a sub-acute metformin dose in later development in the male offspring from the prenatal control group. In the group exposed to prenatal metformin no such activation of this gene was observed. Finally, we demonstrated that GLUT4 mRNA expression in epididymal fat tissue was down-regulated in the metformin exposed male offspring. These principal findings strongly suggest that prenatal metformin exposure does have long-term effects and the altered gene expression profile in the liver during the early neonatal period might be a significant contributor to the phenotype observed in our study.
The dose (300 mg/kg) for metformin administration was selected based on previously published studies of the effects of metformin in rodents 
. In order to calculate the human dose, an allometric body surface area method (BSA) 
was used. 300 mg/kg for a mouse corresponds to 1.7 g of metformin to a 70 kg man. This correlates well to human studies, where the passage of metformin through placenta has previously been studied with the dose of 850 mg twice a day in pregnant women 
. The concentrations measured from the maternal and umbilical vein serum, in the study of Vanky et al. 
, correlates to the concentrations measured in our study. The dose used was also evidently well tolerated since there were no observable side-effects on the dams (such as changes in the body weight development or food intake during pregnancy) and thus the obesity-prone phenotype of the offspring is most probably not a result of altered maternal behaviour or unspecific toxic effects of metformin. Metformin administration was stopped at embryonic day 17.5 to avoid excess stress prior to parturition.
Metformin exposed offspring had a phenotype which resembled that of offspring from maternal undernourishment models 
. In our study, the female offspring showed transient increase in body weight already on regular diet, however, the main difference in the body weight development between the prenatal groups occurred during the high fat diet. The food intake was not measured during the high fat diet, therefore, we cannot determine if the increased body weight originated from the excess intake of calories or decreased metabolic rate or a combination of these two factors.
Body composition measurements revealed that increased body weight was principally due to an increased amount of fat tissue in both sexes. By surgical excision, the most prominent weight change in the fat depots was observed in the mesenteric fat depot. We also observed that the liver weight was increased in metformin exposed offspring. The liver triglycerides were not measured in this study, however the increase in the liver weight coupled with increased amount of mesenteric fat strongly imply that the liver weight arises from increased fat accumulation in the liver 
The metabolic changes did not significantly concern blood lipid profile as at the end of our follow-up time for the offspring there were only minor changes observed. At this point the offspring had been on the high fat diet for 11 weeks and it might be that potential differences had already been blunted by the extensive time of high fat diet consumption. Moreover, as in other published studies related to prenatal programming 
sexual dimorphism was also present in our model. Only the metformin exposed female offspring were shown to have higher cholesterol levels and the metformin exposed male offspring had impaired glucose metabolism.
Prenatal and early neonatal periods have been shown to be critical time periods for the development of energy balance and metabolic circuits 
. Predisposition to an atypical environment during this time window can result in sustained alterations in the phenotype 
. Therefore, we investigated the gene expression profile in the liver and brain from 4-day old offspring. There were significant changes in the hepatic gene expression profile according to the microarray analysis. The most consistent change in hepatic gene expression was seen with insulin induced gene-1 (Insig-1) which showed approximately a 3-fold up-regulation in the mRNA expression in response to prenatal metformin exposure in the microarray analysis. Subsequent qRT-PCR analyses confirmed significant increase in Insig-1 mRNA expression in the neonatal male offspring. Our result, showing the increase in Insig-1 mRNA expression in response to metformin is the first time this gene has been linked to the mode of action of metformin in the liver. Insig-1 is an endoplasmic reticulum (ER) protein involved in lipogenesis. It works through the inactivation of SREBPs 
, a transcription factor family for lipogenic enzymes, through binding to SREBP cleavage activating protein or SCAP and retaining the complex in the ER. Metformin on the other hand has previously been shown to decrease the expression of many lipogenic enzymes in liver 
. Interestingly, metformin exposure has also been demonstrated to induce a fasting-like state in adult mice 
. Moreover, Onken and Driscoll 
have shown that metformin exposure mimics a dietary restriction-like state in C.elegans
by activating insulin independent caloric restriction pathways through AMPK and LKB1. There has also been shown to be a direct connection of Insig-1 and AMPK expression in liver cells 
, although in our study, no difference in AMPK expression was detected either by microarray or qRT-PCR at the age of 4 days (data not shown). Gene set enrichment analyses of the microarray data from the liver showed pathways which are involved in glycerolipid and lipoprotein metabolism to be in enriched in the male offspring exposed to metformin. Moreover, several pathways involved in cell growth and proliferation were enriched in the control group when compared to the metformin exposed offspring. These findings are most probably connected to the direct effects of metformin as it is known that it affects lipid metabolism 
and as there is growing evidence of the anti-proliferative actions of metformin 
. As the fetus develops under the influence of this environment, these pathways might play a role in the later phenotype.
There was minimal differences in the gene expression within the neonatal brain following metformin exposure, only two genes with yet an undefined role in the metabolism were significantly changed. As metformin is a water-soluble agent, its access across the blood-brain barrier is limited and therefore this finding might be expected. There is also a possibility that the gene expression profile of the whole brain conceals subtle changes occurring in energy balance regulating regions such as in hypothalamic nuclei. However, gene set enrichment analyses revealed that processes connected to respiratory electron transport are enriched in the metformin exposed male offspring. Based on the current information we can only hypothesise the importance of the finding. The possible effect of metformin on the function of neuronal mitochondria and its contribution to central regulation of energy balance could be linked to the observed programming effect of metformin. It should be noted that the microarray analyses were based on a relatively small number of animals and therefore should be interpreted cautiously. Further studies are needed to elucidate the findings in this study.
The elevated fasting blood glucose of metformin exposed male offspring could not be explained by increased gluconeogenesis as analyzed by the mRNA expression of gluconeogenic marker genes GLUT2, G6pc, GCK, Pck1 and PPARγ1a (data not shown). However, what we did see was a down-regulation of GLUT4 mRNA expression in epididymal fat tissue. Previous studies have shown that intrauterine growth restriction can down-regulate the expression of GLUT4 by epigenetic mechanisms in the muscle of rats 
and this potentially contributes to the development of metabolic disturbances in later development 
. However, although the muscle is the primary tissue that responses to insulin, there are some indications that fat tissue expression of GLUT4 might actually be more important for the development of glucose intolerance 
and decreased GLUT4 expression would be an indication of insulin-resistant state.
We investigated the effects of prenatal metformin exposure using pregnant inbred C57/BL6NHsd mice on a regular diet. Clinically there are two different type of pregnancy-related conditions for which metformin treatment is used for: polycystic ovary syndrome (PCOS) and gestational diabetes mellitus (GDM). PCOS affects approximately 10% of women of reproductive age and the main features include hyperandrogenemia, hirsutism, oligoanovulation and polycystic ovaries 
. Furthermore, these women are also often characterised by insulin resistance and hyperinsulinemia. One of the clinical problems in PCOS arises from infertility and there are encouraging data for the use of metformin in PCOS, as demonstrated by Morin-Papunen 
. As previously stated, the long-term effects of prenatal exposure to metformin are not known in humans. Interestingly, van Houten et al. (2012) 
have recently published a mouse model mimicking human PCOS. However, because these mice were reported to be anestrous, this mouse model would not be optimal in our current study plan. Thus, our regular diet model has an opportunity to provide essential data before the corresponding long-term human data would be collected.
A clinical follow-up study of the offspring of GDM mothers treated with metformin showed, that when the offspring were 2 years old, there was no difference in the total fat content in the offspring exposed to metformin (alone or combined with insulin) compared to those exposed to insulin 
. Interestingly, the distribution of fat was changed, with a hypothetical protective effect of an increase in subcutaneous fat in metformin/insulin group on the later accumulation of ectopic fat 
. Our own preliminary data also shows that prenatal metformin exposure might protect weight gain during a high fat diet in the offspring when the dams have been on a high fat diet prior and during gestation (unpublished data
Salomäki et al.). Based on different views on the use of metformin during pregnancy 
, we claim that it is justified to study the long-term effects of prenatal metformin exposure on the metabolic phenotype of the offspring in different nutritional and metabolic situations.
In summary, we observed a programming effect of prenatal metformin exposure that became evident during high fat diet at later stage of development. This was characterised by greater increases in body weight and mesenteric fat in both sexes. In addition to this, we observed impaired glucose tolerance in the male offspring. Importantly, the decreased expression of GLUT4 mRNA in epididymal fat at the end of the HFD-phase partly explained the metabolically disturbed phenotype of the male offspring which were prenatally exposed to metformin. Further experiments are required but we do suggest that the potential programming effect of metformin on metabolism should be taken into consideration in clinical situations.