Maternal tadalafil therapy for fetal growth restriction prevents non-alcoholic fatty liver disease and adipocyte hypertrophy in the offspring

We aimed to investigate the effects of maternal tadalafil therapy on fetal programming of metabolic function in a mouse model of fetal growth restriction (FGR). Pregnant C57BL6 mice were divided into the control, L-NG-nitroarginine methyl ester (L-NAME), and tadalafil + L-NAME groups. Six weeks after birth, the male pups in each group were given a high-fat diet. A glucose tolerance test (GTT) was performed at 15 weeks and the pups were euthanized at 20 weeks. We then assessed the histological changes in the liver and adipose tissue, and the adipocytokine production. We found that the non-alcoholic fatty liver disease activity score was higher in the L-NAME group than in the control group (p < 0.05). Although the M1 macrophage numbers were significantly higher in the L-NAME/high-fat diet group (p < 0.001), maternal tadalafil administration prevented this change. Moreover, the epididymal adipocyte size was significantly larger in the L-NAME group than in the control group. This was also improved by maternal tadalafil administration (p < 0.05). Further, we found that resistin levels were significantly lower in the L-NAME group compared to the control group (p < 0.05). The combination of exposure to maternal L-NAME and a high-fat diet induced glucose impairment and non-alcoholic fatty liver disease. However, maternal tadalafil administration prevented these complications. Thus, deleterious fetal programming caused by FGR might be modified by in utero intervention with tadalafil.

Fetal growth restriction (FGR) is a condition in which the fetus does not reach its growth potential for a given gestational age. Studies have shown that the postnatal environment and lifestyle contribute to metabolic disease in adulthood; however, the prenatal environment is also important 1 . According to the fetal programming theory, metabolic diseases such as diabetes, hypertension, and dyslipidemia can be ascribed to the prenatal environment because injuries that occur during important developmental periods cause physiological and metabolic changes 2 . Thus, an inadequate intrauterine environment may lead to long-term adverse effects in adult life. Accordingly, infants with FGR are susceptible to type 2 diabetes mellitus and fatty liver disease in adulthood 3 . FGR is caused by a dysfunction in early placental development 4 , and subsequent postnatal rapid catch-up growth results in metabolic disease development.
L-NG-nitroarginine methyl ester (L-NAME) decreases the vasodilatory effect of nitric oxide (NO) and induces hypertensive disorders in pregnancy (HDP). Similar conditions can be produced in mice, and we previously reported that mice treated with L-NAME are suitable candidates for an FGR mouse model 4 . In that study, we speculated that tadalafil would indirectly promote fetal growth by causing dilatation of the maternal blood sinuses of the placenta 4 . The association between HDP and FGR has been recognized because it results from failed trophoblast invasion and uterine spiral artery remodeling, which leads to blood flow reduction, placental ischemia, and FGR 5 . Furthermore, NO produced by NO synthases regulates the placental vascular tone. Placental vessels express molecular mediators for the NO-dependent pathway, including cyclic guanosine monophosphatespecific phosphodiesterase 5 (PDE5) 6 . Recently, PDE5 inhibitors, which act by dilating arteries and increasing maternal intervention administered. These groups were as follows: control (C), treatment with L-NAME only (L), and treatment with both L-NAME and tadalafil (TL). The control group was not treated with L-NAME or tadalafil. In addition, since each offspring group received a high-fat diet (HFD) after 6 weeks, the term " + H" was added after the three group names (C + H, n = 6; L + H, n = 4; and TL + H, n = 7). There were no significant differences in the weights of the mice who were given a HFD from 6 weeks until the day they were euthanized ( Fig. 1). Further, the whole epididymal adipose tissue, liver, and pancreas weights per body weight (g/kg) did not differ significantly among the groups ( Table 1). The blood pressures in the dams 16 days post-coitum (d.p.c.) were 102.3 ± 7.7 mmHg, 23.7 ± 10.7 mmHg, and 114.2 ± 6.8 mmHg in the C, L, and TL groups, respectively. A significant difference was noted between the C and L, and L and TL groups. The sex of the offspring in the C, L, and TL groups were 20, 14, and 23 male and 10, 21, and 8 female, respectively. The weights of the offspring within 48 h after birth were 1.56 ± 0.11 g, 1.38 ± 0.15 g, and 1.46 ± 0.11 g in the C, L, and TL groups, respectively (C-L, p = 0.0003; C-TL, p = 0.0322; and L-TL, p = 0.13). The weights of the offspring within 3 weeks of birth were 8.51 ± 0.68 g, 8.23 ± 0.59 g, and 8.04 ± 1.15 g in the C, L, and TL groups, respectively (C-L, p = 0.88; C-TL, p = 0.62; L-TL, p = 0.93). Within 5 weeks after the birth, the offspring weighed 19.26 ± 0.64 g, 18.82 ± 1.03 g, and 19.47 ± 1.21 g in the C, L, and TL groups, respectively (C-L p = 0.78, C-TL p = 0.92, L-TL p = 0.56).
GTTs. In terms of the fasting glucose levels, there were no significant differences noted between the groups (Fig. 2). However, the blood glucose levels 30 min after glucose administration were significantly lower in the TL + H group than in the L + H group (p < 0.05). Furthermore, blood glucose levels after 90 min were higher in the L + H group than those in the other groups (C + H, p < 0.01 and TL + H, p < 0.05).

Figure 1.
Postnatal body weights of the pups from different treatment groups. At the time of initial sampling, there were no significant differences in the bodyweights between the high-fat diet groups. The birth weight was not measured to reduce maternal stress. Data are presented as mean ± standard deviation, n = 4-7/group. A two-way repeated analysis of variance with a post hoc Tukey test was used for multiple comparisons. C + H control + high-fat diet, HFD high-fat diet, L + H L-NG-nitroarginine methyl ester + high-fat diet, L-NAME L-NG-nitroarginine methyl ester, TL + H L-NG-nitroarginine methyl ester + tadalafil + high-fat diet. Table 1. Analyses of tissues from mice of different treatment groups. C + H control + HFD, L + H L-NAME + HFD, TL + H L-NAME + tadalafil + HFD, L-NAME L-NG-nitroarginine methyl ester, HFD high-fat diet. Morphological changes in the livers of the offspring. The mean non-alcoholic fatty liver disease (NAFLD) activity score (NAS) was higher in the L + H group than in the C + H group (p < 0.05), whereas there were no significant differences between the C + H and TL + H groups (Fig. 3B). Additionally, the F4/80-positive cell count was higher in the L + H group than in those in the other groups (p < 0.001; Fig. 3C).

Enzyme-linked immunosorbent assay (ELISA).
The serum resistin levels in the L + H group were lower than those in the C + H and TL + H groups (p < 0.05, Fig. 4). However, the serum adiponectin and serum leptin levels did not differ significantly between the groups (Fig. 4).

Discussion/comment
Principal findings. Our study found that maternal L-NAME administration affected both glucose tolerance, and liver and adipose tissue morphology in adult mice offspring. Furthermore, these changes could be prevented through the administration of maternal tadalafil therapy.

Results.
In this study, fatty liver disease was observed in the offspring of mice with L-NAME-induced FGR.
However, the maternal tadalafil therapy results suggested that in utero interventions can be used to modify fetal programming. In addition, we found that these changes could cause glucose intolerance in the L-NAME group but this outcome was partially prevented by tadalafil treatment. Furthermore, mice with L-NAME-induced FGR can be used as a model for preeclampsia as they result in abnormal metabolism in adulthood. An insulin-resistant preeclampsia model (performed at 40 weeks) that used angiotensin 2 type 1 receptor antibodies and a highsugar diet at 40-48 weeks found that fasting insulin levels were also high. In this model, the researchers observed a remarkable increase in the fasting blood glucose levels compared to the controls 13 . Moreover, offspring with FGR undergo catch-up growth and develop insulin resistance with elevated insulin-like growth factor 1 levels during adulthood as a result of these changes 14 . Our results suggest that maternal tadalafil administration can potentially prevent FGR-related glucose intolerance. NAFLD occurs as a result of dysregulated lipid homeostasis, which is primarily attributed to an increase in hepatic fatty acid lipogenesis, and a decrease in fatty acid β-oxidation and lipid output 15 . A previous report showed that the lipid content and messenger ribonucleic acid (mRNA) expression of lipogenesis-associated genes are enhanced in growth-restricted fetuses and neonates 16 . These results suggest that FGR might lead to dyslipidemia and obesity in adulthood 17 . Furthermore, the prevalence of type 2 diabetes is increased in obese and non-obese patients who have been diagnosed with NAFLD 18 . Other features of NAFLD include hyperglycemia 2-h after glucose loading, even when the fasting blood glucose levels are normal, and hyperinsulinemia with decreased hepatic insulin clearance (confirmed with a large patient cohort). Furthermore, glucose intolerance is correlated with the NAS 19 . In this study, the combination of in utero FGR and postnatal administration of an HFD induced histological changes in the livers of the offspring, whereas tadalafil suppressed these changes. Moreover, we showed that the mean NAS in the L + H group alone exceeded 4 points, which indicated severe NAFLD 20 .
In children with NAFLD, numerous activated macrophages are located in the spaces between the damaged hepatocytes. Moreover, macrophages can be used to predict progressive NAFLD before the onset of inflammation or fibrosis 21 . Here, we observed an increase in the F4/80-positive cell count, an M1 macrophage marker, in the L + H group. However, maternal tadalafil ameliorated this change.
Otherwise, adipocytokines are one of the most important contributing factors to glucose intolerance 22 . We used ELISA to show that L-NAME could induce adipocytokine changes. Although an increase in the serum Figure 2. Glucose tolerance test results at 16 weeks in the treatment groups. At 15 weeks, glucose was administered intraperitoneally (1 mg/kg body weight) after an overnight fast. The blood glucose levels 30 min after glucose administration were significantly lower in the TL + H group than in the L + H group. Furthermore, the blood glucose levels at 90 min were higher in the L + H group than those in the other groups. Data are presented as the mean ± standard deviation, n = 4-7/group; **p < 0.01, *p < 0.05, based on a two-way repeated analysis of variance with a post hoc Tukey test for multiple comparisons. C + H control + high-fat diet, HFD high-fat diet, L + H L-NG-nitroarginine methyl ester + high-fat diet, L-NAME L-NG-nitroarginine methyl ester, TL + H L-NG-nitroarginine methyl ester + tadalafil + high-fat diet.  Morphologic changes in the liver and adipose tissue due to tadalafil and L-NAME administration. C + C indicates treatment naïve mice to whom no maternal intervention or HFD was given. Data are presented as the mean ± standard deviation, n = 4-7/ group. ***p < 0.001, **p < 0.01, *p < 0.05, based on a one way-analysis of variance with a post hoc Tukey test for multiple comparisons. C + C control + control diet, C + H control + HFD, HE hematoxylin and eosin, HFD high-fat diet, L + H L-NG-nitroarginine methyl ester + high-fat diet, L-NAME L-NG-nitroarginine methyl ester, NAFLD non-alcoholic fatty liver disease, TL + H L-NAME + tadalafil + HFD, TL + H L-NG-nitroarginine methyl ester + tadalafil + high-fat diet.  www.nature.com/scientificreports/ resistin levels can induce insulin resistance 23 , L-NAME administration decreased serum resistin levels. Previous research has reported that L-NAME induces resistin mRNA expression and improves insulin sensitivity in adults, which may be the reason for this result 24 . In this case, maternal L-NAME administration may change resistin mRNA expression in both the mothers and the pups. Moreover, these levels returned to control levels following maternal tadalafil administration. Despite these results, the glucose tolerance in the offspring improved. In a previous study, obese mice showed a decrease in their serum resistin levels; however, these results were controversial 25 . Thus, further investigation on the relationship between resistin and glucose tolerance in obese individuals is needed. In this study, the area of the adipocytes in the epididymal tissue was lower in the maternal tadalafil group than in the L-NAME group. In addition, L-NAME administration aggravated adipocyte enlargement. Previous studies found that giant adipocytes in in vivo visceral adipose tissue were resistant to the antilipolytic effects of insulin 26 . Increased adipocyte size may be caused by decreased adiponectin secretion, increased free fatty acid release, and imbalanced proinflammatory and inflammatory cytokine production. This is called adipose tissue dysfunction and it can be deleterious in terms of changes in insulin sensitivity [27][28][29] . In addition to regulating systemic energy storage, adipose tissue secretes a number of adipokines that significantly affect lipid homeostasis and insulin resistance 30 . The decrease in the adipocyte size in the maternal tadalafil group suggests that this drug could be used to improve insulin resistance.

Strengths and limitations.
Our study found that maternal tadalafil can alter the metabolism of adult mice offspring. This pharmacological agent modulated the liver and adipose tissue morphology and improved glucose intolerance. Previously, we focused on the effects of maternal tadalafil on birth weight and neurological defects, but we considered the need to address its effects on the metabolism of adult mice offspring. However, this study has several limitations. First, we did not investigate gene expression. We propose that maternal tadalafil therapy alters gene expression in the pre-and postnatal periods. However, such alterations and the mechanism associated with the effects of tadalafil have not been studied. These topics should be addressed in future studies. Second, we did not provide adequate investigation into the effects on the pancreas and, in our next study, we plan to address pancreatic function. Third, there has been no investigation into cases without a second hit. It is crucial that we develop an understanding of this point as tadalafil can potentially induce abnormal gene expressions that might have unfavorable effects. Hence, the clinical applicability of this study for humans should be considered with caution.
Alterations to the metabolic profiles caused by a combination of FGR and HFD were prevented through maternal tadalafil treatment, which suggests that in utero intervention could modify fetal metabolic programming to yield favorable results.
Clinical implications. Although the mortality rate for fetuses with FGR has improved, this condition complicates fetal development and future adult diseases, resulting in significant problems. Therefore, the prevention of such issues by intervening during the fetal period is of great importance. In this study, our results suggest that maternal tadalafil therapy could minimize the effects of HFD as a postnatal second hit by preventing alterations in fetal programming, which is the inherent first hit.

Methods
Nonhuman experimentation. The experimental protocol was approved by the Ethics Committee for Animal Research of the Mie University Graduate School of Medicine (Institutional Review Board number; . All methods were carried out in accordance with the relevant guidelines and regulations.

Animals.
The experimental protocol is shown in Fig. 5. We used the same protocol as the one that we previously described for a mouse model for L-NAME-induced FGR using a C57BL6 background 12 . C57BL6 mice (CLEA, Tokyo, Japan) were purchased 9 d.p.c. The animals were housed individually in a temperature-and humidity-controlled facility with automatically controlled 12-h light and dark cycles. The mice were provided with food and water ad libitum, and the intake and body weight of the pregnant mice were evaluated daily.
The pregnant mice were allocated to the control or L-NAME groups at 11 d.p.c. The groups were matched by weight. The control group received 0.5% carboxymethylcellulose (CMC; Wako Pure Chemical Industries, Osaka, Japan) dissolved into their drinking water. The L-NAME group received 1 mg/ml of L-NAME (Cayman Chemical Company, Ann Arbor, MI, USA) dissolved into 0.5% CMC. The dams that were treated with L-NAME were allocated to the two subgroups at 14 d.p.c. The subgroups were matched by their weight and systolic blood pressure (SBP). SBP was measured using a tail-cuff microsensor connected to a non-invasive blood pressure monitor for mice (MK-2000A; Muromachi Kikai, Tokyo, Japan).
One subgroup continued to only receive L-NAME, whereas the other subgroup received L-NAME with 0.08 mg/ml of tadalafil (Cayman Chemical Company, Ann Arbor, MI, USA) suspended in 0.5% CMC. Maternal SBP measurement was repeated at 16 d.p.c.
The litters were standardized to six to eight pups per litter after 48-h post-partum to ensure that the animals received equal quantities of milk and maternal pup care. To minimize human interference and prevent the hinderance of lactation and care, the birth weights were not measured. All dams were provided with normal drinking water during lactation. After 6 weeks, the male pups from each group were chosen randomly to receive an HFD (4.73 kcal/g, with fat comprising 45% of the total calories, and consisting of soybean oil [5.6%], lard [39.4%], and a 20% protein formula; D12451, Research Diets, New Brunswick, NJ, USA). Thus, the three groups of offspring based on the maternal interventions were as follows: control + HFD, L-NAME + HFD, and L-NAME + tadalafil + HFD (C + H, n = 6; L + H, n = 4; and TL + H, n = 7, respectively). Only the male pups were Scientific Reports | (2021) 11:1186 | https://doi.org/10.1038/s41598-020-80643-0 www.nature.com/scientificreports/ used for the experiments because, with this model, HFD-related metabolic disorders are more prevalent in males than in females 31 . The weights of the pups were recorded and the animals underwent GTTs at 15 weeks. They were euthanized at 20 weeks, after which blood collection and tissue sampling were performed. Figure 5. Experimental protocol. The litters were standardized to 6-8 pups per litter 48-h post-partum, allowing the pups to receive equal amounts of milk and maternal pup care. All dams were given normal drinking water during lactation. After 6 weeks, the male pups from each group were fed an HFD and treated as follows: control + HFD (C + H, n = 6), L-NAME + HFD (L + H, n = 4), and L-NAME + tadalafil + HFD (TL + H, n = 7). HFD high-fat diet, L-NAME L-NG-nitroarginine methyl ester.