Brief high fat high sugar diet results in altered energy and fat metabolism during pregnancy in mice

During pregnancy several maternal adaptations occur in order to support the growing fetus which are further exacerbated by gestational diabetes mellitus (GDM). Previously we developed a mouse model of GDM, however we did not evaluate alterations to energy and fat metabolism. We have also shown that alterations in lipid metabolism are mediated by adrenomedullin (ADM) in normal and GDM pregnancies. Our objectives were: (1) evaluate energy and fat homeostasis in our GDM mouse model and (2) determine if ADM may play a role in these changes. Female mice were placed on either control (P-CD) or high fat, high sucrose diet (P-HFHS) 1 week prior to and throughout pregnancy. Mice were placed into comprehensive lab animal monitoring system (CLAMS) chambers throughout pregnancy. Visceral adipose tissue (VAT) was collected at d17.5 of pregnancy for analysis. Energy Expenditure was significantly increased (p < 0.05) in P-HFHS dams compared to all other groups. VAT ex-vivo lipolysis was increased (p < 0.05) in P-HFHS compared to P-CD dams. VAT gene expression of ADM receptors Crlr, Ramp2, and Ramp3 was increased (p < 0.05) in P-HFHS dams. ADM dose dependently increased ex vivo lipolysis. This data further validates our animal model of GDM and is usefulness in investigating the pathophysiology of GDM.


Scientific Reports
| (2020) 10:20866 | https://doi.org/10.1038/s41598-020-77529-6 www.nature.com/scientificreports/ women 25,26 as well as in the serum and amniotic fluid of GDM women 16,27 Together this data supports a role for ADM in lipid metabolism during GDM. Gaps remain in our understanding of the mechanisms regulating the pathophysiology of GDM, specifically with regards to adipose tissue dysfunction and homeostasis along with the role Adm may play. Here our objectives were two-fold: (1) evaluate energy and fat homeostasis in our GDM mouse model and (2) assess the possible role of Adm in mediating these changes. We hypothesized that GDM dams would have altered energy and fat metabolism and that Adm signaling would be involved in these changes. To test this we analyzed energy expenditure, visceral fat gene expression, ex vivo lipolysis as well as the Adm signaling system in our mouse model of GDM.

Methods
Animals and experimental design. All animal procedures were approved by the Baylor College of Medicine institutional animal care and use committee and performed in accordance with NIH Guide for the Care and Use of Laboratory Animals.
Seven week old C57BL/6J female mice were placed on either a 10% kcal/fat, 0% kcal/sucrose control diet (Research Diets Inc., New Brunswick, NJ, cat#D12451; CD, n = 16) or a 45% kcal/fat, 17% kcal/sucrose diet (Research Diets Inc, cat #D12450K; HFHS, n = 16) 1 week prior to and throughout pregnancy to induced GDM like symptoms as previously described 40 . All females were placed with a proven breeder male for one night and then examined for copulatory plugs in the morning. Plug positive females were considered pregnant (P) and the morning of plug positive was designated as day 0.5 of pregnancy. Females who were plug negative were considered non pregnant (NP) and remained on their respective diets through-out the experiment to determine pregnancy vs diet specific effects. In total we had n = 7 NP-CD, 9 P-CD, 11 NP-HFHS, and 5 P-HFHS. Mice were placed into comprehensive lab animal monitoring system (CLAMS) chambers from day 0.5 to 17.5 of pregnancy to measure food consumption, activity, and energy expenditure (EE). Body composition was analyzed on day 0, 7.5, 11.5, 14.5 and 17.5. On day 17.5 animals were sacrificed using CO 2 inhalation. NP animals were day matched to P animals and evaluated at the equivalent time points. Visceral adipose tissue (VAT) from the periovarian fat pad was collected for gene expression analysis and ex-vivo culture with and without ADM. See Fig. 1 for experimental timeline.
Comprehensive Lab Animal Monitoring System (CLAMS). Mice were placed into CLAMS (Columbus Instruments) in the Mouse Metabolic Research Unit at the USDA/ARS Children's Nutrition Research Center from gestational day (d) 0.5 to 17.5, during which food intake, activity, and energy expenditure (EE) were measured as described previously [41][42][43] . Mice were first acclimated to the CLAMS for 3 days cages prior to breed- Figure 1. Experimental design. Mice were acclimated to powdered food and CLAMS chambers beginning 12 days prior to mating. Seven days prior to mating mice were randomly assigned to either CD or HFHS. On day − 1 mice were mated with proven breeder males for one night, mice where a copulatory plug was detected were considered pregnant and those where no copulatory plug was detected were designated non-pregnant. The day of copulatory plug detection (day 0) mice were placed in CLAMS cages for EE, food intake, and activity level measurements until day 17.5 pregnancy. At days 0, 6. 5, 10.5, 13.5 and 17.5 mice were briefly taken out of the cages, weighed, cages were cleaned, and then re-sealed. On day 17.5 mice were sacrificed and VAT was collected for further experiments. Adipose tissue explant culture. VAT obtained from mice was finely diced and transferred to wells of 24-well plates containing 1 ml of DMEM with 4.5 g/L d-glucose (Gibco, Life technology, Gaithersburg, MD), and cultured in a humidified atmosphere of 21% O 2 and 5% CO 2 at 37 C for 1 h as previously described 17,45 . After refreshing the medium, the tissues were incubated with or without increasing doses of Adm for 24 h and culture medium was collected for glycerol analysis. The glycerol level in culture medium was assessed using Free Glycerol Reagent (Sigma Aldrich, St. Louis, MO, USA) according to manufacture instructions. The absorbance at A540 were read and recorded by a Spectrophotometer CLARIO STAR (BMG Labtech, Inc., Cary, NC, USA) as previously described 17 . Statistical analysis. EE, activity, and food intake were analyzed using a repeated measures function in SAS (SAS Institute, Cary, NC, USA) with pregnancy status and diet as factors. Body weights, lean mass, and fat mass were analyzed with a two-way ANOVA with diet and time as factors with a Bonferroni post hoc analysis for comparison between groups using GraphPad Prism Software. Qiagen RT 2 Profiler PCR array statistics were performed on calculated dCT using a two-way ANOVA with diet and time as factors with a Bonferroni post hoc analysis for comparison between groups using GraphPad Prism. Lipolysis and Adm signaling mRNA gene expression was analyzed by student t-test using GraphPad Prism. ADM gene expression statistical analysis was performed on calculated dCT. A one-way ANOVA was used to analyze ADM dose response effect on lipolysis using GraphPad Prism. Statistical significance was defined as p < 0.05 and data are presented as mean ± SEM.

Results
GDM results in increased energy expenditure. CLAMS was used to measure energy expenditure (EE), activity and food intake throughout pregnancy. There was a significant interaction of diet*pregnancy*day (p = 0.0015) in EE ( Fig. 2A). Pregnant females had increased (p < 0.001) EE compared to non-pregnant females, regardless of diet. EE was increased in P-HFHS dams compared to all other groups (p < 0.001).
Overall activity was decreased in pregnant females compared to non-pregnant females (p < 0.0001) and in HFHS fed females compared to CD females (p < 0.01, Fig. 2B). Activity was decreased in NP-HFHS females compared to NP-CD females (p = 0.005), but not different between P-HFHS and P-CD females (Fig. 2B).
There was a strong interaction of diet*pregnancy on food intake (p < 0.0007) as measured by kcal (Fig. 2C). Food intake was decreased in P-HFHS dams compared to P-CD dams (p = 0.0005), but food intake was not different between NP-CD and NP-HFHS females. Food intake was significantly decreased in NP-CD females compared to P-CD dams (Fig. 2C).
Notably body weight (Fig. 3A) and lean mass (Fig. 3B) were increased (p < 0.05) in pregnant vs non-pregnant females regardless of diet on days 13.5 and 17.5 of pregnancy (Fig. 3A) however there was no difference in body weight between P-CD and P-HFHS dams at any time point. Fat mass (Fig. 3C) was significantly increased (p < 0.05) in P-HFHS dams compared to all other groups on days 6.5, 13.5 and 17.5 of pregnancy. On day 6.5 NP-HFHS females had increased (p < 0.05) fat mass compared to NP-CD and P-CD females, while on days 13.5 and 6.5 NP-HFHS and P-CD had increased (p < 0.05) fat mass compared to NP-CD (Fig. 3C).

Lipolysis is increased in
As we have previously demonstrated ADM plays a role in increased lipolysis associated with GDM in human VAT 15,16 , we sought to determine if Adm may play a role in increased lipolysis in our mouse model. First, we assessed if ADM signaling system is increased in VAT. mRNA expressions for Crlr (Fig. 6B), Ramp2 (Fig. 6C), and Ramp3 (Fig. 6D) were increased in P-HFHS dams compared to P-CD dams, while ADM expression was not different between groups (Fig. 6A). We then assessed if Adm can induce lipolysis in VAT from P-CD and P-HFHS dams. Glycerol release was measured in media of VAT from P-CD and P-HFHS dams incubated ex vivo with increasing doses of Adm for 24 h (Fig. 6E). Adm increased (p < 0.05) lipolysis in a dose dependent manner in both P-CD and P-HFHS dams. Overall, P-HFHS dams had increased (p < 0.01) lipolysis compared to P-CD dams. Taken together this data indicates that Adm may play a role in the mechanisms regulating increased lipolysis observed in GDM dams.

Discussion
Previously we showed that a brief HFHS diet 1 week before and during pregnancy resulted in glucose intolerance, decreased beta cell numbers and serum insulin levels, and increased leptin and triglyceride levels. Here we demonstrate that brief exposure to HFHS diet 1 week before and during pregnancy results in increased maternal EE possibly due to alterations in fat metabolism as indicated by alterations to VAT gene expression as well as Figure 2. Brief HFHS diet exposure results increased energy exposure (EE). EE (A), locomotor activity (B), and food intake (C) were measured from day 1.5 through day 17.5 of pregnancy in NP-CD, P-CD, NP-HFHS, and P-HFHS dams. For EE (A) there was a significant interaction of diet*pregnancy*day (p = 0.0015). Activity (B) was significantly decreased in P vs NP females (p < 0.0001) and in HFHS vs CD fed females (p < 0.01). For food intake there was an interaction of diet*pregnancy (p < 0.0007). n = 7(NP-CD), 9 (P-CD), 11 (NP-HFHS) and 5 (P-HFHS); error bars ± SEM. www.nature.com/scientificreports/ increased lipolysis. These findings are consistent with observations in women with GDM 31,32 further validating this as a valuable and novel animal model of GDM. Furthermore we demonstrate that ADM, as shown in human studies 15,16 , may play a role in altered lipid homeostasis during pregnancy as well as in GDM as indicated by increased receptor mRNA expression and an ability to dose dependently increase lipolysis. During pregnancy energy expenditure increases and energy intake also increases to meet this increased demand 33 . To our knowledge this is the first study that evaluated energy expenditure throughout pregnancy in mice using CLAMS cages. Our data shows that in mice, both energy expenditure and energy intake are increased during pregnancy, indicating that mice are a useful model in evaluating energy balance during pregnancy. Furthermore, our data indicate that while P-HFHS dams had increased energy expenditure compared to all other groups, their energy intake was not different from P-CD dams. This data would indicate that P-HFHS dams are in a negative energy balance compared to P-CD dams, with increased EE but not energy intake.
There are several possible reasons for altered energy homeostasis in P-HFHS dams. Mice with glucose intolerance are often found to have increased energy expenditure 34 . However altered glucose metabolism alone does not completely explain the observed differences in P-HFHS dams. Another major contributor to energy homeostasis is lipid metabolism 35 . Lipid metabolism is known to be increased in pregnancy and even further increased in GDM 5 . We therefore sought to examine possible changes in lipid metabolism to determine if, altered lipid metabolism may also contribute to increased EE in P-HFHS dams.
Analysis of VAT gene expression showed that several genes involved in lipid metabolism were altered in normal pregnancy (P-CD dams) and that these alterations were diminished in GDM (P-HFHS dams). These findings indicated that normal pregnancy increased the gene expression of overall lipid metabolism regulators, Angptl4, Ilk, Klf10, Pck2, Rxrb, Rxrg, and Tgs1, while these factors were not increased in GDM dams. Interestingly a recent study linked Angptl4 to increasing triglyceride levels in pregnancy, and also found that diet induced obese pregnant mice had impaired Angptl4 expression 36 . This data corroborates our observation that Angptl4 was increased in P-CD dams but not P-HFHS dams and potentially plays a role in altered lipid metabolism observed in P-HFHS dams. www.nature.com/scientificreports/ Another key group of factors that were altered in P-CD were genes that play a specific role in fatty acid metabolism including, Apoe, Cd36, Fabp4, and Fads2, however these genes were not altered in P-HFHS dams. These same findings were not observed between NP-CD and NP-HFHS females indicating that these changes were specific to pregnancy and not diet. Decreases in Cd36, which is involved in fatty acid uptake have previously been reported in omental fat tissue from healthy, non-obese and normal glucose tolerant women 37 . To date there are no published reports on adipose expression of Cd36 in women with GDM, however it has been reported that placental expression of Cd36 is reduced in GDM compared to normal pregnancies 38 . Taken together the mRNA expression data indicates that normal pregnancy alters adipose tissue metabolism and that GDM like conditions, as observed in P-HFHS dams diminish these changes leading to altered pregnancy associated lipid metabolism compensation. It should also be noted that our data indicate these changes are specific to pregnancy and not diet alone.
Women with GDM are known to have increased lipolysis later in pregnancy [3][4][5][6] . Here we show that P-HFHS dams late in pregnancy also have increased lipolysis. Previously we showed that P-HFHS have increased triglycerides as well as leptin levels 14 . Together with our previous reports 14 the results presented here clearly indicate that exposure to brief HFHS diet just before and during pregnancy is able to induce adipose tissue dysfunction and altered lipid metabolism as observed in women with GDM.
Previously we have shown that ADM plays a role in mediating adipose tissue dysfunction in GDM 15,16 . Here we showed that ADM also may play a similar role in the altered adipose tissue dysfunction and lipid metabolism observed in P-HFHS dams. In women with GDM, expression of ADM and it's receptor components, CRLR,  (E) Glycerol release was measured in ex vivo culture media of visceral fat from P-CD and P-HFHS dams w/o Adm after 24hrs as a measure lipolysis. Adm increased lipolysis in a dose dependent manner in both P-CD and P-GDM dams. *p < 0.05, different subscripts represent differences among groups (p < 0.01); n = 6 P-CD and 5 P-HFHS, error bars ± SEM. www.nature.com/scientificreports/ RAMP2 and RAMP3 is elevated in term omental adipose tissue 15,16 . We showed that this increase in ADM signaling expression is regulated by glucose and tumor necrosis alpha 15 . ADM is also increased in the serum of GDM women 39 . Our previous studies also showed that ADM dose dependently stimulates lipolysis in human adipocytes and that ADM increased the expression of leptin and resistin in adipose tissue from normal pregnant women 15,16 . We also showed that increases in leptin and resistin observed in adipose tissue from GDM can be blocked by ADM antagonists 15 . Furthermore, ADM inhibited phosphorylation of insulin receptor β 15 . The data presented here also indicates that brief HFHS diet feeding 1 week before and during pregnancy results in increased Adm signaling in adipose tissue. Moreover, Adm was able to induce lipolysis in a dose dependent manner in VAT from pregnant mice further supporting a role for Adm in lipid metabolism during pregnancy. However, the more detailed signaling mechanisms of Adm induced lipolysis require additional studies, and our animal model can be a useful tool in understanding the mechanistic role Adm may play in the altered lipid metabolism associated with GDM. Taken together we demonstrated that exposure to brief HFHS diet 1 week before and during pregnancy results in altered energy balance and lipid metabolism as well as adipose tissue dysfunction. We further demonstrated that increased Adm signaling may play a role in this altered adipose dysfunction and that this animal model maybe useful in further understanding the role of Adm in the pathophysiology of GDM. Further work is needed to elucidate the mechanisms through which Adm is regulating increased lipolysis in GDM. The data presented here further strengthens our mouse model as useful tool for GDM research. Future work will also focus on screening Adm antagonists as well as other compounds that can be useful to develop more effective treatment options for GDM.

Data availability
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