https://doi.org/10.5061/dryad.280gb5mzf

We have submitted a description of the data and file structure, sharing/access information, and computational scripts.

Description of the data and file structure

Figure 1: mPRε is sufficiently expressed in white adipose tissues

(A) mPRε mRNA expression in mouse tissues (postnatal day 49: P49) measured by quantitative RT-PCR (n = 3–4). WATs: white adipose tissues (epididymal adipose tissue); BAT: brown adipose tissue. Control 18S rRNA expression. NC, normal chow; HFD, high-fat diet. Statistical analysis was performed using Student’s t-test.
(B) Expression of mPR family subtypes in mouse WAT (n = 4; P49).
(C) mPRε expression in mature adipocytes (MA) and a stromal vascular fraction (SVF) of mice fed NC or HFD (n = 4).
(D) mPRε mRNA expression in MEF-derived adipocytes during adipogenesis (n = 6–10).
(E) Expression of the progesterone nuclear receptor PGR in mouse WATs (left, n = 4). PGR expression in the MA and SVF of mice fed NC or HFD (right, n = 3–8). (P49). PGR, progesterone receptor. *p < 0.05, **p < 0.01 (Student’s t-test). Results are presented as the mean ± standard error of the mean (SE).

Figure 2: Progesterone enhances insulin resistance via adipose mPRε

(A) Changes in body weight of WT and mPRε-/- mice under high-fat diet (HFD) feeding (n = 4–6). 
(B) Plasma steroid levels at 16 weeks of age (n = 4–6).
(C, E) An oral glucose tolerance test (OGTT) in male (C) or female (E) WT and mPRε-/- mice was performed at 15 min post-progesterone subcutaneous injection. (n = 7–10, *p < 0.05, vs. WT: progesterone). 
(D, F) Insulin tolerance test (ITT) in male (D) or female (F) WT and mPRε-/- mice was performed at 15 min post-progesterone subcutaneous injection (n = 7–10, *p < 0.05 vs WT: progesterone). *p < 0.05, **p < 0.01 (Student’s t-test). 
(H) Inhibitory effects of progesterone on insulin signaling. After pretreatment with progesterone for 30 min, a bolus of insulin (0.5 U/kg, i.p.) with or without progesterone (5 mg/kg, s.c.) was administered. Akt phosphorylation of Ser473 in WAT of wild-type mice after a 5 h of fast (n = 7–9). 
(I) Effect of progesterone on glucose uptake in mouse embryonic fibroblast (MEF)-derived adipocytes from WT or mPRε-/- mice. Glucose uptake was determined by measuring 2-deoxyglucose uptake using an enzymatic photometric assay (n = 4–10); P4: progesterone. *p < 0.05, **p < 0.01 (Dunn’s post hoc test). The results are presented as the mean ± standard error of the mean (SE). N.S.: not significant. See also Figures S1, S2, and S3.

Figure 3: Maternal progesterone-adipose mPRε control blood glucose in embryos

(A) Plasma progesterone levels in mice (male, non-pregnant, GD13.5, and GD16.5 pregnant female mice) (n = 4–6). 
(B) Oral glucose tolerance test (OGTT) in WT and mPRε-/- GD16.5 female mice (n = 4–8, *p < 0.05, **p < 0.01 vs WT). 
(C) Blood glucose levels in WT and mPRε-/- GD16.5 female mice (left) and E16.5 embryos (right) (n = 9–10, **p < 0.01 vs WT). 
(D) OGTT in non-pregnant female mice n = 5–8, *p < 0.05, **p < 0.01 vs WT) 
(E) OGTT in HFD-induced gestational diabetes mellitus model WT and mPRε-/- GD16.5 female mice (n = 4–8). *p < 0.05, **p < 0.01 (Mann–Whitney U test). Results are presented as the mean ± standard error of the mean (SE). See also Figures S4 and S5.

Figure 4: Offspring from mPRε-deficient female mice exhibits metabolic dysfunctions

(B) Body weight change during the growth of offspring from homozygous crosses or heterozygous crosses (n = 7–8). *p < 0.05, **p < 0.01 (Dunn’s post hoc test). 
(C) Changes in body weight of WT and mPRε-/- offspring mice under high-fat diet (HFD) feeding in males (left) or females (right) (n = 4–10). 
(D) Tissue weights of 16-week-old mPRε-/- offspring mice under high-fat diet feeding in males (left) or females (right) (n = 8–10). *p < 0.05, **p < 0.01 (Mann–Whitney U test). Results are presented as the mean ± standard error of the mean (SE). N.S.: not significant. See also Figures S6 and S7.

Figure 5: Prostaglandin production is increased during pregnancy via mPRε in WATs

(A) Beta diversity via the principal component analysis (PCA) based on genes from KEGG (ID mmu00590; mmu04064; mmu04910) in the WATs of WT and mPRε-/- mice with non-pregnant or GD16.5 (n = 5). The compositional similarity was compared using the permutational multivariate analysis of variance. 
(B) KEGG enrichment analysis related to molecular function in WATs of GD16.5 mPRε-/- females. p-values adjusted based on the false discovery rate (FDR). 
(C) Heat map of prostaglandin synthesis pathway, chronic inflammation, insulin resistance-related gene profiles of the WATs from WT and mPRε-/- non-pregnant or GD16.5 female mice (n = 5). Among non-pregnant vs GD16.5 females, 66 identified genes are differentially expressed (absolute log2 fold change > 0.5, p < 0.05: red open square). 
(D) KEGG pathway analysis related to arachidonic acid metabolism in WATs of mPRε-/- GD16.5 females. P-values were adjusted based on the FDR. 
(E) Comprehensive analysis of lipid mediators in WATs of mPRε-/- GD16.5 females; heat map of the top 40% relative lipid metabolite profiles of the WATs of WT and mPRε-/- non-pregnant or GD16.5 female mice (n = 5). Among non-pregnant vs GD16.5 females, 75 genes were differentially expressed (absolute log2 fold change > 0.5, p < 0.05: red open square). 
(F) Arachidonic acid (left) and PGE2 (right) in the WATs of non-pregnant or GD16.5 females. (n = 6–7 per group for arachidonic acid; n = 6–7 per group for PGE2). 
(G) Arachidonic acid levels in response to progesterone (100 nM) in Flp-In mPRε T-REx HEK293 cells (n = 6–8). Cells were induced mPRε expression by treatment with doxycycline (10 μg/mL) for 24 hr. Dox; doxycycline. *p < 0.05, **p < 0.01 (Mann–Whitney U test). The results are presented as the mean ± standard error of the mean (SE). N.S.: not significant. See also Figures S8, S9, S10, and S11.

Figure 6: Progesterone-induced activation of mPRε increases insulin resistance via adipose PGE2 production

(A) Arachidonic acid (left) and PGE2 (right) levels in the white adipose tissues (WATs) of WT and mPRε-/- female mice; 15 min after subcutaneous progesterone injection mice are treated with or without ibuprofen (100 mg/kg) (n = 4–5 per group for arachidonic acid; n = 3–5 per group for PGE2) *p < 0.05, **p < 0.01 (Dunn’s post-hoc). #p < 0.05 (Mann–Whitney U test). 
(B) Oral glucose tolerance test (OGTT) in WT and mPRε-/- female mice was performed at 15 min post-progesterone subcutaneous injection with or without ibuprofen (100 mg/kg). (n = 4–5). P4, progesterone. #p < 0.05, ##p < 0.01 between WT_P4 and WT_P4+ibuprofen (Dunn’s post-hoc test). NS: not significant.

Figure S1: Schematic representation of the mPRε gene structure and expression, related to Figure 2

(C) Expression of mPRε mRNA in mouse adipose tissues of wild-type and mPRε-/- mice (n = 3–4). The expression of 18S rRNA was used as an internal control.

Figure S2: Metabolic parameters in mPRε-/- offspring from mPRε+/- male and mPRε+/- female mice, related to Figure 2

(A) Tissue weight after 12 weeks in mPRε-/- male mice under high-fat diet (HFD) feeding (n = 4–6 from four litters; independent experiments). Epi, epididymal; peri, perirenal; sub, subcutaneous; BAT, brown adipose tissue; WATs, white adipose tissues.
(B) Blood glucose, plasma triglyceride, non-esterified fatty acid (NEFA), and total cholesterol levels in WT and mPRε-/- male mice after HFD intervention for 12 weeks (n = 4–6).
(C) Plasma insulin levels in male mice (n = 4–6 from four litters; independent experiments).
(D) Tissue weight after 12 weeks in mPRε-/- female mice under HFD feeding (n = 4–5 from four litters; independent experiments). Gona, gonadal; par, parametrial.
(E) Blood glucose, plasma triglyceride, NEFAs, and total cholesterol levels in WT and mPRε-/- female mice after HFD intervention for 12 weeks (n = 4–5).
(F) Plasma insulin levels in female mice (n = 4–5 from four litters; independent experiments). All data are presented as the mean ± standard error of the mean (SE).

Figure S3: Functional analysis of adipose mPRε on glucose homeostasis, related to Figure 2

(A) Plasma progesterone levels 30 min after progesterone administration (s.c.) in male (left) and female (right) mice (n = 6–8).
(B) Plasma insulin levels 30 min after progesterone administration (s.c.) in male (left) and female (right) mice (n = 6–9).
(C, D) Expression of mPR family members in mouse liver (C, n = 4) and muscle (D, n = 4) tissues (postnatal day 49, P49).
(E) Oil Red O staining of differentiated adipocytes derived from mPRε-/- mouse embryonic fibroblasts (MEFs) treated with 10 μM pioglitazone and inducer during adipogenesis (n = 3–4).
(F) Oil Red O staining of MEF-derived adipocytes of mPRε-/- mice with progesterone addition (n = 3–10). *p < 0.05, **p < 0.01 (Dunn’s test). Results are presented as the mean ± standard error of the mean (SE). N.S.: not significant.

Figure S4: Functional analysis of maternal adipose mPRε in pregnancy, related to Figure 3

(A) Plasma progesterone levels in GD16.5 females (n = 9–11).
(B) Plasma insulin levels in GD16.5 females (n = 9–11).
(C) Comparison of adipose mPRε expression between non-pregnant and GD16.5 female mice (n = 6). Results are presented as the mean ± standard error of the mean (SE).

Figure S5: mPRs expression and RNA sequencing in the placenta of mPRε-/- mice, related to Figure 3

(A) Expression of mPR subtypes in GD16.5 placenta (n = 4).
(B) Placenta progesterone levels in GD16.5 females (n =4).
(C) Beta diversity determined via principal component analysis (PCA) based on genes from KEGG (ID mmu00590; mmu04064; mmu04910) in the placenta of WT and mPRε-/- GD16.5 female mice (n = 4). Compositional similarity is compared using the permutational multivariate analysis of variance.
(D) KEGG enrichment analysis related to molecular function in the placenta of mPRε-/- GD16.5 female mice. p-values adjusted based on the false discovery rate (FDR). N.S.: not significant.

Figure S6: Body and tissue weights in offspring from mPRε-/- mothers, related to Figure 4

(A, B) Changes in body weight (left), tissue weights (middle), and lean mass (right) of 4-week-old male (A) or female (B) offspring from mPRε-/- mothers (n = 5–8).
(C, D) Normalization of tissue weight by body weight of 16-week-old males (left) and females (right) offspring from mPRε-/- mothers under high-fat diet feeding (n = 8–10). *p < 0.05, **p < 0.01 (Dunn’s test: A and B; Student’s t-test: C and D). All data are presented as the mean ± standard error of the mean (SE). N.S.: not significant.

Figure S7: Metabolic parameters in mPRε-/- offspring from mPRε-/- male and mPRε-/- female mice, related to Figure 4

(A) Blood glucose, plasma triglyceride, non-esterified fatty acid (NEFA), and total cholesterol levels in WT and mPRε-/- male offspring mice after HFD intervention for 12 weeks (n = 8–10).
(B) Plasma insulin levels in male offspring mice (n = 8–10 from three litters; independent experiments).
(C) Plasma steroid levels in 16-week-old male offspring mice (n = 8–9).
(D) Blood glucose, plasma triglyceride, NEFA, and total cholesterol levels in WT and mPRε-/- female offspring mice after HFD intervention for 12 weeks (n = 9–10).
(E) Plasma insulin levels in female offspring mice (n = 10 from three litters; independent experiments).
(F) Plasma steroid levels in 16-week-old female offspring mice (n = 11–12). *p < 0.05, **p < 0.01 (Mann–Whitney U test). All data are presented as the mean ± standard error of the mean (SE).

Figure S8: RNA sequencing in the liver of pregnant mPRε-/- mice, related to Figure 5

(A) Beta diversity via principal component analysis (PCA) based on genes from KEGG (ID mmu00590; mmu04064; mmu04910) in the liver of WT and mPRε-/- GD16.5 female mice (n = 5). Compositional similarity is compared using the permutational multivariate analysis of variance.
(B) KEGG enrichment analysis related to molecular function in the liver of mPRε-/- GD16.5 females. p-values adjusted based on the false discovery rate (FDR).

Figure S9: Progesterone-stimulated mPRε activation promotes PLA2 activity, related to Figure 5

(A) Membrane phospholipids of phosphatidylserine in the WATs of non-pregnant and GD16.5 female mice (n = 4 per group).
(B) PLA2 activity in WATs of mPRε-/- GD16.5 female mice (n = 9–10).
(C) PLA2 activity in response to progesterone (100 nM) in Flp-In mPRε T-REx HEK293 cells treated with or without doxycycline (Dox) (n = 4). Dox; doxycycline. *p < 0.05, **p < 0.01 (Mann–Whitney U test). The results are presented as the mean ± standard error of the mean (SE).

Figure S10: Phospholipid analysis in WATs from pregnant mPRε-/- mice, related to Figure 5

Quantitation of membrane phospholipids in WATs of mPRε-/- GD16.5 female mice (n = 4). All data are presented as the mean ± standard error of the mean (SE).

Figure S11: mPRε overexpressing HEK293 cells, related to Figure 5

(A) mRNA expression of mPRε in Flp-In mPRε T-REx HEK293 cells (n = 4).
(B) cAMP levels (n = 3–6) and
(C) ERK1/2 phosphorylation (n = 4) in response to progesterone in a dose-dependent manner in Flp-In mPRε T-REx HEK293 cells treated with or without doxycycline (Dox). Intracellular cAMP levels were determined using a cAMP assay kit, and each data point is presented relative to the forskolin-induced cAMP levels. Cells were induced mPRε expression by treatment with Dox (10 μg/mL) for 24 hr. Dox; doxycycline. All data are presented as means ± standard error of the mean (SE).

Sharing/Access information

All data supporting the findings of this study are available within the article and its supplemental information. Source data, including images, have been deposited in DRYAD: https://doi.org/10.5061/dryad.280gb5mzf and the DNA Data Bank of Japan (DDBJ) under the accession nos. E-GEAD-852, E-GEAD-853, E-GEAD-854, and E-GEAD-894.