Thermoneutral Housing Enables Studies of Vertical Transmission of Obesogenic Diet-Driven Metabolic Diseases

Abstract

Vertical transmission of obesity is a critical contributor to the unabated obesity pandemic and the associated surge in metabolic diseases. Existing experimental models insufficiently recapitulate "human-like" obesity phenotypes, limiting the discovery of how severe obesity in pregnancy instructs vertical transmission of obesity. Here, via utility of thermoneutral housing and obesogenic diet feeding coupled to syngeneic mating of WT obese female and lean male mice on a C57BL/6 background, we present a tractable, more "human-like" approach to specifically investigate how maternal obesity contributes to offspring health. Using this model, we found that maternal obesity decreased neonatal survival, increased offspring adiposity, and accelerated offspring predisposition to obesity and metabolic disease. We also show that severe maternal obesity was sufficient to skew offspring microbiome and create a proinflammatory gestational environment that correlated with inflammatory changes in the offspring in utero and adulthood. Analysis of a human birth cohort study of mothers with and without obesity and their infants was consistent with mouse study findings of maternal inflammation and offspring weight gain propensity. Together, our results show that dietary induction of obesity in female mice coupled to thermoneutral housing can be used for future mechanistic interrogations of obesity and metabolic disease in pregnancy and vertical transmission of pathogenic traits.

Keywords: amniotic fluid; developmental origin of health and disease (DOHaD); high-fat diet; inflammation; intrauterine programming; metabolic dysfunction-associated steatotic liver disease (MASLD); pregnancy; stillbirth; type 2 diabetes.

Figure 1

Maternal obesity at thermoneutrality leads to adverse neonatal outcomes. (A) Approach used to study maternal obesity in female C57BL/6 mice housed at thermoneutral temperature (red background) and fed chow (CD) or high-fat diet (HFD). Maternal groups are CD-fed lean (CL) and HFD-fed obese (HO). (B) Number of pups per litter (CL n = 6, HO n = 5). (C) Pup weight at birth (CL n = 26, HO n = 16). (D) Pup survival from 24–96 h after birth (CL n = 29, HO n = 39). (E) Male pup weight at weaning (CL n = 6, HO n = 3). (B,C,E) Unpaired t-test. ** p < 0.01; ns = not significant. (D) Log-rank test, curves are significantly different, * p < 0.0001.

Figure 2

Offspring of HFD-fed mothers weaned to CD have changes in adiposity but no metabolic disease. (A) Schematic of approach. Red background denotes thermoneutral housing temperature, and blue background denotes thermo-stressed housing. (B) Weight of CD-fed offspring at 8 weeks of age. Groups are male offspring of dams that were CD-fed lean (CL, n = 5) or HFD-fed obese (HO, n = 4). (C) Lean mass percentage of offspring (CL n = 8, HO n = 5). (D) Fat percentage of offspring (CL n = 8, HO n = 5). (E) eWAT mass of offspring (CL n = 4, HO n = 4). (F) iWAT mass of offspring (CL n = 4, HO n = 4). (G) Area under curve (AUC) from two-hour glucose tolerance test (GTT) (CL n = 4, HO n = 5). (H) Liver weight of offspring (CL n = 4, HO n = 4). (I) ALT of offspring (CL n = 4, HO n = 4). (B–I) Unpaired t-test. ** p < 0.01, *** p < 0.001, ns = not significant.

Figure 3

HFD challenge causes more severe metabolic disease in offspring of HFD-fed obese mothers. (A) Schematic of approach. Red background denotes thermoneutral housing temperature, and blue background denotes thermo-stressed housing. Unless otherwise noted, CD-CL, n = 4; HFD-CL, n = 4; HFD-HO, n = 3. (B) Weight gain of male offspring after 12 weeks on diet. Groups are offspring of dams that were CD-fed lean (CL) or HFD-fed obese (HO). CD-CL, n = 8. (C) Lean mass percentage (lean mass divided by total body weight) of offspring. (D) Fat percentage of offspring. (E) eWAT mass of offspring. (F) iWAT mass of offspring. (G) Area under curve (AUC) from two-hour glucose tolerance test (GTT). CD-CL, n = 3. (H) Liver weight of offspring. CD-CL, n = 7. (I) ALT of offspring. (B–I) One-way ANOVA with Dunnett’s multiple comparisons test. * p < 0.05; ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns = not significant.

Figure 4

Microbiome manipulation does not influence response to HFD challenge in offspring of HFD-fed mothers. (A) Schematic of approach. Red background denotes thermoneutral housing temperature, and blue background denotes thermo-stressed housing. Groups are male offspring of CD-fed or HFD-fed dams. Unless otherwise noted, CD-CD-Abx−, n = 4; HFD-HFD-Abx−, n = 5; HFD-HFD-Abx+, n = 6. (B) Weight gain of offspring after 12 weeks on diet. CD-CD-Abx−, n = 6. (C) Fat percentage of offspring. HFD-HFD-Abx+, n = 5. (D) Lean mass percentage of offspring. HFD-HFD-Abx+, n = 5. (E) eWAT mass of offspring. (F) iWAT mass of offspring. (G) Area under curve (AUC) from two-hour glucose tolerance test (GTT). CD-CD-Abx−, n = 3. (H) Liver weight of offspring. CD-CD-Abx−, n = 7. (I) ALT of offspring. (B–I) One-way ANOVA with Dunnett’s multiple comparisons test. * p < 0.05; ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns = not significant.

Figure 5

Maternal obesity-associated inflammation transfers to the fetus in utero. (A) Schematic of approach. Red background denotes thermoneutral housing temperature. (B) Maternal endotoxin at day 16 of pregnancy. Dams are CD-fed lean (CL, n = 6) or HFD-fed obese (HO, n = 18). (C) Maternal serum IL-6 depicted as % change from the average lean female value. CL, n = 4; HO, n = 5. (D) Amniotic fluid endotoxin concentration at GD16. CL, n = 2; HO, n = 3. (E) MEF IL-6 production in response to LPS stimulation. CL, n = 2; HO, n = 2. (F) Proteins differentially present in lean or obese amniotic fluid, represented by peak intensity. (G) Most significantly differentially regulated pathways based on protein expression data in obese amniotic fluid compared to lean. (H,I) Log fold change of proteins in obese amniotic fluid depicted as fold change compared to lean, involved in (H) acute inflammation pathway or (I) negative regulation of inflammation pathway. (B–E) Two-tailed t-test. * p < 0.05.

Figure 6

Human mothers with metabolic disease have altered inflammation, and their infants experience differential weight gain after birth. (A) Schematic of clinical approach. (B) Cytokine levels in maternal serum in third trimester of pregnancy (Lean, n = 4; Obese, n = 12). (C) Fetal subcutaneous fat thickness in third trimester of pregnancy, as measured by MRI (Lean, n = 11; Obese, n = 30). (D) Infant weight-for-length Z-score change from birth to the second study visit at approximately 9 months of age. Lean Mother: r² = 0.01, m = −0.05, non-zero slope p = 0.78; n = 9. Obese Mother: r² = 0.13, m = 0.12, non-zero slope p = 0.03; n = 22. (C,D) Mean ± SEM. (C) Two-tailed t-test. (D) Linear regression.