2.1. Animals and experimental design

An animal model of maternal liquid fructose intake was developed as previously described (Fauste et al., 2020, Rodrigo et al., 2018, Rodríguez et al., 2013). Female Sprague-Dawley rats weighing 200–240 g were fed ad libitum, a standard rat chow diet (Teklad Global 14 % Protein Rodent Maintenance Diet, Envigo, USA), and housed under controlled light and temperature conditions (12-h light–dark cycle; 22 ± 1 °C). The experimental protocol was approved by the Animal Research Committee of the University San Pablo-CEU, Madrid, Spain (ref. numbers 10/206458.9/13 and 10/042445.9/19). The experimental design was separated into two protocols.

In the first protocol, pregnant animals were randomly separated into a control group, a fructose-supplemented group (Fructose), and a glucose-supplemented group (Glucose) (five to six rats per group) (Rodríguez et al., 2013). Fructose and glucose were supplied as a 10 % (wt/vol) solution in drinking water throughout gestation. The concentration used here (10 % wt/vol) is very close to that of sugar-sweetened beverages (SSB). Control animals received no supplementary sugar. Pregnant rats were allowed to deliver and on the day of birth, each suckling litter was reduced to nine pups per mother. After delivery, both mothers and their pups were maintained with water and food ad libitum. At 21 days of age, pups were separated by gender and male progeny were kept fed on a standard rat chow diet (Teklad Global 14 % Protein Rodent Maintenance Diet, Envigo, USA) and water without additives. Animals within each experimental group were born to different dams to minimize the “litter effect”. In order to know the effects in adult progeny at 240 days of age, one half of the male progeny were randomly separated. When the progeny were 261-days-old, they were sacrificed and blood and livers were collected. Remarkably, these animals had received no subsequent additive in the drinking water for their entire lives (Rodrigo et al., 2018) (Fig. 2 A). The other half of the male progeny were subjected to the next protocol: independently from the experimental group of mothers to which they had been born, they were maintained on solid pellets and supplied with drinking water containing 10 % (wt/vol) fructose. Thus, three experimental groups were formed: C/F, F/F, G/F, the first letter indicating whether the mothers had been supplied with tap water during pregnancy (C: control), or water containing a carbohydrate (F: fructose; G: glucose); and the second letter indicating the period with fructose (F), when they were adults. When the progeny were 261-days-old, they were sacrificed and livers were immediately removed, placed in liquid nitrogen and kept at −80 °C until analysis. In parallel, a fourth experimental group was used, C/C: male progeny from control mothers supplied with water without any additives when adult. The period with fructose was selected to last 21 days (from 240 to 261 days of age) (Fauste et al., 2020) (Fig. 3 A).

Fructose in pregnancy affects hepatic cell surface receptors and cofactors facilitating SARS-CoV-2 entry in adult male progeny. (A) Experimental design. Hepatic levels of specific mRNA for (B) TMPRSS2, (C) neuropilin-1, (D) furin, (E) ADAM17, (F) cathepsin L, and (G) SRB1 genes of 261-day-old male progeny from control (empty bar), fructose-fed (black bar) and glucose-fed (grey bar) pregnant rats. Relative target gene mRNA levels were measured by Real Time PCR as explained in Materials and Methods, normalized to Rps29 levels and expressed in arbitrary units (a.u.). Data are means ± S.E. from 5 to 6 litters. Values not sharing a common letter are significantly different (P < 0.05). TMPRSS2: transmembrane protease serine 2; ADAM17: ADAM metallopeptidase domain 17; SRB1: HDL-scavenger receptor B type 1. E: embryonic/fetal days (E21: delivery); P: postnatal days.

Liquid fructose in gestation exacerbates fructose-induced augmentation of hepatic TMPRSS2 expression in adult male progeny. (A) Experimental design. Hepatic levels of specific mRNA for (B) TMPRSS2, (C) neuropilin-1, (D) furin, (E) ADAM17, (F) cathepsin L, and (G) SRB1 genes. Liver (mRNA) expression of fructose-fed male adult progeny from control (C/F, light grey bar), fructose- (F/F, black bar), and glucose-supplemented (G/F, dark grey bar) mothers. C/C: Control 261-day-old male offspring from control pregnant rats (empty bar, C/C). Relative target gene mRNA levels were measured by Real Time PCR as explained in Materials and Methods, normalized to Rps29 levels and expressed in arbitrary units (a.u.). Data are means ± S.E. from 5 to 6 litters. Values not sharing a common letter are significantly different (P < 0.05). TMPRSS2: transmembrane protease serine 2; ADAM17: ADAM metallopeptidase domain 17; SRB1: HDL-scavenger receptor B type 1. E: embryonic/fetal days (E21: delivery); P: postnatal days.

In the second protocol, pregnant rats were randomly separated into a control group (no supplementary sugar) and a fructose-supplemented group (fructose 10 % wt/vol in drinking water) (seven to eight rats per group) throughout gestation (Rodríguez et al., 2013). Pregnant rats were allowed to deliver and on the day of birth, each suckling litter was reduced to nine pups per mother. After delivery, both mothers and their pups were maintained with water and food ad libitum. At 21 days of age, pups were separated by gender and male progeny were kept fed on a standard rat chow diet (Teklad Global 14 % Protein Rodent Maintenance Diet, Envigo, USA) and water without additives. When the offspring were 3 months old, they were subjected to a new treatment for 21 days regardless of the group of mothers they were born. Male progeny from Control or Fructose-fed mothers were randomly separated into four experimental groups (animals within each experimental group were born to different dams to minimize the “litter effect”): control (C, tap water), fructose (F, fructose 10 % wt/vol in drinking water), fructose and cholesterol diet (FCho, fructose 10 % wt/vol in drinking water and solid food with 2 % added cholesterol; Tecklad Custome Diets TD.07841, Envigo, USA) and tagatose (T, tagatose 10 % wt/vol in drinking water). After 21 days, they were sacrificed and liver and ileum were immediately removed, placed in liquid nitrogen and kept at −80 °C until analysis (Fig. 4 A and 6A).

Fructose and fructose plus cholesterol affect hepatic cell surface receptors and cofactors facilitating SARS-CoV-2 entry in young male progeny. (A) Experimental design. Hepatic levels of specific mRNA for (B) TMPRSS2, (C) neuropilin-1, (D) furin, (E) ADAM17, (F) cathepsin L, and (G) SRB1 genes. Liver (mRNA) expression from control (C, empty bar), fructose- (F, light grey bar), and fructose plus cholesterol-supplemented (FCho, dark grey bar) young male progeny from Control (left panel) or Fructose-fed (right panel) mothers. Relative target gene mRNA levels were measured by Real Time PCR as explained in Materials and Methods, normalized to Rps29 levels and expressed in arbitrary units (a.u.). Data are means ± S.E. from 7 to 8 litters. Asterisks denote a significant difference (*, P < 0.05; **, P < 0.01; ***, P < 0.001) between the groups under the crossbar (groups with a different diet but the same motheŕs diet). Hash symbols denote a significant difference (#, P < 0.05; ##, P < 0.01; ###, P < 0.001) as compared to the Control mothers (groups with the same diet but different motheŕs diet). TMPRSS2: transmembrane protease serine 2; ADAM17: ADAM metallopeptidase domain 17; SRB1: HDL-scavenger receptor B type 1. E: embryonic/fetal days (E21: delivery); P: postnatal days.