Animals

The Institutional Animal Care and Use Committee at the University of Texas Medical Branch, Galveston, TX, approved the study protocol and all procedures. C57BL/6J mice were obtained from Charles River (Wilmington, MA). All animals were individually housed in a temperature- and humidity-controlled facility, with automatically controlled 12-hour light and dark cycles. Room temperature was kept at 26°C, which is within the thermoneutral range of our animal model. Certified personnel and veterinary staff provided regular maintenance and animal care according to institutional animal care and use committee guidelines. The animals were sacrificed by carbon-dioxide inhalation per the American Veterinary Medical Association guidelines.

Experimental protocol

Pregnant C57BL/6J dams were randomly allocated to fructose solution (10% wt/vol, n = 10) or water (n = 10) as the only drinking fluid from day 1 of pregnancy until delivery. Both groups were then allowed to consume regular chow ad libitum. During weaning, dams from both groups used standard chow and drinking tap water. After weaning, the pups were fed standard chow and drinking water. Our dams consumed an average of 7 mL/d fructose solution equivalent to 12 kJ/d/dam (10-15% from total kilocalories consumed daily). Fructose and drinking water were changed every 48 hours. Culling was performed as needed to limit the litter size to a maximum of 6 pups/litter. Pups were regularly weighed. Male and female offspring were randomly identified from each litter early after weaning (day of life 18-21) and placed in different cages for later testing at 12 months age for blood pressure measurements, serum analytes, and computed tomography (CT). All animals were humanely sacrificed, and blood samples were collected by cardiac puncture for further analysis. One male and one female pup from each litter were randomly selected at weaning time for these evaluations at 1 year of age.

Blood pressure measurement

CODA mouse tail-cuff system (Kent Scientific Corp, Torrington, CT) was used for accurate measurement of mouse blood pressure. It utilizes volume pressure recording sensor technology for blood pressure measurement. This technique has been validated with telemetry with 99% correlation. Briefly, a cuff was placed on the mouse’s tail to occlude the blood flow and a noninvasive blood pressure sensor was placed distal to the occlusion cuff for blood pressure measurements. Mice were first acclimated to restraints for 10-20 min/d for at least 3 days, followed by 20 cycles of blood pressure measurement before the study measurement was performed. Multiple measurements of blood pressure were performed and then averaged.

In vivo imaging

Percent visceral adipose tissue (VAT) and liver fat infiltration in rodents were assessed as described previously. Briefly, after sedation with intraperitoneally injected ketamine/xylazine (80-100 mg/kg and 5-10 mg/kg, respectively), images were obtained using a small animal micro-CT scanner (Inveon; Siemens Preclinical Solutions, Knoxville, TN). Imaging parameters were set as follows: voltage 70 kV, current 500 A, resolution 0.107 mm, exposure time 1000 milliseconds, 520 steps, and 360 degrees of rotations. During the scanning, mice were supplemented with nasal cannula oxygen. Each session lasted an average of 15 minutes.

The transverse views of CT images (1 per animal) at the level of the fifth lumbar vertebra were selected for analysis of VAT. The cross-sectional total body area and adipose tissue area were measured using software (Inveon Research Workplace). Percent VAT was calculated as the proportion of VAT to the cross-sectional total body area.

Liver and spleen radiodensity values in a transverse section between the 13th thoracic and first lumbar vertebrae were expressed as liver to spleen density ratio and compared between the groups. The radiodensity of the liver normalized to the radiodensity of the spleen (liver to spleen radiodensity ratio) indicated fat infiltration in the liver. Individuals interpreting VAT and liver radiodensity were blinded to the group allocation.

Serum analysis

Fasting glucose levels were measured using OneTouch Ultra (LifeScan, Milpitas, CA). Mice were then challenged with an intraperitoneal injection of glucose followed by blood collection and glucose level monitoring at 15, 30, 60, 90, and 120 minutes after glucose challenge. Insulin levels were determined using the Ultra-Sensitive mouse insulin enzyme-linked immunosorbent assay kit (Crystal Chem Inc, Downers Grove, IL). Insulin sensitivity was determined using the homeostatic model assessment of insulin resistance (HOMA-IR) and calculated by: (fasting glucose level [mg/dL] * fasting insulin level [mU/L])/405. Triglyceride and total cholesterol levels were measured using commercially available kits (Bioassay Systems, Hayward, CA).

Leptin and adiponectin were measured in mouse sera using the Milliplex mouse adipokine or mouse bone magnetic bead panel (Millipore, Billerica, MA). Briefly, mouse serum was incubated overnight with premixed beads coated with specific capture antibodies in a 96-well plate. The fluid in the plate was gently removed by vacuum and the plate was washed twice with wash buffer provided by the kit using the ELx405 plate washer (BioTek, Winooski, VT) with magnetic platform. Biotinylated detection antibodies were then added and the plate was incubated for 1 hour at room temperature. Streptavidin-phycoerythrin conjugate was added to each well and the plate was further incubated for 30 minutes at room temperature. The fluid in the plate was removed and the plate was then washed again with wash buffer and the beads were resuspended in sheath fluid. The plate was read on the Luminex LX 200, which uses a laser to excite and identify each bead. Xponent software (Millipore, Billerica, MA) was used to process the data according to the kit settings. Data were then interpreted using the Milliplex Analyst Version 3.4. Levels of the serum analyte were quantified based on standard curves created with known concentrations of the analyte of interest. Quality controls were provided with each kit to ensure proper technical procedure was maintained. All samples were run in duplicates.

Data analysis

All measurements were obtained blinded as to group assignment. Analysis was performed using software (STATA 14.0; StataCorp, Dallas, TX). Kolmogorov-Smirnov test was used to check for normal distribution of the data. Data are reported as mean ± SEM or median with interquartile range as appropriate. Statistical analysis was performed using an unpaired Student t test or Mann-Whitney test for nonparametric data. For the intraperitoneal glucose tolerance testing a 2-way repeated measures analysis of variance with Bonferroni correction was used. We did not correct for multiple testing since this is an exploratory study. A P value <.05 was considered statistically significant.

Animals

The Institutional Animal Care and Use Committee at the University of Texas Medical Branch, Galveston, TX, approved the study protocol and all procedures. C57BL/6J mice were obtained from Charles River (Wilmington, MA). All animals were individually housed in a temperature- and humidity-controlled facility, with automatically controlled 12-hour light and dark cycles. Room temperature was kept at 26°C, which is within the thermoneutral range of our animal model. Certified personnel and veterinary staff provided regular maintenance and animal care according to institutional animal care and use committee guidelines. The animals were sacrificed by carbon-dioxide inhalation per the American Veterinary Medical Association guidelines.

Experimental protocol

Pregnant C57BL/6J dams were randomly allocated to fructose solution (10% wt/vol, n = 10) or water (n = 10) as the only drinking fluid from day 1 of pregnancy until delivery. Both groups were then allowed to consume regular chow ad libitum. During weaning, dams from both groups used standard chow and drinking tap water. After weaning, the pups were fed standard chow and drinking water. Our dams consumed an average of 7 mL/d fructose solution equivalent to 12 kJ/d/dam (10-15% from total kilocalories consumed daily). Fructose and drinking water were changed every 48 hours. Culling was performed as needed to limit the litter size to a maximum of 6 pups/litter. Pups were regularly weighed. Male and female offspring were randomly identified from each litter early after weaning (day of life 18-21) and placed in different cages for later testing at 12 months age for blood pressure measurements, serum analytes, and computed tomography (CT). All animals were humanely sacrificed, and blood samples were collected by cardiac puncture for further analysis. One male and one female pup from each litter were randomly selected at weaning time for these evaluations at 1 year of age.

Blood pressure measurement

CODA mouse tail-cuff system (Kent Scientific Corp, Torrington, CT) was used for accurate measurement of mouse blood pressure. It utilizes volume pressure recording sensor technology for blood pressure measurement. This technique has been validated with telemetry with 99% correlation. Briefly, a cuff was placed on the mouse’s tail to occlude the blood flow and a noninvasive blood pressure sensor was placed distal to the occlusion cuff for blood pressure measurements. Mice were first acclimated to restraints for 10-20 min/d for at least 3 days, followed by 20 cycles of blood pressure measurement before the study measurement was performed. Multiple measurements of blood pressure were performed and then averaged.

In vivo imaging

Percent visceral adipose tissue (VAT) and liver fat infiltration in rodents were assessed as described previously. Briefly, after sedation with intraperitoneally injected ketamine/xylazine (80-100 mg/kg and 5-10 mg/kg, respectively), images were obtained using a small animal micro-CT scanner (Inveon; Siemens Preclinical Solutions, Knoxville, TN). Imaging parameters were set as follows: voltage 70 kV, current 500 A, resolution 0.107 mm, exposure time 1000 milliseconds, 520 steps, and 360 degrees of rotations. During the scanning, mice were supplemented with nasal cannula oxygen. Each session lasted an average of 15 minutes.

The transverse views of CT images (1 per animal) at the level of the fifth lumbar vertebra were selected for analysis of VAT. The cross-sectional total body area and adipose tissue area were measured using software (Inveon Research Workplace). Percent VAT was calculated as the proportion of VAT to the cross-sectional total body area.

Liver and spleen radiodensity values in a transverse section between the 13th thoracic and first lumbar vertebrae were expressed as liver to spleen density ratio and compared between the groups. The radiodensity of the liver normalized to the radiodensity of the spleen (liver to spleen radiodensity ratio) indicated fat infiltration in the liver. Individuals interpreting VAT and liver radiodensity were blinded to the group allocation.

Serum analysis

Fasting glucose levels were measured using OneTouch Ultra (LifeScan, Milpitas, CA). Mice were then challenged with an intraperitoneal injection of glucose followed by blood collection and glucose level monitoring at 15, 30, 60, 90, and 120 minutes after glucose challenge. Insulin levels were determined using the Ultra-Sensitive mouse insulin enzyme-linked immunosorbent assay kit (Crystal Chem Inc, Downers Grove, IL). Insulin sensitivity was determined using the homeostatic model assessment of insulin resistance (HOMA-IR) and calculated by: (fasting glucose level [mg/dL] * fasting insulin level [mU/L])/405. Triglyceride and total cholesterol levels were measured using commercially available kits (Bioassay Systems, Hayward, CA).

Leptin and adiponectin were measured in mouse sera using the Milliplex mouse adipokine or mouse bone magnetic bead panel (Millipore, Billerica, MA). Briefly, mouse serum was incubated overnight with premixed beads coated with specific capture antibodies in a 96-well plate. The fluid in the plate was gently removed by vacuum and the plate was washed twice with wash buffer provided by the kit using the ELx405 plate washer (BioTek, Winooski, VT) with magnetic platform. Biotinylated detection antibodies were then added and the plate was incubated for 1 hour at room temperature. Streptavidin-phycoerythrin conjugate was added to each well and the plate was further incubated for 30 minutes at room temperature. The fluid in the plate was removed and the plate was then washed again with wash buffer and the beads were resuspended in sheath fluid. The plate was read on the Luminex LX 200, which uses a laser to excite and identify each bead. Xponent software (Millipore, Billerica, MA) was used to process the data according to the kit settings. Data were then interpreted using the Milliplex Analyst Version 3.4. Levels of the serum analyte were quantified based on standard curves created with known concentrations of the analyte of interest. Quality controls were provided with each kit to ensure proper technical procedure was maintained. All samples were run in duplicates.

Data analysis

All measurements were obtained blinded as to group assignment. Analysis was performed using software (STATA 14.0; StataCorp, Dallas, TX). Kolmogorov-Smirnov test was used to check for normal distribution of the data. Data are reported as mean ± SEM or median with interquartile range as appropriate. Statistical analysis was performed using an unpaired Student t test or Mann-Whitney test for nonparametric data. For the intraperitoneal glucose tolerance testing a 2-way repeated measures analysis of variance with Bonferroni correction was used. We did not correct for multiple testing since this is an exploratory study. A P value <.05 was considered statistically significant.