Objective: Examine the effects of metformin supplementation and caloric restriction on lifespan and survival in C57BL/6 and B6C3F1 mice
Gather these items before starting the experiment. Check off items as you prepare.
Not specified • Not specified • Not specified • Not specified
BioServ • Not specified • Diet No. F05312 • Not specified
Purina Mills • Not specified • Diet # 5001 • Not specified
Farmhispania S.A. (for C57BL/6 groups); Caraco Pharmaceutical Laboratories (for B6C3F1 groups) • Not specified • Not specified • Not specified
Dyets Inc. • Not specified • Not specified • Not specified
BioServ • Not specified • Not specified • Not specified
Not specified (statistical method) • Not specified
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Male C57BL/6 mice were purchased at either 6 months of age (for 0.1% metformin and CR groups) or 11 months of age (for 1% metformin group) from the National Institute on Aging Aged Rodent Colony from Charles Rivers. B6C3F1 male mice were obtained from Harlan Breeders (Indianapolis).
Note: All procedures followed NIH Intramural Research Program protocol number 352TGB2013
“Male C57BL/6 mice at either 6 months of age (for 0.1% metformin and CR) or 11 months of age (for 1% metformin dose) were purchased from the National Institute on Aging Aged Rodent Colony from Charles Rivers”
C57BL/6 mice were maintained on standard purified AIN-93G diet until they reached one year of age when treatments would begin.
Note: Light:dark 12:12-h schedule maintained
“The C57BL/6 mice were maintained on a standard purified mouse diet (AIN-93G) until they reached one year of age when the treatments started”
B6C3F1 mice were randomly assigned to treatment groups at 12 months of age and shifted from ad libitum chow feeding (Diet #5001, Purina Mills) to daily feeding with 13.3 kcal/day/mouse of control diet (AIN-93M).
Note: Random assignment to treatment groups
“male mice (Harlan Breeders; Indianapolis) were randomly assigned to treatment groups at 12 months of age. In the control group, mice were shifted from ad lib chow feeding (Diet # 5001, Purina Mills, Richmond, IN) to daily feeding with 13.3 kcal/day/mouse of control diet”
At one year of age, C57BL/6 mice were divided into treatment groups: standard diet (SD), SD plus 0.1% metformin, SD plus 1% metformin (all ad libitum), or caloric restriction (CR) with 60% of ad libitum food allotment on AIN-93G diet.
Note: CR animals had been on restricted diet starting at 27 weeks of age
“the C57BL/6 groups in this study were fed the standard AIN-93G diet (SD) or AIN-93G plus 0.1% metformin or 1% metformin ad lib for the remainder of their lives”
For C57BL/6 groups: Pure metformin from Farmhispania was mixed to homogeneity with AIN-93G diet during manufacturing by Dyets Inc. For B6C3F1 groups: Metformin from Caraco Pharmaceutical Laboratories was mixed with powdered AIN-93M diet and cold-pressed into 1-g pellets by BioServ at 100 mg/kg diet (0.1% w/w).
Note: Chow produced every 3 months; kept away from light to ensure metformin stability
“Pure metformin was obtained from Farmhispania (Farmhispania S.A. Barcelona, Spain) and mixed to homogeneity during manufacturing of the diets (Dyets Inc., Bethlehem, PA)”
Stock metformin and all chow were stored in the dark at 4°C. Chow was produced every 3 months and never permitted to exceed 50°C. Light/dark cycle in mouse facility was not altered.
Note: Storage in dark to ensure metformin stability
“Stock metformin and all chow were stored in the dark at 4 °C. The chow was produced every 3 months during the length of the study, never was permitted to exceed 50 °C and was kept away from light whenever possible”
All mice were fed daily. For ad libitum groups, food was provided without restriction. For CR and controlled B6C3F1 groups, specific daily food allotments were provided (60% of ad libitum for CR; 13.3 kcal/day for B6C3F1 control). With rare exceptions, all food was eaten each day.
Note: Food consumption monitored to ensure complete consumption
“All mice were fed daily and, with rare exceptions, all food was eaten each day”
Food intake and body weight were measured on a biweekly or bimonthly basis for the duration of the study.
Note: Measurements continued for entire lifespan
“Food intake and body weight were measured on a biweekly or bimonthly basis for the duration of the study”
Mice were monitored throughout the study. Euthanasia criteria were based on independent assessment by a veterinarian according to AAALAC guidelines, with euthanasia only performed when animal condition was incompatible with continued survival.
Note: All animals found dead or euthanized were necropsied for pathology score
“The criteria for euthanasia was based on an independent assessment by a veterinarian, according to AAALAC guidelines and only cases, where the condition of the animal was considered incompatible with continued survival, are represented in the curves”
Every animal found dead or euthanized was necropsied for pathology score.
Note: Pathology scores recorded for all animals
“Every animal found dead or euthanized was necropsied for pathology score”
Survival curves were plotted using the Kaplan-Meier method, which includes all available animals at each time point.
Note: All available animals included at each time point
“Survival curves were plotted using the Kaplan-Meier method, which includes all available animals at each time point”
C57BL/6 mice purchased from National Institute on Aging Aged Rodent Colony from Charles Rivers. B6C3F1 mice from Harlan Breeders (Indianapolis)