Acarbose improves health and lifespan in aging HET3 mice

2.2. Body composition

Acarbose at 1,000 ppm was fed to separate groups of mice starting at 4 months of age, and weights and body composition then evaluated at two sites. At TJL and UT, fat and lean weights were measured by NMR body composition analysis, using the echo MRI^TM 3‐in‐1 (Table ​(Table2),2), in which grams of fat are the mass of the body's fat molecules expressed as g of canola oil, while grams of lean are muscle tissue mass equivalent of all body parts containing water, excluding fat, bone, and substances that do not contribute to the NMR signal, such as hair, teeth, and claws. Testing the same groups of ACA‐fed and age‐ and sex‐matched control mice at 16–17, 22–23 and 29–30 months of age, ACA reduced body and fat weights significantly in females, but had no significant effects on these measures in males. Lean mass was not affected by ACA in either sex (Table ​(Table2).2). At UT, ACA diets also had greater effects in females, reducing body weights by 8–12 g in females at 12–22 months of age (Figure ​(Figure2a),2a), and by 6–8 g in males over the same age range (Figure ​(Figure2d).2d). The decrease in % female body fat (Figure ​(Figure2b)2b) was about twice as great as in males (Figure ​(Figure2e),2e), while the increase in % lean mass in females fed ACA (Figure ​(Figure3c)3c) was two to three times greater than in males (Figure ​(Figure22f).

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Figure 2

Changes with age in body composition due to dietary ACA. Effects of 1,000 ppm ACA diet starting at 8 months of age on weights and body composition from 8 to 22 months of age at UT. Giving numbers of mice at 8–22 months of age: females (32–28 controls, 32–28 ACA‐treated); males (32–14 controls, 33–30 ACA‐treated). The same groups of mice were tested at 8, 12, 16, 20, and 22 months. ACA‐treated have blue dots; controls black dots. Data are shown as mean ± SE, and weights and body fat were significantly lower, while lean mass was significantly higher in both males and females fed the ACA diet as measured by 2‐way ANOVA using GraphPad version 7.03, which accounted for the missing mice

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Figure 3

Effects of ACA on fat depends on specific fat type. Effects of ACA on amount of fat relative to body weight in 12‐month‐old HET3 mice at UM. Numbers of male ACA and controls are 9 each, while 8 females received ACA and 6 were controls. In subscapular fat, ACA effects are sex‐specific: females, p = 0.71; males, p < 0.001. In mesenteric fat, ACA results in a strong reduction in both sexes: p < 0.001. In gonadal fat, ACA effects are similar in both sexes, but weak: p = 0.074. In Inguinal fat, ACA has no effect. p values, from analysis of covariance, reflect differences in the intercept term, which measures whether ACA alters fat pad weight after adjustment for body weight. HET3 mice were fed diet with 1,000 ppm ACA starting at 4 months; controls were fed the base diet. Mice were fasted for 18 hr prior to dissection. This was part of a larger study in which mice had a sham‐operation procedure at age 3 months. They were anesthetized, gonads were exteriorized through an incision and then returned to the abdominal cavity, and the wound closed

A separate set of data from UM defined the effects of ACA feeding on four fat depots using HET3 mice at 12 months of age (Figure ​(Figure3).3). In subscapular fat, there were no ACA effects in females, but highly significant effects in males (p < 0.001). In mesenteric fat, ACA caused highly significant reductions in both sexes (both p < 0.001). In gonadal fat, ACA effects were also similar in both sexes, but did not reach statistical significance, and in inguinal fat, ACA had no effect (Figure ​(Figure33).

2.3. Pathology

As illustrated in Figure ​Figure4,4, males fed ACA had about half as many lung tumors as controls, but ACA feeding did not reduce the already low frequency of lung tumors in females. The frequency of adrenal medullary vasodilation was reduced to an equivalent extent in both sexes, reaching statistical significance in females, and in the pooled data, though not in males (Figure ​(Figure4).4). Replicating results reported by Strong et al. (2016), ACA reduced the incidence of liver degeneration in both male and female UM‐HET3 mice, but only results in males and the pooled population were significant. Finally, females fed ACA had less glomerulosclerosis, but not males in which glomerulosclerosis was rare even in controls (Figure ​(Figure4).4). Lesions that were not affected by ACA feeding, to a significant degree, included endometrial hyperplasia, lymphoma, pancreatic atrophy, renal mineralization, adrenal hyperplasia, adrenal cortical degeneration, hepatic microgranuloma, uterine cysts, uterine cystic endometrial hyperplasia, ovarian atrophy, and ovarian pigment/lipofuscin; inferences about these negative findings would require confirmation using a larger number of test cases.

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Figure 4

Microhistopathology effects of ACA. Lesions are compared in controls and mice fed the 1,000 ppm ACA diet starting at 4 months of age. Numbers of mice: ACA old – 57 F and 54 M. The Control group consisted of 41 male and 43 female animals, of which 11 males and 14 females were contemporaneous with the current ACA population, and the remainder consisted (as a preplanned strategy) of controls from earlier cohorts also evaluated at 22 months of age. About a third of the mice came from each site. The values shown are percentage of cases with the indicated lesions, and p‐values reflect differences between two proportions using an asymptotically, normally distributed z statistic, as documented in the STATA program

2.4. Blood glucose after refeeding

Figure ​Figure55 shows young (4 month old) males and females that were fed either control diets (0 ppm ACA) or diets containing 400, 1,000, or 2,500 ppm ACA for 6 weeks. Food was removed at 6 p.m., and returned at 9 a.m. the next day. Blood glucose levels were measured just before food was returned, and 30, 60, 180, and 360 min after it was returned. There were no significant differences in females, but highly significant (p < 0.002) differences in males, in which higher levels of ACA in diets reduced blood glucose after refeeding.

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Figure 5

ACA effects on blood glucose after refeeding. Postprandial blood glucose is reduced in males but not in females by 6 weeks on ACA diets. Male and female UM‐HET3 mice were given one of four diets for 6 weeks starting at 4 months of age. Each point represents the mean ± SEM of 10 mice, tested at the indicated times. Green symbols indicate diets formulated with ACA at 2,500 ppm; blue 1,000 ppm; red 400 ppm; and white 0 ppm (control). Mice were fasted from 18:00 until 9:00 the next day. Blood glucose was measured at “0” min (before food was returned), and at 30, 60, 180, and 360 min after the food was returned to their cages. All measures were made in each sex in a single session, but in different sessions for each sex. Data were analyzed using RM one‐way analysis of variance (ANOVA) for male and female mice separately. In both sexes, we compared the differences between the four groups using GraphPad Prism 7.03. In females, the different diets did not affect blood glucose levels significantly (p = 0.092). In males, the different diets had significant effects on blood glucose (p = 0.002)

4.2. Removal of mice from the longevity population

Mice were removed from the study because of fighting or accidental death (e.g., during chip implantation) or chip failure, or because they were used for another experimental purpose. For survival analyses, all such mice were treated as alive at the date of their removal from the protocol and lost to follow‐up thereafter. These mice were not included in calculations of median longevity. Overall, <3% of the mice were removed from the longevity populations reported here, with no significant site differences. For details, see Methods to censor mice for lifespan statistics, Supporting Information Appendix S1 .

4.3. Estimation of age at death (lifespan)

At UM and UT, mice were examined daily for signs of ill health from the time they were set up in the experiment. At TJL, once mice were marked as ill, they were examined daily for signs of ill health. Mice were euthanized for humane reasons if so severely moribund that they were considered, by an experienced technician, unlikely to survive for more than an additional 48 hr. The TJL definitive endpoint criterion is the nonresponsiveness of a mouse to being touched, and which is usually accompanied by one or more of the following: slow respiration, feeling cold to the touch, a hunched‐up appearance with matted fur, and signs of sudden weight loss, failure to eat and drink, prominent appearing ribs and spine, and sunken hips.

The age at which a moribund mouse was euthanized was taken as the best available estimate of its natural lifespan. Mice found dead were also noted at each daily inspection.

4.4. Control and experimental diets

Studies with diets done at similar times are reported here, as even the diets with no effects act as useful controls. TestDiet^®, Inc., a division of Purina Mills (Richmond, IN, USA), prepared batches of LabDiet^® 5LG6 food containing each test substance, as well as control diets, at intervals of approximately 4 months, and shipped each batch of food to the three test sites.

1. ACA or Acarbose was purchased from Spectrum Chemical Mfg. Corp., Gardena, CA, product # A3965, CAS # 56180‐94‐0. It was fed continuously at a concentration of 400, 1,000, or 2,500 mg of ACA per kilogram of diet (ppm) to a test group of mice from cohort 2013 starting at 8 months of age. In independent groups of mice, it was fed at 1,000 mg/kg starting at 4 or 8 months of age, as indicated. Probably amounts of ACA were about a third of expected in the diet, as average concentration of acarbose in 5 batches of food pellets was 231 ± 109 (SD) ppm (intended dose 1,000 ppm). A primary effect of acarbose is to reduce postprandial glucose plasma levels in humans (Ruppin et al., 1988). We observed the same result as an outcome measure in this study (Figure ​(Figure5).5). The combination of these results indicates that the same pharmacokinetic and pharmacodynamic effects of acarbose were achieved with our study design. For details, see: Experimental diets—Acarbose, Supporting Information Appendix S1.
2. UA or Ursolic acid was obtained from Wilshire Technologies, Princeton NJ, CAS # 77‐52‐1. It was fed continuously at a concentration of 2,000 ppm to a test group of mice from cohort 2013 starting at 10 months of age.
3. HBX or 2‐(2‐hydroxyphenyl) benzothiazole was obtained from Sigma Aldrich, Inc., St Louis, MO, USA, product # {"type":"entrez-nucleotide","attrs":{"text":"H50802","term_id":"990643","term_text":"H50802"}}H50802, CAS # 835‐64‐3. It was fed continuously at a concentration of 1 ppm to a test group of mice from cohort 2012, starting at 15 months of age. In pilot studies, diets designed to contain 1 and 10 ppm HBX had 85% and 83% of expected amounts, respectively. Serum of mice fed 1 ppm HBX for 8 weeks had serum levels of 89–252 ng/ml HBX, pilot mice fed 10 ppm HBX for 8 weeks had <50 to 316 ppm, with no significant differences in serum levels between the two groups, so 1 ppm HBX was used in the full lifespan study.
4. I767d, INT‐767 or 6α‐ethyl‐24‐nor‐5β‐cholane‐3α,7α,23‐triol‐23 sulfate sodium salt was obtained from WIL Research, Inc., Ashland OH (in 2016 it was renamed Charles River Laboratories, Ashland), and the material was a proprietary product. It was fed continuously at a concentration of 180 ppm to a test group of mice from cohort 2012 starting at 10 months of age. In pilot studies, the amount of INT‐767 in the diet was 73% of the 180 ppm expected. After 8 weeks of diet containing INT‐767, amounts in serum ranged from 254 to 869 ng/ml while control serum contained less than 1.5 ng/ml.

4.5. Measuring amounts of interventions

Assays for acarbose, HBX, and I767 were done at UT. The initial drug material, food pellets, and mouse serum were sampled, prepared, and assayed as detailed in the material published here on line. Samples were quantified using HPLC with ultra violet detection. For details, see: Measuring amounts of Interventions, Supporting Information Appendix S1 .

4.6. Measures of body composition

At TJL, a Nuclear Magnetic Resonance (NMR) Imaging Instrument (EchoMRI™ 3‐in‐1, EchoMRI LLC, Houston, TX, USA) was used to measure body composition of fat, lean, free water, and total water masses. Subjects were placed in a restrainer tube (nonanesthetized), and three consecutive scans, 2 min in duration, were taken sequentially, and data were averaged for each subject. The fat calculation is measured as total body fat inclusive of organ and tissue fat and fatty acids. Lean mass is muscle plus all organs. At UT, mice were placed in a plastic cylinder (4.7 cm ID, 0.15 cm thick) in a qMRI machine (EchoMRI, Echo Medical Systems) and scanned once for 2 min. Data at both sites are expressed as % of body mass.

4.7. Pathology

Using standard microhistopathological methods, J. Erby Wilkinson, a board‐certified veterinary pathologist, examined 22‐ to 24‐month‐old UM‐HET3 mice: 57 females and 54 males had been fed a 1,000 ppm ACA diet since they were 4 months old. There were 14 female and 11 male controls set up at the same time, plus two previous groups of age‐matched UM‐HET3 controls, for a total of 43 females and 41 males not fed ACA. Evaluation of the ACA mice and the new set of age‐matched controls was conducted in a blinded fashion, and the data from the old control mice combined with prior records compiled by Dr. Wilkinson on earlier cohorts of untreated UM‐HET3 mice for statistical purposes.

4.8. Refeeding effects on blood glucose

Ten HET3 mice in each group were given one of four diets for 6 weeks starting at 4 months of age; the diets contained 2,500 ppm, 1,000 ppm, 400 ppm, or 0 ppm of ACA. Mice were then fasted from 18:00 to 09:00 the next morning. Tail vein blood was sampled (around 50 microliters) at “0” minutes before returning food, and again 30, 60, 180, and 360 min after the food was returned. Amounts of glucose were measured in each blood sample using a blood glucose meter One Touch Ultra Blue (Life‐Scan, Milpitas, CA, USA).

4.9. Rotarod

Rotarod performance was tested using the Rotamex‐5 (Columbus Instruments, Columbus, OH, USA) for 6 consecutive days by a technician who was blinded to the treatment groups. Testing on each day consisted of eight trials with a 10‐min rest between trials 4 and 5. Each trial began with the rotarod set at an initial rate of 4 rpm, accelerating to a maximum 40 rpm within 300 s. The latency to fall was recorded by the Rotamex‐5 software, and the average latency to fall was calculated for each day.

4.10. Statistical methods

Significance tests about survival effects are based upon the two‐tailed log‐rank test at p < 0.05, stratified by test site, with censored mice included up until their date of removal from the longevity population. Data from male and female mice are considered separately. In statistical tests described in the text, p values are two‐tailed and reported without adjustment for multiple comparisons, except in Figure ​Figure6.6. Statistical claims related to maximum lifespan are based on Wang et al., (2004), using the Fisher's exact test to compare the proportions of surviving mice, in control and test groups, at the age corresponding to the 90th percentile for survival in the joint distribution of the control and test groups. For the pooled data sets, surviving mice were enumerated at the 90th percentile age for each site separately, and these counts were combined for the overall Fisher's exact test.

This work was funded in part by NIA grants AG022308 (D.E.H.), AG022303 (R.A.M.), and AG022307 and AG013319 (R.S.), with important facilities supported by NCI grant CA034196 (JAX). Some of the studies at UM were supported by the Glenn Foundation for Medical Research. RS is supported by a Senior Research Career Scientist Award from the Department of Veterans Affairs Office of Research and Development. We wish to thank Peter Reifsnyder, Pam J. Krason, Vicki Ingalls, Natalie Perry, Lori Roberts, Roxann Alonzo, Ilkim Erturk, Nelson Durgin, and Vivian Diaz for reliable technical assistance, and The Jackson Laboratory's Center for Biometric Analysis for core services including generation of the NMR data.