Table 1.

Summaries of studies cited in the review with a focus on the effects of metformin on lifespan.

Study	Protocol	Results
Bannister et al. (69)	• T2DM patients treated with metformin or sulfonylurea monotherapy were compared to age- and sex-matched non-diabetic control groups in a retrospective observational analysis from the UK Clinical Practice Research Datalink.	• Patients prescribed sulfonylureas had lower survival rates than non-diabetic controls and diabetic patients prescribed metformin. • Diabetic patients taking metformin had more co-morbidities, but their survival rates were comparable to the non-diabetic control group. • Conclusion: Metformin extends healthspan, but not lifespan in humans.
Willcox and Willcox (70)	• Okinawans have long lifespans. Epidemiological data on older Okinawans, on a caloric restriction-like diet for approximately half their lives, (caloric restriction (10-15%), consumption of foods that mimic biological effects of calorie restriction, and phenotypic evidence consistent with caloric restriction (low body weight, and BMI).	• Caloric restriction likely contributed to the extended healthspan and lifespan of the Okinawans.
Onken and Driscoll (71)	• The effects of metformin on the healthspan and lifespan of the nematode Caenorhabdatis elegans and linked to activation of the serine-threonine kinase LKB1 and AMPK. Benefits of metformin were dependent on expression of the stress-responsive SKN-1/Nrf2, but independent of the insulin-signaling pathway?	• Exposure to 50 mM, but not 1 or 10 mM, metformin significantly enhanced survival of C. elegans by 27%, significantly right-shifted the survival curve and promoted ‘youthful’ mobility. The effects were not observed in the EAT-2 DR model of calorie restriction or in models with deficient AMPK, LKB1 or SKN-1/Nrf2. • Conclusion: Metformin via AMPK/LB1/SKN-1/NrF2 axis extends lifespan in C. elegans.
Espada et al. (72)	• The effects of metformin on lifespan were studied in different age groups of young C.elegans: AD1 -young adults AD4 –adults declining in reproductive potential AD8 –Middle aged AD10 -Old	• Exposure to 10, 25 and 50 mM metformin in AD1 and AD4, 25 and 50 mM decreased life expectancy in AD8, and in old C.elegans (AD10) all concentrations proved toxic. Toxicity was linked to a decrease in mitochondria and lower levels of ATP in the older worms that resulted in enhanced toxicity to metformin. Mutants that were resistant to metformin toxicity had higher mitochondria content and expression of complex 1.
Anisimov et al. (66); Also see Anisimov et al. (73) and Anisimov et al. (74)	• Anisimov et al. (66): Studies in old vs. young mice • 160 female Swiss-H Rappolovo (SHR) mice were in [100 mg/kg] daily, vs. tap water without metformin [control]. • Anisimov et al. (74): Comparison of treatment with metformin: • a) started at 3 months of age. • b) started at 9 months of age. • c) started at 15 months of age.	• When added to the diet of SHR mice, metformin slowed aging and increased lifespan, but did not lower incidence of spontaneous tumors. The anti-aging effectiveness of metformin was reduced in older mice. • Conclusion: Metformin effects on lifespan in mice are age-dependent. • Anisimov et al. (74): Metformin extended lifespan by 14% when started at 1 month but at 9 months only by an insignificant 6%, and at 15 months was ineffective. • In the 2003 paper Anisimov et al demonstrate a comparable lifespan extending effects in mice and rats with the biguanides, phenformin and busoformin that also attenuated tumor development suggesting to the authors that they are potential geroprotetors.
Alfaras et al. (75)	• Intermittent [either every other week (EOW) or two weeks out of 4 (2WM)] treatment of aged male C57/BL6 mice for 17 weeks with 1% metformin in diet	• Intermittent metformin treatment did not lead to early mortality. • EOW metformin resulted in weight loss and improved insulin sensitivity, but not lifespan extension. • Compared to controls evidence of increase in renal lesions in EOW and 2WM mice.
Martin-Montalvo et al. (76)	• Cohorts of middle-aged mice were fed either a normal diet or a standard diet supplemented with 0.1% (w/w) or 1% (w/w) metformin, for the remainder of their lives.	• In male mice, long-term treatment with 0.1% metformin w/w resulted in serum levels of 450 μM increased lifespan by 4.15%, and reduced NF-κB in the liver. • A higher dose of 1% metformin w/w resulted in serum levels of 5 mM and was toxic and resulted in a 14.4% reduction in the average lifespan of mice. • Parallel studies in mouse embryonic fibroblasts demonstrated activation of AMPK without affecting mitochondria electron transport activity. • Conclusion: Metformin extends lifespan in mice, but dose-dependent.. • Note: Comments on the western blot data are available on pubpeer.
Strong et al. (77)	• The National Institute on Aging Interventions Testing Program (ITP) dataset evaluated 0.1% metformin and rapamycin (14 ppm) effects on lifespan in mice.	• Metformin alone did not increase lifespan, but in combination with rapamycin, a benefit was reported. The authors speculate that the ‘benefit’ of metformin is via offsetting the negative effects of rapamycin on metabolism. • Conclusion: Metformin does not extend lifespan in mice.
Smith et al. (78)	• Male Fischer rats from 6 months of age were subjected to either a calorie restricted (CR) diet (70%), or dietary metformin (300 mg/kg/day) versus a control group. Metabolic parameters, body weight and lifespan, were determined.	• Based on Kaplan-Meier survival plot analysis, metformin did not extend lifespan versus control, whereas CR delayed early mortality. • Conclusion: Metformin is not a calorie-restriction mimetic (CRM)
Kulkarni et al. (79)	• MILES Trial commenced October 2014. A crossover, double-blinded, study with 14 elderly subjects with impaired glucose control and each serving as their own control. Subjects were treated with 1700 mg/day metformin for 6 weeks and transcriptomic studies of biopsies from skeletal muscle and subcutaneous adipose tissue were conducted.	• 647 genes were differentially expressed in muscle versus 146 in adipose tissue affecting both metabolic and non-metabolic pathways • Changes in DNA repair and, since repair function declines with age, data suggests an anti-aging action. Similar results for changes in collagen gene expression. • In adipose tissue changes were observed in fatty acid and lipid metabolism as reflected by PPAR and SREBP signaling. • In skeletal muscle changes in pyruvate metabolism, NAD biosynthesis, and down regulation of PARP1 were observed. The latter suggesting an effect on mitochondria function. • Conclusion: Preliminary evidence implying metformin effects on transcription and pathways affecting healthspan and lifespan.
ai. Gerstein et al. (80)	• Analysis of 237 biomarkers from 8401 participants with diabetes or impaired glucose tolerance in the ORIGIN trial (Outcome Reduction with Initial Glargine Intervention).	• ai. Analysis identified 10 biomarkers that identified dysglycemic subjects at higher versus lower CV risk.
aii. Gerstein et al. (81)	• Analysis of the biomarker profile of the 28% of the 8,401 participants in the ORIGIN trial who were receiving metformin.	• aii. Subjects taking metformin also had higher GDF15 (Growth Differentiation Factor 15) levels and lower CV outcomes.
b. Tanaka et al. (82)	• b. Proteomic analysis of plasma from 240 healthy, disease-free, subjects in the age range of 23-93 years.	• b. Plasma levels of GDF15 correlated with chronological age.
c. Coll et al. (83)	• Metformin vs. placebo treatment of fat fed mice either expressing GDF15 or lacking the receptor, GRAL, or treated with a GRAL antagonist.	• Weight loss effect shown to be dependent on expression of the GDF15 receptor, GRAL, (glial cell-derived neurotrophic factor family receptor alpha-like) whereas the antihyperglycemic effect of metformin was independent of the GDF15-GRAL pathway.
d. Modi et al. (84); Wischhusen et al. (85)	• d. Review of literature re. expression levels of GDF15 in various cancers and signaling pathways via EGFR and PI3K, Akt pathways.	• GDF15 a putative prognostic indicator of tumor progression and therapeutic target and raising the question as to whether GDF15 serves as a tumor suppressor, or as a promoter and is a target for the treatment of cancer.