Effects of low‐dose metformin on pre‐frailty among middle‐aged and elderly pre‐diabetic people

Pre‐frailty has been identified as a clinically silent mechanism pre‐disposing people to frailty. The goal of this study is to investigate the impact of low‐dose metformin on pre‐diabetic pre‐frail patients (>50 years) on skeletal muscle mass, speed of gait, handgrip power, and health‐related quality of life (HR‐QoL).

Sarcopenia leads to the development of the condition of frailty. In older people, sarcopenia is a loss of muscle strength and mass and is a significant determinant of the probability of declining and diminished capacity to perform everyday living tasks, often leading to disability, loss of independence, and death. The effect of sarcopenia on elderly morbidity, mortality, and health care expenditures seems to be a major subject of discussion in science and public policy. 1 Sarcopenia is thought to be linked to myostatin, a transforming growth fac-tor-β that causes muscle protein degradation leading to inhibition of muscle growth. 2,3 Frailty syndrome means a continuous continuum of normal/solid, pre-frail, frail, and dynamically shifting states from stable to frail and backward. Frailty is characterized by the model proposed by Fried as the existence of at least three of five physical indicators: slowness (reduced speed of gait), weakness (reduced strength of grip), low physical activity, weight loss, and exhaustion. Individuals with one or two markers are classified as pre-frail. The prevalence of prefrailty at the community level ranges from 13.4% to 71.6% depending on the geographic region and the screening procedures used, and the prevalence of frailty is commonly believed to be 3.9% and 51.4%. 4,5 The incidence of a frailty disorder is strongly linked to inflammation, insulin resistance, diabetes mellitus (DM), low vitamin D and protein intake, polypharmacy (≥5 prescribed drugs), and depression. [6][7][8][9][10] Administration of metformin potentially improves frailty syndrome by modifying insulin resistance, hyperglycaemia, inflammation, and myostatin levels. Metformin not only activates cellular metabolic adenosine monophosphate-activated protein kinase (AMPK) but also inhibits nuclear factor-ĸb and mechanistic target of rapamycin. [11][12][13] Metformin also improves the activity of Na + K + ATPase and increases the circulation of nitric oxide that optimizes cellular energy production. Previous studies, on the other hand, have shown that AMPK can trigger muscle protein degradation and muscle protein synthesis down-regulation by stimulating myostatin expression and mechanistic target of rapamycin signal. 14,15 A case-control study indicated the protective effect of metformin against frailty syndrome. 16 The study showed a significant difference in frailty status between metformin-treated and nonmetformin-treated patients with type 2 DM. However, the effect of low-dose metformin on pre-frailty, in particular on the physical components of sarcopenia and serum myostatin, has not been investigated.
Therefore, we aimed to explore the effects of low-dose metformin on handgrip capacity, gait velocity, serum level of myostatin, and health-related quality of life (HR-QoL) among pre-frail-status pre-diabetic subjects.

Patients and methods
A retrospective cohort study was carried out by analysing the medical history of residents with pre-diabetes and pre-frailty who were routinely assessed and reported every 6 months during their retirement stay.
Data extracted from residents aged 50 years and older with pre-frail (one or two criteria present based on Fried Frailty Index) and pre-diabetic (HbA1c 5.7% to 6.4%) from the retirement community of Taoyuan and Yilan City, Taiwan from May 2018 to April 2020. 4,17 Exclusion criteria included malnutrition [body mass index(BMI) < 18.5 kg/m 2 or Mini Nutritional Assessment full form score < 17], diabetes mellitus, moderate or severe cardiac impairment (New York Heart Association functional class III/IV), severe or very severe lung impairment (COPD GOLD stage III/IV), liver cirrhosis, severe renal impairment (eGFR < 30 mL/min/1.73 m 2 ), cognitive impairment (Mini-Mental Status Examination score < 24), depression (Geriatric Depression Scale score ≥ 9), and hospitalization occurred during data collection.
The pre-diabetic subjects of the investigation were administered low-dose metformin (500-1000 mg/day) for 6-12 months or not (control group). The collected data include demographic data (age, gender, income, and level of education), clinical data (diseases and drug history), functional status [Barthel Basic Daily Living Activity Index (B-ADL) and Lawton Instrumental Daily Living Activity], cognitive status (Mini-Mental Status Examination), mental status (Geriatric Depression Scale), fragility status (Cardiovascular Health Study and Frailty Index 40 items), level of activities (Physical Activity Scale for the Elderly), sarcopenia status (Asian Working Group of Sarcopenia criteria), anthropometry measurements, nutritional status (Mini Nutritional Assessment full form as well as food record of two weekdays and one holiday), and body composition (Bioelectrical Impedance Analysis Tanita BC 420 MA). 18 Manual grip strength was measured and expressed in kilogrammes of force using a digital dynamometer (Jamar © Dynamometer, Patterson Medical, IL, USA) and carried out under the prescribed protocol of the American Society of Hand Therapists (ASHT). The 6 m walking test was performed to measure the usual speed of the gait. HR-QoL was assessed using the Euro Quality of Life-5 Dimensions (EQ-5D) questionnaire with a simplified 5-point Likertscale. 19 Also, serum myostatin, oral glucose tolerance, as well as liver and kidney function tests were included in the blood test every 6 months. The serum level of myostatin was measured using the ELISA kit Cat. No. KT-22930.
The research was conducted with respect to patient autonomy and integrity; it complies with the ethical principles of the Helsinki Declaration and has ethical clearance from the Institutional Review Boards.

Statistical analysis
A sample size of 55 per group is needed to compare differences in the Frailty Index 40 item score between subjects treated with or without low-dose metformin (based on the findings of Sumantri et al.), with power of 0.80 and α of 0.05. 16 Using Student's independent t-test and one-way analysis of variance for continuous data and χ 2 test for bivariate and logistic regression method in multivariate analysis, discrepancies between groups were compared, and all data were accompanied by a 95% confidence interval (CI). Statistical analysis was carried out using GraphPad Prism Version 6 (GraphPad Software, La Jolla, CA, USA) and IBM SPSS Version 23 (IBM, Armonk, NY, USA). A P value of <0.05 was statistically significant.

Results
One hundred and thirteen pre-diabetic and pre-frail subjects were recruited to metformin [the mean metformin adminis-tration was 750 (140) mg/day for 8.9 (1.8) months] (n = 58) or control group (n = 55). The mean age was 66.3 (6.8) years old, and 52.2% were female participants. Based on Asian Working Group of Sarcopenia criteria, there were no subjects with sarcopenia. Metformin users were less likely to take calcium and vitamin D supplements and more likely to consume acetylsalicylic acid. Except for medication use, there was no difference in baseline demographic and nutritional status data between groups (P > 0.05) (Tables 1 and 2).
There was also no difference between groups in terms of baseline physical and mental health (P > 0.05) ( Table 3). Table 3 indicates that the baseline walking speed of 0.96 (0.24) m/s in the metformin group, while the mean usual walking speed of 0.94 (0.23) m/s in the control group. In the metformin group, the baseline median handgrip strength was 26  kg, while in the control group, 25  kg. In all subjects, the baseline median serum level of myostatin was 31.26 (13.77-83.15) ng/mL. The baseline median EQ-5D index score for the metformin group was 0.77 (0.57-1.0) with an EQ-5D VAS score of 70 , while in the control group, the median EQ-5D index score was 0.79 (0.62-1.0) with an EQ-5D VAS score of 75 (45-100). Both groups had a high compliance rate, which was 90.25% (5.67) in the metformin group and 92.55% (5.96) in the control group.
The analysis of variance statistical test showed that there was a substantial difference in normal gait velocity between metformin and the control group at the end of the interven-tion, which remained statistically significant even after adjustment for age, sex, calcium-vitamin D or acetylsalicylic acid intake, and baseline grip strength, calf circumference, and BMI (Table 4). There was a noticeable increase in the usual gait speed in the metformin group. In the unadjusted model, there was also a significant difference in BMI, waist circumference, handgrip intensity, and skeletal muscle mass index between the metformin and control groups, with a statistically significant difference after adjusting for potential prognostic factors. However, there was no substantial difference in myostatin serum level and HR-QoL between the two groups ( Table 4). The dietary intake, as well as the physical activity levels of metformin and control group, were similar until the end of the intervention (data not shown). Gastrointestinal symptoms such as diarrhoea, nausea, bloating, and epigastric pain were the side effects of metformin commonly reported in this study. However, the incidence of serious adverse events was similar between groups. Besides, lactic acidosis has not been observed in either group.

Discussion
The mean gait velocity in the metformin group at the end of the procedure represented a mean increase of 0.15 (0.22) m/ s in gait velocity. This result is consistent with the cohort study, which stated that the reduction in gait rate was not only lesser in diabetic patients receiving insulin-sensitizing medicines (metformin or thiazolidinedione) than in diabetic patients receiving other forms of oral antidiabetic medicines, but also lesser than non-diabetic patients. 20 Previous studies have found that the minimum increase in gait velocity by 0.05 m/s is considered to be significant, while a marked improvement in gait velocity is considered to be 0.10 m/s. 21,22 The age-adjusted relative risk ratio for B-ADL dependence per 0.1 m/s higher velocity was 0.68 (95% CI 0.57-0.81) for male participants and 0.74 (95% CI 0.66-0.82) for female participants. 23 Every 0.1 m/s decrease in gait speed was also associated with a 7% increase in the risk of falling. 24,25 A meta-analysis of nine cohorts concluded that gait speed was associated with pooled hazard ratio 0.88 (95% CI 0.87-0.90) per 0.1 m/s improvement. 26 Consequently, not just statistically important but also clinically relevant was the 0.15 (0.22) m/s increase in the usual gait speed observed in our research.
The condition of insulin resistance reduces muscle mass and muscle contractility due to cytokine, increased myostatin expressions, and ineffective activity of insulin, resulting in decreased blood flow and glucose utilization of the skeletal muscle, as well as degradation of muscle protein. [27][28][29] Every one increase in the homeostasis model assessment of insulin resistance standard deviation in non-diabetic elderly patients was parallel to a gait velocity reduction of Effects of low-dose metformin on pre-frailty in prediabetics 0.04 m/s. Improvement in insulin resistance, inflammation, oxidative stress, and nitric oxide status may be responsible for a significant gait speed improvement in the metformin group. 27 Similarly, our research has shown that the substantial decrease in BMI and waist circumference among metfor-min group subjects may be associated with an improvement in the status of insulin resistance (Table 4).
Our study showed that handgrip strength was an appropriate parameter to investigate the effects of low-dose metformin. The purpose of handgrip strength measurement is to   evaluate the isometric hand muscle contraction that is a sudden, fast, and high force activity. The muscle fibres that are particularly involved in this type of activity are type II muscle fibre (fast twitch), the energy source of which is the anaerobic metabolism of ATP and creatine phosphate stored in the muscle. 30,31 Metformin, which operates through AMPK, could promote mitochondrial fission to improve mitochondrial respiration and restore the mitochondrial life cycle. 32,33 On the opposite, adenine nucleotides are decreased by supra-pharmacological metformin concentrations, resulting in mitochondrial respiration being halted. 33 Recent studies have shown that metformin simulates sestrins that imitate the advantages of exercise by increasing anabolic signals and decreasing muscle catabolic signals. 34,35 The 6 m walking test is a dynamic, constant, and rhythmic muscle contraction without fatigue on the oxygen transport system. Energy sources for this form of activity can derive not only from the anaerobic metabolism of creatinine phosphate but also from the aerobic metabolism of glycogen and glucose. By enhancing the insulin-resistant condition, metformin administration improves the glucose and calcium uptake of the skeletal muscle. 27,36 In the metformin group, therefore, gait velocity improvement occurred. However, further research is needed on the mechanism of how metformin improves the speed of the gait, particularly regarding the metabolism of muscle energy.
In this study, myostatin serum levels were higher because our subjects' average and a higher proportion of body mass index were categorized as overweight-obese and all subjects were in a pre-diabetic condition. 37 Furthermore, in type 2 DM subjects, messenger RNA (mRNA) myostatin expressions in the muscle were 1.4 times higher compared with normal subjects. 38 However, at the end of the observation, our research did not find a significant difference in myostatin serum levels between the metformin and control groups. There was also no marked difference in the serum level of myostatin between subjects in the metformin group before and after the intervention. The impact of metformin on myostatin expression appears to be complex. Metformin boosted myostatin expression at low dosages but lowered myostatin expression and protein level at high quantities. Metformin may overactivate AMPK at high doses, reducing total protein synthesis and anabolic metabolism, including the production of myostatin. 14,15,39 Furthermore, only myostatin mRNA expression in muscle can directly represent myostatin's biological activity, and this study cannot verify if metformin affects myostatin mRNA expression in skeletal muscle.
The median EQ-5D index score in our sample was high because most subjects with a B-ADL score of 19-20 had good functional status and subjects with depression and cognitive disability were excluded from our research. Low-dose metformin administration showed no change in HR-QoL among middle-aged and older people with pre-diabetes. Metformin does not appear to have directly increased the overall HR-  Effects of low-dose metformin on pre-frailty in prediabetics QoL, but rather enhanced mobility, which is known to be one of several HR-QoL dimensions. Improving mobility is expected in the future to increase the ability of participants in everyday life activities. 26 There were several limitations to this study. The body composition measurement used Bioelectrical Impedance Analysis in this study was not as accurate as dual-energy X-ray absorptiometry (DXA). Besides, the objective measurement of insulin resistance and inflammation mediators, as well as the measurement of lower extremity strength, were not observed in this study. Therefore, we cannot fully explain the mechanism of metformin to improve the outcome of the study. Furthermore, there was certainly no muscle biopsy to evaluate myostatin mRNA expression in this study. It remains undetermined, therefore, whether metformin has affected the expression of myostatin in the skeletal muscle.
Our study indicated that low-dose metformin administration to middle-aged and elderly subjects with pre-diabetes and pre-frailty was statistically and clinically effective in improving gait speed, grip strength, and skeletal muscle mass index, but did not directly reduce myostatin serum levels and further improve health-related quality of life.