The Effect of Empagliflozin and Metformin on Oxidative Capacity and Microvascular Reactivity of Skeletal Muscle in Patients With Type 2 Diabetes Mellitus

Type 2 diabetes mellitus is associated with impaired skeletal muscle mitochondrial oxidative capacity and microvascular dysfunction, both of which contribute to reduced exercise tolerance and may precede overt clinical complications. Sodium-glucose co-transporter 2 (SGLT2) inhibitors, such as empagliflozin, have demonstrated cardiovascular and renal benefits in patients with type 2 diabetes through mechanisms that extend beyond glycemic control, including possible effects on mitochondrial function and vascular reactivity. Metformin, the first-line pharmacological treatment for type 2 diabetes, has also been proposed to influence mitochondrial metabolism, though its comparative effect on skeletal muscle oxidative capacity remains incompletely characterized.

This is a prospective, randomized, open-label, interventional study comparing the effects of empagliflozin, metformin, and their combination on skeletal muscle oxidative capacity and microvascular reactivity in individuals with newly diagnosed type 2 diabetes. A total of 54 participants will be randomized in a 1:1:1 ratio (n=18 per group) to: (1) empagliflozin monotherapy, (2) metformin monotherapy, or (3) combination therapy with empagliflozin and metformin.

Study visits and procedures

Baseline visit (Day 0): Participants will be informed of the study procedures and purpose at the diabetes outpatient clinic and invited to participate voluntarily. Those who agree will sign informed consent. Medical records will be reviewed (duration of type 2 diabetes, comorbidities and their treatment, current medications). A clinical examination will be performed, including height, weight, body mass index, blood pressure, and heart rate. Participants will complete a physical activity questionnaire (adapted in part from the International Physical Activity Questionnaire, IPAQ). Maximal handgrip strength will be measured bilaterally using a dynamometer, and a 6-minute walk test will be performed. Blood will be drawn and urine collected for laboratory parameters.

Participants will be instructed to attend each testing visit between 7:00 and 9:00 AM, fasting (12-hour overnight fast), having abstained from caffeine, tobacco, and alcohol, and having avoided moderate-to-vigorous physical activity for at least 24 hours beforehand. To minimize the confounding effect of increased limb use, all measurements will be performed on the non-dominant arm.

Whole-body dual-energy X-ray absorptiometry (DXA) will be used to quantitatively assess body composition. An interstitial glucose monitoring sensor will be applied for the first month of follow-up. Near-infrared spectroscopy (NIRS) measurements will be performed over the flexor digitorum superficialis muscle of the non-dominant forearm at rest, after submaximal muscular work, and during transient brachial artery occlusion, allowing assessment of skeletal muscle oxidative capacity and microvascular reactivity (protocol below). Pharmacological antidiabetic treatment will then be initiated (empagliflozin, metformin, or the combination, per randomization).

Follow-up visits (Day 14, Month 1, Month 3, Month 6): The same protocol will be repeated at each follow-up visit. Body composition will be reassessed at the Month 3 and Month 6 visits.

NIRS sub-protocol Tissue oxygen saturation (StO2) will be measured over the flexor digitorum superficialis muscle using a tissue spectrometer. Three sequential measurements will be obtained: (A) resting StO2 after 5 minutes of rest; (B) StO2 during submaximal handgrip exercise (60% of maximal grip force) interspersed with brief arterial occlusions, used to derive an index of mitochondrial oxidative capacity from the StO2 recovery rate; and (C) StO2 during and following a transient brachial artery occlusion, used to assess microvascular reactivity (peak StO2 and recovery rate after cuff release).

Sample size Sample size was estimated from published values of skeletal muscle oxidative capacity in healthy individuals (2.0 ± 0.3 min-¹), assuming a 20% reduction at baseline in participants with diabetes and a shift toward normal values with empagliflozin treatment (1.7 ± 0.3 min-¹ vs. 2.0 ± 0.3 min-¹), with no effect expected from metformin. With alpha = 0.05 and power = 0.8, at least 16 participants per group are required; 18 per group will be enrolled to allow for dropout.

Statistical analysis Treatment-related changes will be analyzed using one-way repeated measures ANOVA with appropriate post-hoc testing. A P-value less than 0.05 will be considered statistically significant. Statistical analysis will be performed using MedCalc v14 (MedCalc Software bvba).

This study is conducted as a defined sub-study within a larger parent trial evaluating the effects of empagliflozin, metformin, and their combination on skeletal muscle function in individuals with type 2 diabetes (ethics approval by the Republic of Slovenia National Medical Ethics Committee, No. 0120-32/2024-2711-3). This sub-study applies more restrictive inclusion criteria to a homogeneous subgroup of participants (male, aged 40-60 years, BMI 25-35 kg/m²) to minimize confounding related to sex, age, and body composition when assessing the primary outcome of skeletal muscle oxidative capacity.

Inclusion criteria

• Male sex
• Age 18-75 years
• Newly diagnosed type 2 diabetes mellitus, confirmed no more than 2 years prior to enrollment
• Body mass index (BMI) 25-35 kg/m²
• Currently managed at a diabetes outpatient clinic
• No known chronic diabetes-related complications
• No prior antidiabetic pharmacological treatment, with the exception of sulfonylureas

Exclusion criteria

• Other forms of diabetes mellitus (i.e., not type 2)
• Type 2 diabetes mellitus known for more than 2 years
• Comorbidities known to independently affect skeletal muscle oxidative capacity, including: spinal cord injury, multiple sclerosis, amyotrophic lateral sclerosis, cystic fibrosis, local joint immobility, or hemiplegia
• Known mitochondrial disease
• Heart failure, NYHA class II or higher
• Chronic kidney disease, stage 3 or higher
• Poorly controlled chronic medical conditions (e.g., arterial hypertension, hypothyroidism)
• Recurrent urinary tract infections
• Active malignancy currently undergoing oncologic treatment
• Life expectancy less than 3 months
• Symptomatic hyperglycemia with fasting plasma glucose above 18 mmol/L, HbA1c ≥ 10%
• Unwillingness to provide informed consent