Baseline characteristics of the obese type 2 diabetes patients

Demographic characteristics, including concomitant medications, adiposity, blood pressure, glucose control, systemic inflammation, renal function, and hepatic steatosis, were not significantly different among the three treatment groups (Table 1). No serious adverse effects or hypoglycemia over the 12 weeks of intervention were found.

Table 1 Baseline characteristics of participants. Full size table

Primary endpoints: the amount of visceral adiposity, glucose control, and insulin resistance

Effects of MES + HS over time compared with the baseline

From randomly selected 6 subjects, HSP72 expression after the treatment was increased approximately 1.5 times in isolated monocytes compared to before the treatment (Fig. 1. lower right panels) Table 2. This indicates that the activation of HSR was similar levels in our previous study6.

Table 2 Effects of MES + HS over time compared with baseline. Full size table

Figure 1 Outcomes of MES + HS treatment depending on intervention frequency. The absolute changes (average with standard error of the mean) of the markers such as VFA (visceral fat area), SBP (systolic blood pressure), DBP (Diastolic blood pressure), HbA1c (glycated hemoglobin), FPG (fasting plasma glucose), HOMA-IR (homeostasis model assessment as an index of insulin resistance), GA (glycated albumin), adiponectin, TNF-α (tumor necrosis factor- α), WBC (white blood cell count), ACR (albumin creatinine ratio), L-FABP (liver-type fatty acid binding protein), LDL-C (low density lipoprotein cholesterol) and FFA (free fatty acid) on the MES + HS treatment frequency (two, four or seven/w) from each baseline were indicated. The numbers in group: two/w: n = 22, four/w: n = 19, seven/w: n = 19. *p < 0.05. compared to baseline. **p < 0.01. compared to baseline. †p < 0.05. Four/w v.s. Seven/w. #p < 0.05. Two/w v.s. Four/w. ¶p < 0.05. Two/w v.s. Seven/w. ¶¶p < 0.01. Two/w v.s. Seven/w. Lower right panels indicate HSP72 protein expression in monocytes isolated from pre (MES + HS (−)) and after (MES + HS (+)) the treatment of MES + HS (seven times per week). Full size image

Adiposity

The visceral fat area (VFA), measured by computed tomography scan, decreased by 11.69 cm2 (from 166.12 ± 7.75 to 154.44 ± 6.76 cm2; p < 0.001) following MES + HS treatment compared with the baseline value, while the subcutaneous fat area (SFA) was not changed (from 207.11 ± 11.95 to 204.04 ± 12.14 cm2; −3.07 cm2; p = 0.205). As a result, the total fat area decreased significantly by 14.75 cm2 following MES + HS treatment. Body mass index (BMI: from 29.14 ± 0.56 to 28.88 ± 0.58 kg/m2; −0.25 kg/m2; p = 0.001) and waist circumference (Wc: from 100.10 ± 1.33 to 97.86 ± 1.23; −2.24 cm. p < 0.001) both also decreased.

Glucose control and insulin resistance

Fasting plasma glucose (FPG) reduced from 159.38 ± 5.30 to 144.45 ± 4.21 mg/dL (−14.93 mg/dL, p < 0.001). Fasting immune-reactive insulin also decreased, from 9.77 ± 0.74 to 7.91 ± 0.59 μIU/mL (−1.86 μIU/mL, p < 0.001). Hence, HOMA-IR improved from 4.08 ± 0.42 to 2.99 ± 0.29 (−1.09, p < 0.001). HbA1c declined from 7.64 ± 0.08% to 7.28 ± 0.08% (−0.36 ± 0.07%, p < 0.001) and glycated albumin (GA) also dropped from 19.15 ± 0.41 to 18.40 ± 0.39% (−0.75%, p = 0.004). As a result, the clinical target of HbA1c less than 7.0% was achieved by 38.3% (n = 23) of participants after MES + HS treatment. Adiponectin increased from 7.54 ± 0.47 to 8.47 ± 0.54 μg/mL (+0.93 μg/mL, p < 0.001). Furthermore, multiple regression analysis indicated that the decrease of VFA was significantly correlated with the amount of VFA before the treatment (r = 0.565, p < 0.001) and the reduction of HbA1c was significantly correlated with baseline HbA1c (r = 0.485, p = 0.002).

Effects of MES + HS depending on treatment frequency

MES + HS treatment does not have appropriate placebo control because this apparatus transmits heat and electric stimulation simultaneously, and these different frequencies of intervention may be necessary to identify the exact clinical effects of MES + HS (Fig. 1 and Table 3).

Table 3 Effects of MES + HS depending on frequency of treatment. Full size table

Adiposity

VFA was compared among two, four, and seven treatments per week, and the amplitude of reduction was −5.37, −14.24, and −16.45 cm2, respectively (Fig. 1). The trend of VFA decreases in seven treatments per week group was observed compared with that in two treatments per week group (p = 0.054). This trend was also observed in four treatments per week group compared with the two treatments per week (p = 0.071). There were no significant differences between the four and seven treatments per week groups. The changes in Wc showed similar trends, but SFA did not.

Glucose control and insulin resistance

FPG indicated a trend toward decreasing in the seven treatments per week group compared with that in the two treatments per week group (−8.32 vs. −20.47 mg/dL, p = 0.093. Fig. 1). HOMA-IR showed a significant decline in the four treatments per week group compared with that in the two treatments per week group (p = 0.043). Reductions in HbA1c following MES + HS in two, four, and seven treatments per week were −0.10 ± 0.11%, −0.36% ± 0.12 and −0.65% ± 0.10, respectively (Fig. 1). The decrease of HbA1c in the seven treatments per week group was significantly greater than that in the two or four treatments per week group (2 vs. 7, p = 0.001; 4 vs. 7, p = 0.036). GA showed a similar trend in reduction, but did not reach a significant difference. Adiponectin levels were increased over time in all groups, but were not different among the three groups.

Effects of MES + HS in sex differences

Adiposity

VFA significantly decreased from 177.28 ± 10.25 to 161.72 ± 8.87 cm2 (−15.55 cm2, p < 0.001, n = 48) in male but not female participants (−2.66 cm2, p = 0.262, n = 22). The amplitude of VFA reduction was significantly larger in male than in female participants (p = 0.017). SFA showed a trend toward reduction in female participants (−10.59 cm2, p = 0.068), but not in male participants (+0.16 cm2, p = 0.485). BMI (−0.30 kg/m2, p = 0.003) and Wc (−2.67 cm, p < 0.001) were both decreased only in men, but the sex differences were not obvious (Table 4).

Table 4 Effects of MES + HS in sex differences. Full size table

Glucose control and insulin resistance

FPG (from 167.50 ± 6.86 to 145.31 ± 5.27 mg/dL, −22.19 mg/dL, p < 0.001) and HOMA-IR (from 4.23 ± 0.56 to 2.92 ± 0.37, −1.31, p < 0.001) both significantly improved only in males. The sex difference in FPG was significant (p < 0.001). HbA1c showed a minor trend toward reduction in female participants (Δ = −0.17 ± 0.15%, p = 0.140) and a significant decrease in male participants (Δ = −0.44 ± 0.07%, p < 0.001). The difference in the reduction of HbA1c was obvious (p = 0.033). GA indicated a similar change of +0.12% (p = 0.384) in female and −1.12% (p < 0.001) in male participants, and the difference was significant (p = 0.017). However, the level of adiponectin increased both in female (+1.07 μg/mL; p = 0.011) and male participants (+0.87 μg/mL; p < 0.001).

Secondary outcomes: blood pressure, systemic inflammation, renal function, hepatic steatosis, and lipids

Effects of MES + HS over time compared with baseline

Systemic inflammation

Tumor necrosis factor (TNF)-α levels in serum decreased from 1.64 ± 0.11 to 1.24 ± 0.08 pg/mL (−0.40 pg/mL; p < 0.001). Interleukin (IL)-6 was not changed significantly (from 3.17 ± 0.29 to 2.78 ± 0.31 pg/mL, −0.44 pg/mL; p = 0.105). C-reactive protein (CRP) decreased from 1968.77 ± 442.90 to 1305.17 ± 246.04 ng/mL (−663.60 ng/mL; p = 0.008) and the counts of white blood cells (WBC) also decreased from 6751.67 ± 204.86 to 6315.00 ± 183.01/μL (−436.67/μL; p < 0.001) (Table 2).

Renal function

The estimated glomerular filtration rate (eGFR) was elevated from 76.52 ± 2.23 to 79.48 ± 2.35 mL/min/1.73 m2 (+2.96 mL/min/1.73 m2; p < 0.001). Renal albumin excretion, estimated by urinary albumin creatinine ratio (ACR) was reduced from 75.30 ± 20.87 to 56.47 ± 15.82 mg/g Cre (−18.73 mg/g Cre; p = 0.015). Oxidative stress marker in renal tubules, evaluated by liver-type fatty acid-binding protein (L-FABP), was decreased from 5.42 ± 0.53 to 4.38 ± 0.49 μg/g Cre (−1.04 μg/g Cre; p = 0.007) as well.

Hepatic steatosis and lipids

The AST/ALT ratio increased from 0.86 ± 0.03 to 0.92 ± 0.04 (+0.06; p = 0.007). Uric acid (UA) slightly, but significantly, decreased from 5.58 ± 0.19 to 5.43 ± 0.18 mg/dL (−0.15 mg/dL; p = 0.042). Low-density lipoprotein cholesterol (LDL-C) showed a trend toward reduction (from 109.30 ± 3.54 to 105.80 ± 3.45 mg/dL, −3.50 mg/dL, p = 0.055). High-density lipoprotein cholesterol (HDL-C) and free fatty acid (FFA) were not changed by MES + HS treatment. Triglyceride (TG) levels were significantly reduced from 183.40 ± 18.75 to 153.38 ± 12.46 mg/dL (−30.02 mg/dL; p = 0.015).

Effects of MES + HS depending on frequency of treatment

Blood pressure

A decrease in diastolic blood pressure (DBP) but not systolic (SBP) was observed in the seven treatments per week compared with that in the two or four treatments per week group (p = 0.050 and 0.033, respectively. Figure 1) (Fig. 1 and Table 3).

Systemic inflammation

Although chronic inflammatory markers such as TNF-α, IL-6, CRP, and the WBC count decreased over time in all intervention groups, the differences among groups were not statistically significant (Fig. 1).

Renal function

eGFR was elevated and ACR and L-FABP were decreased in every intervention group, but a significant difference was observed only in L-FABP in the seven treatments per week group compared with that of two treatments per week group (p = 0.046 Fig. 1).

Hepatic steatosis and lipids

Almost all markers of hepatic steatosis (AST/ALT ratio), UA, and lipid profiles showed favorable trends of changes in all groups. Significant reductions in LDL-C were observed in the seven treatments per week compared with that in the two treatments per week group (p = 0.028. Fig. 1).

Effects of MES + HS in sex differences

Blood pressure

The decreases in SBP (−4.38 mmHg; p = 0.012) and DBP (−2.74 mmHg; p = 0.006) were observed only in male participants, and the sex differences were statistically significant (p = 0.030 and p = 0.029, respectively). Heart rates were also reduced only in male participants (−2.31 beats/min; p = 0.044) (Table 4).

Systemic inflammation

Although TNF-α decreased in both female (−0.36 pg/mL; p = 0.020) and male participants (−0.42 pg/mL; p < 0.001), IL-6 (−0.87 pg/mL; p < 0.001), CRP (−907.26 ng/mL; p = 0.001), and WBC (−478.57/μL; p < 0.001) were reduced only in males. The sex difference in reduction of IL-6 was significant (p = 0.008).

Renal function

eGFR (+2.33 mL/min/1.73 m2 in women, p = 0.034; +2.66 mL/min/1.73 m2 in men, p = 0.001) and L-FABP (−0.97 μg/g Cre in women, p = 0.025; −1.06 μg/g Cre in men, p = 0.032) were ameliorated in female as well as male participants. A sex difference was not observed.

Hepatic steatosis and lipids

Amelioration of the AST/ALT ratio (+0.07; p = 0.019), UA (−0.18 mg/dL; p = 0.043), HDL-C (+2.14 mg/dL; p = 0.037), TG (−22.07 mg/dL; p = 0.036), and free fatty acid (FFA: −70.40 μEq/L; p = 0.038) was observed only in male participants. Significant sex differences were observed in the increases of HDL-C (p = 0.011).

Sub-analysis with or without DPP-4 inhibitors

In most cases, the favorable changes in biophysical and biochemical parameters were not influenced by concomitant anti-diabetic medications. However, FPG was significantly reduced from 162.16 ± 6.96 to 142.29 ± 5.41 mg/dL (−19.87 mg/dL; p < 0.001) in subjects prescribed with DPP-4 inhibitors, but only a trend in those without DPP-4 inhibitors (−6.41 mg/dL; p = 0.082). The reduction in FPG was significantly greater in subjects with DPP-4 inhibitors than those in without DPP-4 inhibitors (p = 0.030). A significant difference was observed in the decrease of L-FABP (−1.56 μg/g Cre; p = 0.049 vs. without DPP-4 inhibitors) and in the increase of the AST/ALT ratio (+0.09; p = 0.031 vs. without DPP-4 inhibitors) in subjects with DPP-4 inhibitors (Supplementary Table 3).