Changes in lipid biomarkers, liver stiffness and steatosis following sodium-glucose cotransporter two inhibitor therapy in type two diabetes

Table 1 Basic sociodemographic characteristics and comorbidities of study participants (n = 67)

Characteristic		n (%)
	Median age and range in years	66 (37-89)
Gender	Male	40 (59.7)
	Female	27 (40.3)
Duration of diabetes mellitus type 2 (T2DM)	< 5 years	40 (59.7)
	5-10 years	16 (23.9)
	11-15 years	5 (7.5)
	> 15 years	6 (9.0)
Comorbidities (other than T2DM)	Hypertension	53 (79)
	Kidney disease	9 (13.4)
	Hyperlipidemia	42 (62.7)
	Hypothyroidism	3 (4.5)
	Hyperthyroidism	1 (1.5)
	Depression	4 (6.0)
	Anxiety	2 (3.0)
	Liver disease	6 (9.0)
	Gallbladder and bile duct disease	5 (7.5)
Other comorbidities		14 (20.9)

Table 2 Changes in alcohol consumption, smoking status, and lifestyle characteristics of the study participants between baseline and 6 months of sodium-glucose cotransporter 2 inhibitor therapy

Parameter		n (%) or n/N1		P value2
		Baseline	6 months
Frequency of alcohol consumption	No alcohol consumption	31 (46.3)	29 (43.9)	0.36
	Occasional alcohol consumption	32 (47.8)	31 (47.0)
	Regular alcohol consumption	4 (6.0)	6 (9.1)
1Regular drinkers-frequency (N = 4) (e.g., 250 mL of beer, 125 mL of wine or 30 mL of spirits)	Up to 2 drinks per week	1/4	1/4	0.323
	Up to 2 drinks per day	2/4	3/4
	Up to 3 drinks per day	1/4	0
Cigarette smoking status	Never smoker	28 (42.4)	23 (36.5)	0.25
	Former smoker	23 (34.8)	24 (38.1)
	Current smoker	15 (22.7)	16 (25.4)
1Daily cigarette consumption among smokers (N = 15)	≤ 20 cigarettes/day	5/15	7/15	0.183
	21-40 cigarettes/day	9/15	8/15
	> 40 cigarettes/day	1/15	0
1Duration of smoking (N = 15)	1–10 years	1/15	1/15	> 0.99
	More than 20 years	14/15	14/15
Main daily activity lasting more than 6 hours (N = 65)	Sitting	30 (46.2)	30 (40.2)	0.90
	Standing	10 (15.4)	8 (12.3)
	Walking	26 (38.5)	27 (41.5)
Number of main meals consumed per day (N = 66)	One	4 (6.1)	3 (4.5)	0.51
	Two	25 (37.9)	21 (31.8)
	Three	30 (45.5)	38 (57.6)
	Four or more	7 (10.6)	4 (6.1)
Dietary supplement use		10 (14.9)	16 (26.7)	0.06

¹n denotes the number of participants, and N denotes the total number of participants in the relevant subgroup (current smokers or regular drinkers).

²McNemar-Bowker’s test.

³Marginal Homogeneity test.

Table 3 Changes in biochemical and anthropometric parameters from baseline to 6 months after initiation of sodium-glucose cotransporter 2 inhibitor therapy

Parameter	Baseline, median (IQR)	6 months, median (IQR)	Difference1	95%CI	P value2
BMI (kg/m2)	31.46 (28.35-34.67)	30.12 (28.09-34.34)	-0.79	-1.1 to -0.5	< 0.001a
WC (cm)	107 (102-115)	104 (97.75-107)	-4	-5 to -2.5	< 0.001a
PG (mmol/L)	6.9 (6.3-8.6)	6.4 (5.5-7.6)	-0.6	-1 to -0.2	0.006a
HbA1c (%)	7 (6.3-8)	6.7 (6.3-7.3)	-0.25	-0.5 to -0.05	0.008a
HbA1c (mmol/mol)	52.5 (44.3-63.8)	50 (45-56)	-2	-4.5 to 0	0.04a
ALT (U/L)	28 (19-36)	26 (19-35)	-0.5	-3.5 to 1	0.41
AST (U/L)	25 (18-29)	24 (20-30)	0.5	-1.5 to 2	0.63
ALP (U/L)	67.5 (55-83.3)	67 (56.8-84.3)	0.5	-2 to 3.5	0.61
GGT (U/L)	26 (20-43)	25 (19-40)	-0.5	-2 to 1.5	0.68
Erythrocytes (1012/L)	4.7 (4.5-5.1)	5 (4.6-5.4)	0.24	0.15 to 0.32	< 0.001a
Hemoglobin (g/L)	141 (133-149)	146 (134-158)	4.5	2.5 to 6.5	< 0.001a
Hematocrit (L/L)	0.4 (0.4-0.4)	0.4 (0.4-0.5)	0.02	0.009 to 0.02	< 0.001a
MCV (fL)	88.3 (85.6-91.8)	88 (84.8-91.1)	-0.8	-1.5 to -0.2	0.02a
MCH (pg)	29.6 (28.7-30.5)	29.5 (27.8-30.8)	-0.4	-0.7 to -0.2	0.001a
MCHC (g/L)	335 (330-341)	334 (327-342)	-2	-4 to 0	0.06
RDW (%)	13.3 (12.7-14.1)	13.5 (13-14.5)	0.3	0.15 to 0.6	< 0.001a
Platelet count (109/L)	237 (202-270)	232 (193-264)	-3.5	-11.5 to 5	0.42
Leukocytes (109/L)	8 (6.3-9.3)	7.9 (6.9-9.6)	0.3	-0.05 to 0.55	0.09
Albumin (g/L)	44.7 (43-46.7)	45.3 (43.2-47.2)	0.3	-0.35 to 0.9	0.32
Total protein (g/L)	72 (69-75)	74 (70-77)	1.9	0.95 to 2.65	< 0.001a
Prothrombin time (ratio)	1.1 (1-1.2)	1.1 (1.1-1.2)	0.05	0.02 to 0.08	0.002a
Prothrombin time (INR)	1 (0.9-1.1)	1 (0.9-1)	-0.02	-0.05 to 0	0.001a
Fibrinogen-activity (g/L)	3.5 (2.9-4)	3.7 (3.1-4.3)	0.2	0 to 0.4	0.05
Thrombin time (second)	19.4 (18.4-21.5)	19.8 (19.2-20.8)	0.45	-0.4 to 1.3	0.33
Total bilirubin (µmol/L)	12 (9-14)	12 (9.8-16.3)	1.0	0 to 2	0.02a
Conjugated bilirubin (µmol/L)	2 (1-3)	2 (2-3)	0	0 to 0.5	0.27
Cholesterol (mmol/L)	4.4 (3.7-5.2)	4.2 (3.5-5.3)	0	-0.2 to 0.2	0.86
HDL (mmol/L)	1.2 (1-1.5)	1.3 (1.1-1.5)	0.05	-0.001 to 0.1	0.15
LDL (mmol/L)	2.4 (2-3.2)	2.3 (1.8-3.3)	-0.05	-0.25 to 0.1	0.42
Triglycerides (mmol/L)	1.9 (1.3-2.5)	1.8 (1.3-2.6)	0.03	-0.2 to 0.2	0.79

¹Hodges-Lehmann difference of median.

²Wilcoxon test.

^aP < 0.05.

ALT: Alanine aminotransferase; ALP: Alkaline phosphatase; AST: Aspartate aminotransferase; BMI: Body mass index; CI: Confidence interval; GGT: Gamma-glutamyl transferase; HbA1c: Glycated hemoglobin; HDL: High-density lipoprotein; INR: International normalized ratio; IQR: Interquartile range; LDL: Low-density lipoprotein; MCV: Mean corpuscular volume; MCH: Mean corpuscular hemoglobin; MCHC: Mean corpuscular hemoglobin concentration; RDW: Red cell distribution width; PG: Plasma glucose; WC: Waist circumference.

Table 4 Changes in liver stiffness assessed by two-dimensional shear wave elastography, steatosis assessed by ultrasound-guided attenuation parameter, and lipogenesis biomarkers measured by enzyme-linked immunosorbent assay from baseline to 6 months after initiation of sodium-glucose cotransporter 2 inhibitor therapy

Parameter	Baseline, median (IQR)	6 months, median (IQR)	Difference1	95%CI	P value2
Ultrasound imaging
E Median 2D-SWE (kPa)	6.2 (5.7-7.5)	6.1 (5.4-6.8)	-0.51	-0.93 to -0.21	< 0.001a
Med UGAP (dB/cm/MHz)	0.7 (0.7-0.8)	0.7 (0.6-0.8)	-0.02	-0.04 to 0.01	0.14
E IQR 2D-SWE (kPa)	1.3 (0.8-2.1)	0.1 (0-0.1)	-0.37	-0.69 to -0.09	0.005a
IQR UGAP (dB/cm/MHz)	0.05 (0.04-0.07)	0.06 (0.04-0.07)	0.01	-0.005 to 0.02	0.24
ELISA
PPAR-γ (ng/L)	895 (727.8-1105.6)	565.4 (336.7-839.8)	-297.03	-399.7 to -196.7	< 0.001a
PPAR-α (ng/L)	1667 (1133.7-3223.7)	1294 (779-2514)	-274.8	-818.5 to 308.7	0.38
SREBP-1c (ng/L)	5151.5 (3037.4-6969.7)	4900 (4100-5700)	174.3	-729.2 to 1062.0	0.72
MTTP (ng/L)	198.4 (103.8-301.3)	428.9 (351.6-532.1)	233.9	183.1 to 277.4	< 0.001a

¹Hodges-Lehmann difference of median.

²Wilcoxon test.

^aP < 0.001.

UGAP: Ultrasound-guided attenuation parameter; ELISA: Enzyme-linked immunosorbent assay; PPAR-γ: Peroxisome proliferator-activated receptor gamma; PPAR-α: Peroxisome proliferator-activated receptor alpha; SREBP-1c: Sterol regulatory element-binding protein-1c; MTTP: Microsomal triglyceride transfer protein; IQR: Interquartile range; CI: Confidence interval; 2D-SWE: Two-dimensional shear wave elastography.

Table 5 Distribution of participants by fibrosis stage, steatosis stage, nonalcoholic fatty liver disease fibrosis score and Fibrosis-4 index risk categories at baseline and after 6 months of sodium-glucose cotransporter 2 inhibitor therapy, n (%)

Parameter		Baseline	6 months	P value
Fibrosis stage	F0/F1	54 (80.6)	60 (89.6)	0.161
	F2	2 (3)	3 (4.5)
	F3	3 (4.5)	1 (1.5)
	F4	8 (11.9)	3 (4.5)
Steatosis stage	S0	13 (21.3)	16 (26.2)	0.891
	S1	15 (24.6)	15 (24.6)
	S2	18 (29.5)	15 (24.6)
	S3	15 (24.6)	15 (24.6)
NFS risk category	Low	9 (14.3)	9 (14.3)	0.852
	Intermediate	42 (66.7)	40 (63.5)
	High	12 (19.0)	14 (22.2)
FIB-4 risk category	Low	53 (79.1)	52 (77.6)	0.812
	Intermediate	11 (16.4)	11 (16.4)
	High	3 (4.5)	4 (6.0)

¹McNemar-Bowker test.

²Marginal homogeneity test.

FIB-4: Fibrosis-4 index; NFS: Nonalcoholic fatty liver disease fibrosis score.

Table 6 Spearman correlation between laboratory parameters and lipogenesis-related biomarker concentrations after 6 months of sodium-glucose cotransporter 2 inhibitor therapy

Parameter	PPAR-γ	PPAR-α	SREBP-1c	MTTP
Cholesterol (mmol/L)	0.109 (0.38)	0.026 (0.84)	0.037 (0.77)	0.301 (0.01)a
HDL (mmol/L)	0.001 (0.99)	0.125 (0.32)	-0.097 (0.44)	0.049 (0.70)
LDL (mmol/L)	0.065 (0.60)	-0.034 (0.79)	0.075 (0.55)	0.284 (0.02)a
Triglycerides (mmol/L)	0.081 (0.52)	0.069 (0.58)	0.199 (0.11)	0.075 (0.55)
ALT (U/L)	-0.097 (0.43)	0.222 (0.07)	0.210 (0.09)	0.089 (0.48)
AST (U/L)	-0.205 (0.10)	0.250 (0.04)a	0.069 (0.58)	0.007 (0.95)
ALP (U/L)	-0.092 (0.46)	0.082 (0.52)	-0.073 (0.56)	0.021 (0.87)
AST/ALT ratio	-0.032 (0.80)	-0.093 (0.45)	-0.274 (0.02)a	-0.141 (0.25)
GGT (U/L)	0.041 (0.74)	0.074 (0.56)	-0.090 (0.47)	0.085 (0.50)
Albumin (g/L)	0.028 (0.82)	-0.023 (0.86)	0.279 (0.02)a	-0.069 (0.58)
Prothrombin time INR	-0.144 (0.26)	-0.256 (0.04)a	-0.157 (0.22)	-0.177 (0.17)
Total bilirubin (µmol/L)	0.072 (0.56)	-0.105 (0.40)	0.004 (0.98)	-0.198 (0.11)
Conjugated bilirubin (µmol/L)	0.009 (0.95)	-0.025 (0.84)	-0.013 (0.92)	-0.300 (0.01)a

^aP < 0.05.

Values are Spearman’s correlation coefficients (ρ) with corresponding P values in parentheses. Concentrations of lipogenesis-related biomarkers were measured in ng/L. ALT: Alanine aminotransferase; ALP: Alkaline phosphatase; AST: Aspartate aminotransferase; GGT: Gamma-glutamyl transferase; HDL: High-density lipoprotein; INR: International normalized ratio; MTTP: Microsomal triglyceride transfer protein; PPAR-γ: Peroxisome proliferator-activated receptor gamma; PPAR-α: Peroxisome proliferator-activated receptor alpha; SREBP-1c: Sterol regulatory element-binding protein 1c.

Table 7 Spearman correlations of clinical, laboratory, and lipogenesis-related biomarkers with liver stiffness and hepatic steatosis (ultrasound-guided attenuation parameter) at baseline and after 6 months of sodium-glucose cotransporter 2 inhibitor therapy

Parameter	Baseline		6 months
	2D-SWE	UGAP	2D-SWE	UGAP
Age (year)	0.079 (0.53)	-0.288 (0.02)a	-0.028 (0.82)	-0.254 (0.04)a
BMI (kg/m2)	0.563 (< 0.001)a	0.467 (< 0.001)a	0.467 (< 0.001)a	0.538 (< 0.001)a
WC (cm)	0.582 (< 0.001)a	0.467 (< 0.001)a	0.652 (< 0.001)a	0.551 (< 0.001)a
ALT (U/L)	0.172 (0.16)	0.458 (< 0.001)a	0.326 (0.01)a	0.465 (< 0.001)a
AST (U/L)	0.138 (0.27)	0.379 (< 0.001)a	0.258 (0.03)a	0.311 (0.01)a
ALP (U/L)	-0.072 (0.56)	-0.012 (0.93)	0.079 (0.53)	0.001 (0.99)
AST/ALT ratio	-0.104 (0.40)	-0.324 (0.01)a	-0.215 (0.08)	-0.423 (< 0.001)a
GGT (U/L)	0.167 (0.18)	0.513 (< 0.001)a	0.285 (0.02)a	0.517 (< 0.001)a
Cholesterol (mmol/L)	-0.125 (0.31)	0.110 (0.40)	-0.249 (0.04)a	-0.085 (0.50)
HDL (mmol/L)	-0.125 (0.31)	-0.132 (0.31)	-0.250 (0.04)a	-0.323 (0.01)a
LDL (mmol/L)	-0.063 (0.61)	0.048 (0.71)	-0.249 (0.05)	-0.05 (0.69)
Triglycerides (mmol/L)	0.057 (0.65)	0.369 (< 0.001)a	0.161 (0.20)	0.371 (< 0.001)a
UGAP	0.357 (0.005)a	-	0.422 (< 0.001)a	-
PPAR-γ	-0.068 (0.58)	-0.047 (0.72)	-0.233 (0.06)	0.045 (0.72)
PPAR-α	-0.016 (0.90)	-0.183 (0.16)	0.023 (0.85)	0.143 (0.25)
SREBP-1c	-0.080 (0.52)	-0.037 (0.78)	-0.127 (0.31)	0.050 (0.69)
MTTP	0.055 (0.66)	0.017 (0.89)	-0.268 (0.03)a	0.066 (0.60)
NFS	0.334 (0.01)a	0.050 (0.71)	0.236 (0.06)	-0.032 (0.80)
FIB-4	0.057 (0.65)	-0.141 (0.28)	0.014 (0.91)	-0.201 (0.10)

^aP < 0.05.

Values are Spearman’s correlation coefficients (ρ) with corresponding P-values in parentheses. Concentrations of lipogenesis-related biomarkers were measured in ng/L, two-dimensional shear wave elastography in kPa; ultrasound-guided attenuation parameter in dB/cm/MHz. 2D-SWE: Two-dimensional shear wave elastography; ALT: Alanine aminotransferase; ALP: Alkaline phosphatase; AST: Aspartate aminotransferase; BMI: Body mass index; FIB-4: Fibrosis-4 index; GGT: Gamma-glutamyl transferase; HDL: High-density lipoprotein; LDL: Low-density lipoprotein; MTTP: Microsomal triglyceride transfer protein; NFS: Non-alcoholic fatty liver fibrosis score; PPAR-γ: Peroxisome proliferator-activated receptor gamma; PPAR-α: Peroxisome proliferator-activated receptor alpha; SREBP-1c: Sterol regulatory element-binding protein 1c; UGAP: Ultrasound-guided attenuation parameter; WC: Waist circumference.

Table 8 Comparison of selected laboratory parameters across advanced fibrosis risk categories defined by the nonalcoholic fatty liver disease fibrosis score and Fibrosis-4 index after 6 months of sodium-glucose cotransporter 2 inhibitor therapy

Parameter	Low risk	Intermediate risk	High risk	P value1
NFS	n = 9	n = 41	n = 15
ALT (U/L)	38 (33.5-84)	35 (29.5-59)	28 (19.5-35.5)	0.01a
AST (U/L)	34 (24.5-54)	26 (23.5-40.5)	23 (18-28)	0.18
AST/ALT ratio	0.7 (0.6-0.9)	0.7 (0.7-0.9)	0.9 (0.7-1.1)	< 0.001b
Erythrocytes	4.8 (4.6-5.3)	5.3 (4.9-5.8)	4.9 (4.4-5.1)	0.01c
MCV (fL)	84.5 (82.6-92.7)	84.7 (81.7-87.4)	88 (85.8-92)	0.04a
Platelet count (109/L)	282 (264.5-355.5)	318 (277.5-391)	240 (207-260.5)	< 0.001b
Leukocytes (109/L)	9.4 (7.4-10)	9.2 (6.9-10.6)	7.8 (6.6-9.1)	0.20
Albumin (g/L)	44.7 (44-46.3)	45.3 (44.1-48.7)	45 (43.4-47)	0.04d
Total protein (g/L)	74 (70-77.5)	77 (74-78)	72 (69-74.8)	0.02a
FIB-4	n = 52	n = 11	n = 4
ALT (U/L)	26 (20.3-35)	21 (17-29)	22.5 (15-38.3)	0.44
AST (U/L)	23.5 (20-28)	23 (21-31)	34 (22-44.5)	0.30
AST/ALT ratio	0.9 (0.7-1.1)	1.2 (0.7-1.4)	1.3 (1.2-1.7)	0.004a
Erythrocytes	5 (4.6-5.4)	5 (4.7-5.5)	4.3 (4.1-5)	0.15
MCV (fL)	88 (84.7-91.3)	88 (85.9-91.5)	88 (85.6-88.9)	0.90
Platelet count (109/L)	248 (214-283)	193 (174-221)	145.5 (139.3-177.3)	< 0.001a
Leukocytes (109/L)	8.3 (7.3-9.5)	7.6 (6.9-9.8)	5.5 (4.7-6)	0.01d
Albumin (g/L)	45.3 (43.1-47)	47.4 (43.1-47.7)	44.4 (41.3-46.2)	0.47
Total protein (g/L)	74 (70.4-77)	76 (71-77)	70 (70-72.3)	0.24

¹Kruskal-Wallis test (post hoc Conover test).

^aP < 0.05 were observed between the low-risk group and the intermediate- and high-risk groups.

^bP < 0.05 were observed between all three risk groups.

^cP < 0.05 were observed between the low- and high-risk groups.

^dP < 0.05 were observed between the high-risk group and the low- and intermediate-risk groups.

n denotes the number of patients in each fibrosis risk category. Values are presented as median (interquartile range). ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; MCV: Mean corpuscular volume; NFS: Nonalcoholic fatty liver disease fibrosis score; FIB-4: Fibrosis-4 index.