Effects of isomaltulose on insulin resistance and metabolites in patients with non‑alcoholic fatty liver disease: A metabolomic analysis.

Insulin resistance is associated with a poor prognosis in non‑alcoholic fatty liver disease (NAFLD) patients. Isomaltulose, a naturally‑occurring disaccharide, is reported to improve glucose and lipid metabolisms in obese patients. The present study aimed to investigate the effects of isomaltulose on insulin resistance and various metabolites in NAFLD patients. Five male patients with NAFLD consumed 20 g isomaltulose or sucrose (control). Changes in insulin resistance and metabolites were evaluated by alterations of serum C‑peptide immunoreactivity (CPR) and metabolomic analysis from baseline to 15 min after the administration, respectively. There was no significant difference in changes of blood glucose level; however, the CPR level was significantly decreased in the Isomaltulose group compared to the control group (0.94±0.89 vs. ‑0.12±0.31, P=0.0216). In a metabolomic analysis, a significant alteration was seen in 52 metabolites between the control and Isomaltulose groups. In particular, the taurodeoxycholic acid level significantly increased approximately 12.5‑fold, and the arachidonic acid level significantly decreased approximately 0.01‑fold. Together, it present study demonstrated that isomaltulose improved insulin resistance in NAFLD patients. It was also revealed that isomaltulose affects taurodeoxycholic acid and arachidonic acid. Thus, isomaltulose may have a beneficial effect on insulin resistance through alterations of bile acid and fatty acid metabolisms in NAFLD patients.

RCT Entities:   Population Interventions Outcomes

Introduction

Non-alcoholic fatty liver disease (NAFLD) is a pandemic disease, and one-third of the population worldwide is affected (1,2). Since the liver is one of the target organs of insulin, a high prevalence of insulin resistance is seen in patients with NAFLD (3,4). An increase in insulin resistance is associated with the development of not only type 2 diabetes mellitus but also life-threatening diseases such as liver cirrhosis, hepatocellular carcinoma, and cardiovascular diseases in patients with NAFLD (5). Thus, improved insulin resistance is an important therapeutic target in patients with NAFLD. Anti-diabetic agents including pioglitazone (6), glucagon-like peptide-1 (GLP-1) analogue (7), and sodium-glucose cotransporter 2 inhibitor (8) lead to metabolic and histologic improvement in patients with NAFLD complicated by diabetes mellitus. However, no effective medication has been approved yet for patients with NAFLD.

Isomaltulose (6-0-α-D-glucopyranosyl-D-fructose) is a naturally-occurring disaccharide found in honey (9) and is composed of glucose and fructose, similar to sucrose (10). Both isomaltulose and sucrose are digested into glucose and fructose by α-glucosidase in the small intestine and contribute the same caloric value of 4 kcal/g (11,12). However, the digestive rate of isomaltulose is significantly slower than that of sucrose because the structural feature of isomaltulose is that glucose and fructose are linked by an alpha-1,6-glycosidic bond (12,13). In addition, isomaltulose affects the secretion of gut hormones such as glucose-dependent insulinotropic polypeptide (GIP) and GLP-1 (14,15), leading to an improvement of insulin resistance. Thus, isomaltulose is a low glycemic index sweetener as well as a functional disaccharide and is currently used in various medical food and drink applications instead of sucrose (16).

In a rat model of metabolic syndrome, isomaltulose is reported to reduce visceral fat mass and improves glucose intolerance, resulting in inhibition of increase in blood pressure and progression of diabetic nephropathy (17). Furthermore, in healthy subjects, isomaltulose ingestion inhibits an increase in insulin resistance and blood pressure (11,18). The beneficial effects of isomaltulose on visceral fat, insulin resistance, and blood pressure in patients who were obese have been reported in double-blind, placebo-controlled interventional studies (19,20). However, the effect of isomaltulose on insulin resistance has never been investigated in patients with NAFLD.

An increase in insulin resistance can be caused by various factors including amino acid, fatty acid, and bile acid metabolism (3,21,22). Metabolomic analysis is a systematic examination of metabolites in a given biological sample and can reveal novel pathways (23). Metabolomic analysis has recently been applied to the identification of the pharmacological mechanisms of berberine, an isoquinoline alkaloid, for nonalcoholic steatohepatitis (NASH) treatment and revealed metabolic disruption involving phospholipids and unsaturated fatty acids in a rat model of NASH (24). However, metabolomic analysis has not been used to identify the pharmacological mechanisms of isomaltulose in patients with NAFLD.

The aim of this study was to investigate effects of isomaltulose on insulin resistance in patients with NAFLD. In addition, using metabolomic analysis, we investigated the effect of isomaltulose on various metabolisms in patients with NAFLD.

Subjects and methods

Study design and ethics

This was a randomized, single-blinded controlled interventional study, and our study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki, as reflected by the prior approval of the institutional review board of The Ethic Committee of Kurume University. All experiments were performed in accordance with relevant guidelines and regulations. All subjects provided written informed consent to participate in this study.

Subjects

A total of 5 male patients diagnosed with NAFLD were enrolled in this study. The following patient inclusion criteria were used: Patients with i) NAFLD, ii) age >20 years, and iii) written informed consent. The exclusion criteria were as follows: Patients with i) hemoglobin A1c >8.0%, ii) insulin or α-glucosidase inhibitor treatment, iii) liver cirrhosis, iv) renal failure, v) a history of cardiovascular disease, or vi) participation in any other clinical trial.

The subjects were randomly assigned into the sucrose (Control) or isomaltulose group. After a 14-day wash-out term, each subject was assigned to the other group.

Diagnosis of NAFLD

NAFLD was diagnosed according to the Clinical Practice Guidelines for NAFLD/NASH as follows (25): i) hepatic steatosis evaluated by liver biopsy, ultrasonography, computed tomography, or magnetic resonance imaging; ii) ethanol intake <30 g/day; and iii) exclusion of other liver diseases, including chronic hepatitis B, chronic hepatitis C, autoimmune hepatitis, drug-induced liver disease, primary biliary cholangitis, primary sclerosing cholangitis, biliary obstruction, Wilson's disease, and hemochromatosis.

Isomaltulose or sucrose administration and blood collection

After a 12-hour overnight fast, the subjects consumed 20 g isomaltulose in 200 ml of water or 20 g sucrose (Control) in 200 ml of water in 1 min at 8:30 a.m. During the procedure, the subjects were instructed to rest on a bed and fast. Venous blood samples were collected before and 15 min after administration. For plasma GIP and GLP-1 measurements, blood samples were collected into a BD™ P100 Blood Collection System containing protein stabilizers (BD Biosciences, Franklin Lakes, NJ, USA). Blood samples were centrifuged at 3,000 × g for 15 min at 4°C and stored at −20°C until analysis.

Laboratory determinations

Serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), γ-glutamyl transpeptidase (GGT), total bilirubin, total cholesterol, low-density lipoprotein (LDL)-cholesterol, blood urea nitrogen (BUN), creatinine, nonesterified fatty acid, C-peptide immunoreactivity (CPR), and blood glucose level were measured using standard clinical methods (SRL Inc., Tokyo, Japan) as previously described (4,26,27).

Measurement of serum GIP and GLP-1 levels

As previously described (28), plasma GIP and GLP-1 levels were measured by an enzyme-linked immunosorbent assay using a Human GIP ELISA kit (YK253; Yanaihara Institute Inc., Fujimiya, Japan) and a Human GLP1 (7–36) ELISA kit (ab184857; Abcam, Cambridge, UK), respectively, according to each manufacturer's instructions.

Metabolomic analysis

Metabolomic analysis was performed twice using serum samples taken before (0 min) and 15 min after isomaltulose or sucrose treatment.

Preparation

Serum samples taken before and 15 min after isomaltulose or sucrose treatment from a representative subject were applied to a metabolomic analysis. Metabolome measurements were performed at a service facility of LSI Medience Corporation (Tokyo, Japan). Briefly, serum (200 µl) was added to methanol (800 µl) and then mixed for 15 min with a shaker at room temperature. After it was centrifuged by 10,000 × g for 10 min, the supernatant was dried up with nitrogen gas, and the residue was dissolved with 10% acetonitrile aqueous solution (200 µl). After adding internal standards, they were analyzed with both liquid chromatography mass spectrometry (LC-MS) and capillary electrophoresis coupled to mass spectrometry (CE-MS). Tuning and calibration were performed with a standard solution provided by Agilent Technology, and the resolution errors were controlled within 3 ppm. The order of measurement was randomized to minimize the specific error in each group. Quality control samples were prepared by pooling samples and were analyzed every 5 samples to verify the measurement accuracy.

LC-time-of-flight mass spectrometry (TOFMS) analysis

LC-MS datasets were acquired on a liquid chromatography system (Agilent HP1200; Agilent Technologies, Inc., Santa Clara, CA, USA) equipped with a C18 column (2 µm, 50×2.0 mm ID, CAPCELL PAK C18 IF; Shiseido, Tokyo, Japan) and coupled with an electrospray ionization quadrupole TOFMS (Agilent 6520; Agilent Technologies, Inc.). Solvent A was composed of a 5 mM ammonium acetate aqueous solution, while solvent B was acetonitrile. Metabolites were eluted at a flow rate of 0.2 ml/min at 40°C with a linear gradient of 10–90% solvent B over 10 min, followed by a further 5 min hold at 100% solvent B. The mass spectrometer was operated in positive and negative scan mode (m/z 60 to 1,200) with a capillary voltage of 3,500 V. The nebulizing gas pressure was 40 psi, and the dry gas flow was 8 l/min at 350°C.

CE-TOFMS analysis

Ionic metabolites were measured in the positive mode of CE-TOFMS (Agilent CE-TOFMS 6520; Agilent Technologies, Inc.,). Metabolites were separated in a fused-silica capillary (50 µm i.d.x100 cm total length; GL Science, Tokyo, Japan) filled with 1 mol/l formic acid aqueous solution (cation mode), or 20 mM ammonium formate and 20 mM ammonium acetate aqueous solution (pH 10, anion mode) as an electrolyte. The sample solution was injected at 5 kPa for 15 sec (approximately 15 nl), and a voltage of 30 kV was applied. The temperatures of capillary and sample trays were maintained at room temperature and 5°C, respectively. The sheath liquid was methanol/water (50%v/v) containing 5 mM ammonium acetate. CE-TOFMS was operated in both positive and negative scan mode (m/z 60 to 1,200). The capillary voltage was set at 3,500 V, and the nitrogen gas (heater temperature 250°C) flow rate was set at 10 l/min.

Data analysis

A data file of mass spectrometry was converted to csv format with an Agilent csv converter. All peak positions (retention time and m/z) and areas were calculated by Markeranalysis (LSI Medience, Tokyo, Japan) (29). All peak areas were aligned into one data sheet, and the errors of peak intensities were corrected by internal standards. Noise peaks were deleted compared with the peaks detected in blank samples. Metabolites were identified by comparing the retention time and m/z with standard data set established by LSI Medience Corporation.

Effects of isomaltulose on insulin resistance, GIP, GLP-1 secretion, and metabolites

Insulin resistance was evaluated by an increment of serum CPR as previously described (30). ΔCPR was the change of serum CPR level from baseline to 15 min after sucrose or isomaltulose administration. Similarly, changes in GIP and GLP-1 secretions were evaluated by ΔGIP and ΔGLP-1, which were changes of plasma GIP and GLP-1 levels from baseline to 15 min after sucrose or isomaltulose administration, respectively. The effects of isomaltulose on metabolites were also evaluated by change of each metabolite level from baseline (0 min) to 15 min after sucrose or isomaltulose administration (Δ each metabolite). Variable that increased or decreased after the administration was shown as positive or negative expression level (∆ variable or -∆ variable) (Fig. 1).

Figure 1.

Changes in variable after administration of sucrose or isomaltulose. Effects of sucrose or isomaltulose on variables were evaluated by change of each variable level from baseline (0 min) to 15 min after administration. Variable that increased or decreased after the administration was shown as positive or negative expression level (∆ variable or -∆ variable).

Statistical analysis

Data are expressed as numbers or means ± standard deviations. Differences between the two groups were analyzed using the Wilcoxon signed-rank test. In metabolome analysis, mea n fold-change and t-test for all detected peaks were performed between the two groups. P<0.05 was considered to indicate a statistically significant difference.

Results

Baseline patient characteristics

The baseline patient characteristics are summarized in Table I. No significant difference was seen in the serum levels of AST, ALT, GGT, or total bilirubin between the sucrose (Control) and Isomaltulose groups. There was no significant difference in fasting blood glucose or serum CPR levels between the 2 groups (Table I).

Table I.

Patients' characteristics at baseline.

Characteristic	Control	Isomaltulose	P-value
Age (yeare)	48.6±11.8	48.6±11.8	1.0000
Body mass index	35.1±7.0	35.1±7.0	1.0000
Red blood cells (×104/µl)	509.6±70.6	514.4±59.7	0.9105
Hemoglobin (g/dl)	15.5±1.8	15.6±1.3	0.9540
White blood cells (/µl)	8,500±2,515	7,600±2,531	0.5882
Platelet count (×104/µl)	29.5±4.1	28.7±4.2	0.7634
Aspartate aminotransferase (IU/l)	31.2±13.2	30.0±14.7	0.8953
Alanine aminotransferase (IU/l)	45.6±36.9	40.2±28.5	0.8024
Lactate dehydrogenase (IU/l)	227.2±109.8	237.2±108.4	0.8884
γ-glutamyl transpeptidase (IU/l)	55.6±39.9	54.4±36.0	0.9614
Total bilirubin (mg/dl)	0.52±0.18	0.46±0.18	0.6130
Total cholesterol (mg/dl)	207.0±26.4	197.2±21.1	0.5350
Low-density lipoprotein cholesterol (mg/dl)	138.6±17.6	125.0±23.8	0.3362
Triglycerides (mg/dl)	144.2±51.4	154.2±56.5	0.7772
Blood urea nitrogen (mg/dl)	14.7±2.9	14.7±4.2	0.9864
Creatinine (mg/dl)	0.85±0.16	0.80±0.20	0.6998
Fasting blood glucose (mg/dl)	132.0±38.7	145.0±50.0	0.6586
C-peptide immunoreactivity (ng/dl)	2.38±0.99	2.61±1.71	0.8025
Nonesterified fatty acid (µEq/l)	675.0±316.8	574.4±221.0	0.5782

Data are expressed as mean ± SD. Differences between the two groups were analyzed using Wilcoxon signed-rank test. P-values of <0.05 are considered significant.

Effect of isomaltulose on changes in blood glucose and serum CPR levels

The effect of isomaltulose on blood glucose and serum CPR levels was evaluated by changes in these variables from baseline to 15 min after sucrose or isomaltulose administration. Although there was no significant difference in changes of blood glucose levels, the serum CPR level was significantly decreased in the Isomaltulose group compared to that in the Control group (Fig. 2).

Figure 2.

Effect of isomaltulose on changes in blood glucose and serum CPR levels. The effect of isomaltulose on blood glucose (A) and serum CPR levels (B) was evaluated by changes in these variables from baseline to 15 min after sucrose (Control) or isomaltulose administration. P-values of <0.05 were considered significant. N.S., not significant; CPR, C-peptide immunoreactivity.

Effect of isomaltulose on changes in plasma GIP and GLP-1 levels

The effect of isomaltulose on plasma GIP and GLP-1 levels was evaluated by changes in these variables from baseline to 15 min after sucrose or isomaltulose administration. There was no significant difference in changes of plasma GIP and GLP-1 levels between the Control and Isomaltulose groups (Table II).

Table II.

Effects of isomaltulose on changes in plasma GIP and GLP-1 levels.

Level	Control	Isomaltulose	P-value
∆GIP	23.1±25.6	14.0±9.5	0.8345
∆GLP-1	−51.5±98.0	166.7±66.6	0.1437

Data are expressed as mean ± SD. ∆GIP and ∆GLP-1 are changes of plasma GIP and GLP-1 levels from baseline to 15 min after sucrose (Control) or isomaltulose administration, respectively. Differences between the 2 groups were analyzed using the Wilcoxon signed-rank test. P-values of <0.05 are considered significant. GIP, glucose-dependent insulinotropic polypeptide; GLP-1, glucagon-like peptide-1.

Effect of isomaltulose on serum metabolite levels

With metabolomic analysis, the effects of isomaltulose on 201 metabolite levels were evaluated by changes in these variables from baseline to 15 min after sucrose or isomaltulose administration. A significant alteration was seen in 52 metabolites between the Control and Isomaltulose groups (Table III). Many of these 52 altered metabolites were categorized as bile acid (6 metabolites), fatty acid (6 metabolites), or glycine/serine metabolism (4 metabolites) (Fig. 3).

Table III.

Metabolites significantly altered by isomaltulose in patients with NAFLD.

			Control		Isomaltulose
	Metabolite	Category	Mean	SD	Mean	SD	Ratio	P-value
1	L-Arginine	Amino acid	−507	80	−2,570	180	5.00	0.0001
2	L-Ornithine	Urea cycle	−62	64	1,390	137	−25.00	0.0001
3	N-Acetylornithine	Arginin, proline metabolism	−60	157	−1,832	111	33.33	0.0001
4	Glycodeoxycholate	Bile acid	29	3	134	12	4.55	0.0001
5	Urea	Urea cycle	−79	12	115	23	−1.45	0.0002
6	Betaine	Glycine, serin metabolism	−446	398	2,613	307	−5.88	0.0005
7	D-Xylulose	Sugar	51	6	88	2	1.72	0.0006
8	Glycocholate	Bile acid	1,423	93	25,816	1,114	16.67	0.0006
9	6-Aminohexanoate	Fatty acid metabolism	14	3	42	5	2.94	0.0008
10	Indole acetate	Tryptophan metabolism	−170	25	41	32	−0.24	0.0008
11	Theobromine	Caffeine metabolism	−440	139	350	80	−0.79	0.0010
12	Taurodeoxycholic acid	Bile acid	2,848	49	36,895	1,997	12.50	0.0011
13	Uridine	Pyrimidine metabolism	416	135	1,917	323	4.55	0.0017
14	L-Lactic acid	Glycolysis	5,159	744	2,058	178	0.40	0.0022
15	Eicosadienoic acid (20:2)	Fatty acid	−18,121	2,263	2,174	4,642	−0.12	0.0024
16	D-Glycerate	Glycine, serin metabolism	3	11	239	60	100.00	0.0026
17	1-6-Anhydro-beta-d-Glucose	Sugar metabolism	642	39	1,409	202	2.17	0.0030
18	N-N-dimethylarginine	Arginin, proline metabolism	−19	20	102	26	−5.26	0.0032
19	Tridecanoic acid	Fatty acid	51	76	719	181	14.29	0.0041
20	Taurocholate	Bile acid	16	23	210	54	12.50	0.0046
21	Creatinine	Arginin, proline metabolism	−994	580	1,401	494	−1.41	0.0055
22	Choline	Glycine, serin metabolism	−2,423	443	1,316	1,183	−0.54	0.0069
23	Gramine	Tryptophan metabolism	15	20	−136	47	−9.09	0.0070
24	5-Oxoproline	Glutathione metabolism	−77	41	523	213	−6.67	0.0087
25	Pseudouridine	Pyrimidine metabolism	51	37	−59	20	−1.15	0.0103
26	Eicosenoic acid (20:1)	Fatty acid	18,075	4,264	−2,476	6,533	−0.14	0.0103
27	(R)-3-Hydroxybutanoate	Butanoate metabolism	13	62	190	30	14.29	0.0113
28	Taurocyamine	Taurine and hypotaurine metabolism	−233	112	153	103	−0.66	0.0118
29	Hydroxypropionic acid	Pyrimidine metabolism	−59	23	−137	22	2.33	0.0131
30	D-Glyceraldehyde	Sugar metabolism	14,027	292	4,661	2,058	0.33	0.0143
31	Docosapentaenoate (n3 DPA; 22:5n3)	Fatty acid	−14,387	2,318	10,570	10,482	−0.74	0.0158
32	4-Methyl-2-oxopentanoate	Valine, leucine and isoleucine metabolism	72	70	252	39	3.45	0.0178
33	Hyodeoxycholate	Bile acid	332	83	128	37	0.38	0.0179
34	L-Histidine	Amino acid	−41	49	215	104	−5.26	0.0183
35	L-Methionine S-oxide	Cystein metabolism	19	12	−19	12	−1.02	0.0190
36	Undecanoate (11:0)	Fatty acid	−22	62	343	155	−16.67	0.0191
37	beta-D-Glucose	Sugar	2,496	550	3,920	372	1.56	0.0206
38	Abscisate	Plant metabolites	−4	17	36	9	−10.00	0.0216
39	N2,N2-Dimethylguanosine	Nucleotide metabolism	−75	33	13	25	−0.18	0.0218
40	1-5-anhydro-D-glucitol	Sugar	−2	8	15	2	−7.69	0.0223
41	1-Methylhistidine	Histidine metabolism	−132	123	255	148	−1.92	0.0250
42	beta-hydroxyisovaleric acid	Valine, leucine and isoleucine metabolism	−254	130	508	355	−2.00	0.0251
43	Arachidonate	Fatty acid	48,499	15,330	−473	20,184	−0.01	0.0287
44	N,N-Dimethylglycine	Glycine, serin metabolism	−23	21	127	75	−5.56	0.0290
45	(±)-1,2-Diphenylethylenediamine	Unclassified	26	18	−53	37	−2.04	0.0301
46	Caffeine	Caffeine metabolism	−1,724	485	110	895	−0.06	0.0355
47	α-D-Glucose 6-phosphate	Glycolysis	2	43	122	52	50.00	0.0364
48	(S)(+)-Allantoin	Purine metabolism	955	82	718	109	0.75	0.0398
49	Chenodeoxycholate	Bile acid	3,201	854	1,629	332	0.51	0.0411
50	Homogentisate	Phenylalanine tyrosine metabolism	−54	39	33	34	−0.62	0.0424
51	Ranitidine	Unclassified	6	10	−14	6	−2.17	0.0432
52	L-Tryptophan	Amino acid	−144	45	282	255	−1.96	0.0463

SD, standard deviation.

Figure 3.

Effect of isomaltulose on serum metabolite levels. With metabolomic analysis, significant alteration was seen in 52 metabolites between the Control and isomaltulose groups. Many of the 52 altered metabolites were seen in categories of bile acid (6 metabolites), fatty acid (6 metabolites), and glycine/serine metabolism (4 metabolites).

A representative change of metabolite in each category was an increased taurodeoxycholic acid level in the bile acid category, a decreased arachidonic acid level in the fatty acid category, and an increased betaine level in the glycine/serine category after isomaltulose treatment (Fig. 4A-C). In addition, a significant decrease was seen in the serum L-arginine level in the isomaltulose group compared to that in the Control group (Fig. 4D), while the serum L-ornithine level was significantly increased in the isomaltulose group compared to that in the Control group (Fig. 4E).

Figure 4.

Effect of isomaltulose on serum taurodeoxycholic acid, arachidonic acid, betaine, L-arginine, and L-ornithine levels. The effect of isomaltulose on serum (A) taurodeoxycholic acid, (B) arachidonic acid, (C) betaine, (D) L-arginine, and (E) L-ornithine levels was evaluated by changes in these variables from baseline (0 min) to 15 min after sucrose (Control) or isomaltulose administration. P-values of <0.05 were considered significant.

Discussion

In this study, we demonstrated that isomaltulose improved insulin resistance in patients with NAFLD. Although isomaltulose did not have a significant effect on the changes in serum GIP and GLP-1 levels, we showed that isomaltulose significantly affects various metabolites, in particular, taurodeoxycholic acid, arachidonic acid, and betaine. Thus, isomaltulose may have a beneficial effect on insulin resistance through alterations in various metabolisms, in particular bile acid, fatty acid, and glycine/serine metabolisms.

Isomaltulose has been reported to improve insulin resistance in rats and in patients who are obese (19,31). In agreement with these previous reports, the serum CPR level was significantly decreased in the Isomaltulose group compared to that in the Control group in this study. Thus, we demonstrated that isomaltulose improved insulin resistance in patients with NAFLD. In this study, serum CPR was used for evaluation of insulin resistance instead of immunoreactive insulin (IRI). This was because DCPR is the most significant determinant of insulin secretion increments (30). Moreover, in patients with chronic liver disease, the hepatic insulin degradation rate is decreased, and CPR is more accurate than IRI for the evaluation of insulin secretion (32). Furthermore, the IRI level is affected by hemolysis during blood collection (33) and is inaccurate in the presence of an anti-insulin antibody (34).

The slow digestive rate of isomaltulose is reported to affect secretion of GIP and GLP-1, resulting in a reduction of insulin resistance (14,15). However, in this study, no significant change was seen in GIP or GLP-1 levels. The reason for the discrepancy between previous studies and our study remains unclear, and following are possible explanations (1): The number of enrolled patients may not have had enough power to detect significant changes or (2) since plasma dipeptidyl peptidase-4 activity is accelerated in patients with NAFLD (35), degradation of GIP and GLP-1 could be increased in patients with NAFLD.

To investigate possible mechanisms for isomaltulose-induced suppression of insulin resistance, we performed a metabolomic analysis and demonstrated that isomaltulose had significant effects on various metabolites in patients with NAFLD. A large number of altered metabolites were categorized into bile acid, fatty acid, or glycine/serine metabolisms. A representative change in each category was an increased taurodeoxycholic acid level, a decreased arachidonic acid level, and an increased betaine level in bile acid, fatty acid, and glycine/serine metabolisms, respectively. The causal relationships between these changes in metabolites and the improvement of insulin resistance remain unclear. However, Qi et al reported that taurodeoxycholic acid may reduce the increase of phospholipids, sphingomyelins, and ceramides induced by a high-fat diet, leading to an improvement of insulin resistance in a mouse model (36). Arachidonic acid is reported to down-regulate the insulin-dependent glucose transporter gene, resulting in an increase in insulin resistance (37). In addition, betaine was recently reported to improve insulin resistance through an activation of forkhead box O1-induced NLRP3 inflammasomes (38). These previous reports support that isomaltulose may have improved insulin resistance potentially through alterations in bile acid, fatty acid, and glycine/serine metabolisms in this study.

The metabolomic analysis also revealed a significant decrease in the serum L-arginine level and a significant increase in the serum L-ornithine level in the isomaltulose group. L-arginine is reported to inhibit gene expression of insulin receptor substrate-1, phosphatidylinositol 3-kinase, and Akt in the insulin signaling pathway, leading to an increase in insulin resistance (39). L-ornithine is reported to regulate growth hormone/insulin-like growth factor-1/insulin-like growth factor-binding protein 3 complex in muscle tissue, leading to improved insulin resistance (40). Taken together, along with alterations in bile acid, fatty acid, and glycine/serine metabolisms, alterations in L-arginine and L-ornithine may contribute to improvement of insulin resistance in patients with NAFLD.

A major limitation of this study is small sample size, and further study is required to verify of our results. In this study, changes in metabolisms were evaluated by a global metabolomic analysis. Since environmental factors including outdoor temperature are known to be associated with insulin resistance and other metabolisms (41–43), isomaltulose or sucrose was administered to the all subjects at the same time on the same day by personal stuffs. Thus, sample size was limited because of the equalization for environmental factors in this study. Another limitation is that taste of isomaltulose is slightly different from that of sucrose and subjects might recognize the difference between isomaltulose and sucrose. However, taste correlates with chemical structure (44,45), and it is impossible to have a control disaccharide with same taste of isomaltulose. In this study, a random assignment was performed just before the administration and then, subjects were at rest on a bed and fast during the procedure. Thus, the taste difference between isomaltulose and sucrose is considered to have no influence on the results.

In conclusion, we showed that isomaltulose improved insulin resistance in patients with NAFLD. In addition, we revealed that isomaltulose significantly affect various metabolites, including taurodeoxycholic acid, arachidonic acid, and betaine. Thus, isomaltulose may improve insulin resistance mainly through alterations in various metabolisms in patients with NAFLD.