Skip to main content

Targeting hepatic sulfane sulfur/hydrogen sulfide signaling pathway with α-lipoic acid to prevent diabetes-induced liver injury via upregulating hepatic CSE/3-MST expression

Abstract

Background

Diabetes-induced liver injury is a complication of diabetes mellitus of which there are no approved drugs for effective treatment or prevention. This study investigates possible hepatoprotective effect of alpha-lipoic acid (ALA), and sulfane sulfur/hydrogen sulfide pathway as a novel protective mechanism in a rat model of type 2 diabetes-induced liver injury.

Methods

Thirty Sprague–Dawley rats underwent fasting for 12 h after which fasting blood glucose was measured and rats were randomly assigned to diabetic and non-diabetic groups. Type 2 diabetes mellitus (T2DM) was induced in diabetic group by administration of nicotinamide (110 mg/kg) and streptozotocin (55 mg/kg). Diabetic rats were treated daily with ALA (60 mg/kg/day p.o.) or 40 mg/kg/day DL-propargylglycine (PPG, an inhibitor of endogenous hydrogen sulfide production) for 6 weeks and then sacrificed. Liver, pancreas and blood samples were collected for analysis. Untreated T2DM rats received distilled water.

Results

Hypoinsulinemia, hyperglycemia, hepatomegaly and reduced hepatic glycogen content were observed in untreated T2DM rats compared to healthy control group (p < 0.001). Also, the pancreas of untreated T2DM rats showed severely damaged pancreatic islets while liver damage was characterized by markedly increased hepatocellular vacuolation, sinusoidal enlargement, abnormal intrahepatic lipid accumulation, severe transaminitis, hyperbilirubinemia, and impaired hepatic antioxidant status and inflammation compared to healthy control rats (p < 0.01). While pharmacological inhibition of hepatic sulfane sulfur/hydrogen sulfide with PPG administration aggravated these pathological changes (p < 0.05), ALA strongly prevented these changes. ALA also significantly increased hepatic expression of hydrogen sulfide-producing enzymes (cystathionine γ-lyase and 3-mecaptopyruvate sulfurtransferase) as well as hepatic sulfane sulfur and hydrogen sulfide levels compared to all groups (p < 0.01).

Conclusions

To the best of our knowledge, this is the first experimental evidence showing that ALA prevents diabetes-induced liver injury by activating hepatic sulfane sulfur/hydrogen sulfide pathway via upregulation of hepatic cystathionine γ-lyase and 3-mecaptopyruvate sulfurtransferase expressions. Therefore, ALA could serve as a novel pharmacological agent for the treatment and prevention of diabetes-induced liver injury, with hepatic sulfane sulfur/hydrogen sulfide as a novel therapeutic target.

Background

Diabetes-induced liver injury is a common complication of diabetes mellitus due to impaired metabolism of glucose and lipids. As the liver is the hub for the metabolism of these essential biomolecules, intrahepatic alterations in their metabolism either dependent or independent of extrahepatic conditions lead to their abnormal accumulation in the liver [1]. Both type 1 (T1DM) and type 2 (T2DM) diabetic patients are at high risk of having abnormal deposition of free fatty acids (FFA) and triglycerides in the liver parenchyma, as well as elevated liver enzymes, hepatic fibrosis and an increase in morbidity and mortality due to diabetes-induced liver injury [2,3,4,5]. Also, non-alcoholic fatty liver disease (NAFLD; characterized by abnormal accumulation of lipids in liver cells) and its progressive form, non-alcoholic steatohepatitis (NASH), are common causes of hepatic dysfunction particularly in T2DM patients, even if they drink little or no alcohol. If left untreated or uncontrolled, NAFLD and NASH progress to cirrhosis (advanced scarring) and end-stage liver disease or hepatocellular carcinoma, hallmarked by hepatomegaly as one of the most common physical examination findings [6, 7]. Such hepatic damage is similar to that caused by heavy alcohol use. Early manifestations of NAFLD and NASH in diabetic patients include glycogenated nuclei usually in periportal hepatocytes, as well as steatosis, and the presence of immune cells such as lymphocytes and neutrophils in the liver [8].

There are studies showing that oxidative stress due to over-production of reactive oxygen species (ROS), and activation of inflammatory pathways play central roles in the development and progression of NAFLD and NASH [9, 10]. Oxidative damage to hepatocytes due to hepatic glucotoxicity activates a cascade of inflammatory events including the recruitment of immune cells in response to acute hepatic injury. ROS-mediated damage to subcellular structures such as the mitochondria causes impaired β-oxidation of FFA in the liver, leading to its accumulation, a condition known as steatosis [9, 10]. In addition, there is an increase in apoptotic hepatocytes and caspase-3 activation in the progression of NAFLD and NASH as a result of the cumulative effect of ROS-mediated damage and inflammation [11,12,13]. Despite all these intensive studies, there are currently no approved pharmacological agents for effective treatment of patients with NAFLD and NASH including diabetic patients. Besides, existing anti-diabetic and anti-hyperlipidemic drugs only retard the disease progression but do not prevent or reverse the pathology. Moreover, some of these conventional drugs have adverse effects ranging from bladder cancer, hepatotoxicity to lactic acidosis [14,15,16]. Therefore, identification of novel drugs or development new pharmacotherapeutic regimens aimed at effective treatment or prevention of the pathology with less severe adverse effects is “a holy grail” in diabetology.

Burgeoning evidence from human and animal studies including ours shows that alpha-lipoic acid (ALA), an organosulfur antioxidant synthesized from cysteine (an endogenous source of hydrogen sulfide) [17, 18], improves other diabetic complications when used alone or in combination with other agents [19,20,21,22]. In humans and animals, ALA is synthesized in the mitochondria of hepatocytes and other cell types, where it helps mitochondrial enzymes to convert nutrients including glucose and fatty acids into energy, thus functioning as a cofactor for mitochondrial enzymes in cellular bioenergetics [23]. As the liver is the main organ in energy homeostasis by converting nutrients absorbed by the intestine into glucose, fatty acids and other substrates, and storage of glycogen and lipids for the body’s use, it suggests that ALA plays a significant part in the homeostatic function of the liver in the metabolism of these biomolecules. ALA is also administered exogenously after which it is absorbed and converted enzymatically to dihydrolipoic acid (DHLA) [24, 25]. Results from two recent preclinical studies have shown that administration of ALA produces DHLA, which releases hydrogen sulfide (H2S) from sulfane sulfur (a reactive and labile divalent sulfur atom covalently bonded to another sulfur atom) in the homogenate of rat liver tissue [26, 27]. H2S, a gas recognized by its characteristic obnoxious odour of rotten eggs, is the third established member of gasotransmitter family next to nitric oxide and carbon monoxide, with biological usefulness and therapeutic potential at low nanomolar to middle micromolar concentrations [28, 29]. In the liver, H2S is principally synthesized by cystathionine γ-lyase (CSE) and 3-mecaptopyruvate sulfurtransferase (3-MST), a cytosolic and mitochondrial enzyme respectively, and their inhibition leads to various forms of chronic liver disease [30, 31]. Interestingly, these enzymes have been recently reported to also produce sulfane sulfur, suggesting that inhibitors of endogenous H2S production may also inhibit cellular sulfane sulfur levels. Just like ALA, H2S is also administered exogenously using H2S-containing compounds [32,33,34].

While there is a substantial body of evidence on the therapeutic benefits of H2S in various pathologies including diabetic conditions [35,36,37], it is unknown whether a relationship exists between H2S and ALA in diabetic liver disease. In this present study, we sought to investigate for the first time the effect of ALA on hepatic sulfane sulfur/H2S signaling pathway in a rat model of type 2 diabetes-induced liver injury.

Methods

Ethical statement

All the animal work in this study was conducted according to relevant national and international guidelines and was approved by the University of Ghana Institutional Animal Care and Use Committee (Protocol ID: UG-IACUC 002/19-20), adhering to protocol and maintaining quality assurance in accordance with good laboratory practice and ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments).

Experimental animals

Thirty male Sprague–Dawley rats (Hsd:SD stock) weighing 180–220 g were purchased at 6–8 weeks of age from Noguchi Memorial Institute for Medical Research, University of Ghana, Legon, Accra, Ghana. The animals were housed in stainless steel cages (34 cm × 47 cm × 18 cm) with softwood shavings as bedding, under controlled ambient temperature (23 ± 2 °C), relative humidity (50 ± 10%) and light/dark condition (L:D cycle 12:12 h) in the Department of Animal Experimentation of the same Institute with ad libitum access to standard rat chow (Agricare Ltd, Kumasi, Ashanti Region, Ghana) and tap water.

Experimental grouping and procedures

The experimental procedure is illustrated in Fig. 1. In brief, rats underwent fasting for 12 h prior to induction of type 2 diabetes mellitus (T2DM), and blood samples were obtained by tail snipping for measurement of fasting blood glucose (FBG) with a portable hand-held glucometer (One Touch Select Plus®; LifeScan Inc., Zug, Switzerland). Seven of the rats were used as non-diabetic healthy control (HC; received distilled water orally) while T2DM was induced in the remaining 23 rats by intraperitoneal (i.p.) administration of 55 mg/kg streptozotocin (Sigma Aldrich, Missouri, USA) after i.p. injection of 110 mg/kg nicotinamide (Nanjing Yasnt Bio-Tech Co. Ltd—Nanjing, China). Only rats with FBG above 13.9 mmol/L (250 mg/dL) were considered as diabetic [38]. Following this, the diabetic rats were randomly divided into 3 groups being T2DM (untreated diabetic control [n = 9]; received distilled water), T2DM + ALA (received 60 mg/kg/day ALA by oral gavage; n = 7) and T2DM + PPG (received 40 mg/kg/day i.p; n = 7). Alpha-lipoic acid (ALA) was purchased from Nanjing Yasnt Bio-Tech Co. Ltd—Nanjing, China in powdered form, while DL-propargylglycine (PPG; an inhibitor of endogenous H2S production) from Sigma Aldrich Co, St. Louis, MO, USA. Clinical parameters such as body weight (BW) and food intake were measured every week in all groups till the day of sacrifice. All rats received their respective treatments (distilled water, ALA and PPG) daily for 6 weeks after which they were sacrificed under ketamine (60 mg/kg)/xylazine (10 mg/kg) anesthesia (i.p.) and blood samples were collected by cardiac puncture into EDTA tubes. The liver was harvested and weighed and relative liver weight [liver weight (LW)/BW ratio (%)] was computed. A piece of the liver tissue from each rat was snap-frozen and stored in a −80 °C freezer for western blot analysis, biochemical assays and measurement of sulfane sulfur and H2S production while another piece and pancreas were fixed in 10% neutral-buffered formalin for histological analysis. Glycosylated hemoglobin (HbA1c; a reliable marker and important index of average glycemia under diabetic conditions) was measured from whole blood using an automatic biochemical analyzer (Nycocard Reader, Axis Shield, Oslo, Norway).

Fig. 1
figure 1

The rat model of type 2 diabetes mellitus (T2DM)-induced liver injury. Following 12 h of fasting, fasting blood glucose (FBG) was measured and T2DM was induced by nicotinamide (NIC; 110 mg/kg) and streptozotocin (STZ; 55 mg/kg) administration. T2DM was confirmed on day 3 after its induction. Diabetic rats received either oral administration of alpha-lipoic acid (T2DM + ALA; 60 mg/kg/day) or intraperitoneal injection of DL-propargylglycine (T2DM + PPG; 40 mg/kg/day) for 6 weeks. Untreated diabetic and non-diabetic rats received distilled water orally and served as diabetic (T2DM) and healthy control (HC) groups respectively. The rats were sacrificed after 6 weeks and samples were collected for analysis

Plasma preparation and measurement of biochemical parameters

Whole blood sample was spun in a centrifuge at 3000 rpm for 15 min at 4 °C to obtain plasma. Upon obtaining plasma samples from the centrifugation step, plasma triglycerides, total cholesterol, low-density lipoproteins-cholesterol (LDL) and high-density lipoproteins-cholesterol (HDL) were measured with Mindray BS-200 Biochemistry Auto-analyzer, Shenzhen, China, while plasma insulin levels were measured using a radioimmunoassay (RIA) kit from Atom High-Tech Co., Ltd., Beijing, China, following the manufacturer's instructions. Plasma content of non-esterified free fatty acids (FFA) was measured using a non-esterified FFA colorimetric kit (Elabscience, USA) on an i-Mark microplate reader. Plasma levels of liver function markers such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and total bilirubin were measured with a Biochem-Immuno Autoanalyzer (TBA-40FR, Toshiba, Tokyo, Japan) while interleukin-1β (IL-1β), IL-6 and tumor necrosis factor-alpha (TNF-α) in plasma and liver tissue were measured by ELISA with the help of a DuoSet kit and in accordance with the manufacturer’s guide (Quantikine, R&D Systems, Minneapolis, MN, USA). Pancreatic β-cell function was assessed by computing HOMA-β using the formula: 20 × Insulin (μ IU/mL) ÷ Glucose (mmol/L) − 3.5).

Measurement of intrahepatic and plasma antioxidant status

Determination of intrahepatic and plasma malondialdehyde levels

Hepatic malondialdehyde (MDA; a by-product of lipid peroxidation and an indicator of ROS production) was measured using thiobarbituric acid reactive substances (TBARS) method following the manufacturer’s guide (Abcam, Toronto, ON, Canada). Concisely, about 50 mg of liver tissue was homogenized in 100 μL phosphate-buffered saline (PBS) containing butylated hydroxytoluene (Cell Biolabs, Netherlands) and centrifuged at 10,000 g for 5 min at 4 °C. About 50 mL of the supernatant was collected and 50 mL SDS-Lysis solution (Cell Biolabs) was then added to MDA standards and incubated for 5 min at room temperature. Following this step, 125 mL of TBA reagent (Cell Biolabs) was added and incubated for 60 min at 95 °C and then cooled to room temperature for 5 min after which it was centrifuged at 1000 g for 15 min at 4 °C. The supernatant was collected and 150 μL of 2-Butanol (Merck, Darmstadt, Germany) was added to it, mixed for 2 min followed by centrifugation at 10,000 g for 5 min at 4 °C. Lipid peroxidation was determined from 200 μL of the butanol (organic) fraction by measuring optical density at 532 nm and expressed as ng/mg of liver tissue. The method was repeated using 200 μL of plasma samples, and the lipid peroxidation was measured at the same optical density.

Determination of intrahepatic and plasma glutathione content and superoxide dismutase activity

The content of glutathione (GSH) in liver tissue was measured using a spectrophotometric method following instructions from the test kit from Promega (Madison, WI, USA). In a nutshell, about 50 mg of frozen-kept liver tissue was homogenized in 1 mL of ice-cold 0.5% potassium chloride and sonicated for 1 min. The homogenate was centrifuged at 3000 rpm for 10 min at 4 °C and the supernatant was collected and used together with the GSH standard provided in the test kits. Hepatic GSH was quantified by spectophotometrically with the help of a SpectraMax 2 plate reader. The procedure was repeated using 150 μL of plasma samples. Measurement of SOD activity in liver and plasma was also done using 50 mg of frozen-kept liver tissue and 300 μL of plasma sample according to a previously described method by Xu et al. [39] and following the instructions from the test kit (Nanjing Kaiji Bio, Nanjing, China).

Extraction and measurement of intrahepatic triglyceride, cholesterol, FFA and glycogen contents

Triglyceride

Extraction of triglyceride and total cholesterol was done using the Folch method described by Qasem et al. [40]. Summarily, lipids were extracted in a 2:1 mixture of chloroform and methanol, with a total volume of 5 mL after 50 mg of liver tissue was weighed and homogenized. Next, the extract was then centrifuged at 2500 g for 15 min and the supernatant was collected and placed in a chamber filled with nitrogen to allow evaporation to dryness, leaving a residue. About 5 mL of isopropyl alcohol containing 10% Triton X was added to the residue and centrifuged at 10 000 g for 10 min. The supernatant was collected and used for the measurement of triglyceride and cholesterol in the liver tissue with the help of a commercially available test kit and following the manufacturer’s guide (E1013, Applygen Technologies Inc., Beijing, China).

Free fatty acid

Extraction and determination of free fatty acid (FFA) in the liver were done as previously described [41]. In brief, 200 mg of liver tissue was homogenized in a 1 mL of lysis buffer containing 2 mmol/L NaCl, 200 μL of 20 mmol/L EDTA, and 50 mmol/L sodium phosphate buffer with a pH of 7.4. Following the homogenization step, 100 μL of the supernatant was mixed with 100 μL of tert-butyl alcohol and 50 μL of Triton X-100/methyl alcohol mixture (1:1 vol/vol) to extract the FFA and then measured using a colorimetric assay kit (Sigma-Aldrich, Ontario, Canada).

Glycogen

Measurement of hepatic glycogen content was done using a commercially available colorimetric glycogen assay kit adhering to the manufacturer’s instructions (Abcam, Toronto, Ontario, Canada). Briefly, 10 mg of liver tissue was rapidly homogenized with 200 µL ddH2O for 10 min on ice and then boiled for another 10 min to inactivate enzymes. The homogenate was centrifuged at 18,000 rpm for 10 min and the supernatant was collected. The supernatant (30 μL) was added to a 96-well plate and the volume was brought to 50 µL with glycogen hydrolysis buffer provided in the kit. A volume of 2 µL of hydrolysis enzyme mix was added to standard and samples, and mixed well, followed by incubation at room temperature for 30 min. Next, 48 µL of reaction mix was added to the standard and samples, and incubated at room temperature for 30 min, and absorbance of the solution was determined at 450 nm with a microplate reader.

Measurement of intrahepatic sulfane sulfur and H2S contents

Measurement of hepatic sulfane sulfur

Hepatic sulfane sulfur was measured using cold cyanolysis method with colorimetric detection as previously described [42]. This is a method that involves a nucleophilic attack on a sulfur-sulfur bond by cyanide. Briefly, following cold homogenization on ice using 50 mg of liver tissue, a solution containing 720 μL of distilled water, 80 μL of 1 mol/L NH3, and 100 μL of 0.5 mol/L KCN was added to 100 μL of the tissue homogenate and mixed thoroughly. Next, the mixture was incubated at room temperature for 45 min after which 20 μL of 38% formaldehyde solution and 200 μL of Goldstein reagent containing Fe3+ cation were added and centrifuged at 12,000 g for 10 min. The supernatant was collected and the absorbance was measured at a wavelength of 460 nm. The pool of sulfane sulfur was determined from a calibration curve with 1 mmol/L KSCN.

Measurement of hepatic and plasma H 2 S production

The content of H2S in liver tissue was measured spectrophotometrically as previously described [43]. In short, about 50 mg of liver tissue was homogenized in cold KHPO4 buffer (1:10). A volume of 200 μL of the homogenate was mixed with 200 μL of 2 mmol/L pyridoxal 5′-phosphate, 200 μL of 10 mM L-cysteine and 200 μL of 10% trichloroacetic acid. Next, the mixture was incubated at 37 °C for 30 min after which zinc acetate (1% w/v, 250 μL 1%) was added to trap H2S. Following this step, N,N-dimethyl-p-phenylenediamine sulfate (20 mmol/L, 133 μL) in 7.2 mol/L HCl and FeCl3 (30 mmol/L, 133 μL) in 1.2 mol/L HCl were added. The resulting solution was transferred to a 96-well plate and the absorbance of the solution was determined at 670 nm. The H2S production was calculated against a standard curve of NaHS (0.1–100 μmol/L). Plasma H2S was measured using a sulfide ion selective electrode (Lazar Research Laboratories, Inc., Los Angeles, CA, USA) and a sulfide antioxidant buffer as we previously described [44].

Western blotting

The expressions of hepatic CSE and 3-MST proteins and β-actin (house-keeping protein) were determined by western blot technique. To be brief, hepatic expression of these proteins on the nitrocellulose membranes were detected using CSE (1:1000; Abnova, USA), 3-MST (1:1000; Santa Cruz, The Netherlands) and β-actin (1:5000; Abcam, Canada) primary antibodies following an overnight incubation at 4 °C. Next, the membranes were washed three times with a washing buffer (Tris-buffered saline [TBS] with 0.004% Tween) and incubated with their respective HRP-linked secondary antibodies (1:1000) in TBS + Tween-20 supplemented with 3% bovine serum albumin (w/v) at room temperature for 1 h. The blots were developed with SuperSignal West Dura Extended Duration Substrate (Thermo Scientific, USA) and protein bands were visualized with a Gene Genome. The intensities of the target protein bands were quantified using GeneTools analysis software (Westburg B.V., Leusden, The Netherlands) and normalized against the intensity of β-actin.

Histopathology

Hematoxylin/Eosin stain

Formalin-fixed liver tissue samples were processed for histological examination as we previously described [45]. Concisely, the samples were dehydrated in alcohol series (70%, 80%, 90% and 100%) followed by further dehydration in xylene. Following the dehydration step, the samples were embedded in molten paraffin wax and then cut from a similar site of the tissue and in a similar plane at 4 μm thick. Next, samples were rehydrated in a decreasing series of alcohol and distilled water and then stained with hematoxylin/eosin (HE) to evaluate morphological changes.

Oil red O stain

To visualize and examine triglyceride and other lipid deposits in diabetic liver tissue, frozen sections of liver tissue from each group were stained using Oil red O staining protocol. In brief, samples were cut at 8 mm thickness and air-dried on glass slides. The sections were then fixed in formalin and washed under running tap water for 5 min after which they were rinsed in 60% isopropanol. Next, the liver samples were stained with freshly prepared Oil red O solution for 15 min and then rinsed again in 60% isopropanol. Following this step, the samples were dipped briefly in hematoxylin and then rinsed with distilled water. The samples were mounted in an aqueous mounting medium and examined under a light microscope (Leica DM1000, Leica Microsystems, Morrisville, NC, USA) at 100 × magnification by two experienced hepatic pathologists in a double-blinded fashion independently. Fatty degeneration was scored by following the classification system according to Toblli et al. [46]. Thus, 0 (absent), 1 (mild; 10% of microscopic field), 2 (moderate: 25% of microscopic field), 3 (severe; 50% of microscopic field) and 4 (very severe; > 50% of microscopic field).

Periodic acid-Schiff stain

Pancreas tissue samples were dewaxed and stained with periodic acid-Schiff (PAS) reagent and counterstained briefly with Meyer’s hematoxylin. Both liver and pancreas tissue sections were examined under a light microscope (Leica DM1000, Leica Microsystems, Morrisville, NC, USA) at 400 × magnifications and the liver sections were scored on the degree of hepatocellular vacuolation, sinusoidal enlargement and liver fatty degeneration in a double-blinded fashion independently by two experienced hepatic pathologists as previously described [47].

Statistical analysis

All results are shown as mean ± standard error of the mean (SEM). One-way analysis of variance (ANOVA) with Tukey post-hoc comparison test was used to evaluate differences between groups. Statistical calculations were done using GraphPad Prism analysis software (Prism 8, GraphPad Software, Inc., San Diego, CA, USA). The criterion for statistical significance between groups was set at p < 0.05.

Results

Maintenance of body weight and preservation of glycemic and insulin levels and intrahepatic glycogen content by ALA

We measured clinical parameters such as body weight, food intake, blood glucose, hepatic glycogen content, pancreatic β-cell function and plasma insulin in addition to pancreas histology under diabetic conditions and the effect of ALA. Induction of T2DM resulted in a progressive decline in body weight, food consumption and persistent hyperglycemia over the 6-week period, which correlated with marked increase in liver weight and relative liver weight compared to healthy control (HC) rats (Fig. 2A–F). In addition, untreated T2DM rats also showed hypoinsulinemia, significantly decreased β-cell function and hepatic glycogen content and severely damaged pancreatic islets in comparison with rats in the HC group (Fig. 2G–J;). Whereas administration of DL-propargylglycine (PPG) further aggravated these changes in T2DM + PPG rats although not significantly (Fig. 2A, D–G, I and J; p > 0.05) with the exception of HOMA-β, food intake and fasting blood glucose, which increased significantly over time (Fig. 2B, C and H), treatment with ALA in T2DM + ALA group of rats prevented these changes over the 6-week period, maintaining the levels of these parameters at HC levels (Fig. 2A–J; p > 0.05). Thus, ALA maintained body weight and preserved glycemic and insulin levels as well as hepatic glycogen content and pancreatic islet structure under diabetic conditions.

Fig. 2
figure 2

Body weight, glycemic status, plasma insulin and pancreas histology. A Body weight, B food consumption C fasting blood glucose D liver weight E relative liver weight, F HbA1c, G plasma insulin, H HOMA-β, I hepatic glycogen content and J representative micrographs of PAS-stained images of pancreatic tissues at 400 × magnification. Black arrows point to pancreatic islets. HC = Healthy control; T2DM = Untreated diabetic control; T2DM + ALA = Diabetic rats treated with alpha-lipoic acid; DT2M + PPG = Diabetic rats treated with propargylglycine. #p < 0.0001 vs. T2DM, $p < 0.001 vs. T2DM, *p < 0.05 vs. T2DM

Preservation of hepatic integrity by ALA

As abnormal liver morphology and function under uncontrolled diabetes are usually due to fatty changes in the diabetic liver, we investigated the effect of ALA on hepatic structure and function following induction of T2DM by performing liver histology, liver function test (ALT, AST, ALP, total bilirubin), and by determining hepatic lipid profile (triglyceride, total cholesterol, FFA, LDL and HDL), inflammation (IL-1β; IL-6 and TNF-α) and antioxidant status (MDA, GSH and SOD). 6 weeks of untreated T2DM resulted in severe liver injury evidenced by hepatocellular vacuolation, sinusoidal enlargement and liver fatty degeneration (Fig. 3A–C). This positively correlated with severe transaminitis, hyperbilirubinemia and impaired plasma and hepatic lipid profile characterized by hyperlipidemia and hepatic steatosis compared to HC rats (Fig. 3D–O). Consistent with these pathological changes are significantly increased levels of plasma and hepatic pro-inflammatory cytokines and impaired hepatic antioxidant status marked by increased levels of IL-1β, IL-6, TNF-α and MDA and decreased GSH content and SOD activity relative to rats in HC group (Fig. 4A–L). While these pathological changes became worse in PPG-treated diabetic rats (Figs. 3A–O, 4A–L), ALA treatment protected against diabetes-induced liver injury by strongly preserving hepatic integrity, which was not statistically significant compared to that in HC rats with the exception of ALP and hepatic GSH which were significantly higher in ALA-treated rats than HC rats (Fig. 3A–O, 4A–L). Taken together, administration of ALA preserved hepatic integrity under T2DM conditions.

Fig. 3
figure 3

Liver histology, parameters of liver function and lipid profile A Representative photomicrographs of hematoxylin and eosin, and oil red O staining at 400 × and 100 × magnification respectively. Yellow arrows in HE images point to hepatocellular vacuolation and sinusoidal enlargement while red stains in oil red O images indicate triglyceride and other lipid accumulation. B and C Quantification of liver histology, D ALT, E AST, F ALP, G total bilirubin, H plasma triglyceride, I hepatic triglyceride, I plasma triglyceride, J plasma total cholesterol, K hepatic total cholesterol, L plasma FFA, M hepatic FFA, N plasma LDL and O plasma HDL. HC = Healthy control; T2DM = Untreated diabetic control; T2DM + ALA = Diabetic rats treated with alpha-lipoic acid; DT2M + PPG = Diabetic rats treated with propargylglycine

Fig. 4
figure 4

Inflammation and antioxidant status. A Plasma IL-1β B plasma IL-6, C plasma TNF-α, D hepatic IL-β, E hepatic IL-6, F hepatic TNF-α, G plasma MDA, H plasma GSH, I plasma SOD, J hepatic MDA, K hepatic GSH and L hepatic SOD. HC = Healthy control; T2DM = Untreated diabetic control; T2DM + ALA = Diabetic rats treated with alpha-lipoic acid; DT2M + PPG = Diabetic rats treated with propargylglycine

Activation of intrahepatic sulfane sulfur/H2S signaling pathway by ALA

To investigate the potential involvement of hepatic sulfane sulfur/H2S as a mechanism in diabetes-induced liver injury and the effect of ALA, we measured these two sulfur species and H2S-producing enzymes (CSE and 3-MST) in the liver tissue. Untreated T2DM for 6 weeks significantly downregulated the expression of hepatic CSE and 3-MST enzymes, resulting in markedly reduced levels of hepatic sulfane sulfur and endogenous H2S in plasma and liver tissue in comparison with HC rats (Fig. 5A–F; p < 0.05). PPG administration further reduced plasma H2S and hepatic sulfane sulfur levels, with a significant reduction in hepatic H2S (Fig. 5D–F) while it also reduced hepatic CSE and 3-MST expression levels but not significantly (Fig. 5A–C; p > 0.05). However, treatment with ALA strongly upregulated the expression of these enzymes and markedly increased hepatic sulfane sulfur and H2S levels even above the levels in HC rats (Fig. 5A–F; p < 0.01). In summary, ALA administration activated hepatic sulfane sulfur/H2S signaling pathway, and thereby contributed to the prevention of diabetes-induced hepatic injury in ALA-treated rats.

Fig. 5
figure 5

Western blot and hepatic sulfane sulfur/H2S system. A Image of a Western blot showing hepatic expression of CSE and 3-MST. Quantification of B CSE and C 3-MST. Measurement of D plasma H2S, E hepatic H2S and F hepatic sulfane sulfur. HC = Healthy control; T2DM = Untreated diabetic control; T2DM + ALA = Diabetic rats treated with alpha-lipoic acid; DT2M + PPG = Diabetic rats treated with propargylglycine

Discussion

Several lines of preclinical and clinical evidence have shown that the liver is one of the organs greatly affected by diabetes mellitus. However, there are currently no approved pharmacological agents for effective treatment or prevention of diabetes-induced liver injury. In this study, we report that 6 weeks of untreated type 2 diabetes mellitus (T2DM) in rats causes liver injury similar to the clinical version of non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH; a progressive and aggressive form of NAFLD), both of which are characterized principally by hyperlipidemia and abnormal intrahepatic lipid accumulation without excessive alcohol intake. Hepatic histopathological alterations were evidenced by hepatocellular vacuolation, sinusoidal enlargement and liver fatty degeneration. Gross examination revealed hepatomegaly while functional changes were evidenced by severe transaminitis, hyperbilirubinemia, and impaired lipid profile. Other pathological changes include glycogen storage disruption, impaired hepatic antioxidant status and activation of pro-inflammatory pathway. We present evidence from a murine model of T2DM (one of the most common etiologies of NAFLD and NASH in humans) suggesting that suppression of hepatic sulfane sulfur/hydrogen sulfide (H2S) signaling pathway contributes to diabetes-induced liver injury, and that the activation of this pathway by alpha-lipoic acid (ALA) mediates hepatoprotection under diabetic condition. Importantly, pharmacological blockage of sulfane sulfur/H2S signaling pathway with DL-propargylglycine (PPG) resulted in further liver injury, with more lipid accumulation in the liver and plasma as seen in patients with NAFLD and NASH.

In the present study, treatment of T2DM rats with ALA for 6 weeks upregulated hepatic expression of the H2S-producing enzymes, CSE and 3-MST, and increased the levels of hepatic H2S and sulfane sulfur (the precursor of H2S), and thus contributed to hepatoprotection against diabetes-induced liver injury. Our observation is consistent with two previous studies in which administration of ALA increased sulfane sulfur and H2S levels in rat liver homogenates [26, 27]. Moreover, ALA treatment has been reported to restore bioenergetic effect of CSE and 3-MST and protected vascular smooth muscle cells in diabetic models similar to ours, in which hyperglycemia impaired CSE/H2S and 3-MST/H2S pathways [48, 49]. As observed in the present study, untreated T2DM as well as pharmacological inhibition of sulfane sulfur/H2S pathway strongly impaired hepatic H2S production, characterized by downregulation of hepatic CSE and 3-MST expressions and reduced levels of sulfane sulfur and endogenous H2S, and contributed to the development of NAFLD and NASH in diabetic rats, while ALA prevented this pathology. This finding corroborates those of several preclinical models of NAFLD and NASH in which hepatic CSE and 3-MST protein and gene expressions were markedly downregulated while activation of H2S pathway by H2S donor compounds protected the liver and prevented further progression of NAFLD and NASH [50,51,52]. Beyond the boundaries of these exciting preclinical findings, a placebo-controlled randomized clinical trial involving daily administration of ALA either alone or in combination with vitamin E for 6 months provided hepatoprotection in patients with NAFLD and NASH by reducing the levels of plasma triglyceride and tumor necrosis factor-alpha (TNF-α; a pro-inflammatory cytokine), and improved steatosis scores compared to placebo-control patients [19]. ALA also improved serum insulin levels in patients with NAFLD by reducing insulin resistance in two recent double-blind, placebo-controlled, randomized clinical trials [53, 54], and increased serum H2S levels, leading to improved glycemic status in T2DM patients [48]. In addition, ALA is currently under phase 4 clinical trial for the treatment of NAFLD (clinicaltrials.gov).

It is important to note that the hepatoprotective effect of ALA under diabetic conditions is linked to its therapeutic properties including anti-hyperglycemic, anti-apoptotic, anti-hyperlipidemic, antioxidant, anti-inflammatory and anti-fibrotic properties, which can be attributed to its ability to activate sulfane sulfur/H2S pathway. As shown in our study, impaired insulin secretion due to the destruction of cells in the pancreatic islet of Langerhans, particularly insulin-secreting pancreatic β-cells, resulted in hypoinsulinemia and persistent hyperglycemia in T2DM rats over the 6-week period, and further worsened following inhibition of sulfane sulfur/H2S pathway with PPG administration. However, ALA administration protected the β-cells and activated insulin secretion, thereby maintaining normoglycemia. As ALA activated sulfane sulfur/H2S pathway in our study, Takahashi et al. [55] observed that reactive sulfur species such as sulfane sulfur and H2S activate insulin secretory pathway via regulation of tRNA methylthiolation, and inhibited high glucose- and hyperglycemia-induced apoptosis of insulin-secreting pancreatic β-cells [56, 57]. These observations including ours, refute previous reports suggesting that H2S in pancreatic islets inhibits insulin secretion by activating adenosine triphosphate (ATP)-dependent potassium (KATP) channels [58, 59]. Although H2S is known to activate and open KATP channels under various conditions, it is possible that these contradictory reports on the effect of H2S on insulin secretion could be due to the different doses of different H2S donor compounds used in these studies, thus resulting in different concentrations of total H2S in the insulin-secreting β-cells. This assertion is supported by the findings of Wu and colleagues [60], who attributed the inhibition of insulin secretion by H2S to its over-production. It is also possible that ALA has other mechanisms for increased insulin secretion besides its interaction with KATP channels such as increased phosphorylation of insulin receptor-β and insulin receptor substrate-1 as well as upregulation of glucose-stimulated insulin secretion (GSIS) genes glucokinase, glucose transporter 2 and Kir6.2 in the insulin signaling pathway, which have been reported to restore insulin secretion and protected islet cells under high glucose exposure [61]. Moreover, a phase 4 clinical trial is currently underway to determine whether ALA administration prevents or attenuates fatty acid-induced impairment of insulin secretion and insulin sensitivity (clinicaltrails.gov).

The anti-hyperglycemic effect of ALA as observed in our study, is also linked to its ability to activate insulin receptors and enhance their activity, hence ALA could be considered an insulin-mimetic agent. Moreover, ALA has been shown to be actively involved in glucose-regulating mechanisms by functioning as an insulin sensitizer in skeletal muscles [62], facilitating translocation of glucose transporter protein subtype 4 (GLUT 4) to the plasma [45] and activating adenosine monophosphate-activated protein kinase (AMPK) [62], a widely-recognized enzyme that regulates hepatic gluconeogenesis and cellular energy homeostasis by inhibiting gluconeogenic genes and enzymes which are abnormally regulated in gluconeogenesis pathway in T2DM [63]. In addition, as ALA activated sulfane sulfur/H2S system, H2S has been reported to attenuate hyperglycemia-induced vascular inflammation via AMPK activation [64], raising the possibility that ALA could serve a therapeutic purpose in other complications of chronic hyperglycemia of diabetes. Our result in the present study also includes increased intrahepatic glycogen content in ALA-treated T2DM rats, which was significantly reduced in untreated and PPG-treated T2DM rats, similar to findings from previous rat models of T2DM [65, 66]. This implies glycogen storage disruption in our T2DM model, which is commonly observed in diabetic patients with glycogen hepatopathy or liver cirrhosis (the advanced stage of NAFLD and NASH). It further suggests that ALA could maintain glucose homoeostasis under diabetic conditions by reducing gluconeogenesis and increasing glycogenesis, a pathway for disposal of excess glucose. Although our study did not include molecular mechanisms underlying this effect of ALA, inhibition of intrahepatic gluconeogenesis and increased glycogenesis have been associated with activation of AMPK signaling [67], a pathway that is also activated by ALA.

In addition to its anti-hyperglycemic effect, ALA also ameliorated hepatic steatosis and hypertriglyceridemia under diabetic conditions by stimulating triacylglycerol clearance and downregulating hepatic triacylglycerol secretion [68]. This aligns with our observation, in which ALA prevented the accumulation of triglyceride, cholesterol and free fatty acids (FFA) in the liver and plasma of T2DM rats. As ALA increased hepatic sulfane sulfur and endogenous H2S levels, with upregulated hepatic CSE and 3-MST expressions, thereby preventing hepatic lipid accumulation, similar observations have been made in previous studies. For example, it has been shown that downregulation of hepatic CSE and 3-MST and reduced endogenous H2S levels in mice and humans contribute to hypertriglyceridemia and accumulation of other lipids in the liver [69,70,71]. In the same studies, the authors reported that activation of the H2S pathway with H2S donor compounds including ALA reduced lipid accumulation in hepatocytes and improved NAFLD and NASH by stimulating hepatic autophagy through activation of AMPK signaling pathway dependent or independent of mammalian target of rapamycin (mTOR; a central regulator of cellular homeostasis) as well as via SIRT1/LKB1/AMPK pathway [69,70,71]. While these pathways were beyond the scope of our study, it can be inferred that activation of the hepatic sulfane sulfur/H2S pathway by ALA in the present study may have activated these molecular pathways and possibly other potential lipid-lowering pathways in the diabetic liver, thus reducing intrahepatic lipid content and improving NAFLD and NASH. The anti-hyperlipidemic effect of ALA is also associated with upregulation of short-, medium- and long-chain fatty acid metabolic processes (Acot1, Acot2, Acsf2, and Crat) and downregulation of lipogenic genes such as Pnpla3, Pnpla5, Elovl6, Acly and Gpam, which are major genetic risk factors for developing NAFLD [72]. These transcriptional changes may be partly responsible for the result in the present study, suggesting that inhibition of these genes by gene therapy or pharmacotherapy may be a novel approach for the treatment or prevention of NAFLD in diabetic patients as well as in patients with other metabolic diseases involving hyperlipidemia. Other mechanisms such as alterations in triglyceride secretion by liver cells due to malfunction of lipoproteins, and hyperglycemia-induced hepatic upregulation of GLUT2, which transports excess sugar from the blood into the liver and converted into excess fat, have been suggested as contributory factors in the development of NAFLD and NASH in diabetic patients [73]. Therefore, our results raise the possibility that ALA might have inhibited these mechanisms, leading to the improved lipid profile in our T2DM rats.

Besides the anti-hyperglycemic and anti-hyperlipidemic effects of ALA discussed above, we also observed in the present study that its hepatoprotective effect under diabetic conditions is partly due to its antioxidant and anti-inflammatory properties. Induction of T2DM in our model changed the intrahepatic oxidative balance over the 6-week period. This was hallmarked by markedly elevated levels of malondialdehyde (MDA; a by-product of lipid peroxidation and indicator of reactive oxygen species [ROS] production) and significantly reduced glutathione (GSH) content and superoxide dismutase (SOD) activity, thus overwhelming hepatic antioxidant defence system. The impaired intrahepatic antioxidant status correlated with pathological changes in liver morphology and function (increased ALT, AST and ALP, and total bilirubin) and inflammation (increased IL-1β, IL-6 and TNF-α levels). Our observations of the pathological changes in the diabetic liver in the present study are closely similar to those observed in chronic stress in liver cells. It is known that increased lipid peroxidation and free fatty acid oxidation lead to over-production of ROS-induced oxidative stress, which activates several stress-sensitive kinase signaling cascades such as JNK and IKKβ [74]. Also, ROS act on unsaturated fatty acids, and initiates lipid peroxidation, leading to changes in the fluidity and permeability of the liver cell membrane. This creates a suitable local environment for inflammatory infiltration, resulting in liver inflammation and activation of fibrotic and necrotic pathways that favor pathogenesis and progression of NAFLD and NASH [74].

As a potent antioxidant, which is often referred to as the “antioxidant of antioxidants” due to its ability to activate and recruit other antioxidants, ALA in the present study significantly improved hepatic antioxidant status and inflammation by decreasing the levels of ROS and pro-inflammatory cytokines, and increasing GSH and SOD levels, and thus contributing to hepatoprotection similar to previous reports in other liver disease models [75, 76]. Our result provides experimental support for the observations in two clinical trials in which antioxidant supplements significantly reduced transaminitis, hepatic fat accumulation and liver stiffness, and improved lipid profile in patients with NAFLD [77, 78]. It also confirms that induction of oxidative stress and activation of inflammatory cascade are crucial in the development and progression of NAFLD and NASH, and that activation of the H2S system indeed triggers hepatic antioxidant defence against NAFLD and NASH. Moreover, it has been shown that both ALA and other compounds containing H2S activate KATP channels, which in turn activate antioxidant and anti-inflammatory pathways, leading to organ protection [79,80,81]. It is also possible that the activation of antioxidant and anti-inflammatory pathways by ALA may be via other mechanisms besides its interaction with KATP channels such as activation of the transcription factor nuclear erythroid 2-related factor 2 (Nrf2; a regulator of transcription of antioxidants and other cytoprotective genes) as well as inhibition of nuclear factor-kappaB (NF-κB; a major protein complex that mediates inflammation by influencing pro-inflammatory cytokine production) and inhibiting the phosphorylating action of mitogen-activated protein kinase (MAPK; another major pro-inflammatory mediator) [82, 83]. Other studies also reported anti-fibrotic effect of ALA and H2S in animal models of hepatic fibrosis including diabetic animals by inhibiting the expression of the fibrotic proteins, transforming growth factor-beta1 (TGF-β1) and alpha-smooth muscle actin (α-SMA) [84, 85]. Although sulfane sulfur was not measured in these studies, it should be alluded that most of the studies that report increased levels of endogenous H2S production by H2S donor compounds also observe corresponding increases in cellular sulfane sulfur levels [86, 87]. Besides, sulfane sulfur and H2S always co-exist, and recent investigations suggest that some of the physiological processes previously reported to be regulated by H2S are actually mediated by sulfane sulfur [88, 89]. Moreover, our result also suggests that pharmacological inhibition of endogenous H2S also reduces sulfane sulfur levels. Taken together, the hepatoprotective effect of ALA in the present study may, at least in part, be due to its antioxidant, anti-inflammatory and anti-fibrotic properties.

While our results are exciting, we were unable to examine the diabetic liver at the subcellular level due to technical challenges. Such an assessment would have provided additional information on microvascular fatty degeneration as well as ultrastructural progressive impairment in the diabetic liver including mitochondria and other intracytoplasmic organelles of our T2DM rats.

Conclusion

To sum up, we have demonstrated for the first time that ALA prevents diabetes-induced liver injury by activating hepatic sulfane sulfur/H2S pathway via upregulation of hepatic CSE and 3-MST protein expression. This constitutes a novel mechanism in the pharmacological treatment or prevention of NAFLD and NASH under diabetic conditions. Considering that there is significant hepatic involvement in diabetes mellitus coupled with the lack of approved pharmacological agents for diabetes-induced liver injury, our results provide experimental support for the idea that ALA or novel drugs containing sulfane sulfur and H2S with anti-hyperglycemic, anti-hyperlipidemic, antioxidant, anti-inflammatory and anti-fibrotic properties, should be designed and applied for the treatment and prevention of NAFLD and NASH in diabetic patients.

Availability of data and materials

The dataset used and/or analysed during the current study are available from the corresponding author on reasonable request.

Abbreviations

T2DM:

Type 2 diabetes mellitus

T1DM:

Type 1 diabetes mellitus

ALA:

Alpha-lipoic acid

DHLA:

Dihydrolipoic acid

H2S:

Hydrogen sulfide

CSE:

Cystathionine γ-lyase

3-MST:

3-Mecaptopyruvate sulfurtransferase

PPG:

DL-propargylglycine

NAFLD:

Non-alcoholic fatty liver disease

NASH:

Non-alcoholic steatohepatitis

ROS:

Reactive oxygen species

GSH:

Glutathione

SOD:

Superoxide dismutase

IL-1β:

Interleukin-1beta

IL-6:

Interleukin-6

TNF-α:

Tumor necrosis factor-alpha

TGF-β1:

Transforming growth factor-beta1

α-SMA:

Alpha-smooth muscle actin

MDA:

Malondialdehyde

FFA:

Free fatty acid

AMPK:

Adenosine monophosphate-activated protein kinase

mTOR:

Mammalian target of rapamycin

GLUT4:

Glucose transporter protein subtype 4

KATP :

Adenosine triphosphate (ATP)-dependent potassium

LDL:

Low-density lipoprotein

HDL:

High-density lipoproteins

ALT:

Alanine aminotransferase

AST:

Aspartate aminotransferase

ALP:

Alkaline phosphatase

FBG:

Fasting blood glucose

HbA1c:

Glycosylated haemoglobin

References

  1. Adeyemi DO, Ukwenya VO, Obuotor EM, Adewole SO. Anti-hepatotoxic activities of Hibiscus sabdariffa L. in animal model of streptozotocin diabetes-induced liver damage. BMC Complement Altern Med. 2014;14:277.

    Article  PubMed  PubMed Central  Google Scholar 

  2. Targher G, Bertolini L, Padovani R, Rodella S, Zoppini G, Pichiri I, Sorgato C, Zenari L, Bonora E. Prevalence of non-alcoholic fatty liver disease and its association with cardiovascular disease in patients with type 1 diabetes. J Hepatol. 2010;53(4):713–8.

    Article  CAS  PubMed  Google Scholar 

  3. Leite NC, Villela-Nogueira CA, Pannain VL, Bottino AC, Rezende GF, Cardoso CR, Salles GF. Histopathological stages of nonalcoholic fatty liver disease in type 2 diabetes: prevalences and correlated factors. Liver Int. 2011;31(5):700–6.

    Article  PubMed  Google Scholar 

  4. Mandal A, Bhattarai B, Kafle P, Khalid M, Jonnadula SK, Lamicchane J, Kanth R, Gayam V. Elevated liver enzymes in patients with type 2 diabetes mellitus and non-alcoholic fatty liver disease. Cureus. 2018;10(11):e3626.

    PubMed  PubMed Central  Google Scholar 

  5. Chen X, Wen HQ, Li QL, Shen LS, Luo XW, Zhou B, Guo RM. Quantification of liver fat deposition in obese and diabetic patients: a pilot study on the correlation with myocardium and periapical fat content. Liver Res. 2022;6(2):103–10.

    Article  CAS  Google Scholar 

  6. Musso G, Gambino R, Cassader M, Pagano G. Meta-analysis: natural history of non-alcoholic fatty liver disease (NAFLD) and diagnostic accuracy of non-invasive tests for liver disease severity. Ann Med. 2011;43(8):617–49.

    Article  PubMed  Google Scholar 

  7. Yasui K, Hashimoto E, Komorizono Y, Koike K, Arii S, Imai Y, Shima T, Kanbara Y, Saibara T, Mori T, Kawata S, Uto H, Takami S, Sumida Y, Takamura T, Kawanaka M, Okanoue T, Japan NASH Study Group, Ministry of Health Labour and Welfare of Japan. Characteristics of patients with nonalcoholic steatohepatitis who develop hepatocellular carcinoma. Clin Gastroenterol Hepatol. 2011;9(5):428–33.

    Article  PubMed  Google Scholar 

  8. Zang S, Wang L, Ma X, Zhu G, Zhuang Z, Xun Y, Zhao F, Yang W, Liu J, Luo Y, Liu Y, Ye D, Shi J. Neutrophils play a crucial role in the early stage of nonalcoholic steatohepatitis via neutrophil elastase in Mice. Cell Biochem Biophys. 2015;73(2):479–87.

    Article  CAS  PubMed  Google Scholar 

  9. Moreno-Fernandez ME, Giles DA, Stankiewicz TE, Sheridan R, Karns R, Cappelletti M, Lampe K, Mukherjee R, Sina C, Sallese A, Bridges JP, Hogan SP, Aronow BJ, Hoebe K, Divanovic S. Peroxisomal β-oxidation regulates whole body metabolism, inflammatory vigor, and pathogenesis of nonalcoholic fatty liver disease. JCI Insight. 2018;3(6):e93626.

    Article  PubMed Central  Google Scholar 

  10. Zheng F, Cai Y. Concurrent exercise improves insulin resistance and nonalcoholic fatty liver disease by upregulating PPAR-γ and genes involved in the beta-oxidation of fatty acids in ApoE-KO mice fed a high-fat diet. Lipids Health Dis. 2019;18(1):6.

    Article  PubMed  PubMed Central  Google Scholar 

  11. Fealy CE, Haus JM, Solomon TP, Pagadala M, Flask CA, McCullough AJ, Kirwan JP. Short-term exercise reduces markers of hepatocyte apoptosis in nonalcoholic fatty liver disease. J Appl Physiol. 2012;113(1):1–6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. González-Rodríguez A, Mayoral R, Agra N, Valdecantos MP, Pardo V, Miquilena-Colina ME, Vargas-Castrillón J, Lo Iacono O, Corazzari M, Fimia GM, Piacentini M, Muntané J, Boscá L, García-Monzón C, Martín-Sanz P, Valverde ÁM. Impaired autophagic flux is associated with increased endoplasmic reticulum stress during the development of NAFLD. Cell Death Dis. 2014;5(4):e1179.

    Article  PubMed  PubMed Central  Google Scholar 

  13. Nasiri-Ansari N, Nikolopoulou C, Papoutsi K, Kyrou I, Mantzoros CS, Kyriakopoulos G, Chatzigeorgiou A, Kalotychou V, Randeva MS, Chatha K, Kontzoglou K, Kaltsas G, Papavassiliou AG, Randeva HS, Kassi E. Empagliflozin Attenuates Non-Alcoholic Fatty Liver Disease (NAFLD) in high fat diet fed ApoE(-/-) Mice by activating autophagy and reducing ER stress and apoptosis. Int J Mol Sci. 2021;22(2):818.

    Article  CAS  PubMed Central  Google Scholar 

  14. Athyros VG, Tziomalos K, Gossios TD, Griva T, Anagnostis P, Kargiotis K, Pagourelias ED, Theocharidou E, Karagiannis A, Mikhailidis DP, GREACE Study Collaborative Group. Safety and efficacy of long-term statin treatment for cardiovascular events in patients with coronary heart disease and abnormal liver tests in the Greek Atorvastatin and Coronary Heart Disease Evaluation (GREACE) Study: a post-hoc analysis. Lancet. 2010;376(9756):1916–22.

    Article  CAS  PubMed  Google Scholar 

  15. Chalasani N, Younossi Z, Lavine JE, Diehl AM, Brunt EM, Cusi K, Charlton M, Sanyal AJ, American Gastroenterological Association; American Association for the Study of Liver Diseases; American College of Gastroenterology. The diagnosis and management of non-alcoholic fatty liver disease: practice guideline by the American Gastroenterological Association, American Association for the study of liver diseases, and American College of Gastroenterology. Gastroenterology. 2012;142(7):1592–609.

    Article  PubMed  Google Scholar 

  16. Sato K, Gosho M, Yamamoto T, Kobayashi Y, Ishii N, Ohashi T, Nakade Y, Ito K, Fukuzawa Y, Yoneda M. Vitamin E has a beneficial effect on nonalcoholic fatty liver disease: a meta-analysis of randomized controlled trials. Nutrition. 2015;31(7–8):923–30.

    Article  CAS  PubMed  Google Scholar 

  17. Padmalayam I, Hasham S, Saxena U, Pillarisetti S. Lipoic acid synthase (LASY): a novel role in inflammation, mitochondrial function and insulin resistance. Diabetes. 2000;58:600–8.

    Article  Google Scholar 

  18. Szelag M, Mikulski D, Molski M. Quantum-chemical investigation of the structure and the antioxidant properties of α-lipoic acid and its metabolites. J Mol Model. 2012;18:2907–16.

    Article  CAS  PubMed  Google Scholar 

  19. Basu PP, Shah J, Aloysius M, Aloysius M, Jnr Brown RS. Effect of Vitamin E and alpha lipoic acid in nonalcoholic fatty liver disease: a randomized, placebo-controlled, open-label, prospective clinical trial (VAIN Trial). Open J Gastroenterol. 2014;4(5):199–207.

    Article  Google Scholar 

  20. Agathos E, Tentolouris A, Eleftheriadou I, Katsaouni P, Nemtzas I, Petrou A, Papanikolaou C, Tentolouris N. Effect of α-lipoic acid on symptoms and quality of life in patients with painful diabetic neuropathy. J Int Med Res. 2018;46(5):1779–90.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Dugbartey GJ, Alornyo KK, N’guessan BB, Atule S, Mensah SD, Adjei S. Supplementation of conventional anti-diabetic therapy with alpha-lipoic acid prevents early development and progression of diabetic nephropathy. Biomed Pharmacother. 2022;149:112818.

    Article  CAS  PubMed  Google Scholar 

  22. Dugbartey GJ, Wonje QL, Alornyo KK, Robertson L, Adams I, Boima V, Mensah SD. Combination therapy of alpha-lipoic acid, gliclazide and ramipril protects against development of diabetic cardiomyopathy via inhibition of TGF-β/Smad pathway. Front Pharmacol. 2022;13:850542.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Bustamante J, Lodge JK, Marcocci L, Tritschler HJ, Packer L, Rihn BH. Alpha-lipoic acid in liver metabolism and disease. Free Radic Biol Med. 1998;24(6):1023–39.

    Article  CAS  PubMed  Google Scholar 

  24. Shay KP, Moreau RF, Smith EJ, Smith AR, Hagen TM. Alpha-lipoic acid as a dietary supplement: molecular mechanisms and therapeutic potential. Biochim Biophys Acta. 2009;1790(10):1149–60.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Bast A, Haenen GR. Lipoic acid: a multifunctional antioxidant. BioFactors. 2003;17:207–13.

    Article  CAS  PubMed  Google Scholar 

  26. Bilska A, Dudek M, Iciek M, Kwiecień I, Sokołowska-Jezewicz M, Filipek B, Włodek L. Biological actions of lipoic acid associated with sulfane sulfur metabolism. Pharmacol Rep. 2008;60(2):225–32.

    CAS  PubMed  Google Scholar 

  27. Bilska-Wilkosz A, Iciek M, Kowalczyk-Pachel D, Górny M, Sokołowska-Jeżewicz M, Włodek L. Lipoic acid as a possible pharmacological source of hydroge sulfide/sulfane sulfur. Molecules. 2017;22(3):388.

    Article  PubMed Central  Google Scholar 

  28. Abe K, Kimura H. The possible role of hydrogen sulfide as an endogenous neuromodulator. J Neurosci. 1996;16:1066–71.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Blackstone E, Morrison M, Roth MB. H2S induces a suspended animation-like state in mice. Science. 2005;308(5721):518.

    Article  CAS  PubMed  Google Scholar 

  30. Panagaki T, Randi EB, Szabo C. Role of hydrogen sulfide and 3-mercaptopyruvate sulfurtransferase in the regulation of the endoplasmic reticulum stress response in hepatocytes. Biomolecules. 2020;10(12):1692.

    Article  CAS  PubMed Central  Google Scholar 

  31. Werge MP, McCann A, Galsgaard ED, Holst D, Bugge A, Albrechtsen NJW, Gluud LL. The role of the transsulfuration pathway in non-alcoholic fatty liver disease. J Clin Med. 2021;10(5):1081.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Li L, Whiteman M, Guan YY, Neo KL, Cheng Y, Lee SW, Zhao Y, Baskar R, Tan CH, Moore PK. Characterization of a novel water-soluble hydrogen sulfide-releasing molecule (GYY4137): new insight into the biology of hydrogen sulfide. Circulation. 2008;117:2351–60.

    Article  CAS  PubMed  Google Scholar 

  33. Juriasingani S, Jackson A, Zhang MY, Ruthirakanthan A, Dugbartey GJ, Sogutdelen E, Levine M, Mandurah M, Whiteman M, Luke P, Sener A. Evaluating the effects of subnormothermic perfusion with AP39 in a novel blood-free model of ex vivo kidney preservation and reperfusion. Int J Mol Sci. 2021;22:7180.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Zhang MY, Dugbartey GJ, Juriasingani S, Akbari M, Liu W, Haig A, McLeod P, Arp J, Sener A. Sodium thiosulfate-supplemented UW solution protects renal grafts against prolonged cold ischemia-reperfusion injury in a murine model of syngeneic kidney transplantation. Biomed Pharmacother. 2022;145:112435.

    Article  PubMed  Google Scholar 

  35. Dugbartey GJ. Diabetic nephropathy: a potential savior with “rotten-egg” smell. Pharmacol Rep. 2017;69(2):331–9.

    Article  CAS  PubMed  Google Scholar 

  36. Ye P, Gu Y, Zhu YR, Chao YL, Kong XQ, Luo J, Ren XM, Zuo GF, Zhang DM, Chen SL. Exogenous hydrogen sulfide attenuates the development of diabetic cardiomyopathy via the FoxO1 pathway. J Cell Physiol. 2018;233(12):9786–98.

    Article  CAS  PubMed  Google Scholar 

  37. Li H, Liu S, Wang Z, Zhang Y, Wang K. Hydrogen sulfide attenuates diabetic neuropathic pain through NO/cGMP/PKG pathway and μ-opioid receptor. Exp Biol Med (Maywood). 2020;245(9):823–34.

    Article  CAS  Google Scholar 

  38. Dugbartey GJ, Wonje QL, Alornyo KK, Adams I, Diaba DE. Alpha-lipoic acid treatment improves adverse cardiac remodelling in the diabetic heart—the role of cardiac hydrogen sulfide-synthesizing enzymes. Biochem Pharmacol. 2022;203:115179.

    Article  CAS  PubMed  Google Scholar 

  39. Xu G, Zhao X, Fu J, Wang X. Resveratrol increase myocardial Nrf2 expression in type 2 diabetic rats and alleviate myocardial ischemia/reperfusion injury (MIRI). Ann Palliat Med. 2019;8(5):565–75.

    Article  PubMed  Google Scholar 

  40. Qasem RJ, Cherala G, D’mello AP. Maternal protein restriction during pregnancy and lactation in rats imprints long-term reduction in hepatic lipid content selectively in the male offspring. Nutr Res. 2010;30(6):410–7.

    Article  CAS  PubMed  Google Scholar 

  41. Liu J, Han L, Zhu L, Yu Y. Free fatty acids, not triglycerides, are associated with non-alcoholic liver injury progression in high fat diet induced obese rats. Lipids Health Dis. 2016;15:27.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Wood JL. Sulfane sulfur. Methods Enzymol. 1987;143:25–9.

    Article  CAS  PubMed  Google Scholar 

  43. Ohta J, Ubuka T, Kodama H, Sugahara K, Yao K, Masuoka N, Kinuta M. Increase in cystathionine content in rat liver mitochondria after D L-propargylglycine administration. Amino Acids. 1995;9(2):111–22.

    CAS  PubMed  Google Scholar 

  44. Dugbartey GJ, Talaei F, Houwertjes MC, Goris M, Epema AH, Bouma HR, Henning RH. Dopamine treatment attenuates acute kidney injury in a rat model of deep hypothermia and rewarming—the role of renal H2S-producing enzymes. Eur J Pharmacol. 2015;15(769):225–33.

    Article  Google Scholar 

  45. Khamaisi M, Potashnik R, Tirosh A, Demshchak E, Rudich A, Tritschler H, Wessel K, Bashan N. Lipoic acid reduces glycemia and increases muscle GLUT4 content in streptozotocin-diabetic rats. Metabolism. 1997;46(7):763–8.

    Article  CAS  PubMed  Google Scholar 

  46. Toblli JE, Ferder L, Stella I, Angerosa M, Inserra F. Enalapril prevents fatty liver in nephrotic rats. J Nephrol. 2002;15(4):358–67.

    CAS  PubMed  Google Scholar 

  47. Horai Y, Akatsuka A, Mizukawa M, Niishina H, Nishikawa S, Ono Y, Takemoto K, Mochida H. Current status and prospects for quantitative analysis of digital image of pathological specimen using image processing software including artificial intelligence. Translat Regulat Sci. 2020;2(3):72–9.

    Google Scholar 

  48. Qiu X, Liu K, Xiao L, Jin S, Dong J, Teng X, Guo Q, Chen Y, Wu Y. Alpha-lipoic acid regulates the autophagy of vascular smooth muscle cells in diabetes by elevating hydrogen sulfide level. Biochim Biophys Acta Mol Basis Dis. 2018;1864(11):3723–38.

    Article  CAS  PubMed  Google Scholar 

  49. Coletta C, Módis K, Szczesny B, Brunyánszki A, Oláh G, Rios EC, Yanagi K, Ahmad A, Papapetropoulos A, Szabo C. Regulation of vascular tone, angiogenesis and cellular bioenergetics by the 3-mercaptopyruvate sulfurtransferase/H2S pathway: functional impairment by hyperglycemia and restoration by DL-α-Lipoic acid. Mol Med. 2015;21(1):1–14.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Luo ZL, Tang LJ, Wang T, Dai RW, Ren JD, Cheng L, Xiang K, Tian FZ. Effects of treatment with hydrogen sulfide on methionine-choline deficient diet-induced non-alcoholic steatohepatitis in rats. J Gastroenterol Hepatol. 2014;29(1):215–22.

    Article  CAS  PubMed  Google Scholar 

  51. Peh MT, Anwar AB, Ng DS, Atan MS, Kumar SD, Moore PK. Effect of feeding a high fat diet on hydrogen sulfide (H2S) metabolism in the mouse. Nitric Oxide. 2014;41:138–45.

    Article  CAS  PubMed  Google Scholar 

  52. Li M, Xu C, Shi J, Ding J, Wan X, Chen D, Gao J, Li C, Zhang J, Lin Y, Tu Z, Kong X, Li Y, Yu C. Fatty acids promote fatty liver disease via the dysregulation of 3-mercaptopyruvate sulfurtransferase/hydrogen sulfide pathway. Gut. 2018;67(12):2169–80.

    Article  CAS  PubMed  Google Scholar 

  53. Amirkhizi F, Hamedi-Shahraki S, Hosseinpour-Arjmand S, Ebrahimi-Mameghani M. The effect of alpha-lipoic acid on liver function and metabolic markers in obese patients with non-alcoholic fatty liver disease: a double-blind randomized controlled trial. Iran Red Crescent Med J. 2018;20(3):1–11.

    Article  Google Scholar 

  54. Rahmanabadi A, Mahboob S, Amirkhizi F, Hosseinpour-Arjmand S, Ebrahimi-Mameghani M. Oral α-lipoic acid supplementation in patients with non-alcoholic fatty liver disease: effects on adipokines and liver histology features. Food Funct. 2019;10(8):4941–52.

    Article  CAS  PubMed  Google Scholar 

  55. Takahashi N, Wei FY, Watanabe S, Hirayama M, Ohuchi Y, Fujimura A, Kaitsuka T, Ishii I, Sawa T, Nakayama H, Akaike T, Tomizawa K. Reactive sulfur species regulate tRNA methylthiolation and contribute to insulin secretion. Nucleic Acids Res. 2017;45(1):435–45.

    Article  CAS  PubMed  Google Scholar 

  56. Kaneko Y, Kimura T, Taniguchi S, Souma M, Kojima Y, Kimura Y, Kimura H, Niki I. Glucose-induced production of hydrogen sulfide may protect the pancreatic beta-cells from apoptotic cell death by high glucose. FEBS Lett. 2009;583(2):377–82.

    Article  CAS  PubMed  Google Scholar 

  57. Taniguchi S, Kang L, Kimura T, Niki I. Hydrogen sulphide protects mouse pancreatic β-cells from cell death induced by oxidative stress, but not by endoplasmic reticulum stress. Br J Pharmacol. 2011;162(5):1171–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Yang W, Yang G, Jia X, Wu L, Wang R. Activation of KATP channels by H2S in rat insulin-secreting cells and the underlying mechanisms. J Physiol. 2005;569(Pt 2):519–31.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Ali MY, Whiteman M, Low CM, Moore PK. Hydrogen sulphide reduces insulin secretion from HIT-T15 cells by a KATP channel-dependent pathway. J Endocrinol. 2007;195(1):105–12.

    Article  CAS  PubMed  Google Scholar 

  60. Wu L, Yang W, Jia X, Yang G, Duridanova D, Cao K, Wang R. Pancreatic islet overproduction of H2S and suppressed insulin release in Zucker diabetic rats. Lab Invest. 2009;89(1):59–67.

    Article  CAS  PubMed  Google Scholar 

  61. Wang Y, Dong W, Ding X, Wang F, Wang Y, Chen X, Yu L, Li X, Zhang A, Peng Y. Protective effect of α-lipoic acid on islet cells co-cultured with 3T3L1 adipocytes. Exp Ther Med. 2012;4(3):469–74.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Lee WJ, Song KH, Koh EH, Won JC, Kim HS, Park HS, Kim MS, Kim SW, Lee KU, Park JY. Alpha-lipoic acid increases insulin sensitivity by activating AMPK in skeletal muscle. Biochem Biophys Res Commun. 2005;332(3):885–91.

    Article  CAS  PubMed  Google Scholar 

  63. Lee JM, Seo WY, Song KH, Chanda D, Kim YD, Kim DK, Lee MW, Ryu D, Kim YH, Noh JR, Lee CH, Chiang JY, Koo SH, Choi HS. AMPK-dependent repression of hepatic gluconeogenesis via disruption of CREB.CRTC2 complex by orphan nuclear receptor small heterodimer partner. J Biol Chem. 2010;285(42):32182–91.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Manna P, Jain SK. L-cysteine and hydrogen sulfide increase PIP3 and AMPK/PPARγ expression and decrease ROS and vascular inflammation markers in high glucose treated human U937 monocytes. J Cell Biochem. 2013;114:2334–45.

    Article  CAS  PubMed  Google Scholar 

  65. Magalhães DA, Kume WT, Correia FS, Queiroz TS, Allebrandt Neto EW, Santos MPD, Kawashita NH, França SA. High-fat diet and streptozotocin in the induction of type 2 diabetes mellitus: a new proposal. An Acad Bras Cienc. 2019;91(1):e20180314.

    Article  PubMed  Google Scholar 

  66. Govindarajan S, Babu SN, Vijayalakshmi MA, Manohar P, Noor A. Aloe vera carbohydrates regulate glucose metabolism through improved glycogen synthesis and downregulation of hepatic gluconeogenesis in diabetic rats. J Ethnopharmacol. 2021;281:114556.

    Article  CAS  PubMed  Google Scholar 

  67. Yu X, Meng Z, Fang T, Liu X, Cheng Y, Xu L, Liu X, Li X, Xue M, Li T, Sun B, Chen L. Empagliflozin inhibits hepatic gluconeogenesis and increases glycogen synthesis by AMPK/CREB/GSK3β signalling pathway. Front Physiol. 2022;13:817542.

    Article  PubMed  PubMed Central  Google Scholar 

  68. Butler JA, Hagen TM, Moreau R. Lipoic acid improves hypertriglyceridemia by stimulating triacylglycerol clearance and downregulating liver triacylglycerol secretion. Arch Biochem Biophys. 2009;485(1):63–71.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Park KG, Min AK, Koh EH, Kim HS, Kim MO, Park HS, Kim YD, Yoon TS, Jang BK, Hwang JS, Kim JB, Choi HS, Park JY, Lee IK, Lee KU. Alpha-lipoic acid decreases hepatic lipogenesis through adenosine monophosphate-activated protein kinase (AMPK)-dependent and AMPK-independent pathways. Hepatology. 2008;48(5):1477–86.

    Article  CAS  PubMed  Google Scholar 

  70. Yang Y, Li W, Liu Y, Sun Y, Li Y, Yao Q, Li J, Zhang Q, Gao Y, Gao L, Zhao J. Alpha-lipoic acid improves high-fat diet-induced hepatic steatosis by modulating the transcription factors SREBP-1, FoxO1 and Nrf2 via the SIRT1/LKB1/AMPK pathway. J Nutr Biochem. 2014;25(11):1207–17.

    Article  CAS  PubMed  Google Scholar 

  71. Sun L, Zhang S, Yu C, Pan Z, Liu Y, Zhao J, Wang X, Yun F, Zhao H, Yan S, Yuan Y, Wang D, Ding X, Liu G, Li W, Zhao X, Liu Z, Li Y. Hydrogen sulfide reduces serum triglyceride by activating liver autophagy via the AMPK-mTOR pathway. Am J Physiol Endocrinol Metab. 2015;309(11):E925–35.

    Article  CAS  PubMed  Google Scholar 

  72. Pashaj A, Yi X, Xia M, Canny S, Riethoven JJM, Moreau R. Characterization of genome-wide transcriptional changes in liver and adipose tissues of ZDF (fa/fa) rats fed R-α-lipoic acid by next-generation sequencing. Physiol Genomics. 2013;45(23):1136–43.

    Article  PubMed  Google Scholar 

  73. Im SS, Kang SY, Kim SY, Kim HI, Kim JW, Kim KS, Ahn YH. Glucose-stimulated upregulation of GLUT2 gene is mediated by sterol response element-binding protein-1c in the hepatocytes. Diabetes. 2005;54(6):1684–91.

    Article  CAS  PubMed  Google Scholar 

  74. Carter-Kent C, Zein NN, Feldstein AE. Cytokines in the pathogenesis of fatty liver and disease progression to steatohepatitis: implications for treatment. Am J Gastroenterol. 2008;103:1036–42.

    Article  CAS  PubMed  Google Scholar 

  75. Dulundu E, Ozel Y, Topaloglu U, Sehirli O, Ercan F, Gedik N, Sener G. Alpha-lipoic acid protects against hepatic ischemia-reperfusion injury in rats. Pharmacology. 2007;79(3):163–70.

    Article  CAS  PubMed  Google Scholar 

  76. Fayez AM, Zakaria S, Moustafa D. Alpha lipoic acid exerts antioxidant effect via Nrf2/HO-1 pathway activation and suppresses hepatic stellate cells activation induced by methotrexate in rats. Biomed Pharmacother. 2018;105:428–33.

    Article  CAS  PubMed  Google Scholar 

  77. Lirussi F, Azzalini L, Orando S, Orlando R, Angelico F. Antioxidant supplements for non-alcoholic fatty liver disease and/or steatohepatitis. Cochrane Database Syst Rev. 2007;24:004996.

    Google Scholar 

  78. Abenavoli L, Greco M, Milic N, Accattato F, Foti D, Gulletta E, Luzza F. Effect of mediterranean diet and antioxidant formulation in non-alcoholic fatty liver disease: a randomized study. Nutrients. 2017;9(8):870.

    Article  PubMed Central  Google Scholar 

  79. Zhao W, Zhang J, Lu Y, Wang R. The vasorelaxant effect of H(2)S as a novel endogenous gaseous K(ATP) channel opener. EMBO J. 2001;20(21):6008–16.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  80. Dudek M, Bilska-Wilkosz A, Knutelska J, Mogilski S, Bednarski M, Zygmunt M, Iciek M, Sapa J, Bugajski D, Filipek B, Włodek L. Are anti-inflammatory properties of lipoic acid associated with the formation of hydrogen sulfide? Pharmacol Rep. 2013;65(4):1018–24.

    Article  CAS  PubMed  Google Scholar 

  81. Dudek M, Knutelska J, Bednarski M, Nowiński L, Zygmunt M, Bilska-Wilkosz A, Iciek M, Otto M, Żytka I, Sapa J, Włodek L, Filipek B. Alpha lipoic acid protects the heart against myocardial post ischemia-reperfusion arrhythmias via KATP channel activation in isolated rat hearts. Pharmacol Rep. 2014;66(3):499–504.

    Article  CAS  PubMed  Google Scholar 

  82. Maciejczyk M, Żebrowska E, Nesterowicz M, Żendzian-Piotrowska M, Zalewska A. α-Lipoic acid strengthens the antioxidant barrier and reduces oxidative, nitrosative, and glycative damage, as well as inhibits inflammation and apoptosis in the hypothalamus but not in the cerebral cortex of insulin-resistant rats. Oxid Med Cell Longev. 2022;2022:7450514.

    Article  PubMed  PubMed Central  Google Scholar 

  83. Lee SJ, Kang JG, Ryu OH, Kim CS, Ihm SH, Choi MG, Yoo HJ, Kim DS, Kim TW. Effects of alpha-lipoic acid on transforming growth factor beta1-p38 mitogen-activated protein kinase-fibronectin pathway in diabetic nephropathy. Metabolism. 2009;58(5):616–23.

    Article  CAS  PubMed  Google Scholar 

  84. El-Maadawy WH, Hammam OA, Seif El-Din SH, El-Lakkany NM. α-Lipoic acid modulates liver fibrosis: a cross talk between TGF-β1, autophagy, and apoptosis. Hum Exp Toxicol. 2020;39(4):440–50.

    Article  CAS  PubMed  Google Scholar 

  85. Xi YX, Wen X, Jiao LJ, Wei YX, Chang GQ, Wu R, Sun FQ, Hao JH, Li HZ. Effects of exogenous H2S on hepatic fibrosis in diabetic mice and its mechanism. Zhongguo Ying Yong Sheng Li Xue Za Zhi. 2020;36(4):318–20.

    PubMed  Google Scholar 

  86. Ezeriņa D, Takano Y, Hanaoka K, Urano Y, Dick TP. N-acetyl cysteine functions as a fast-acting antioxidant by triggering intracellular H2S and sulfane sulfur production. Cell Chem Biol. 2018;25(4):447-459.e4.

    Article  PubMed  PubMed Central  Google Scholar 

  87. Jurkowska H, Wróbel M. Inhibition of human neuroblastoma cell proliferation by N-acetyl-L-cysteine as a result of increased sulfane sulfur level. Anticancer Res. 2018;38(9):5109–13.

    Article  CAS  PubMed  Google Scholar 

  88. Kimura Y, Mikami Y, Osumi K, Tsugane M, Oka J, Kimura H. Polysulfides are possible H2S-derived signaling molecules in rat brain. FASEB J. 2013;27(6):2451–7.

    Article  CAS  PubMed  Google Scholar 

  89. Kimura Y, Toyofuku Y, Koike S, Shibuya N, Nagahara N, Lefer D, Ogasawara Y, Kimura H. Identification of H2S3 and H2S produced by 3-mercaptopyruvate sulfurtransferase in the brain. Sci Rep. 2015;5:14774.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Funding

This work was partly funded with a seed research grant from Carnegie Corporation of New York under the “Building a New Generation of Academics in Africa” (BANGA-Africa) project, awarded to Dr. George J. Dugbartey (Grant # UG-BA/PD-015/2017-2018) and partly by his personal support.

Author information

Authors and Affiliations

Authors

Contributions

GJD conceived and designed the experiment; GJD, KKA and SA performed the experiment; SA supervised the technical aspect of the animal experimentation work; ROK and DA provided technical assistance; GJD provided the overall direction of the study; GJD, KKA and IA analysed the data; IA and GJD prepared the figures. GJD and KKA drafted the manuscript. GJD and SA reviewed the manuscript. All authors have read and approved the final manuscript.

Corresponding author

Correspondence to George J. Dugbartey.

Ethics declarations

Ethics approval and consent to participate

All experiments involving animals were reviewed and approved by the University of Ghana Institutional Animal Care and Use Committee (Protocol ID: UG-IACUC 002/19-20).

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Dugbartey, G.J., Alornyo, K.K., Adams, I. et al. Targeting hepatic sulfane sulfur/hydrogen sulfide signaling pathway with α-lipoic acid to prevent diabetes-induced liver injury via upregulating hepatic CSE/3-MST expression. Diabetol Metab Syndr 14, 148 (2022). https://doi.org/10.1186/s13098-022-00921-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s13098-022-00921-x

Keywords

  • Alpha-lipoic acid (ALA)
  • Type 2 diabetes mellitus (T2DM)
  • Diabetes-induced liver injury
  • Non-alcoholic fatty liver disease (NAFLD)
  • Non-alcoholic steatohepatitis (NASH)
  • Sulfane sulfur
  • H2S pathway