Yhhu981, a novel compound,stimulates fatty acid oxidation via the activation of AMPK and ameliorates lipid metabolism disorder in ob/ob mice

Aim:

Defects in fatty acid metabolism contribute to the pathogenesis of insulin resistance and obesity. In this study, we investigated the effects of a novel compound yhhu981 on fatty acid metabolism in vitro and in vivo.

Methods:

The capacity to stimulate fatty acid oxidation was assessed in C2C12 myotubes. The fatty acid synthesis was studied in HepG2 cells using isotope tracing. The phosphorylation of AMPK and acetyl-CoA carboxylase (ACC) was examined with Western blot analysis. For in vivo experiments, ob/ob mice were orally treated with yhhu981 acutely (300 mg/kg) or chronically (150 or 300 mg·kg⁻¹·d⁻¹ for 22 d). On the last day of treatment, serum and tissue samples were collected for analysis.

Results:

Yhhu981 (12.5–25 μmol/L) significantly increased fatty acid oxidation and the expression of related genes (Sirt1, Pgc1α and Mcad) in C2C12 myotubes, and inhibited fatty acid synthesis in HepG2 cells. Furthermore, yhhu981 dose-dependently increased the phosphorylation of AMPK and ACC in both C2C12 myotubes and HepG2 cells. Compound C, an AMPK inhibitor, blocked fatty acid oxidation in yhhu981-treated C2C12 myotubes and fatty acid synthesis decrease in yhhu981-treated HepG2 cells. Acute administration of yhhu981 decreased the respiratory exchange ratio in ob/ob mice, whereas chronic treatment with yhhu981 ameliorated the lipid abnormalities and ectopic lipid deposition in skeletal muscle and liver of ob/ob mice.

Conclusion:

Yhhu981 is a potent compound that stimulates fatty acid oxidation, and exerts pleiotropic effects on lipid metabolism by activating AMPK.     

Acute and chronic administration of SHR117887, a novel and specific dipeptidyl peptidase-4 inhibitor,improves metabolic control in diabetic rodent models

Aim:

Dipeptidyl deptidase-4 (DPP-4) inhibitors are a new class of anti-diabetic agents. The purpose of this study was to assess the acute and chronic effects of SHR117887, a novel DPP-4 inhibitor, on metabolic control and pancreatic β-cell function in normal or diabetic rodent models.

Methods:

In the acute experiments, ICR mice, diet-induced obese (DIO) rats and ob/ob mice were subjected to an oral glucose tolerance test (OGTT) following a single oral administration of SHR117887 (0.1, 0.3, 1 or 3 mg/kg). Blood samples were collected to measure glucose, insulin, DPP-4 activity and active GLP-1 level. In the chronic experiments, ob/ob mice was administered SHR117887 (3, 10 or 30 mg/kg) twice daily for 33 d to assess the effects on metabolic control and pancreatic β-cell function. Vildagliptin (LAF237) was used as a positive control in all the experiments.

Results:

Acute oral administration of SHR117887 dose-dependently decreased the serum DPP-4 activity and improved glucose tolerance in ICR mice, DIO rats and ob/ob mice. This was accompanied by significant increases in the serum active GLP-1 and insulin levels. Chronic administration of SHR117887 significantly decreased fasting blood glucose level and improved the lipid profiles in ob/ob mice by reducing the serum triglyceride and free fatty acid levels, and its efficacy was comparable with that of vildagliptin at the same molarity. Moreover, chronic administration of SHR117887 increased the insulin staining of islet cells, which is suggestive of improved β-cell function.

Conclusion:

SHR117887 is a potent DPP-4 inhibitor that improves metabolic control and β-cell function in diabetic rodent models, suggesting that it could be a new therapeutic agent for the treatment of type 2 diabetes.     

Antihyperglycaemic effect of saccharin in diabetic ob/ob mice

1. The effect of chronic saccharin (benzosulphimide) consumption on glucose homeostasis was examined in normal lean +/+ mice and genetically obese hyperglycaemic insulin-resistant ob/ob mice.
2. Consumption of a 5% (w/v) sodium saccharin solution for 7 weeks prevented the development of hyperglycaemia, improved glucose tolerance (area under curve decreased by 51%), reduced the extent of hyperinsulinaemia (by 21%), and reduced excessive weight gain (by 18%) in ob/ob mice.
3. Consumption of 5% (w/v) sodium saccharin temporarily decreased hyperphagia at the beginning of treatment, decreased hepatic glycogen content (by 47%), increased abdominal muscle glycogen content (by 82%), but did not significantly alter the hypoglycaemic response to exogenous insulin in ob/ob mice.
4. Consumption of a 1% (w/v) sodium saccharin solution did not prevent the development of hyperglycaemia in ob/ob mice.
5. Normal lean +/+ mice consuming 5% (w/v) sodium saccharin solution showed a marginal decrease (by 8%) in glycaemia, and glucose tolerance was improved (area under curve decreased by 30%) without a significant change in the insulin response to glucose or the hypoglycaemic effect of exogenous insulin.
6. The results suggest that chronic consumption of saccharin can defer the development of hyperglycaemia and improve glucose homeostasis in insulin-resistant ob/ob mice through a mechanism that is independent of insulin.

     

Emodin,an 11β-hydroxysteroid dehydrogenase type 1 inhibitor,regulates adipocyte function in vitro and exerts anti-diabetic effect in ob/ob mice

Aim:

Emodin (1,3,8-trihydroxy-6-methylanthraquinone) is a potent and selective inhibitor of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) with the ability to ameliorate metabolic disorders in diet-induced obese mice. In the present study, we investigated the effects of emodin on adipocyte function and the underlying mechanisms in vitro, and its anti-diabetic effects in ob/ob mice.

Methods:

3T3-L1 adipocytes were used for in vitro studies. 11β-HSD1A activity was evaluated with a scintillation proximity assay. The adipogenesis, glucose uptake, lipolysis and adiponectin secretion were investigated in 3T3-L1 adipocytes treated with emodin in the presence of active (corticosterone) or inactive glucocorticoid (11-dehydrocorticosterone). For in vivo studies, ob/ob mice were administered emodin (25 and 50 mg·kg⁻¹·d⁻¹, ip) for 26 d. On the last day of administration, the serum was collected and the mesenteric and perirenal fat were dissected for analyses.

Results:

Emodin inhibited the 11β-HSD1 activity in 3T3-L1 adipocytes in concentration- and time-dependent manners (the IC₅₀ values were 7.237 and 4.204 μmol/L, respectively, after 1 and 24 h treatment. In 3T3-L1 adipocytes, emodin (30 μmol/L) suppressed 11-dehydrocorticosterone-induced adipogenesis without affecting corticosterone-induced adipogenesis; emodin (3 μmol/L) reduced 11-dehydrocorticosterone-stimulated lipolysis, but had no effect on corticosterone-induced lipolysis. Moreover, emodin (3 μmol/L) partly reversed the impaired insulin-stimulated glucose uptake and adiponectin secretion induced by 11-dehydrocorticosterone but not those induced by corticosterone. In ob/ob mice, long-term emodin administration decreased 11β-HSD1 activity in mesenteric adipose tissues, lowered non-fasting and fasting blood glucose levels, and improved glucose tolerance.

Conclusion:

Emodin improves the inactive glucocorticoid-induced adipose tissue dysfunction by selective inhibition on 11β-HSD1 in adipocyte in vitro and improves glycemic control in ob/ob mice.     

Pharmacological inhibition of diacylglycerol acyltransferase 1 reduces body weight gain, hyperlipidemia, and hepatic steatosis in db/db mice

Aim:

To test whether pharmacological inhibition of Diacylglycerol acyltransferase 1 (DGAT1) by a small-molecule inhibitor H128 can improve metabolism disorders in leptin receptor-deficient db/db mice.

Methods:

To investigate the effect of H128 on intestinal fat absorption,db/db mice were acutely given a bolus of corn oil by gavage. The mice were further orally administered H128 (3 and 10 mg/kg) for 5 weeks. Blood glucose, lipids, insulin, ALT, and AST as well as hepatic triglycerides were measured. The insulin tolerance test was performed to evaluate insulin sensitivity. The expression of genes involved in fatty acid oxidation was detected by RT-PCR.

Results:

Oral administration of H128 (10 mg/kg) acutely inhibited intestinal fat absorption following a lipid challenge in db/db mice. Chronic treatment with H128 significantly inhibited body weight gain, decreased food intake, and induced a pronounced reduction of serum triglycerides. In addition, H128 treatment markedly ameliorated hepatic steatosis, characterized by decreased liver weight, lipid droplets, and triglyceride content as well as serum ALT and AST levels. Furthermore, H128 treatment increased the expression of the CPT1 and PPARα genes in liver, suggesting that H128 enhanced fatty acid oxidation in db/db mice. However, neither blood glucose nor insulin tolerance was affected by H128 treatment throughout the 5-week experimental period.

Conclusion:

DGAT1 may be an effective therapeutic target for the treatment of obesity, hyperlipidemia and hepatic steatosis.     

A novel potent synthetic steroidal liver X receptor agonist lowers plasma cholesterol and triglycerides and reduces atherosclerosis in LDLR(-/-) mice

BACKGROUND AND PURPOSE

Potent synthetic nonsteroidal liver X receptor (LXR) agonists like T0901317 induce triglyceridaemia and fatty liver, effects not observed with some natural and synthetic steroidal, relatively weak agonists of LXR. To determine if potency is responsible for the lack of side effects with some steroidal agonists, we investigated the in vivo effects of a novel steroidal LXR agonist, ATI-111, that is more potent than T0901317.

EXPERIMENTAL APPROACH

Eight week old male LDLR^–/– mice fed an atherogenic diet were orally treated with vehicle or ATI-111 at 3 and 5 mg·kg⁻¹·day⁻¹ for 8 weeks, and effects on plasma and liver lipid levels, expression of genes involved in lipid metabolism and on atherogenesis were analysed.

KEY RESULTS

ATI-111 increased the expression of genes involved in lipid transport, such as ABCA1, ABCG1 and ABCG5/G8, in intestine and macrophages; decreased ABCG1, apoE; and slightly increased ABCA1 and ABCG5/G8 expression in liver. ATI-111 markedly increased sterol regulatory element-binding protein (SREBP)-1c mRNA in some tissues, whereas acetyl-coenzyme A carboxylase and fatty acid synthase expression was unaffected or only slightly increased in intestine and liver. ATI-111 inhibited the conversion of SREBP-1c precursor form to its active form. Compared with vehicle-treated mice, the levels of hepatic lipids and liver-secreted nascent lipoproteins were not altered, while a significant decrease in plasma cholesterol and triglyceride levels was observed in ATI-111-treated mice. ATI-111 significantly inhibited atherogenesis in three separate vascular sites.

CONCLUSIONS AND IMPLICATIONS

ATI-111 is a promising candidate for further development as a treatment of certain vascular diseases as it lacks the significant side effects associated with nonsteroidal LXR agonists, the induction of fatty liver and hypertriglyceridaemia.     

Peripheral endocannabinoid dysregulation in obesity: relation to intestinal motility and energy processing induced by food deprivation and re-feeding

Background and purpose:

Endocannabinoids in tissues controlling energy homeostasis are altered in obesity, thus contributing to metabolic disorders. Here we evaluate endocannabinoid dysregulation in the small intestine of mice with diet-induced obesity (DIO) and in peripheral tissues of Zucker and lean rats following food deprivation and re-feeding.

Experimental approach:

Intestinal transit, evaluated using rhodamine-B-labelled dextran, and small intestinal endocannabinoid levels, measured by liquid chromatography mass spectrometry, were measured in mice fed normal or high-fat diets (HFDs). Endocannabinoid levels were measured also in various tissues of lean and Zucker rats fed ad libitum or following overnight food deprivation with and without subsequent re-feeding.

Key results:

After 8 weeks of HFD, baseline intestinal transit was increased in DIO mice and enhanced by cannabinoid CB₁ receptor antagonism less efficaciously than in lean mice. Small intestinal anandamide and 2-arachidonoylglycerol levels were reduced and increased respectively. In Zucker rats, endocannabinoids levels were higher in the pancreas, liver and duodenum, and lower in the subcutaneous adipose tissue. Food deprivation increased endocannabinoid levels in the duodenum and liver of both rat strains, in the pancreas of lean rats and in adipose tissues of Zucker rats.

Conclusions and implications:

Reduced anandamide levels might account for increased intestinal motility in DIO mice. Regulation of endocannabinoid levels in rat peripheral tissues, induced by food deprivation and re-feeding, might participate in food intake and energy processing and was altered in Zucker rats. These data, together with previous observations, provide further evidence for dysregulation of peripheral endocannabinoids in obesity.     

Tumour necrosis factor‐α inhibition with lenalidomide alleviates tissue oxidative injury and apoptosis in ob/ob obese mice

Lenalidomide (Revlimid; Selleck Chemicals, Houston, TX, USA), an analogue of thalidomide, possesses potent cytokine modulatory capacity through inhibition of cytokines such as tumour necrosis factor (TNF)‐α, a cytokine pivotal for the onset and development of complications in obesity and diabetes mellitus. The present study was designed to evaluate the effect of lenalidomide on oxidative stress, protein and DNA damage in multiple organs in an ob/ob murine model of obesity. To this end, C57BL/6 lean and ob/ob obese mice were administered lenalidomide (50 mg/kg per day, p.o.) for 5 days. Oxidative stress, protein and DNA damage were assessed using the conversion of reduced glutathione (GSH) to oxidized glutathione (GSSG), carbonyl formation and Comet assay, respectively. Apoptosis was evaluated using caspase 3 activity, and levels of Bax, Bcl‐2, Bip, caspase 8, caspase 9 and TNF‐α were assessed using western blot analysis. Lenalidomide treatment did not affect glucose clearance in lean or ob/ob mice. Obese mice exhibited a reduced GSH/GSSG ratio in the liver, gastrocnemius skeletal muscle and small intestine, as well as enhanced protein carbonyl formation, DNA damage and caspase 3 activity in the liver, kidney, skeletal muscle and intestine; these effects were alleviated by lenalidomide, with the exception of obesity‐associated DNA damage in the liver and kidney. Western blot analysis revealed elevated TNF‐α, Bax, Bcl‐2, Bip, caspase 8 and caspase 9 in ob/ob mice with various degrees of reversal by lenalidomide treatment. Together, these data indicate that lenalidomide protects against obesity‐induced tissue injury and protein damage, possibly in association with antagonism of cytokine production and cytokine‐induced apoptosis and oxidative stress.     

Cannabidiol improves brain and liver function in a fulminant hepatic failure-induced model of hepatic encephalopathy in mice

BACKGROUND AND PURPOSE

Hepatic encephalopathy is a neuropsychiatric disorder of complex pathogenesis caused by acute or chronic liver failure. We investigated the effects of cannabidiol, a non-psychoactive constituent of Cannabis sativa with anti-inflammatory properties that activates the 5-hydroxytryptamine receptor 5-HT_1A, on brain and liver functions in a model of hepatic encephalopathy associated with fulminant hepatic failure induced in mice by thioacetamide.

EXPERIMENTAL APPROACH

Female Sabra mice were injected with either saline or thioacetamide and were treated with either vehicle or cannabidiol. Neurological and motor functions were evaluated 2 and 3 days, respectively, after induction of hepatic failure, after which brains and livers were removed for histopathological analysis and blood was drawn for analysis of plasma liver enzymes. In a separate group of animals, cognitive function was tested after 8 days and brain 5-HT levels were measured 12 days after induction of hepatic failure.

KEY RESULTS

Neurological and cognitive functions were severely impaired in thioacetamide-treated mice and were restored by cannabidiol. Similarly, decreased motor activity in thioacetamide-treated mice was partially restored by cannabidiol. Increased plasma levels of ammonia, bilirubin and liver enzymes, as well as enhanced 5-HT levels in thioacetamide-treated mice were normalized following cannabidiol administration. Likewise, astrogliosis in the brains of thioacetamide-treated mice was moderated after cannabidiol treatment.

CONCLUSIONS AND IMPLICATIONS

Cannabidiol restores liver function, normalizes 5-HT levels and improves brain pathology in accordance with normalization of brain function. Therefore, the effects of cannabidiol may result from a combination of its actions in the liver and brain.     

Inverse relationship between serum osteocalcin levels and nonalcoholic fatty liver disease in postmenopausal Chinese women with normal blood glucose levels

Aim:

Osteocalcin is involved in the progression of nonalcoholic fatty liver disease (NAFLD) in animal models and humans. In this study we investigated the relationship between serum osteocalcin levels and NAFLD in postmenopausal Chinese women.

Methods:

A total of 733 postmenopausal women (age range: 41–78 years) with normal blood glucose levels were enrolled in this cross-sectional study. Women taking lipid-lowering or anti-hypertensive drugs were excluded. Serum osteocalcin levels were assessed using an electrochemiluminescence immunoassay. The degree of NAFLD progression for each subject was assessed through ultrasonography. The fatty liver index (FLI) of each subject was calculated to quantify the degree of liver steatosis.

Results:

The median level of serum osteocalcin for all subjects enrolled was 21.99 ng/mL (interquartile range: 17.84–26.55 ng/mL). Subjects with NAFLD had significantly lower serum osteocalcin levels (18.39 ng/mL; range: 16.03–23.64 ng/mL) compared with those without NAFLD (22.31 ng/mL; range: 18.55–27.06 ng/mL; P<0.01). Serum osteocalcin levels decreased with incre¬mental changes in the FLI value divided by the quartile (P-value for trend<0.01). The serum osteocalcin levels showed a negative correlation with the FLI values, even after adjusting for confounding factors (standardized β=−0.124; P<0.01). Binary logistic regression analysis identified an individual''s serum osteocalcin level as an independent risk factor for NAFLD (odds ratio: 0.951; 95% confidence interval: 0.911–0.992; P=0.02).

Conclusion:

Serum osteocalcin levels are inversely correlated with NAFLD in postmenopausal Chinese women with normal blood glucose levels.     

PPARδ agonists have opposing effects on insulin resistance in high fat-fed rats and mice due to different metabolic responses in muscle

BACKGROUND AND PURPOSE

The peroxisome proliferator-activated receptor (PPAR)δ has been considered a therapeutic target for diabetes and obesity through enhancement of fatty acid oxidation. The present study aimed to characterize the effects of PPARδ agonists during insulin resistance of the whole body, muscle and liver.

EXPERIMENTAL APPROACH

Wistar rats and C57BL/J6 mice were fed a high fat diet (HF) and then treated with PPARδ agonists NNC61-5920 and GW501516. The effects on insulin resistance were evaluated by hyperinsulinaemic clamp or glucose tolerance tests combined with glucose tracers.

KEY RESULTS

In HF rats, 3 weeks of treatment with NNC61-5920 reduced the glucose infusion rate (by 14%, P < 0.05) and glucose disposal into muscle (by 20–30%, P < 0.01) during hyperinsulinaemic clamp. Despite increased mRNA expression of carnitine palmitoyltransferase-1, pyruvate dehydrogenase kinase 4 and uncoupling protein 3 in muscle, plasma and muscle triglyceride levels were raised (P < 0.01). Similar metabolic effects were observed after extended treatment with NNC61-5920 and GW501516 to 6 weeks. However, HF mice treated with NNC61-5920 improved their plasma lipid profile, glucose tolerance and insulin action in muscle. In both HF rats and mice, NNC61-5920 treatment attenuated hepatic insulin resistance and decreased expression of stearoyl-CoA desaturase 1, fatty acid translocase protein CD36 and lipoprotein lipase in liver.

CONCLUSIONS AND IMPLICATIONS

PPARδ agonists exacerbated insulin resistance in HF rats in contrast to their beneficial effects on metabolic syndrome in HF mice. These opposing metabolic consequences result from their different effects on lipid metabolism and insulin sensitivity in skeletal muscle of these two species.     

Small molecule glucokinase activators disturb lipid homeostasis and induce fatty liver in rodents: a warning for therapeutic applications in humans

Background and Purpose

Small-molecule glucokinase activators (GKAs) are currently being investigated as therapeutic options for the treatment of type 2 diabetes (T2D). Because liver overexpression of glucokinase is thought to be associated with altered lipid profiles, this study aimed at assessing the potential lipogenic risks linked to oral GKA administration.

Experimental Approach

Nine GKA candidates were qualified for their ability to activate recombinant glucokinase and to stimulate glycogen synthesis in rat hepatocytes and insulin secretion in rat INS-1E cells. In vivo activity was monitored by plasma glucose and HbA1c measurements after oral administration in rodents. Risk-associated effects were assessed by measuring hepatic and plasma triglycerides and free fatty acids, as well as plasma aminotransferases, and alkaline phosphatase.

Key Results

GKAs, while efficiently decreasing glycaemia in acute conditions and HbA1c levels after chronic administration in hyperglycemic db/db mice, were potent inducers of hepatic steatosis. This adverse outcome appeared as soon as 4 days after daily oral administration at pharmacological doses and was not transient. GKA treatment similarly increased hepatic triglycerides in diabetic and normoglycaemic rats, together with a pattern of metabolic phenotypes including different combinations of increased plasma triglycerides, free fatty acids, alanine and aspartyl aminotransferases, and alkaline phosphatase. GKAs belonging to three distinct structural families induced hepatic steatosis in db/db mice, arguing in favour of a target-mediated, rather than a chemical class-mediated, effect.

Conclusion and Implications

Given the risks associated with fatty liver disease in the general population and furthermore in patients with T2D, these findings represent a serious warning for the use of GKAs in humans.

Linked Article

This article is commented on by Rees and Gloyn, pp. 335–338 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2012.02201.x     

TS-071 is a novel, potent and selective renal sodium-glucose cotransporter 2 (SGLT2) inhibitor with anti-hyperglycaemic activity

BACKGROUND AND PURPOSE

The renal sodium-glucose cotransporter 2 (SGLT2) plays an important role in the reuptake of filtered glucose in the proximal tubule and therefore may be an attractive target for the treatment of diabetes mellitus. This study characterizes the pharmacological profile of TS-071 ((1S)-1,5-anhydro-1-[5-(4-ethoxybenzyl)-2-methoxy-4-methylphenyl]-1-thio-d-glucitol hydrate), a novel SGLT2 inhibitor in vitro and in vivo.

EXPERIMENTAL APPROACH

Inhibition of glucose uptake by TS-071 was studied in CHO-K1 cells stably expressing either human SGLT1 or SGLT2. Single oral dosing studies were performed in rats, mice and dogs to assess the abilities of TS-071 to increase urinary glucose excretion and to lower plasma glucose levels.

KEY RESULTS

TS-071 inhibited SGLT2 activity in a concentration-dependent manner and was a potent and highly selective inhibitor of SGLT2. Orally administered TS-071 increased urinary glucose excretion in Zucker fatty rats and beagle dogs at doses of 0.3 and 0.03 mg·kg⁻¹ respectively. TS-071 improved glucose tolerance in Zucker fatty rats without stimulating insulin secretion and reduced hyperglycaemia in streptozotocin (STZ)-induced diabetic rats and db/db mice at a dose of 0.3 mg·kg⁻¹.

CONCLUSION AND IMPLICATIONS

These data indicate that TS-071 is a potent and selective SGLT2 inhibitor that improves glucose levels in rodent models of type 1 and 2 diabetes and may be useful for the treatment for diabetes mellitus.     

Alpha-lipoic acid attenuates insulin resistance and improves glucose metabolism in high fat diet-fed mice

Aim:

To investigate whether alpha-lipoic acid (ALA) could attenuate the insulin resistance and metabolic disorders in high fat diet-fed mice.

Methods:

Male mice were fed a high fat diet (HFD) plus ALA (100 and 200 mg·kg⁻¹·d⁻¹) or HFD plus a positive control drug metformin (300 mg·kg⁻¹·d⁻¹) for 24 weeks. During the treatments, the relevant physiological and metabolic parameters of the mice were measured. After the mice were euthanized, blood samples and livers were collected. The expression of proteins and genes related to glucose metabolism in livers were analyzed by immunoblotting and real time-PCR.

Results:

HFD induced non-alcoholic fatty liver disease (NAFLD) and abnormal physiological and metabolic parameters in the mice, which were dose-dependently attenuated by ALA. ALA also significantly reduced HFD-induced hyperglycemia and insulin resistance in HFD-fed mice. Furthermore, ALA significantly upregulated the glycolytic enzymes GCK, HK-1 and PK, and the glycogen synthesis enzyme GS, and downregulated the gluconeogenic enzymes PEPCK and G6Pase, thus decreased glucose production, and promoted glycogen synthesis and glucose utilization in livers. Moreover, ALA markedly increased PKB/Akt and GSK3β phosphorylation, and nuclear carbohydrate response element binding protein (ChREBP) expression in livers. Metformin produced similar effects as ALA in HFD-fed mice.

Conclusion:

ALA is able to sustain glucose homeostasis and prevent the development of NAFLD in HFD-fed mice.     

Palmitic acid induces autophagy in hepatocytes via JNK2 activation

Aim： Free fatty acid-induced lipotoxicity plays a crucial role in the progression of nonalcoholic fatty liver disease （NAFLD）. In the present study we investigated the effects of a high-fat diet and free fatty acids on the autophagic process in hepatocytes in vivo and in vitro and the underlying mechanisms.
Methods： LC3-II expression, a hallmark of autophagic flux, was detected in liver specimens from patients with non-alcoholic steatohepatitis （NASH） as well as in the livers of C57BL/6 mice fed a high-fat diet （HFD） up to 16 weeks. LC3-II expression was also analyzed in human SMMC-7721 and HepG2 hepatoma cells exposed to palmitic acid （PA）, a saturated fatty acid. PA-induced apoptosis was detected by Annexin V staining and specific cleavage of PARP in the presence and absence of different agents.

Results： LC3-II expression was markedly increased in human NASH and in liver tissues of HFD-fed mice. Treatment of SMMC-7721 cells with PA increased LC3-II expression in time- and dose-dependent manners, whereas the unsaturated fatty acid oleic acid had no effect. Inhibition of autophagy with 3MA sensitized SMMC-7721 cells to PA-induced apoptosis, whereas activation of autophagy by rapamycin attenuated PA-induced PARP cleavage. The autophagy-associated proteins Beclin1 and Atg5 were essential for PA-induced autophagy in SMMC-7721 cells. Moreover, pretreatment with SP600125, an inhibitor of JNK, effectively abrogated PA-mediated autophagy and apoptosis. Specific knockdown of JNK2, but not JNK1, in SMMC-7721 cells significantly suppressed PA-induced autophagy and enhanced its pro-apoptotic activity; whereas specific knockdown of JNK1 had the converse effect. Similar results were obtained when HepG2 cells were tested.

Conclusion： JNK1 promotes PA-induced lipoapoptosis, whereas JNK2 activates pro-survival autophagy and inhibits PA lipotoxicity. Our results suggest that modulation of autophagy may have therapeutic benefits in the treatment of lipid-related metabolic diseases.     

Acanthopanax senticosus reverses fatty liver disease and hyperglycemia in ob/ob mice

Non-alcoholic fatty liver disease (NAFLD) is common in obesity. However, weight reduction alone does not prevent the progression of NAFLD to end-stage disease associated with the development of cirrhosis and liver disease. In a previous experiment, 50% ethanol extract of Acanthopanax senticosus stem bark (ASSB) was found to reduce body weight and insulin resistance in high fat diet-induced hyperglycemic and hyperlipidemic ICR mice. To evaluate the anti-steatosis action of ASSB, insulin-resistant ob/ob mice with fatty livers were treated with ASSB ethanol extract for an 8 week-period. ASSB ethanol extract reversed the hepatomegaly, as evident in reduction of % liver weight/body weight ratio. ASSB ethanol extract also specifically lowered circulating glucose and lipids, and enhanced insulin action in the liver. These changes culminated in inhibition of triglyceride synthesis in non-adipose tissues including liver and skeletal muscle. Gene expression studies confirmed reductions in glucose 6-phosphatase and lipogenic enzymes in the liver. These results demonstrate that ASSB ethanol extract is an effective treatment for insulin resistance and hepatic steatosis in ob/ob mice by decreasing hepatic lipid synthesis.     

Novel liver-specific cholic acid-cytarabine conjugates with potent antitumor activities: Synthesis and biological characterization

Aim:

Cytarabine is an efficient anticancer agent for acute myelogenous leukemia, but with short plasma half-life and rapid deamination to its inactive metabolite. The aim of this study was to design and synthesize novel cholic acid-cytarabine conjugates to improve its pharmacokinetic parameters.

Methods:

The in vitro stability of novel cholic acid-cytarabine conjugates was investigated in simulated gastric and intestinal fluid, mouse blood and liver homogenate using HPLC. The portacaval samples of the conjugates were examined in male Sprague-Dawley rats using LC/MS, and in vivo distribution was examined in male Kunming mice using LC/MS. Antitumor activities were tested in HL60 cells using MTT assay.

Results:

Cholic acid-cytarabine compounds with four different linkers were designed and synthesized. All the four cholic acid-cytarabine conjugates could release cytarabine when incubated with the simulated gastric and intestinal fluid, mouse blood and liver homogenate. The conjugates 6, 12, and 16 were present in the portacaval samples, whereas the conjugate 7 was not detected. The conjugates 6 and 16 showed high specificity in targeting the liver (liver target index 34.9 and 16.3, respectively) and good absorption in vivo, as compared with cytarabine. In cytarabine-sensitive HL60 cells, the conjugates 6, 12, and 16 retained potent antitumor activities.

Conclusion:

Three novel cholic acid-cytarabine conjugates with good liver-targeting properties and absorption were obtained. Further optimization of the conjugates is needed in the future.     

Insulin therapy stimulates lipid synthesis and improves endocrine functions of adipocytes in dietary obese C57BL/6 mice

Aim:

To evaluate whether insulin intervention could affect the metabolic and endocrine functions of adipose tissue.

Methods:

C57BL/6 mice were fed on a high-fat-diet for 12−16 weeks to induce insulin resistance. Insulin intervention was administered in the high-fat-diet mice for 4 weeks at 12 weeks (early insulin treatment) or 16 weeks (late insulin treatment). Intraperitoneal glucose tolerance tests were performed before and after insulin treatment. Expression levels of factors involved in the triglyceride synthesis and endocrine functions of adipose tissue including phosphoenolpyruvate carboxykinase (PEPCK-C), fatty acid synthase (FAS), aquaporin 7 (AQP7), adiponectin, visfatin, and interleukin-6 (IL-6) were determined by Western blot.

Results:

In the obese mice, glucose tolerance was impaired; triglyceride content was increased in the liver tissue; protein expression of FAS and adiponectin was decreased; expression of visfatin was increased in adipose tissue. After 4-week insulin treatment, glucose tolerance was improved; triglyceride content was decreased in the liver and skeletal muscle; expression of PEPCK-C, FAS, and adiponectin was increased in the adipose tissue; IL-6 and AQP7 expression was reduced in the fat. Early insulin treatment had better effect in increasing the expression of FAS and PEPCK-C and decreasing the expression of IL-6.

Conclusion:

These results indicate that insulin can target adipocytes for improvement of insulin sensitivity through stimulating triglyceride synthesis and partly improving endocrine functions.     

Serum vitamin D is associated with non-alcoholic fatty liver disease in Chinese males with normal weight and liver enzymes

Aim:

Considering the characterization of vitamin D deficiency as a risk factor of ectopic fat deposition, the association of serum 25-hydroxy vitamin D₃ [25(OH)D₃] levels with non-alcoholic fatty liver disease (NAFLD) was evaluated in Chinese men with normal body mass index (BMI) and enzyme markers of liver function.

Methods:

A total of 514 participants (22 to 79 years old) with normal BMI and liver enzymes were identified for analysis. Abdominal ultrasound was performed to diagnose NAFLD, and the fatty liver index (FLI) was calculated to quantify liver steatosis. Serum 25(OH)D₃ levels were determined by an electrochemiluminescence immunoassay.

Results:

Among the entire study population, the mean levels of serum 25(OH)D₃ were 15.32±5.77 ng/mL. However, when serum 25(OH)D₃ levels were compared between non-NAFLD subjects (n=438) and NAFLD subjects (n=76), the latter showed significantly lower levels (15.65±5.89 ng/mL vs 13.46±4.65 ng/mL, P=0.002). In addition, serum 25(OH)D₃ levels were found to be significantly correlated with FLI after adjustment for age and BMI (r=−0.108, P=0.014). Logistic regression showed that serum 25(OH)D₃ levels were independently correlated with NAFLD (OR: 0.937, 95% CI: 0.884–0.993, P=0.028). Furthermore, stepwise regression analysis revealed that serum 25(OH)D₃ levels were inversely associated with FLI (β=−0.055, P=0.040).

Conclusion:

The present study demonstrated that serum 25(OH)D₃ levels were inversely associated with NAFLD, even in subjects with normal total body fat, suggesting a potential role of lower levels of vitamin D in the occurrence and development of NAFLD.     

Transformation and action of extracellular NAD+ in perfused rat and mouse livers

Aim:

Transformation and possible metabolic effects of extracellular NAD⁺ were investigated in the livers of mice (Mus musculus; Swiss strain) and rats (Rattus novergicus; Holtzman and Wistar strains).

Methods:

The livers were perfused in an open system using oxygen-saturated Krebs/Henseleit-bicarbonate buffer (pH 7.4) as the perfusion fluid. The transformation of NAD⁺ was monitored using high-performance liquid chromatography.

Results:

In the mouse liver, the single-pass metabolism of 100 μmol/L NAD⁺ was almost complete; ADP-ribose and nicotinamide were the main products in the outflowing perfusate. In the livers of both Holtzman and Wistar rats, the main transformation products were ADP-ribose, uric acid and nicotinamide; significant amounts of inosine and AMP were also identified. On a weight basis, the transformation of NAD⁺ was more efficient in the mouse liver. In the rat liver, 100 μmol/L NAD⁺ transiently inhibited gluconeogenesis and oxygen uptake. Inhibition was followed by a transient stimulation. Inhibition was more pronounced in the Wistar strain and stimulation was more pronounced in the Holtzman strain. In the mouse liver, no clear effects on gluconeogenesis and oxygen uptake were found even at 500 μmol/L NAD⁺.

Conclusion:

It can be concluded that the functions of extracellular NAD⁺ are species-dependent and that observations in one species are strictly valid for that species. Interspecies extrapolations should thus be made very carefully. Actually, even variants of the same species can demonstrate considerably different responses.