Yinhua Ni

Metformin Prevents and Reverses Inflammation in a Non-Diabetic Mouse Model of Nonalcoholic Steatohepatitis

Background Optimal treatment for nonalcoholic steatohepatitis (NASH) has not yet been established, particularly for individuals without diabetes. We examined the effects of metformin, commonly used to treat patients with type 2 diabetes, on liver pathology in a non-diabetic NASH mouse model. Methodology/Principal Findings Eight-week-old C57BL/6 mice were fed a methionine- and choline-deficient plus high fat (MCD+HF) diet with or without 0.1% metformin for 8 weeks. Co-administration of metformin significantly decreased fasting plasma glucose levels, but did not affect glucose tolerance or peripheral insulin sensitivity. Metformin ameliorated MCD+HF diet-induced hepatic steatosis, inflammation, and fibrosis. Furthermore, metformin significantly reversed hepatic steatosis and inflammation when administered after the development of experimental NASH. Conclusions/Significance These histological changes were accompanied by reduced hepatic triglyceride content, suppressed hepatic stellate cell activation, and the downregulation of genes involved in fatty acid metabolism, inflammation, and fibrogenesis. Metformin prevented and reversed steatosis and inflammation of NASH in an experimental non-diabetic model without affecting peripheral insulin resistance.

Roles of Chemokines and Chemokine Receptors in Obesity-Associated Insulin Resistance and Nonalcoholic Fatty Liver Disease

Abundant evidence has demonstrated that obesity is a state of low-grade chronic inflammation that triggers the release of lipids, aberrant adipokines, pro-inflammatory cytokines, and several chemokines from adipose tissue. This low-grade inflammation underlies the development of insulin resistance and associated metabolic comorbidities such as type 2 diabetes mellitus (T2DM) and nonalcoholic fatty liver disease (NAFLD). During this development, adipose tissue macrophages accumulate through chemokine (C-C motif) receptor 2 and the ligand for this receptor, monocyte chemoattractant protein-1 (MCP-1), is considered to be pivotal for the development of insulin resistance. To date, the chemokine system is known to be comprised of approximately 40 chemokines and 20 chemokine receptors that belong to the seven-transmembrane G protein-coupled receptor family and, as a result, chemokines appear to exhibit a high degree of functional redundancy. Over the past two decades, the physiological and pathological properties of many of these chemokines and their receptors have been elucidated. The present review highlights chemokines and chemokine receptors as key contributing factors that link obesity to insulin resistance, T2DM, and NAFLD.

Effects of light cues on re-entrainment of the food-dominated peripheral clocks in mammals

Although the light/dark (LD) cycle is a weak Zeitgeber in peripheral clocks compared with food stimuli, whether the effect of light cues on the re-entrainment of peripheral clocks can be masked by that of the dominating food cue remains unknown. In the present study, the individual reversal of LD cycle for 7 days could not obviously affect circadian patterns of core clock genes (Bmal1, Cry1, Per1, and Dec1) in the liver and heart of restricted-fed rats. In contrast, reversing the feeding schedule together with the LD cycle markedly enhanced the re-entrainment of peripheral clocks compared with reversal of the feeding regimen alone. In addition, LD reversal alone for 7 days contrarily regulated the expression levels of Cry1, Per1, and Dec1 in the liver and heart. Moreover, daytime restricted feeding not only induced different phase shift rates of the four examined clock genes but also led to reversed phase shift direction in their resetting processes in these two peripheral clocks. Furthermore, the resetting sequences of these genes were also disparate between these two peripheral clocks. These observations suggest that the mechanisms underlying the liver and heart clocks are distinct, which may distinguish them from each other in the SCN-synchronized peripheral system.

Astaxanthin prevents and reverses diet-induced insulin resistance and steatohepatitis in mice: A comparison with vitamin E.

Hepatic insulin resistance and nonalcoholic steatohepatitis (NASH) could be caused by excessive hepatic lipid accumulation and peroxidation. Vitamin E has become a standard treatment for NASH. However, astaxanthin, an antioxidant carotenoid, inhibits lipid peroxidation more potently than vitamin E. Here, we compared the effects of astaxanthin and vitamin E in NASH. We first demonstrated that astaxanthin ameliorated hepatic steatosis in both genetically (ob/ob) and high-fat-diet-induced obese mice. In a lipotoxic model of NASH: mice fed a high-cholesterol and high-fat diet, astaxanthin alleviated excessive hepatic lipid accumulation and peroxidation, increased the proportion of M1-type macrophages/Kupffer cells, and activated stellate cells to improve hepatic inflammation and fibrosis. Moreover, astaxanthin caused an M2-dominant shift in macrophages/Kupffer cells and a subsequent reduction in CD4+ and CD8+ T cell recruitment in the liver, which contributed to improved insulin resistance and hepatic inflammation. Importantly, astaxanthin reversed insulin resistance, as well as hepatic inflammation and fibrosis, in pre-existing NASH. Overall, astaxanthin was more effective at both preventing and treating NASH compared with vitamin E in mice. Furthermore, astaxanthin improved hepatic steatosis and tended to ameliorate the progression of NASH in biopsy-proven human subjects. These results suggest that astaxanthin might be a novel and promising treatment for NASH.

Feeding-induced rapid resetting of the hepatic circadian clock is associated with acute induction of Per2 and Dec1 transcription in rats.

The synchronization of the master clock to photic cues is associated with a rapid induction of Per1, which plays an important role in initiating light-induced circadian resetting. However, the transcriptional mechanisms of clock gene expression in food-entrainable peripheral clocks have not been fully assessed. To understand how food cues might entrain a mammalian peripheral clock, we examined the responses in the expression of clock genes in rat livers to different feeding stimuli. The food-entrainable liver clock is more flexible than the light-entrainable SCN clock and can be reset rapidly at any time of day. A 30 min feeding stimulus was sufficient to significantly induce the expression of Per2 and Dec1 within 1 h and alter the transcript levels and circadian phases of other selected clock genes (Bmal1, Cry1, Per1, Per3, Dec2, and Rev-erba) in the liver clock at longer time intervals. Moreover, among the examined clock genes, Per2 was most sensitive to food cues, which could be significantly induced by a minimal amount of food. Furthermore, in contrast to the other hepatic clock genes, the feeding reversal-induced 12 h phase shift of Per2 could be rapidly and consistently accomplished, regardless of the shift of the light/dark cycle. In conclusion, the feeding-induced resetting of the circadian clock in the liver is associated with the acute induction of Per2 and Dec1 transcription, which may serve as the main and secondary input regulators that initiate this feeding-induced circadian resetting. (Author correspondence: azwfu2003@yahoo.com.cn)

DPP-4 inhibition by linagliptin attenuates obesity-related inflammation and insulin resistance by regulating M1/M2 macrophage polarization

Dipeptidyl peptidase 4 (DPP-4) cleaves a large number of chemokine and peptide hormones involved in the regulation of the immune system. Additionally, DPP-4 may also be involved in macrophage-mediated inflammation and insulin resistance. Thus, the current study investigated the effect of linagliptin, an inhibitor of DPP-4, on macrophage migration and polarization in white adipose tissue (WAT) and liver of high-fat diet–induced obese (DIO) mice. DPP-4+ macrophages in lean and obese mice were quantified by fluorescence-activated cell sorting (FACS) analysis. DPP-4 was predominantly expressed in F4/80+ macrophages in crown-like structures compared with adipocytes in WAT of DIO mice. FACS analysis also revealed that, compared with chow-fed mice, DIO mice exhibited a significant increase in DPP-4+ expression in cells within adipose tissue macrophages (ATMs), particularly M1 ATMs. Linagliptin showed a greater DPP-4 inhibition and antioxidative capacity than sitagliptin and reduced M1-polarized macrophage migration while inducing an M2-dominant shift of macrophages within WAT and liver, thereby attenuating obesity-induced inflammation and insulin resistance. Loss of macrophage inflammatory protein-1α, a chemokine and DPP-4 substrate, in DIO mice abrogated M2 macrophage-polarizing and insulin-sensitizing effects of linagliptin. Therefore, the inhibition of DPP-4 by linagliptin reduced obesity-related insulin resistance and inflammation by regulating M1/M2 macrophage status.

β-Cryptoxanthin alleviates diet-induced nonalcoholic steatohepatitis by suppressing inflammatory gene expression in mice.

Recent nutritional epidemiological surveys showed that serum β-cryptoxanthin inversely associates with the risks for insulin resistance and liver dysfunction. Consumption of β-cryptoxanthin possibly prevents nonalcoholic steatohepatitis (NASH), which is suggested to be caused by insulin resistance and oxidative stress from nonalcoholic fatty liver disease. To evaluate the effect of β-cryptoxanthin on diet-induced NASH, we fed a high-cholesterol and high-fat diet (CL diet) with or without 0.003% β-cryptoxanthin to C56BL/6J mice for 12 weeks. After feeding, β-cryptoxanthin attenuated fat accumulation, increases in Kupffer and activated stellate cells, and fibrosis in CL diet-induced NASH in the mice. Comprehensive gene expression analysis showed that although β-cryptoxanthin histochemically reduced steatosis, it was more effective in inhibiting inflammatory gene expression change in NASH. β-Cryptoxanthin reduced the alteration of expression of genes associated with cell death, inflammatory responses, infiltration and activation of macrophages and other leukocytes, quantity of T cells, and free radical scavenging. However, it showed little effect on the expression of genes related to cholesterol and other lipid metabolism. The expression of markers of M1 and M2 macrophages, T helper cells, and cytotoxic T cells was significantly induced in NASH and reduced by β-cryptoxanthin. β-Cryptoxanthin suppressed the expression of lipopolysaccharide (LPS)-inducible and/or TNFα-inducible genes in NASH. Increased levels of the oxidative stress marker thiobarbituric acid reactive substances (TBARS) were reduced by β-cryptoxanthin in NASH. Thus, β-cryptoxanthin suppresses inflammation and the resulting fibrosis probably by primarily suppressing the increase and activation of macrophages and other immune cells. Reducing oxidative stress is likely to be a major mechanism of inflammation and injury suppression in the livers of mice with NASH.

Regulation of circadian gene expression in the kidney by light and food cues in rats

Although studies involving the circadian response to external time cues indicate that the peripheral clocks are dominated mainly by food cues, whether and how changes in the light and food cues affect the circadian rhythm of the renal clock is still largely unknown. In the present study, we found that the circadian phases of Bmal1, Clock, Cry1, Per1, and Per2 were altered differently by the stimuli of food and light cues in the kidney. After the individual reversal of the light-dark (LD) cycle for 7 days, Per1 displayed a 4-h phase delay, whereas the peak phases of Bmal1, Clock, Cry1 and Per2 almost remained the same as those in the control condition. With regard to the feeding-induced circadian resetting of the renal clock, we found that the resetting processes of clock genes could not be completed within 7 days, suggesting a weak synchronization effect of the food cue on the renal circadian clock. Moreover, the reentrainment of the clock genes was greatly enhanced after the reversal of both the feeding schedule and the LD cycle. Noticeably, the phases of Per1 and Clock were shifted most rapidly by 12 h within 3 days after the simultaneous reversal of the feeding schedule and the LD cycle, whereas their peak phases were only shifted by 4 h and 8 h, respectively, on the 7th day after the individual reversal of the feeding schedule. Thus Per1 and Clock may play important roles in the light-induced resetting of the circadian rhythms in the kidney.

Prevention and Reversal of Lipotoxicity-Induced Hepatic Insulin Resistance and Steatohepatitis in Mice by an Antioxidant Carotenoid, β- Cryptoxanthin

Excessive hepatic lipid accumulation promotes macrophages/Kupffer cells activation, resulting in exacerbation of insulin resistance and progression of nonalcoholic steatohepatitis (NASH). However, few promising treatment modalities target lipotoxicity-mediated hepatic activation/polarization of macrophages for NASH. Recent epidemiological surveys showed that serum β-cryptoxanthin, an antioxidant carotenoid, was inversely associated with the risks of insulin resistance and liver dysfunction. In the present study, we first showed that β-cryptoxanthin administration ameliorated hepatic steatosis in high-fat diet-induced obese mice. Next, we investigated the preventative and therapeutic effects of β-cryptoxanthin using a lipotoxic model of NASH: mice fed a high-cholesterol and high-fat (CL) diet. After 12 weeks of CL diet feeding, β-cryptoxanthin administration attenuated insulin resistance and excessive hepatic lipid accumulation and peroxidation, with increases in M1-type macrophages/Kupffer cells and activated stellate cells, and fibrosis in CL diet-induced NASH. Comprehensive gene expression analysis showed that β-cryptoxanthin down-regulated macrophage activation signal-related genes significantly without affecting most lipid metabolism-related genes in the liver. Importantly, flow cytometry analysis revealed that, on a CL diet, β-cryptoxanthin caused a predominance of M2 over M1 macrophage populations, in addition to reducing total hepatic macrophage and T-cell contents. In parallel, β-cryptoxanthin decreased lipopolysaccharide-induced M1 marker mRNA expression in peritoneal macrophages, whereas it augmented IL-4-induced M2 marker mRNA expression, in a dose-dependent manner. Moreover, β-cryptoxanthin reversed steatosis, inflammation, and fibrosis progression in preexisting NASH in mice. In conclusion, β-cryptoxanthin prevents and reverses insulin resistance and steatohepatitis, at least in part, through an M2-dominant shift in macrophages/Kupffer cells in a lipotoxic model of NASH.

Nonalcoholic Fatty Liver Disease and Insulin Resistance: New Insights and Potential New Treatments

Nonalcoholic fatty liver disease (NAFLD) is one of the most common chronic liver disorders worldwide. It is associated with clinical states such as obesity, insulin resistance, and type 2 diabetes, and covers a wide range of liver changes, ranging from simple steatosis to non-alcoholic steatohepatitis (NASH), liver cirrhosis, and hepatocellular carcinoma. Metabolic disorders, such as lipid accumulation, insulin resistance, and inflammation, have been implicated in the pathogenesis of NAFLD, but the underlying mechanisms, including those that drive disease progression, are not fully understood. Both innate and recruited immune cells mediate the development of insulin resistance and NASH. Therefore, modifying the polarization of resident and recruited macrophage/Kupffer cells is expected to lead to new therapeutic strategies in NAFLD. Oxidative stress is also pivotal for the progression of NASH, which has generated interest in carotenoids as potent micronutrient antioxidants in the treatment of NAFLD. In addition to their antioxidative function, carotenoids regulate macrophage/Kupffer cell polarization and thereby prevent NASH progression. In this review, we summarize the molecular mechanisms involved in the pathogenesis of NAFLD, including macrophage/Kupffer cell polarization, and disturbed hepatic function in NAFLD. We also discuss dietary antioxidants, such as β-cryptoxanthin and astaxanthin, that may be effective in the prevention or treatment of NAFLD.