Ya Pei

Metformin Ameliorates Hepatic Steatosis and Inflammation without Altering Adipose Phenotype in Diet-Induced Obesity

Non-alcoholic fatty liver disease (NAFLD) is closely associated with obesity and insulin resistance. To better understand the pathophysiology of obesity-associated NAFLD, the present study examined the involvement of liver and adipose tissues in metformin actions on reducing hepatic steatosis and inflammation during obesity. C57BL/6J mice were fed a high-fat diet (HFD) for 12 weeks to induce obesity-associated NAFLD and treated with metformin (150 mg/kg/d) orally for the last four weeks of HFD feeding. Compared with HFD-fed control mice, metformin-treated mice showed improvement in both glucose tolerance and insulin sensitivity. Also, metformin treatment caused a significant decrease in liver weight, but not adiposity. As indicated by histological changes, metformin treatment decreased hepatic steatosis, but not the size of adipocytes. In addition, metformin treatment caused an increase in the phosphorylation of liver AMP-activated protein kinase (AMPK), which was accompanied by an increase in the phosphorylation of liver acetyl-CoA carboxylase and decreases in the phosphorylation of liver c-Jun N-terminal kinase 1 (JNK1) and in the mRNA levels of lipogenic enzymes and proinflammatory cytokines. However, metformin treatment did not significantly alter adipose tissue AMPK phosphorylation and inflammatory responses. In cultured hepatocytes, metformin treatment increased AMPK phosphorylation and decreased fat deposition and inflammatory responses. Additionally, in bone marrow-derived macrophages, metformin treatment partially blunted the effects of lipopolysaccharide on inducing the phosphorylation of JNK1 and nuclear factor kappa B (NF-κB) p65 and on increasing the mRNA levels of proinflammatory cytokines. Taken together, these results suggest that metformin protects against obesity-associated NAFLD largely through direct effects on decreasing hepatocyte fat deposition and on inhibiting inflammatory responses in both hepatocytes and macrophages.

Berberine Ameliorates Hepatic Steatosis and Suppresses Liver and Adipose Tissue Inflammation in Mice with Diet-induced Obesity

Increasing evidence demonstrates that berberine (BBR) is beneficial for obesity-associated non-alcoholic fatty liver disease (NAFLD). However, it remains to be elucidated how BBR improves aspects of NAFLD. Here we revealed an AMP-activated protein kinase (AMPK)-independent mechanism for BBR to suppress obesity-associated inflammation and improve hepatic steatosis. In C57BL/6J mice fed a high-fat diet (HFD), treatment with BBR decreased inflammation in both the liver and adipose tissue as indicated by reduction of the phosphorylation state of JNK1 and the mRNA levels of proinflammatory cytokines. BBR treatment also decreased hepatic steatosis, as well as the expression of acetyl-CoA carboxylase and fatty acid synthase. Interestingly, treatment with BBR did not significantly alter the phosphorylation state of AMPK in both the liver and adipose tissue of HFD-fed mice. Consistently, BBR treatment significantly decreased the phosphorylation state of JNK1 in both hepatoma H4IIE cells and mouse primary hepatocytes in both dose-dependent and time-dependent manners, which was independent of AMPK phosphorylation. BBR treatment also caused a decrease in palmitate-induced fat deposition in primary mouse hepatocytes. Taken together, these results suggest that BBR actions on improving aspects of NAFLD are largely attributable to BBR suppression of inflammation, which is independent of AMPK.

Myeloid Cell-specific Disruption of Period1 and Period2 Exacerbates Diet-induced Inflammation and Insulin Resistance

Background: Circadian clockworks gate macrophage inflammatory responses. Results: Myeloid cell-specific disruption of Period1 and Period2 exacerbates diet-induced adipose and liver inflammation and systemic insulin resistance. Conclusion: Macrophage circadian dysregulation contributes to diet-induced inflammation and metabolic phenotypes in adipose and liver tissues. Significance: Interactions between circadian clocks and pathways mediating adipose tissue inflammation are critical in the development and possibly treatment of obesity-associated metabolic disorders. The circadian clockworks gate macrophage inflammatory responses. Given the association between clock dysregulation and metabolic disorders, we conducted experiments to determine the extent to which over-nutrition modulates macrophage clock function and whether macrophage circadian dysregulation is a key factor linking over-nutrition to macrophage proinflammatory activation, adipose tissue inflammation, and systemic insulin resistance. Our results demonstrate that 1) macrophages from high fat diet-fed mice are marked by dysregulation of the molecular clockworks in conjunction with increased proinflammatory activation, 2) global disruption of the clock genes Period1 (Per1) and Per2 recapitulates this amplified macrophage proinflammatory activation, 3) adoptive transfer of Per1/2-disrupted bone marrow cells into wild-type mice potentiates high fat diet-induced adipose and liver tissue inflammation and systemic insulin resistance, and 4) Per1/2-disrupted macrophages similarly exacerbate inflammatory responses and decrease insulin sensitivity in co-cultured adipocytes in vitro. Furthermore, PPARγ levels are decreased in Per1/2-disrupted macrophages and PPARγ2 overexpression ameliorates Per1/2 disruption-associated macrophage proinflammatory activation, suggesting that this transcription factor may link the molecular clockworks to signaling pathways regulating macrophage polarization. Thus, macrophage circadian clock dysregulation is a key process in the physiological cascade by which diet-induced obesity triggers macrophage proinflammatory activation, adipose tissue inflammation, and insulin resistance.

A role for PFKFB3/iPFK2 in metformin suppression of adipocyte inflammatory responses

Metformin improves obesity-associated metabolic dysregulation, but has controversial effects on adipose tissue inflammation. The objective of the study is to examine the direct effect of metformin on adipocyte inflammatory responses and elucidate the underlying mechanisms. Adipocytes were differentiated from 3T3-L1 cells and treated with metformin at various doses and for different time periods. The treated cells were examined for the proinflammatory responses, as well as the phosphorylation states of AMPK and the expression of PFKFB3/iPFK2. In addition, PFKFB3/iPFK2-knockdown adipocytes were treated with metformin and examined for changes in the proinflammatory responses. The following results were obtained from the study. Treatment of adipocytes with metformin decreased the effects of lipopolysaccharide on inducing the phosphorylation states of JNK p46 and on increasing the mRNA levels of IL-1β and TNFα. In addition, treatment with metformin increased the expression of PFKFB3/iPFK2, but failed to significantly alter the phosphorylation states of AMPK. In PFKFB3/iPFK2-knockdown adipocytes, treatment with metformin did not suppress the proinflammatory responses as did it in control adipocytes. In conclusion, metformin has a direct effect on suppressing adipocyte proinflammatory responses in an AMPK-independent manner. Also, metformin increases adipocyte expression of PFKFB3/iPFK2, which is involved in the anti-inflammatory effect of metformin.

Nutritional approaches for managing obesity-associated metabolic diseases

Obesity is an ongoing pandemic and serves as a causal factor of a wide spectrum of metabolic diseases including diabetes, fatty liver disease, and cardiovascular disease. Much evidence has demonstrated that nutrient overload/overnutrition initiates or exacerbates inflammatory responses in tissues/organs involved in the regulation of systemic metabolic homeostasis. This obesity-associated inflammation is usually at a low-grade and viewed as metabolic inflammation. When it exists continuously, inflammation inappropriately alters metabolic pathways and impairs insulin signaling cascades in peripheral tissues/organs such as adipose tissue, the liver and skeletal muscles, resulting in local fat deposition and insulin resistance and systemic metabolic dysregulation. In addition, inflammatory mediators, e.g., proinflammatory cytokines, and excessive nutrients, e.g., glucose and fatty acids, act together to aggravate local insulin resistance and form a vicious cycle to further disturb the local metabolic pathways and exacerbate systemic metabolic dysregulation. Owing to the critical role of nutrient metabolism in controlling the initiation and progression of inflammation and insulin resistance, nutritional approaches have been implicated as effective tools for managing obesity and obesity-associated metabolic diseases. Based on the mounting evidence generated from both basic and clinical research, nutritional approaches are commonly used for suppressing inflammation, improving insulin sensitivity, and/or decreasing fat deposition. Consequently, the combined effects are responsible for improvement of systemic insulin sensitivity and metabolic homeostasis.

Glucose and Palmitate Differentially Regulate PFKFB3/iPFK2 and Inflammatory Responses in Mouse Intestinal Epithelial Cells

The gene PFKFB3 encodes for inducible 6-phosphofructo-2-kinase, a glycolysis-regulatory enzyme that protects against diet-induced intestine inflammation. However, it is unclear how nutrient overload regulates PFKFB3 expression and inflammatory responses in intestinal epithelial cells (IECs). In the present study, primary IECs were isolated from small intestine of C57BL/6J mice fed a low-fat diet (LFD) or high-fat diet (HFD) for 12 weeks. Additionally, CMT-93 cells, a cell line for IECs, were cultured in low glucose (LG, 5.5 mmol/L) or high glucose (HG, 27.5 mmol/L) medium and treated with palmitate (50 μmol/L) or bovine serum albumin (BSA) for 24 hr. These cells were analyzed for PFKFB3 and inflammatory markers. Compared with LFD, HFD feeding decreased IEC PFKFB3 expression and increased IEC proinflammatory responses. In CMT-93 cells, HG significantly increased PFKFB3 expression and proinflammatory responses compared with LG. Interestingly, palmitate decreased PFKFB3 expression and increased proinflammatory responses compared with BSA, regardless of glucose concentrations. Furthermore, HG significantly increased PFKFB3 promoter transcription activity compared with LG. Upon PFKFB3 overexpression, proinflammatory responses in CMT-93 cells were decreased. Taken together, these results indicate that in IECs glucose stimulates PFKFB3 expression and palmitate contributes to increased proinflammatory responses. Therefore, PFKFB3 regulates IEC inflammatory status in response to macronutrients.

Disruption of adenosine 2A receptor exacerbates NAFLD through increasing inflammatory responses and SREBP1c activity

Adenosine 2A receptor (A2AR) exerts protective roles in endotoxin‐ and/or ischemia‐induced tissue damage. However, the role for A2AR in nonalcoholic fatty liver disease (NAFLD) remains largely unknown. We sought to examine the effects of global and/or myeloid cell‐specific A2AR disruption on the aspects of obesity‐associated NAFLD and to elucidate the underlying mechanisms. Global and/or myeloid cell–specific A2AR‐disrupted mice and control mice were fed a high‐fat diet (HFD) to induce NAFLD. In addition, bone marrow–derived macrophages and primary mouse hepatocytes were examined for inflammatory and metabolic responses. Upon feeding an HFD, both global A2AR‐disrupted mice and myeloid cell–specific A2AR‐defcient mice revealed increased severity of HFD‐induced hepatic steatosis and inflammation compared with their respective control mice. In in vitro experiments, A2AR‐deficient macrophages exhibited increased proinflammatory responses, and enhanced fat deposition of wild‐type primary hepatocytes in macrophage–hepatocyte cocultures. In primary hepatocytes, A2AR deficiency increased the proinflammatory responses and enhanced the effect of palmitate on stimulating fat deposition. Moreover, A2AR deficiency significantly increased the abundance of sterol regulatory element‐binding protein 1c (SREBP1c) in livers of fasted mice and in hepatocytes upon nutrient deprivation. In the absence of A2AR, SREBP1c transcription activity was significantly increased in mouse hepatocytes. Conclusion: Taken together, our results demonstrate that disruption of A2AR in both macrophage and hepatocytes accounts for increased severity of NAFLD, likely through increasing inflammation and through elevating lipogenic events due to stimulation of SREBP1c expression and transcription activity. (Hepatology 2018;68:48‐61).

Expression of STING Is Increased in Liver Tissues from Patients With NAFLD and Promotes Macrophage-mediated Hepatic Inflammation and Fibrosis in Mice

BACKGROUND & AIMS Transmembrane protein 173 (TMEM173 or STING) signaling by macrophage activates the type I interferon-mediated innate immune response. The innate immune response contributes to hepatic steatosis and non-alcoholic fatty liver disease (NAFLD). We investigated whether STING regulates diet-induced in hepatic steatosis, inflammation, and liver fibrosis in mice. METHODS Mice with disruption of Tmem173 (STINGgt) on a C57BL/6J background, mice without disruption of this gene (controls), and mice with disruption of Tmem173 only in myeloid cells were fed a standard chow diet, a high-fat diet (HFD; 60% fat calories), or a methionine- and choline-deficient diet (MCD). Liver tissues were collected and analyzed by histology and immunohistochemistry. Bone marrow cells were isolated from mice, differentiated into macrophages, and incubated with 5,6-dimethylxanthenone-4-acetic acid (DMXAA; an activator of STING) or cyclic guanosine monophosphate-adenosine monophosphate (cGAMP). Macrophages or their media were applied to mouse hepatocytes or human hepatic stellate cells (LX2) cells, which were analyzed for cytokine expression, protein phosphorylation, and fat deposition (by oil red O staining after incubation with palmitate). We obtained liver tissues from patients with and without NAFLD and analyzed these by immunohistochemistry. RESULTS Non-parenchymal cells of liver tissues from patients with NAFLD had higher levels of STING than cells of liver tissues from patients without NAFLD. STINGgt mice and mice with disruption only in myeloid cells developed less severe hepatic steatosis, inflammation, and/or fibrosis after the HFD or MCD than control mice. Levels of phosphorylated c-Jun N-terminal kinase and p65 and mRNAs encoding tumor necrosis factor and interleukins 1B and 6 (markers of inflammation) were significantly lower in liver tissues from STINGgt mice vs control mice after the HFD or MCD. Transplantation of bone marrow cells from control mice to STINGgt mice restored the severity of steatosis and inflammation after the HFD. Macrophages from control, but not STINGgt, mice increased markers of inflammation in response to lipopolysaccharide and cGAMP. Hepatocytes and stellate cells cocultured with STINGgt macrophages in the presence of DMXAA or incubated with the medium collected from these macrophages had decreased fat deposition and markers of inflammation compared with hepatocytes or stellate cells incubated with control macrophages. CONCLUSIONS Levels of STING were increased in liver tissues from patients with NAFLD and mice with HFD-induced steatosis. In mice, loss of STING from macrophages decreased the severity of liver fibrosis and the inflammatory response. STING might be a therapeutic target for NAFLD.

Cyclic GMP-AMP Ameliorates Diet-induced Metabolic Dysregulation and Regulates Proinflammatory Responses Distinctly from STING Activation

Endogenous cyclic GMP-AMP (cGAMP) binds and activates STING to induce type I interferons. However, whether cGAMP plays any roles in regulating metabolic homeostasis remains unknown. Here we show that exogenous cGAMP ameliorates obesity-associated metabolic dysregulation and uniquely alters proinflammatory responses. In obese mice, treatment with cGAMP significantly decreases diet-induced proinflammatory responses in liver and adipose tissues and ameliorates metabolic dysregulation. Strikingly, cGAMP exerts cell-type-specific anti-inflammatory effects on macrophages, hepatocytes, and adipocytes, which is distinct from the effect of STING activation by DMXAA on enhancing proinflammatory responses. While enhancing insulin-stimulated Akt phosphorylation in hepatocytes and adipocytes, cGAMP weakens the effects of glucagon on stimulating hepatocyte gluconeogenic enzyme expression and glucose output and blunts palmitate-induced hepatocyte fat deposition in an Akt-dependent manner. Taken together, these results suggest an essential role for cGAMP in linking innate immunity and metabolic homeostasis, indicating potential applications of cGAMP in treating obesity-associated inflammatory and metabolic diseases.