14 research outputs found
Nutrient Regulation of PFKFB3/iPFK2 and Its Role in Regulating Diet-Induced Inflammation in Intestinal Epithelial Cells
The gene PFKFB3 encodes for inducible 6-phosphofructo-2-kinase (iPFK2), an important regulatory enzyme of glycolysis. It is shown that PFKFB3/iPFK2 links metabolic and inflammatory pathways in adipose tissue; however, whether it functions in the same manner within small intestine, where nutrients are assimilated and first interact with the body, is unknown. Therefore, the present study firstly investigated how diet, macronutrients, e.g. glucose and palmitate, and bacterial metabolites influence PFKFB3/iPFK2 expression, and secondly determined how altered gene expression relates to inflammatory responses in small intestinal epithelial cells (IECs).
HFD feeding and in vitro palmitate treatment were associated with reduced PFKFB3/iPFK2 but increased proinflammatory responses. LFD feeding and glucose treatment showed the opposite result. In vitro overexpression of PFKFB3/iPFK2 lead to reduced proinflammatory responses while inhibition of PFKFB3/iPFK2 was associated with increased inflammatory markers. Treatment with the bacterial metabolite indole stimulated PFKFB3/iPFK2 and reduced the generation of inflammation.
Together these findings indicate that macronutrients differentially regulate PFKFB3/iPFK2 expression in IECs, where carbohydrates stimulate PFKFB3/iPFK2 and saturated fats contribute to proinflammatory mechanisms. Further, results confirm an anti-inflammatory ability of PFKFB3/iPFK2 within IECs and suggest an additional anti-inflammatory mechanism of action of indole in regulating inflammation through PFKFB3/iPFK2
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 mu mol/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.National Institutes of Health [HL108922, HL095556, R01DK095828, R01DK095862]; National Natural Science Foundation of China [81100562/H0711]; Hatch Program of National Institutes of Food and Agriculture (NIFA)SCI(E)[email protected]; [email protected]
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 nonalcoholic 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.National Institutes of Health [HL108922, HL095556, R01DK095828, R01DK095862]; National Natural Science Foundation of China [81100562/H0711]; Hatch Program of the National Institutes of Food and Agriculture (NIFA)SCI(E)[email protected]; [email protected]
Indole Alleviates Diet-induced Hepatic Steatosis and Inflammation in a Manner Involving Myeloid Cell PFKFB3
Background and aims: Indole is a microbiota metabolite that exerts anti-inflammatory responses. However, the relevance of indole to human non-alcoholic fatty liver disease (NAFLD) is not clear. It also remains largely unknown whether and how indole acts to protect against NAFLD. The present study sought to examine the association between the circulating levels of indole and liver fat content in human subjects and explore the mechanisms underlying indole actions in mice with diet-induced NAFLD.
Approach and results: In a cohort of 137 subjects, the circulating levels of indole were reversely correlated with body mass index. In addition, the circulating levels of indole in obese subjects were significantly lower than those in lean subjects and were accompanied with increased liver fat content. At the whole-animal level, treatment of high-fat diet (HFD)-fed C57BL/6J mice with indole caused significant decreases in the severity of hepatic steatosis and inflammation. In cultured cells, indole treatment stimulated the expression of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), a master regulatory gene of glycolysis, and suppressed macrophage proinflammatory activation in a PFKFB3-dependent manner. Moreover, myeloid cell-specific PFKFB3 disruption exacerbated the severity of HFD-induced hepatic steatosis and inflammation and blunted the effect of indole on alleviating diet-induced NAFLD phenotype.
Conclusions: Taken together, our results demonstrate that indole is relevant to human NAFLD and capable of alleviating diet-induced NAFLD phenotypes in mice in a myeloid cell PFKFB3-dependent manner. Therefore, indole mimetic and/or macrophage-specific PFKFB3 activation may be the viable preventive and/or therapeutic approaches for inflammation-associated diseases including NAFLD
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
EUK-134 ameliorates nNOSμ translocation and skeletal muscle fiber atrophy during short-term mechanical unloading
Metformin treatment ameliorates HFD-induced insulin resistance and glucose intolerance.
<p>Male C57BL/6J mice, at 5–6 weeks of age, were fed a high-fat diet (HFD) and treated with metformin (Met, 150 mg/kg body weight/d, in phosphate-buffered saline (PBS)) or PBS for the last 4 weeks of HFD feeding. As an additional control, gender- and age-matched mice were fed a low-fat diet (LFD) for 12 weeks and treated only with PBS for the last 4 weeks. Data are means ± SE, n = 6–10. (A) Body weight was monitored weekly during the feeding/treatment regimen. (B) Food intake was calculated based on food consumption per day per mouse. (C) Insulin tolerance tests (ITT). (D) Glucose tolerance tests (GTT). For C and D, mice were fasted for 4 hr and received an intraperitoneal injection of insulin (1 U/kg body weight) (C) or glucose (2 g/kg body weight) (D). *, <i>P</i><0.05 and **, <i>P</i><0.01 HFD-Met vs. HFD-PBS for the same time point (C and D).</p
Metformin treatment blunts hepatocyte fat deposition, increases AMPK phosphorylation, and decreases inflammatory responses.
<p>(A) Hepatocyte fat deposition. Bottom panels, cells were incubated with palmitate (Pal). (B) Hepatocyte mRNA levels were quantified using real-time PCR. For A and B, H4IIE cells were treated with metformin (500 µM) or PBS in the presence of palmitate (250 µM) or bovine serum albumin (BSA) for 24 hr (B) and stained with Oil-Red-O for the last 1 hr (A). (C) Hepatocyte AMPK signaling and inflammatory signaling were examined using Western blot analyses. H4IIE cells were treated with metformin (500 µM) or PBS for 24 hr in the presence or absence of LPS (100 ng/ml) for the last 30 min. For bar graphs (B and C), data are means ± SE, n = 6–8. <sup>†</sup>, <i>P</i><0.05 and <sup>††</sup>, <i>P</i><0.01 Pal + PBS vs. BSA + PBS (B) or LPS vs. PBS (without LPS) under the same condition (with or without Met) (C); *, <i>P</i><0.05 and **, <i>P</i><0.01 Pal + Met vs. Pal + PBS (B) or Met vs. PBS (without Met) under the same condition (with or without LPS) (C).</p
Metformin treatment ameliorates HFD-induced liver inflammatory responses.
<p>Mice were treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091111#pone-0091111-g001" target="_blank">Figure 1</a>. After the feeding/treatment regimen, mice were fasted for 4 hr prior to collection of tissue samples. (A) Liver sections were stained for F4/80<sup>+</sup> cells. (B) Liver inflammatory signaling. Liver extracts were subjected to Western blot analyses. Ratios of phosphorylated JNK1 to total JNK1 (Pp46/p46) and phosphorylated NF-κB p65 to total p65 (Pp65/p65) were quantified using densitometry and normalized to GAPDH. (C) Liver mRNA levels of proinflammatory cytokines were analyzed using real-time PCR. For bar graphs (B and C), data are means ± SE, n = 6–8. <sup>†</sup>, <i>P</i><0.05 and <sup>††</sup>, <i>P</i><0.01 HFD-PBS vs. LFD-PBS; *, <i>P</i><0.05 and **, <i>P</i><0.01 HFD-Met vs. HFD-PBS.</p
Metformin treatment does not alter HFD-induced adiposity and adipose tissue inflammation.
<p>Mice were treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091111#pone-0091111-g001" target="_blank">Figure 1</a>. After the feeding/treatment regimen, mice were fasted for 4 hr prior to collection of tissue samples. (A) Adipose tissue histology (H&E staining). (B) Adipose tissue sections were stained for F4/80<sup>+</sup> cells. (C) Adipose tissue macrophage infiltration. Percentages of mature macrophages (F4/80<sup>+</sup> CD11b<sup>+</sup> cells) in adipose tissue stromal cells were calculated using FACS analyses (n = 4–6). (D) Adipose tissue AMPK signaling and inflammatory signaling were examined using Western blot analyses (n = 4–6). (E) The mRNA levels of adipose genes were quantified using real-time PCR (n = 4–6). For bar graphs (C and E), data are means ± SE.</p