P-glycoprotein dysfunction contributes to hepatic steatosis and obesity in mice

Although the main role of P-glycoprotein (Pgp) is to extrude a broad range of xenochemicals and to protect the organism against xenotoxicity, it also transports a large range of endogenous lipids. Using mice lacking Pgp, we have investigated the possible involvement of Pgp in lipid homeostasis in vivo. In a long term study, we have followed the food intake, body status and lipid markers in plasma and liver of wild-type and mdr1ab(-/-) mice over 35 weeks. Pgp-deficient mice showed excess weight, hypertrophy of adipose mass, high insulin and glucose levels in plasma. Some of these metabolic disruptions appeared earlier in Pgp-deficient mice fed high-fat diet. Moreover, hepatosteatosis with increased expression of genes involved in liver detoxification and in de novo lipid synthesis occurred in Pgp-deficient mice. Overall, Pgp deficiency clearly induced obesity in FVB genetic background, which is known to be resistant to diet-induced obesity. These data reinforce the finding that Pgp gene could be a contributing factor and possibly a relevant marker for lipid disorder and obesity. Subsequent to Pgp deficiency, changes in body availabilities of lipids or any Pgp substrates may affect metabolic pathways that favour the occurrence of obesity. This is of special concern because people are often facing simultaneous exposition to many xenochemicals, which inhibits Pgp, and an excess in lipid dietary intake that may contribute to the high prevalence of obesity in our occidental societies.     

Linagliptin improves insulin sensitivity and hepatic steatosis in diet-induced obesity

Linagliptin (TRADJENTA?) is a selective dipeptidyl peptidase-4 (DPP-4) inhibitor. DPP-4 inhibition attenuates insulin resistance and improves peripheral glucose utilization in humans. However, the effects of chronic DPP-4 inhibition on insulin sensitivity are not known. The effects of long-term treatment (3-4 weeks) with 3 mg/kg/day or 30 mg/kg/day linagliptin on insulin sensitivity and liver fat content were determined in diet-induced obese C57BL/6 mice. Chow-fed animals served as controls. DPP-4 activity was significantly inhibited (67-89%) by linagliptin (P<0.001). Following an oral glucose tolerance test, blood glucose concentrations (measured as area under the curve) were significantly suppressed after treatment with 3 mg/kg/day (-16.5% to -20.3%; P<0.01) or 30 mg/kg/day (-14.5% to -26.4%; P<0.05) linagliptin (both P<0.01). Liver fat content was significantly reduced by linagliptin in a dose-dependent manner (both doses P<0.001). Diet-induced obese mice treated for 4 weeks with 3 mg/kg/day or 30 mg/kg/day linagliptin had significantly improved glycated hemoglobin compared with vehicle (both P<0.001). Significant dose-dependent improvements in glucose disposal rates were observed during the steady state of the euglycemic-hyperinsulinemic clamp: 27.3 mg/kg/minute and 32.2 mg/kg/minute in the 3 mg/kg/day and 30 mg/kg/day linagliptin groups, respectively; compared with 20.9 mg/kg/minute with vehicle (P<0.001). Hepatic glucose production was significantly suppressed during the clamp: 4.7 mg/kg/minute and 2.1 mg/kg/minute in the 3 mg/kg/day and 30 mg/kg/day linagliptin groups, respectively; compared with 12.5 mg/kg/minute with vehicle (P<0.001). In addition, 30 mg/kg/day linagliptin treatment resulted in a significantly reduced number of macrophages infiltrating adipose tissue (P<0.05). Linagliptin treatment also decreased liver expression of PTP1B, SOCS3, SREBP1c, SCD-1 and FAS (P<0.05). Other tissues like muscle, heart and kidney were not significantly affected by the insulin sensitizing effect of linagliptin. Long-term linagliptin treatment reduced liver fat content in animals with diet-induced hepatic steatosis and insulin resistance, and may account for improved insulin sensitivity.     

Macrophage TNF-alpha contributes to insulin resistance and hepatic steatosis in diet-induced obesity

Obesity is commonly associated with development of insulin resistance and systemic evidence of inflammation. Macrophages contribute to inflammatory amplification in obesity and may contribute directly to insulin resistance and the development of nonalcoholic fatty liver disease through the production of inflammatory cytokines, including tumor necrosis factor (TNF)-alpha. To test this hypothesis, we transplanted male wild-type (WT) and TNF-alpha deficient (KO) mice with either TNF-alpha-sufficient (TNF-alpha(+/+)) or TNF-alpha-deficient (TNF-alpha(-/-)) bone marrow. After consuming a high-fat diet for 26 wk, metabolic and morphometric characteristics of the animals were analyzed. While there were no differences in terms of relative weight gain, body composition analysis yielded a lower relative adipose and higher relative lean mass in mice lacking TNF-alpha, which was partially explained by reduced epididymal fat pad and liver weight. TNF-alpha(-/-) -->KO mice exhibited enhanced insulin sensitivity compared with that observed in TNF-alpha(+/+)-->KO mice; remarkably, no protection against insulin resistance was provided by transplanting TNF-alpha(-/-) bone marrow in WT mice compared with TNF-alpha(+/+)-->WT. The preserved insulin sensitivity seen in TNF-alpha(-/-)-->KO mice provided protection against the development of hepatic steatosis. Taken together, these data indicate that macrophage-derived TNF-alpha contributes to the pattern and extent of fat accumulation and insulin resistance in diet-induced obesity; however, this contribution is negligible in the presence of host-derived TNF-alpha.     

Endocannabinoids and liver disease. IV. Endocannabinoid involvement in obesity and hepatic steatosis

Endocannabinoids are endogenous lipid mediators that interact with the same receptors as plant-derived cannabinoids to produce similar biological effects. The well-known appetitive effect of smoking marijuana has prompted inquiries into the possible role of endocannabinoids in the control of food intake and body weight. This brief review surveys recent evidence that endocannabinoids and their receptors are involved at multiple levels in the control of energy homeostasis. Endocannabinoids are orexigenic mediators and are part of the leptin-regulated central neural circuitry that controls energy intake. In addition, they act at multiple peripheral sites including adipose tissue, liver, and skeletal muscle to promote lipogenesis and limit fat elimination. Their complex actions could be viewed as anabolic, increasing energy intake and storage and decreasing energy expenditure, as components of an evolutionarily conserved system that has insured survival under conditions of starvation. In the era of plentiful food and limited physical activity, pharmacological inhibition of endocannabinoid activity offers benefits in the treatment of obesity and its hormonal/metabolic consequences.     

CGI-58 knockdown in mice causes hepatic steatosis but prevents diet-induced obesity and glucose intolerance

Mutations of Comparative Gene Identification-58 (CGI-58) in humans cause triglyceride (TG) accumulation in multiple tissues. Mice genetically lacking CGI-58 die shortly after birth due to a skin barrier defect. To study the role of CGI-58 in integrated lipid and energy metabolism, we utilized antisense oligonucleotides (ASOs) to inhibit CGI-58 expression in adult mice. Treatment with two distinct CGI-58-targeting ASOs resulted in ∼80–95% knockdown of CGI-58 protein expression in both liver and white adipose tissue. In chow-fed mice, ASO-mediated depletion of CGI-58 did not alter weight gain, plasma TG, or plasma glucose, yet raised hepatic TG levels ∼4-fold. When challenged with a high-fat diet (HFD), CGI-58 ASO-treated mice were protected against diet-induced obesity, but their hepatic contents of TG, diacylglycerols, and ceramides were all elevated, and intriguingly, their hepatic phosphatidylglycerol content was increased by 10-fold. These hepatic lipid alterations were associated with significant decreases in hepatic TG hydrolase activity, hepatic lipoprotein-TG secretion, and plasma concentrations of ketones, nonesterified fatty acids, and insulin. Additionally, HFD-fed CGI-58 ASO-treated mice were more glucose tolerant and insulin sensitive. Collectively, this work demonstrates that CGI-58 plays a critical role in limiting hepatic steatosis and maintaining hepatic glycerophospholipid homeostasis and has unmasked an unexpected role for CGI-58 in promoting HFD-induced obesity and insulin resistance.     

SR-BI deficiency disassociates obesity from hepatic steatosis and glucose intolerance development in high fat diet-fed mice

Scavenger receptor BI (SR-BI) has been suggested to modulate adipocyte function. To uncover the potential relevance of SR-BI for the development of obesity and associated metabolic complications, we compared the metabolic phenotype of wild-type and SR-BI deficient mice fed an obesogenic diet enriched in fat. Both male and female SR-BI knockout mice gained significantly more weight as compared to their wild-type counterparts in response to 12 weeks high fat diet feeding (1.5-fold; P < .01 for genotype). Plasma free cholesterol levels were ~2-fold higher (P < .001) in SR-BI knockout mice of both genders, whilst plasma cholesteryl ester and triglyceride concentrations were only significantly elevated in males. Strikingly, the exacerbated obesity in SR-BI knockout mice was paralleled by a better glucose handling. In contrast, only SR-BI knockout mice developed atherosclerotic lesions in the aortic root, with a higher predisposition in females. Biochemical and histological studies in male mice revealed that SR-BI deficiency was associated with a reduced hepatic steatosis degree as evident from the 29% lower (P < .05) liver triglyceride levels. Relative mRNA expression levels of the glucose uptake transporter GLUT4 were increased (+47%; P < .05), whilst expression levels of the metabolic PPARgamma target genes CD36, HSL, ADIPOQ and ATGL were reduced 39%–58% (P < .01) in the context of unchanged PPARgamma expression levels in SR-BI knockout gonadal white adipose tissue. In conclusion, we have shown that SR-BI deficiency is associated with a decrease in adipocyte PPARgamma activity and a concomitant uncoupling of obesity development from hepatic steatosis and glucose intolerance development in high fat diet-fed mice.     

Quantitative analysis of the murine lipid droplet-associated proteome during diet-induced hepatic steatosis

Hepatic steatosis is characterized by the accumulation of lipid droplets (LDs), which are composed of a neutral lipid core surrounded by a phospholipid monolayer embedded with many proteins. Although the LD-associated proteome has been investigated in multiple tissues and organisms, the dynamic changes in the murine LD-associated proteome in response to obesity and hepatic steatosis have not been studied. We characterized the hepatic LD-associated proteome of C57BL/6J male mouse livers following high-fat feeding using isobaric tagging for relative and absolute quantification. Of the 1,520 proteins identified with a 5% local false discovery rate, we report a total of 48 proteins that were increased and 52 proteins that were decreased on LDs in response to high-fat feeding. Most notably, ribosomal and endoplasmic reticulum proteins were increased and extracellular and cytosolic proteins were decreased in response to high-fat feeding. Additionally, many proteins involved in fatty acid catabolism or xenobiotic metabolism were enriched in the LD fraction following high-fat feeding. In contrast, proteins involved in glucose metabolism and liver X receptor or retinoid X receptor activation were decreased on LDs of high-fat-fed mice. This study provides insights into unique biological functions of hepatic LDs under normal and steatotic conditions.     

Interleukin-38 alleviates hepatic steatosis through AMPK/autophagy-mediated suppression of endoplasmic reticulum stress in obesity models

Interleukin-38 (IL-38), recently recognized as a cytokine with anti-inflammatory properties that mitigate type 2 diabetes, has been associated with indicators of insulin resistance and nonalcoholic fatty liver disease (NAFLD). This study investigated the impact of IL-38 on hepatic lipid metabolism and endoplasmic reticulum (ER) stress. We assessed protein expression levels using Western blot analysis, while monodansylcadaverine staining was employed to detect autophagosomes in hepatocytes. Oil red O staining was utilized to examine lipid deposition. The study revealed elevated serum IL-38 levels in high-fat diet (HFD)-fed mice and IL-38 secretion from mouse keratinocytes. IL-38 treatment attenuated lipogenic lipid accumulation and ER stress markers in hepatocytes exposed to palmitate. Furthermore, IL-38 treatment increased AMP-activated protein kinase (AMPK) phosphorylation and autophagy. The effects of IL-38 on lipogenic lipid deposition and ER stress were nullified in cultured hepatocytes by suppressing AMPK through small interfering (si) RNA or 3-methyladenine (3MA). In animal studies, IL-38 administration mitigated hepatic steatosis by suppressing the expression of lipogenic proteins and ER stress markers while reversing AMPK phosphorylation and autophagy markers in the livers of HFD-fed mice. Additionally, AMPK siRNA, but not 3MA, mitigated IL-38-enhanced fatty acid oxidation in hepatocytes. In summary, IL-38 alleviates hepatic steatosis through AMPK/autophagy signaling-dependent attenuation of ER stress and enhancement of fatty acid oxidation via the AMPK pathway, suggesting a therapeutic strategy for treating NAFLD.     

Shotgun lipidomics: multidimensional MS analysis of cellular lipidomes

Shotgun lipidomics, comprised of intrasource separation, multidimensional mass spectrometry and computer-assisted array analysis, is an emerging powerful technique in lipidomics. Through effective intrasource separation of predetermined groups of lipid classes based on their intrinsic electrical propensities, analyses of lipids from crude extracts of biologic samples can be directly and routinely performed. Appropriate multidimensional array analysis of lipid pseudomolecular ions and fragments can be performed leading to the identification and quantitation of targeted lipid molecular species. Since most biologic lipids are linear combinations of aliphatic chains, backbones and head groups, a rich repertoire of multiple lipid building blocks present in discrete combinations represent experimental observables that can be computer reconstructed in conjunction with their pseudomolecular ions to directly determine the lipid molecular structures from a lipid extract. Through this approach, dramatic increases in the accessible dynamic range for ratiometric quantitation and discrimination of isobaric molecular species can be achieved without any prior column chromatography or operator-dependent supervision. At its current state of development, shotgun lipidomics can analyze over 20 lipid classes, hundreds of lipid molecular species and more than 95% of the mass content of a cellular lipidome. Thus, understanding the biochemical mechanisms underlying lipid-mediated disease states will be greatly facilitated by the power of shotgun lipidomics.     

Hydroxytyrosol ameliorates insulin resistance by modulating endoplasmic reticulum stress and prevents hepatic steatosis in diet-induced obesity mice

Endoplasmic reticulum (ER) is a principal organelle responsible for energy and nutrient management. Its dysfunction has been viewed in the context of obesity and related glucolipid metabolic disorders. However, therapeutic approaches to improve ER adaptation and systemic energy balance in obesity are limited. Thus, we examined whether hydroxytyrosol (HT), an important polyphenolic compound found in virgin olive oil, could correct the metabolic impairments in diet-induced obesity (DIO) mice. Here, we found that HT gavage for 10 weeks significantly ameliorated glucose homeostasis and chronic inflammation and decreased hepatic steatosis in DIO mice. At the molecular level, ER stress indicators, inflammatory and insulin signaling markers demonstrated that high-fat diet (HFD)-induced ER stress and insulin resistance (IR) in insulin sensitive tissue were corrected by HT. In vitro studies confirmed that HT supplementation (100 μM) attenuated palmitate-evoked ER stress, thus rescuing the downstream JNK/IRS pathway. As a result from suppression of ER stress in the liver, HT further decreased hepatic sterol regulatory element-binding protein-1 expression (SREBP1). Additionally, aberrant expression of genes involved in hepatic lipogenesis (SREBP1, ACC, FAS, SCD1) caused by HFD was restored by HT. These findings suggested that HT ameliorated chronic inflammation and IR and decreased hepatic steatosis in obesity by beneficial modulation of ER stress.     

Stable isotope analysis of dynamic lipidomics

Metabolic pathway flux is a fundamental element of biological activity, which can be quantified using a variety of mass spectrometric techniques to monitor incorporation of stable isotope-labelled substrates into metabolic products. This article contrasts developments in electrospray ionisation mass spectrometry (ESI-MS) for the measurement of lipid metabolism with more established gas chromatography mass spectrometry and isotope ratio mass spectrometry methodologies. ESI-MS combined with diagnostic tandem MS/MS scans permits the sensitive and specific analysis of stable isotope-labelled substrates into intact lipid molecular species without the requirement for lipid hydrolysis and derivatisation. Such dynamic lipidomic methodologies using non-toxic stable isotopes can be readily applied to quantify lipid metabolic fluxes in clinical and metabolic studies in vivo. However, a significant current limitation is the absence of appropriate software to generate kinetic models of substrate incorporation into multiple products in the time domain. Finally, we discuss the future potential of stable isotope-mass spectrometry imaging to quantify the location as well as the extent of lipid synthesis. This article is part of a Special Issue entitled: BBALIP_Lipidomics Opinion Articles edited by Sepp Kohlwein.     

Loss of regulator of G protein signaling 5 exacerbates obesity, hepatic steatosis, inflammation and insulin resistance

Background

The effect of regulator of G protein signaling 5 (RGS5) on cardiac hypertrophy, atherosclerosis and angiogenesis has been well demonstrated, but the role in the development of obesity and insulin resistance remains completely unknown. We determined the effect of RGS5 deficiency on obesity, hepatic steatosis, inflammation and insulin resistance in mice fed either a normal-chow diet (NC) or a high-fat diet (HF).

Methodology/Principal Findings

Male, 8-week-old RGS5 knockout (KO) and littermate control mice were fed an NC or an HF for 24 weeks and were phenotyped accordingly. RGS5 KO mice exhibited increased obesity, fat mass and ectopic lipid deposition in the liver compared with littermate control mice, regardless of diet. When fed an HF, RGS5 KO mice had a markedly exacerbated metabolic dysfunction and inflammatory state in the blood serum. Meanwhile, macrophage recruitment and inflammation were increased and these increases were associated with the significant activation of JNK, IκBα and NF-κBp65 in the adipose tissue, liver and skeletal muscle of RGS5 KO mice fed an HF relative to control mice. These exacerbated metabolic dysfunction and inflammation are accompanied with decreased systemic insulin sensitivity in the adipose tissue, liver and skeletal muscle of RGS5 KO mice, reflected by weakened Akt/GSK3β phosphorylation.

Conclusions/Significance

Our data suggest that loss of RGS5 exacerbates HF-induced obesity, hepatic steatosis, inflammation and insulin resistance.     

Shotgun lipidomics: multidimensional MS analysis of cellular lipidomes

Shotgun lipidomics, comprised of intrasource separation, multidimensional mass spectrometry and computer-assisted array analysis, is an emerging powerful technique in lipidomics. Through effective intrasource separation of predetermined groups of lipid classes based on their intrinsic electrical propensities, analyses of lipids from crude extracts of biologic samples can be directly and routinely performed. Appropriate multidimensional array analysis of lipid pseudomolecular ions and fragments can be performed leading to the identification and quantitation of targeted lipid molecular species. Since most biologic lipids are linear combinations of aliphatic chains, backbones and head groups, a rich repertoire of multiple lipid building blocks present in discrete combinations represent experimental observables that can be computer reconstructed in conjunction with their pseudomolecular ions to directly determine the lipid molecular structures from a lipid extract. Through this approach, dramatic increases in the accessible dynamic range for ratiometric quantitation and discrimination of isobaric molecular species can be achieved without any prior column chromatography or operator-dependent supervision. At its current state of development, shotgun lipidomics can analyze over 20 lipid classes, hundreds of lipid molecular species and more than 95% of the mass content of a cellular lipidome. Thus, understanding the biochemical mechanisms underlying lipid-mediated disease states will be greatly facilitated by the power of shotgun lipidomics.     

Genetic determinants of hepatic steatosis in man

Hepatic steatosis is one of the most common liver disorders in the general population. The main cause of hepatic steatosis is nonalcoholic fatty liver disease (NAFLD), representing the hepatic component of the metabolic syndrome, which is characterized by type 2 diabetes, obesity, and dyslipidemia. Insulin resistance and excess adiposity are considered to play key roles in the pathogenesis of NAFLD. Although the risk factors for NAFLD are well established, the genetic basis of hepatic steatosis is largely unknown. Here we review recent progress on genomic variants and their association with hepatic steatosis and discuss the potential impact of these genetic studies on clinical practice. Identifying the genetic determinants of hepatic steatosis will lead to a better understanding of the pathogenesis and progression of NAFLD.