The pathologies associated with functional titration of phosphatidylinositol transfer protein alpha activity in mice

Phosphatidylinositol transfer proteins (PITPs) bind phosphatidylinositol (PtdIns) and phosphatidylcholine and play diverse roles in coordinating lipid metabolism/signaling with intracellular functions. The underlying mechanisms remain unclear. Genetic ablation of PITPalpha in mice results in neonatal lethality characterized by intestinal and hepatic steatosis, spinocerebellar neurodegeneration, and glucose homeostatic defects. We report that mice expressing a PITPalpha selectively ablated for PtdIns binding activity (Pitpalpha(T59D)), as the sole source of PITPalpha, exhibit phenotypes that recapitulate those of authentic PITPalpha nullizygotes. Analyses of mice with graded reductions in PITPalpha activity reveal proportionately graded reductions in lifespan, demonstrate that intestinal steatosis and hypoglycemia are apparent only when PITPalpha protein levels are strongly reduced (>or=90%), and correlate steatotic and glucose homeostatic defects with cerebellar inflammatory disease. Finally, reconstitution of PITPalpha expression in the small intestine substantially corrects the chylomicron retention disease and cerebellar inflammation of Pitpalpha(0/0) neonates, but does not rescue neonatal lethality in these animals. These data demonstrate that PtdIns binding is an essential functional property of PITPalpha in vivo, and suggest a causal linkage between defects in lipid transport and glucose homeostasis and cerebellar inflammatory disease. Finally, the data also demonstrate intrinsic neuronal deficits in PITPalpha-deficient mice that are independent of intestinal lipid transport defects and hypoglycemia.     

Changes in hepatic gene expression upon oral administration of taurine-conjugated ursodeoxycholic acid in ob/ob mice

Nonalcoholic fatty liver disease (NAFLD) is highly prevalent and associated with considerable morbidities. Unfortunately, there is no currently available drug established to treat NAFLD. It was recently reported that intraperitoneal administration of taurine-conjugated ursodeoxycholic acid (TUDCA) improved hepatic steatosis in ob/ob mice. We hereby examined the effect of oral TUDCA treatment on hepatic steatosis and associated changes in hepatic gene expression in ob/ob mice. We administered TUDCA to ob/ob mice at a dose of 500 mg/kg twice a day by gastric gavage for 3 weeks. Body weight, glucose homeostasis, endoplasmic reticulum (ER) stress, and hepatic gene expression were examined in comparison with control ob/ob mice and normal littermate C57BL/6J mice. Compared to the control ob/ob mice, TUDCA treated ob/ob mice revealed markedly reduced liver fat stained by oil red O (44.2±5.8% vs. 21.1±10.4%, P<0.05), whereas there was no difference in body weight, oral glucose tolerance, insulin sensitivity, and ER stress. Microarray analysis of hepatic gene expression demonstrated that oral TUDCA treatment mainly decreased the expression of genes involved in de novo lipogenesis among the components of lipid homeostasis. At pathway levels, oral TUDCA altered the genes regulating amino acid, carbohydrate, and drug metabolism in addition to lipid metabolism. In summary, oral TUDCA treatment decreased hepatic steatosis in ob/ob mice by cooperative regulation of multiple metabolic pathways, particularly by reducing the expression of genes known to regulate de novo lipogenesis.     

Phosphatidylinositol transfer proteins couple lipid transport to phosphoinositide synthesis

Phosphatidylinositol transfer proteins (PITPs) are lipid binding proteins that can catalyse the transfer of phosphatidylinositol (PI) from membranes enriched in PI to PI-deficient membranes. Three soluble forms of PITP of 35--38 kDa (PITP alpha, PITP beta and rdgB beta) and two larger integral proteins of 160 kDa (rdgB alpha I and II), which contain a PITP domain, are found in mammalian cells. PITPs are intimately associated with the compartmentalised synthesis of different phosphorylated inositol lipids. PI is the primary inositol lipid that is synthesised at the endoplasmic reticulum and is further phosphorylated in distinct membrane compartments by many specific lipid kinases to generate seven phosphorylated inositol lipids which are required for both signalling and for membrane traffic. PITPs play essential roles in both signalling via phospholipase C and phosphoinositide 3-kinases and in multiple aspects of membrane traffic including regulated exocytosis and vesicle biogenesis.     

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.     

酒精与肝脏脂质代谢

酒精滥用是一个重大的公共健康问题。酒精通过刺激脂肪酸合成,抑制脂肪酸的氧化导致肝脏脂质积累,进而诱发肝细胞病变,导致脂肪肝的病发。从转录调控脂质代谢的改变,异常甲硫氨酸代谢对内质网应激反应的作用等方面概述酒精与脂质代谢的相互调控机制,并阐述了这些调控机制之间的内在联系以及酒精如何影响肝脏脂质代谢,从而导致脂肪肝形成的最新相关研究进展。     

Phosphorylation of eIF2α signaling pathway attenuates obesity-induced non-alcoholic fatty liver disease in an ER stress and autophagy-dependent manner

Non-alcoholic fatty liver disease (NAFLD) is the most common liver disorder and frequently exacerbates in postmenopausal women. In NAFLD, the endoplasmic reticulum (ER) plays an important role in lipid metabolism, in which salubrinal is a selective inhibitor of eIF2α de-phosphorylation in response to ER stress. To determine the potential mechanism of obesity-induced NAFLD, we employed salubrinal and evaluated the effect of ER stress and autophagy on lipid metabolism. Ninety-five female C57BL/6 mice were randomly divided into five groups: standard chow diet, high-fat (HF) diet, HF with salubrinal, HF with ovariectomy, and HF with ovariectomy and salubrinal. All mice except for SC were given HF diet. After the 8-week obesity induction, salubrinal was subcutaneously injected for the next 8 weeks. The expression of ER stress and autophagy markers was evaluated in vivo and in vitro. Compared to the normal mice, the serum lipid level and adipose tissue were increased in obese mice, while salubrinal attenuated obesity by blocking lipid disorder. Also, the histological severity of hepatic steatosis and fibrosis in the liver and lipidosis was suppressed in response to salubrinal. Furthermore, salubrinal inhibited ER stress by increasing the expression of p-eIF2α and ATF4 with a decrease in the level of CHOP. It promoted autophagy by increasing LC3II/I and inhibiting p62. Correlation analysis indicated that lipogenesis in the development of NAFLD was associated with ER stress. Collectively, we demonstrated that eIF2α played a key role in obesity-induced NAFLD, and salubrinal alleviated hepatic steatosis and lipid metabolism by altering ER stress and autophagy through eIF2α signaling.Subject terms: Obesity, Metabolic syndrome, Outcomes research     

Deletion of tumor necrosis factor-α receptor type 1 exacerbates insulin resistance and hepatic steatosis in aromatase knockout mice

The relevance of estrogen functions in lipid metabolism has been suggested in patients with estrogen-signaling deficiencies. Their importance was further implied by studies in estrogen-deficient mice (ArKO mice), which progressively developed hepatic steatosis. As circulating tumor necrosis factor (TNF)-α levels are known to positively correlate with disturbances in lipid metabolism, we investigated the impact of the loss of TNF-α signaling on carbohydrate and lipid metabolism in ArKO mice. Histological examinations of the livers of mice at 5 months of age revealed that ArKO male mice lacking the TNF-α receptor type 1 (TNFR1) gene (ArKO/TNFR1KO) or both the TNFR 1 and 2 genes (ArKO/TNFR1&2KO) developed more severe hepatic steatosis than ArKO or ArKO/TNFR2KO mice. Serum analyses demonstrated a clear increase in cholesterol and insulin levels in the ArKO/TNFR1KO mice compared with the ArKO mice. Glucose- and insulin-tolerance tests further revealed exacerbation of the systemic insulin resistant phenotype in the ArKO/TNFR1KO mice. Hepatic expression of lipogenic genes including fatty-acid synthase and stearoyl-Coenzyme A desaturase 1 were more markedly upregulated in the ArKO/TNFR1KO mice than the ArKO mice. These findings indicate that under estrogen-deficient physiological conditions, hepatic lipid metabolism would benefit from TNF-α mediated signaling via TNFR1.     

Reduced hepatic fatty acid oxidation in fasting PPARalpha null mice is due to impaired mitochondrial hydroxymethylglutaryl-CoA synthase gene expression

Glucose and fatty acid metabolism (oxidation versus esterification) has been measured in hepatocytes isolated from 24 h starved peroxisome proliferator-activated receptor-alpha (PPARalpha) null and wild-type mice. In PPARalpha null mice, the development of hypoglycemia during starvation was due to a reduced capacity for hepatic gluconeogenesis secondary to a 70% lower rate of fatty acid oxidation. This was not due to inappropriate expression of the hepatic CPT I gene, which was similar in both genotypes, but to impaired mitochondrial hydroxymethylglutaryl-CoA synthase gene expression in the PPARalpha null mouse liver. We also demonstrate that hepatic steatosis of fasting PPARalpha null mice was not due to enhanced triglyceride synthesis.     

SCD1 is nutritionally and spatially regulated in the intestine and influences systemic postprandial lipid homeostasis and gut-liver crosstalk

Stearoyl-CoA desaturase-1 is an endoplasmic reticulum (ER)-membrane resident protein that inserts a double bond into saturated fatty acids, converting them into their monounsaturated counterparts. Previous studies have demonstrated an important role for SCD1 in modulating tissue and systemic health. Specifically, lack of hepatic or cutaneous SCD1 results in significant reductions in tissue esterified lipids. While the intestine is an important site of lipid esterification and assimilation into the body, the regulation of intestinal SCD1 or its impact on lipid composition in the intestine and other tissues has not been investigated. Here we report that unlike other lipogenic enzymes, SCD1 is enriched in the distal small intestine and in the colon of chow-fed mice and is robustly upregulated by acute refeeding of a high-sucrose diet. We generated a mouse model lacking SCD1 specifically in the intestine (iKO mice). These mice have significant reductions not only in intestinal lipids, but also in plasma triacylglycerols, diacylglycerols, cholesterol esters, and free cholesterol. Additionally, hepatic accumulation of diacylglycerols is significantly reduced in iKO mice. Comprehensive targeted lipidomic profiling revealed a consistent reduction in the myristoleic (14:1) to myristic (14:0) acid ratios in intestine, liver, and plasma of iKO mice. Consistent with the reduction of the monounsaturated fatty acid myristoleic acid in hepatic lipids of chow fed iKO mice, hepatic expression of Pgc-1α, Sirt1, and related fatty acid oxidation genes were reduced in chow-fed iKO mice. Further, lack of intestinal SCD1 reduced expression of de novo lipogenic genes in distal intestine of chow-fed mice and in the livers of mice fed a lipogenic high-sucrose diet. Taken together, these studies reveal a novel pattern of expression of SCD1 in the intestine. They also demonstrate that intestinal SCD1 modulates lipid content and composition of not only intestinal tissues, but also that of plasma and liver. Further, these data point to intestinal SCD1 as a modulator of gut-liver crosstalk, potentially through the production of novel signaling lipids such as myristoleic acid. These data have important implications to understanding how intestinal SCD1 may modulate risk for post-prandial lipemia, hepatic steatosis, and related pathologies.     

Differing endoplasmic reticulum stress response to excess lipogenesis versus lipid oversupply in relation to hepatic steatosis and insulin resistance

Mitochondrial dysfunction and endoplasmic reticulum (ER) stress have been implicated in hepatic steatosis and insulin resistance. The present study investigated their roles in the development of hepatic steatosis and insulin resistance during de novo lipogenesis (DNL) compared to extrahepatic lipid oversupply. Male C57BL/6J mice were fed either a high fructose (HFru) or high fat (HFat) diet to induce DNL or lipid oversupply in/to the liver. Both HFru and HFat feeding increased hepatic triglyceride within 3 days (by 3.5 and 2.4 fold) and the steatosis remained persistent from 1 week onwards (p<0.01 vs Con). Glucose intolerance (iAUC increased by ～60%) and blunted insulin-stimulated hepatic Akt and GSK3β phosphorylation (～40-60%) were found in both feeding conditions (p<0.01 vs Con, assessed after 1 week). No impairment of mitochondrial function was found (oxidation capacity, expression of PGC1α, CPT1, respiratory complexes, enzymatic activity of citrate synthase & β-HAD). As expected, DNL was increased (～60%) in HFru-fed mice and decreased (32%) in HFat-fed mice (all p<0.05). Interestingly, associated with the upregulated lipogenic enzymes (ACC, FAS and SCD1), two (PERK/eIF2α and IRE1/XBP1) of three ER stress pathways were significantly activated in HFru-fed mice. However, no significant ER stress was observed in HFat-fed mice during the development of hepatic steatosis. Our findings indicate that HFru and HFat diets can result in hepatic steatosis and insulin resistance without obvious mitochondrial defects via different lipid metabolic pathways. The fact that ER stress is apparent only with HFru feeding suggests that ER stress is involved in DNL per se rather than resulting from hepatic steatosis or insulin resistance.     

E3 ubiquitin ligase Grail promotes hepatic steatosis through Sirt1 inhibition

In obese adults, nonalcoholic fatty liver disease (NAFLD) is accompanied by multiple metabolic dysfunctions. Although upregulated hepatic fatty acid synthesis has been identified as a crucial mediator of NAFLD development, the underlying mechanisms are yet to be elucidated. In this study, we reported upregulated expression of gene related to anergy in lymphocytes (GRAIL) in the livers of humans and mice with hepatic steatosis. Grail ablation markedly alleviated the high-fat diet-induced hepatic fat accumulation and expression of genes related to the lipid metabolism, in vitro and in vivo. Conversely, overexpression of GRAIL exacerbated lipid accumulation and enhanced the expression of lipid metabolic genes in mice and liver cells. Our results demonstrated that Grail regulated the lipid accumulation in hepatic steatosis via interaction with sirtuin 1. Thus, Grail poses as a significant molecular regulator in the development of NAFLD.Subject terms: Cell signalling, Metabolic disorders     

Genetic ablation of phosphatidylinositol transfer protein function in murine embryonic stem cells

Phosphatidylinositol transfer proteins (PITPs) regulate the interface between signal transduction, membrane-trafficking, and lipid metabolic pathways in eukaryotic cells. The best characterized mammalian PITPs are PITP alpha and PITP beta, two highly homologous proteins that are encoded by distinct genes. Insights into PITP alpha and PITP beta function in mammalian systems have been gleaned exclusively from cell-free or permeabilized cell reconstitution and resolution studies. Herein, we report for the first time the use of genetic approaches to directly address the physiological functions of PITP alpha and PITP beta in murine cells. Contrary to expectations, we find that ablation of PITP alpha function in murine cells fails to compromise growth and has no significant consequence for bulk phospholipid metabolism. Moreover, the data show that PITP alpha does not play an obvious role in any of the cellular activities where it has been reconstituted as an essential stimulatory factor. These activities include protein trafficking through the constitutive secretory pathway, endocytic pathway function, biogenesis of mast cell dense core secretory granules, and the agonist-induced fusion of dense core secretory granules to the mast cell plasma membrane. Finally, the data demonstrate that PITP alpha-deficient cells not only retain their responsiveness to bulk growth factor stimulation but also retain their pluripotency. In contrast, we were unable to evict both PITP beta alleles from murine cells and show that PITP beta deficiency results in catastrophic failure early in murine embryonic development. We suggest that PITP beta is an essential housekeeping PITP in murine cells, whereas PITP alpha plays a far more specialized function in mammals than that indicated by in vitro systems that show PITP dependence.     

Hepatocyte-specific deletion of BAP31 promotes SREBP1C activation,promotes hepatic lipid accumulation,and worsens IR in mice

Conditional knockout mice with targeted disruption of B-cell associated protein (BAP)31 in adult mouse liver were generated and challenged with a high-fat diet (HFD) for 36 or 96 days and markers of obesity, diabetes, and hepatic steatosis were determined. Mutant mice were indistinguishable from WT littermates, but exhibited increased HFD-induced obesity. BAP31-deletion in hepatocytes increased the expression of SREBP1C and the target genes, including acetyl-CoA carboxylase 1 and stearoyl-CoA desaturase-1, and increased hepatic lipid accumulation and HFD-induced liver steatosis. Immunoprecipitation assay showed that BAP31 interacts with SREBP1C and insulin-induced gene 1 (INSIG1), and BAP31-deletion reduces INSIG1 expression, suggesting that BAP31 may regulate SREBP1C activity by modulating INSIG1 protein levels. Additionally, BAP31-deletion induced glucose and insulin intolerance, decreased Akt and glycogen synthase kinase 3β phosphorylation, and enhanced hepatic glucose production in mice. Expression of endoplasmic reticulum (ER) stress markers was significantly induced in BAP31-mutant mice. HFD-induced inflammation was aggravated in mutant mice, along with increased c-Jun N-terminal kinase and nuclear factor-κB activation. These findings demonstrate that BAP31-deletion induces SREBP activation and promotes hepatic lipid accumulation, reduces insulin signaling, impairs glucose/insulin tolerance, and increases ER stress and hepatic inflammation, explaining the protective roles of BAP31 in the development of liver steatosis and insulin resistance in HFD-induced obesity in animal models.     

Sar1b transgenic male mice are more susceptible to high-fat diet-induced obesity,insulin insensitivity and intestinal chylomicron overproduction

In the intracellular secretory network, nascent proteins are shuttled from the endoplasmic reticulum to the Golgi by transport vesicles requiring Sar1b, a small GTPase. Mutations in this key enzyme impair intestinal lipid transport and cause chylomicron retention disease. The main aim of this study was to assess whether Sar1b overexpression under a hypercaloric diet accelerated lipid production and chylomicron (CM) secretion, thereby inducing cardiometabolic abnormalities. To this end, we generated transgenic mice overexpressing human Sar1b (Sar1b^+/+) using pBROAD3-mcs that features the ubiquitous mouse ROSA26 promoter. In response to a high-fat diet (HFD), Sar1b^+/+ mice displayed significantly increased body weight and adiposity compared with Sar1b^+/+ mice under the same regimen or with wild-type (WT) mice exposed to chow diet or HFD. Furthermore, Sar1b^+/+ mice were prone to liver steatosis as revealed by significantly elevated hepatic triglycerides (TG) and cholesterol in comparison with WT animals. They also exhibited augmented levels of plasma TG along with alterations in fatty acid composition. Concomitantly, they showed susceptibility to develop insulin insensitivity and they responded abnormally to oral glucose tolerance test. Finally, Sar1b^+/+ mice that have been treated with Triton WR-1330 (to inhibit TG catabolism) and orotic acid (to block secretion of very low-density lipoprotein by the liver) responded more efficiently to fat meal tests as reflected by the rise in plasma TG and CM concentrations, indicating exaggerated intestinal fat absorption. These results suggest that Sar1b^+/+ under HFD can elicit cardiometabolic traits as revealed by incremental weight gain, fat deposition, dyslipidemia, hepatic steatosis, insulin insensitivity and intestinal fat absorption.