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Dysregulation of hepatic very low-density lipoprotein (VLDL) secretion contributes to the pathogenesis of metabolic diseases, such as Non-alcoholic fatty liver disease (NAFLD) and hyperlipidemia. Accumulating evidence have suggested that long non-coding RNAs (lncRNAs) had malfunctioning roles in the pathogenesis of NAFLD. However, the function of lncRNAs in controlling hepatic VLDL secretion remains largely unillustrated. Here, we identified a novel long non-coding RNA, lncRHPL, which was liver-enriched, downregulated upon high fat diet feeding, and inhibited by oleic acid treatment in primary hepatocytes. With genetic manipulation in mice and primary hepatocytes, depletion of lncRHPL induces hepatic VLDL secretion accompanied with decreased hepatic lipid contents. Conversely, lncRHPL restoration reduces VLDL secretion with an increased lipid deposition in hepatocytes. Mechanistic analyses indicate that lncRHPL binds directly to heterogeneous nuclear ribonuclear protein U (hnRNPU), thereby enhances its stability, and that hnRNPU can transcriptional activate Bmal1, leading to inhibition of VLDL secretion in hepatocytes. lncRHPL deficiency accelerates the protein degradation of hnRNPU and suppresses the transcription of Bmal1, which in turn activates VLDL secretion in hepatocytes. Taken together, lncRHPL is a novel suppressor of hepatic VLDL secretion. Activating the lncRHPL/hnRNPU/Bmal1/MTTP axis represent a potential strategy for the maintenance of intrahepatic and plasma lipid homeostasis.

Dysregulation of hepatic very low-density lipoprotein (VLDL) secretion contributes to the pathogenesis of metabolic diseases, such as Non-alcoholic fatty liver disease (NAFLD) and hyperlipidemia. Accumulating evidence have suggested that long non-coding RNAs (lncRNAs) had malfunctioning roles in the pathogenesis of NAFLD. However, the function of lncRNAs in controlling hepatic VLDL secretion remains largely unillustrated. Here, we identified a novel long non-coding RNA, lncRHL, which was liver-enriched, downregulated upon high fat diet feeding, and inhibited by oleic acid treatment in primary hepatocytes. With genetic manipulation in mice and primary hepatocytes, depletion of lncRHL induces hepatic VLDL secretion accompanied with decreased hepatic lipid contents. Conversely, lncRHL restoration reduces VLDL secretion with an increased lipid deposition in hepatocytes. Mechanistic analyses indicate that lncRHL binds directly to heterogeneous nuclear ribonuclear protein U (hnRNPU), thereby enhances its stability, and that hnRNPU can transcriptional activate Bmal1, leading to inhibition of VLDL secretion in hepatocytes. lncRHL deficiency accelerates the protein degradation of hnRNPU and suppresses the transcription of Bmal1, which in turn activates VLDL secretion in hepatocytes. Taken together, lncRHL is a novel suppressor of hepatic VLDL secretion. Activating the lncRHL/hnRNPU/Bmal1/MTTP axis represent a potential strategy for the maintenance of intrahepatic and plasma lipid homeostasis.

Background and aims The regulation of hepatic very-low-density lipoprotein (VLDL) secretion is vital for lipid metabolism whose pathogenetic status is involved in fatty liver disease and dyslipidemia seen in hepatic steatosis. Accumulated evidence suggest that apolipoprotein E (ApoE) is closely related to hepatic VLDL secretion. Here, we report that the expression of patatin-like phospholipase domain containing protein 7 (PNPLA7) is strongly induced by hepatic steatosis and positively correlates with plasma triacylglycerol (TAG) levels in the human subjects, whereas the role of PNPLA7 in hepatic VLDL secretion is unknown. Approach and results Herein, with genetic manipulation in the mice, the deficiency of hepatic PNPLA7 expression resulted in reduced VLDL secretion accompanied by enhanced hepatic lipid accumulation and decreased hepatic ApoE expression. Furthermore, knockdown of PNPLA7 in the livers of the db/db mice also resulted in significant reduction in plasma TAG level but aggravated hepatic steatosis. Importantly, we observed that PNPLA7 interacted with ApoE and presumably at the site of endoplasmic reticulum. Mechanistically, we have shown that PNPLA7 could modulate polyubiquitination and proteasomal-mediated degradation of ApoE. Overexpressed ApoE restored the impaired VLDL-TAG metabolism in PNPLA7-knockdown primary hepatocytes. Conclusion PNPLA7 plays a critical role in regulating hepatic VLDL secretion by modulating ApoE stability through its interaction with ApoE.

Chronic hepatitis C virus (HCV) infection has a close association with type 2 diabetes mellitus. Although the mechanisms of insulin resistance in chronic hepatitis C (CHC) patients have been extensively studied, little attention has been given to the role of β-cell function in HCV-associated diabetes. Here, we analysed β-cell function in CHC patients and HCV-infected mouse model and found in addition to insulin resistance, impaired pancreatic β-cell function occurred in CHC patients and HCV-infected C/OTg mice, not only in diabetic individuals but also in individuals with impaired fasting glucose levels. Both first-phase and second-phase insulin secretion were impaired, at least partially due to the reduction of exocytosis of secretory insulin-containing granules following HCV infection. Up-regulated p38δ in HCV-infected β-cells resulted in inactivation of protein kinase D (PKD), which was responsible for impaired insulin secretory capacity of β-cells. Thus, impaired insulin secretion due to HCV infection in β-cells contributes to HCV-associated type 2 diabetes. These findings provided a new inspiration for the important prognostic and therapeutic implications in the management of CHC patients with impaired fasting glucose.

Lipid metabolic abnormalities in cancer cells are increasingly being studied. Several studies have reported that phosphatidylserine-specific phospholipase A1 (PLA1A) might be involved in the pathogenesis of cancers. Nevertheless, the function and mechanistic details of PLA1A in lung adenocarcinoma (LUAD) progression remain largely undefined. In the present study, low PLA1A expression was correlated with poor prognosis in patients with LUAD. Results from in vitro and in vivo animal studies showed that overexpressed PLA1A suppressed the proliferation of LUAD cells in vitro and tumor growth in vivo through regulation of cyclin abundance, thereby inducing S-phase arrest. Meanwhile, PLA1A overexpression attenuated migration and invasion of LUAD cells, including by inhibiting the epithelial-mesenchymal transition. Mechanistically, PLA1A overexpression inhibited aggressiveness of LUAD cells through elevated lysophosphatidylserine, which acts via G-protein-coupled receptor 174, further activating cAMP/protein kinase A pathway. Activating G-protein-coupled receptor 174/protein kinase A pathway may involve effects on cell cycle regulators and transcription factors-regulated epithelial-mesenchymal transition. The study uncovered the mechanism through which PLA1A regulates LUAD proliferation, invasion, and migration. These results demonstrate the potential use of PLA1A as a biomarker for diagnosing LUAD, which may therefore potentially serve as a therapeutic target for LUAD.

Hepatic gluconeogenesis plays a crucial role in maintaining blood glucose homeostasis in mammals. Globe knockout of suppressor of cytokine signalling-2 (SOCS2), a feedback inhibitor of cytokine signalling, has been shown resistant to high-fat-diet (HFD)-induced hepatic steatosis with impaired glucose tolerance in mice. However, the underlying mechanism of SOCS2 regulates hepatic glucose homeostasis still undefined. In the present study, we demonstrated that the hepatic SOCS2 expression is markedly reduced in fasted C57BL/6 J mice or db/db mice. Moreover, hepatic SOCS2 expression levels are induced by metformin treatment. Ablation of SOCS2 attenuates suppressing effects of metformin on gluconeogenesis in hepatocytes. Gain- and loss-of-function studies indicated that SOCS2 regulates hepatic gluconeogenic genes expression and glucose output by mediating JAK2/STAT5 signalling pathway in db/db mice. Mechanistically, we observed that SOCS2 inactivates STAT5 by attenuating the interaction between JAK2 and STAT5, which in turn reduces hepatic gluconeogenesis. The present study reveals a critical role of SOCS2 in regulating hepatic gluconeogenesis. The inhibitory effect of metformin on gluconeogenesis is mediated, at least in part, by upregulating SOCS2 and therefore reducing hepatic gluconeogenic genes expression. SOCS2 may represent a new therapeutic target for the treatment of diabetes.

How amphipathic phospholipids are shuttled between the membrane bilayer remains an essential but elusive process, particularly at the endoplasmic reticulum (ER). One prominent phospholipid shuttling process concerns the biogenesis of APOB-containing lipoproteins within the ER lumen, which may require bulk trans-bilayer movement of phospholipids from the cytoplasmic leaflet of the ER bilayer. Here, we show that TMEM41B, present in the lipoprotein export machinery, encodes a previously conceptualized ER lipid scramblase mediating trans-bilayer shuttling of bulk phospholipids. Loss of hepatic TMEM41B eliminates plasma lipids, due to complete absence of mature lipoproteins within the ER, but paradoxically also activates lipid production. Mechanistically, scramblase deficiency triggers unique ER morphological changes and unsuppressed activation of SREBPs, which potently promotes lipid synthesis despite stalled secretion. Together, this response induces full-blown nonalcoholic hepatosteatosis in the TMEM41B-deficient mice within weeks. Collectively, our data uncovered a fundamental mechanism safe-guarding ER function and integrity, dysfunction of which disrupts lipid homeostasis.

BackgroundThis study aimed to systematically investigate the associations between serum sphingolipids and insulin sensitivity as well as insulin secretion. This study also aimed to reveal potential predictors for insulin sensitivity or give perceptive insight into disease processes.MethodsWe conducted a lipidomics evaluation of molecularly distinct SPs in the serum of 86 consecutive Chinese adults with or without obesity and diabetes using electrospray ionization mass spectrometry coupled with liquid chromatography. The GIR30 was measured under steady conditions to assess insulin sensitivity by the gold standard hyperinsulinemic-euglycemic clamp. We created the ROC curves to detect the serum SMs diagnostic value and establish the diagnosis of insulin sensitivity.ResultsDifferential correlation network analysis illustrated correlations amongst lipids, insulin sensitivity, insulin secretion and other clinical indexes. Total and subspecies of serum SMs and globotriaosylceramides (Gb3s) were positively related to GIR30, free FAs (FFA 16:1, FFA20:4), some long chain GM3 and complex ceramide GluCers showed strong negative correlations with GIR30. Notably, ROC curves showed that SM/Cer and SM d18:0/26:0 may be good serum lipid predictors of diagnostic indicators of insulin sensitivity close to conventional clinical indexes such as 1/HOMA-IR (all areas under the curve >0.80) based on GIR30 as standard diagnostic criteria.ConclusionsThese results provide novel associations between serum sphingolipid between insulin sensitivity measured by the hyperinsulinemic-euglycemic clamp. We further identify two specific SPs that may represent prognostic biomarkers for insulin sensitivity.

Little is known about how glycogen metabolism is manipulated during exercise. In article number 2001502, John Zhong Li, Tiemin Liu, Xingxing Kong, and coworkers identify that interferon regulatory factor 4 (IRF4) in skeletal muscle regulates glycogen metabolism and subsequent exercise capacity. This work reveals a regulatory pathway including IRF4/protein targeting to glycogen (PTG)/glycogen synthesis on controlling exercise capacity. [Image: see text]

Promoting development and function of brown and beige fat may represent an attractive treatment of obesity. In the present study, we show that fat Klf9 expression is markedly induced by cold exposure and a β-adrenergic agonist. Moreover, Klf9 expression levels in human white adipose tissue (WAT) are inversely correlated with adiposity, and Klf9 overexpression in primary fat cells stimulates cellular thermogenesis, which is Ucp1-dependent. Fat-specific Klf9 transgenic mice gain less weight and have smaller fat pads due to increased thermogenesis of brown and beige fat. Moreover, Klf9 transgenic mice displayed lower fasting blood glucose levels, improved glucose tolerance and insulin sensitivity under the high-fat diet condition. Conversely, Klf9 mutation in brown adipocytes reduces the expression of thermogenic genes, causing a reduction in cellular respiration. Klf9-mutant mice exhibited obesity and cold sensitivity due to impairments in the thermogenic function of fat. Finally, fat Klf9 deletion inhibits the β3-agonist-mediated induction of WAT browning and brown adipose tissue thermogenesis. Mechanistically, Cold-inducible Klf9 stimulates expression of PGC-1α, a master regulator of fat thermogenesis, by a direct binding to its gene promoter region, subsequently promoting energy expenditure. The present study reveals a critical role for Klf9 in mediating thermogenesis of brown and beige fat.

Exercise‐induced fatigue and exhaustion are interesting areas for many researchers. Muscle glycogen is critical for physical performance. However, how glycogen metabolism is manipulated during exercise is not very clear. The aim here is to assess the impact of interferon regulatory factor 4 (IRF4) on skeletal muscle glycogen and subsequent regulation of exercise capacity. Skeletal muscle‐specific IRF4 knockout mice show normal body weight and insulin sensitivity, but better exercise capacity and increased glycogen content with unaltered triglyceride levels compared to control mice on chow diet. In contrast, mice overexpression of IRF4 displays decreased exercise capacity and lower glycogen content. Mechanistically, IRF4 regulates glycogen‐associated regulatory subunit protein targeting to glycogen (PTG) to manipulate glucose metabolism in skeletal muscle. Knockdown of PTG can reverse the effects imposed by the absence of IRF4 in vivo. These studies reveal a regulatory pathway including IRF4/PTG/glycogen synthesis on controlling exercise capacity., Muscle glycogen is critical for exercise‐induced fatigue. However, how glycogen is manipulated during exercise is not well‐known. Interferon regulatory factor 4 (IRF4) was induced by exercise. Mice without IRF4 specific in muscle show increased glycogen content pre‐exercise and subsequently affects exercise capacity. These studies reveal a feedback signaling pathway including IRF4/protein targeting to glycogen (PTG)/glycogen synthesis that controlling exercise capacity.

Continual spermatogenesis relies on the actions of an undifferentiated spermatogonial population that is composed of stem cells and progenitors. Here, using mouse models, we explored the role of RNA binding proteins (RBPs) in regulation of the biological activities of this population. Proteins bound to polyadenylated RNAs in primary cultures of undifferentiated spermatogonia were captured with oligo (dT)-conjugated beads after UV-crosslinking and profiled by proteomics (termed as mRBPome capture), yielding a putative repertoire of 473 RBPs. From this database, the RBP TRIM71 was identified and found to be expressed by stem and progenitor spermatogonia in prepubertal and adult mouse testes. Tissue-specific deletion of TRIM71 in the male germline led to reduction of the undifferentiated spermatogonial population and a block in transition to the differentiating state. Collectively, these findings demonstrate a key role of the RBP system in regulation of the spermatogenic lineage and may provide clues about influence on the biology of progenitor cell populations in other lineages.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Promoting development and function of brown and beige fat may represent an attractive treatment of obesity. In the current study, we show that fat Klf9 expression is markedly induced by cold exposure and a β-adrenergic agonist. Moreover, Klf9 expression levels in human white adipose tissue (WAT) are inversely correlated with adiposity, and Klf9 overexpression in primary fat cells stimulates cellular thermogenesis, which is Ucp1 dependent. Fat-specific Klf9 transgenic mice gain less weight and have smaller fat pads due to increased thermogenesis of brown and beige fat. Moreover, Klf9 transgenic mice displayed lower fasting blood glucose levels and improved glucose tolerance and insulin sensitivity under the high-fat diet condition. Conversely, Klf9 mutation in brown adipocytes reduces the expression of thermogenic genes, causing a reduction in cellular respiration. Klf9-mutant mice exhibited obesity and cold sensitivity due to impairments in the thermogenic function of fat. Finally, fat Klf9 deletion inhibits the β3 agonist–mediated induction of WAT browning and brown adipose tissue thermogenesis. Mechanistically, cold-inducible Klf9 stimulates expression of Pgc1α, a master regulator of fat thermogenesis, by a direct binding to its gene promoter region, subsequently promoting energy expenditure. The current study reveals a critical role for KLF9 in mediating thermogenesis of brown and beige fat.

Dysregulation of gluconeogenesis contributes to the pathogenesis of metabolic disease, such as type-2 diabetes. The role of long non-coding RNAs (lncRNAs) in the pathogenesis of diabetes has recently received increased attention. In the present study, we identified a novel lncRNA, betaine-homocysteine methyltransferase-antisense (Bhmt-AS), and examined its expression patterns under pathophysiological conditions. Our results revealed that the expression of Bhmt-AS was significantly increased in the livers of fasted and db/db mice and was induced by gluconeogenic hormonal stimuli. The Bhmt-AS was also shown to be a concordant regulator of Bhmt expression. Functionally, depletion of Bhmt-AS suppressed hepatic glucose production both in vivo and in vitro. Adenovirus-mediated hepatic knockdown of Bhmt-AS improved pyruvate tolerance, glucose tolerance, and insulin sensitivity. Furthermore, overexpression of Bhmt restored the decreased glucose production caused by knockdown of Bhmt-AS in primary hepatocytes. Taken together, we uncovered a novel antisense lncRNA (Bhmt-AS) that is co-expressed with Bhmt and concordantly and specifically regulates Bhmt expression both in vitro and in vivo to regulate hepatic gluconeogenesis.

SREBPs are master regulators of lipid homeostasis and undergo sterol‐regulated export from ER to Golgi apparatus for processing and activation via COPII‐coated vesicles. While COPII recognizes SREBP through its escort protein SCAP, factor(s) specifically promoting SREBP/SCAP loading to the COPII machinery remains unknown. Here, we show that the ER/lipid droplet‐associated protein Cideb selectively promotes the loading of SREBP/SCAP into COPII vesicles. Sterol deprivation releases SCAP from Insig and enhances ER export of SREBP/SCAP by inducing SCAP‐Cideb interaction, thereby modulating sterol sensitivity. Moreover, Cideb binds to the guanine nucleotide exchange factor Sec12 to enrich SCAP/SREBP at ER exit sites, where assembling of COPII complex initiates. Loss of Cideb inhibits the cargo loading of SREBP/SCAP, reduces SREBP activation, and alleviates diet‐induced hepatic steatosis. Our data point to a linchpin role of Cideb in regulated ER export of SREBP and lipid homeostasis.

Chronic hepatitis C virus (HCV) infection has a close association with type 2 diabetes mellitus (T2DM). Although the mechanisms of insulin resistant in hepatitis C (CHC) patients have been extensively studied, little attention has been given to the role of beta cell function in HCV-associated diabetes. Here, we found HCV could infected the beta cell directly and impair pancreatic beta cell function in CHC and mouse model. Both first-phase and second-phase insulin secretion was impaired, at least partially due to the reduction of exocytosis of secretory insulin-containing granules following HCV infection. Upregulated p38δ in HCV-infected beta cells resulted in inactivation of protein kinase D (PKD), which was responsible for impaired insulin secretory capacity of beta cells. These findings provided a new perspective into mechanism of HCV induced type 2 diabetes and a new inspiration for the important prognostic and therapeutic implications in the management of CHC patients with glucose intolerance. Funding Statement: This work was supported by grants from the National Natural Sciences Foundation of China [grant number 81770859, 81271828, 81471079, 31271268], the National Basic Research Program of China [grant number 2015CB554304], the Youth Innovation Promotion Association, Open Research Fund Program of the State Key Laboratory of Virology of China (grant number 2018IOV003) Declaration of Interests: The authors have declared that no conflict of interest exists. Ethics Approval Statement: Human studies were approved by the Ethics Committee of the First Hospital of Jilin University and the First Affiliated Hospital of Xinjiang Medical University. The written informed consents were received from participants prior to inclusion in the study. All animal experiments were approved by the Institutional Animal Ethical Committee of Wuhan Institute of Virology, Chinese Academy of Sciences (WIV, CAS).

Mitochondria are essential for maintaining skeletal muscle metabolic homeostasis during adaptive response to a myriad of physiologic or pathophysiological stresses. The mechanisms by which mitochondrial function and contractile fiber type are concordantly regulated to ensure muscle function remain poorly understood. Evidence is emerging that the Folliculin interacting protein 1 (Fnip1) is involved in skeletal muscle fiber type specification, function, and disease. In this study, Fnip1 was specifically expressed in skeletal muscle in Fnip1-transgenic (Fnip1Tg) mice. Fnip1Tg mice were crossed with Fnip1-knockout (Fnip1KO) mice to generate Fnip1TgKO mice expressing Fnip1 only in skeletal muscle but not in other tissues. Our results indicate that, in addition to the known role in type I fiber program, FNIP1 exerts control upon muscle mitochondrial oxidative program through AMPK signaling. Indeed, basal levels of FNIP1 are sufficient to inhibit AMPK but not mTORC1 activity in skeletal muscle cells. Gain-of-function and loss-of-function strategies in mice, together with assessment of primary muscle cells, demonstrated that skeletal muscle mitochondrial program is suppressed via the inhibitory actions of FNIP1 on AMPK. Surprisingly, the FNIP1 actions on type I fiber program is independent of AMPK and its downstream PGC-1α. These studies provide a vital framework for understanding the intrinsic role of FNIP1 as a crucial factor in the concerted regulation of mitochondrial function and muscle fiber type that determine muscle fitness., Author summary Mitochondria provide an essential source of energy to drive cellular processes and the function of mitochondria is particularly important in skeletal muscle, a metabolically demanding tissue that depends critically on mitochondria, accounting for ~40% of total body mass. In this study, we discovered an essential function of adaptor protein FNIP1 in the coordinated regulation of the mitochondrial and structural programs controlling muscle fitness. Using both gain-of-function and loss-of-function strategies in mice and muscle cells, we provide clear genetic data that demonstrate FNIP1-dependent signaling is crucial for muscle mitochondrial remodeling as well as type I muscle fiber specification. We also uncover that FNIP1 exerts control upon muscle mitochondrial program through AMPK but not mTORC1 signaling. Furthermore, we demonstrate that FNIP1 acts independently of PGC-1α to regulate fiber type specification. Hence, our study emphasizes FNIP1 as a dominant factor that coordinates mitochondrial and muscle fiber type programs that govern muscle fitness.

BackgroundSPs are a group of ubiquitously produced lipids that are structurally involved in cell membranes and include SMs, ceramides, GM3s, etc. Sphingolipids and their substrates and constituents, FAs, are implicated in the pathogenesis of various metabolic diseases associated with obesity, diabetes, and atherosclerosis. Decreased insulin sensitivity or insulin secretion is a multifactorial condition related to obesity and diabetes. Therefore, it is likely that perturbations in serum SPs are associated with diseases. Identifying these associations may reveal useful predictors or give perceptive insight into disease processes. This study aimed to systematically investigate the associations between serum sphingolipids and insulin sensitivity as well as insulin secretion. This study also aimed to reveal potential predictors for insulin sensitivity or give perceptive insight into disease processes.MethodsWe conducted a lipidomics evaluation of molecularly distinct SPs in the serum of 86 consecutive Chinese adults with or without obesity and diabetes using electrospray ionization mass spectrometry coupled with liquid chromatography. The GIR30 was measured under steady conditions to assess insulin sensitivity by the gold standard hyperinsulinemic-euglycemic clamp, and FPIR was measured to evaluate insulin secretion by the IVGTT. We created the ROC curves to detect the serum SMs diagnostic value and establish the diagnosis of insulin sensitivity based on GIR30 derived from the glucose clamp, which is the standard method, and the cutoff point of GIR30 was set as 5.1 mg/(kg *min).ResultsDifferential correlation network analysis illustrated correlations amongst lipids, insulin sensitivity, insulin secretion and other clinical indexes. Total and subspecies of serum SMs and globotriaosylceramides (Gb3s) were positively related to GIR30, free FAs (FFA 16:1, FFA20:4), some long chain GM3 and complex ceramide GluCers showed strong negative correlations with GIR30. We also found that higher concentrations of serum free FAs (FFA 22:6, FFA 22:5, FFA 18:2, FFA 18:1) were associated with a higher risk of decreased insulin secretion. Notably, ROC curves showed that SM/Cer and SM d18:0/26:0 may be good serum lipid predictors of diagnostic indicators of insulin sensitivity close to conventional clinical indexes such as 1/HOMA-IR (all areas under the curve >0.80) based on GIR30 as standard diagnostic criteria. ConclusionsThese results provide novel associations between serum sphingolipid between insulin sensitivity measured by the hyperinsulinemic-euglycemic clamp. This suggests that the balance of SMs metabolism, rather than ceramide is correlated with the pathology of insulin resistance, obesity and T2DM. We further identify two specific SPs that may represent prognostic biomarkers for insulin sensitivity.

Histone deacetylase 9 (HDAC9) regulates hepatic gluconeogenesis by deacetylating Forkhead box O 1 (FoxO1). HDAC9 upregulation is involved in hepatitis C virus (HCV)-associated exaggerated gluconeogenesis. Herein, we found in addition to FoxO1, HDAC9 also regulates other gluconeogenic transcription factors, including peroxisomeproliferator-activated receptor-γ coactivator-1α (PGC-1α), cyclic AMP-responsive element-binding protein (CREB), and glucocorticoid receptor (GR). Unlike FoxO1, which is regulated by post-translational modification responses to HDAC9, HDAC9 regulates PGC-1α, CREB and GR by altering gene expression. Similar to PGC-1α, CREB and GR were found to be novel regulatory targets of FoxO1 by examination of the FoxO1 binding site in their promoter. PGC-1α, CREB and GR were upregulated in response to HDAC9 via FoxO1 deacetylation. These findings indicate that HDAC9-FoxO1 signalling contributes to gluconeogenesis by modulating the expression of gluconeogenic transcription factors. In particular, metabolic profiling demonstrated a clear shift towards gluconeogenesis metabolism, and HDAC9-FoxO1 signalling can be strongly induced to upregulate gluconeogenic transcription factors following HCV infection. The positive correlation between HDAC9 and gluconeogenic transcription factor expression levels in the livers of both HCV-infected patients and normal individuals further emphasizes the clinical relevance of these results. Thus, HDAC9-FoxO1 signalling axis is involved in regulating gluconeogenic transcription factors, gluconeogenesis, and HCV-induced type 2 diabetes.

For many years, the coexistence of Helicobacter pylori (H. pylori) infection and atherosclerotic diseases in a disproportional number of patients has inspired the presumption that a causal relationship exists between the conditions. This presumption was evidenced by the simultaneously declining epidemics of duodenal ulcer and coronary artery diseases in the United States in the past 40 years, and was further evidenced by the detection of H. pylori and their DNA in atherosclerotic plaques. Suggested mechanisms underlying this possible causality include intensified inflammatory stress, dyslipidemia, abnormal glucose metabolism, arterial stiffness, increased blood pressure and malnutrition. However, a causal relationship was disproved by results from other studies, which indicated that common risk factors shared by H. pylori infection and atherosclerosis, such as aging, cigarette smoking, disadvantageous socioeconomic status and high salt intake, may predestine their coexistence. Understanding the nature of the relationship between H. pylori infection and atherosclerosis is of vital importance, because a causality may indicate a possible strategy for preventing and treating atherosclerosis by eradicating H. pylori infection in selected individuals. This review assessed current evidence for and against a causal relationship between H. pylori infection and atherosclerosis.

Large and high quality user base is an important determinant of firm performance. Firms spend a great amount of resources in building referral programs to grow user base. Yet, substantial variations exist in the effectiveness of these referral programs with various incentive structures. We argue that the extent to which referral programs can grow user base depends on how existing users evaluate the cost and benefit of their referral behavior. Using a large-scale randomized field experiment with around one hundred thousand participants on one of the largest P2P lending platforms in China, we find that referral programs structured as self-benefit (egoistic) appeal or other-benefit (altruistic) appeal significantly reduce the number of new users brought by the existing users but new users from other-benefit appeal made more investment. Our study demonstrates that under certain circumstances, these referral programs structured as egoistic or altruistic incentive structure may backfire.

A genetic variant in PNPLA3 (PNPLA3(I148M)), a triacylglycerol (TAG) hydrolase, is a major risk factor for nonalcoholic fatty liver disease (NAFLD); however, the mechanism underlying this association is not known. To develop an animal model of PNPLA3-induced fatty liver disease, we generated transgenic mice that overexpress similar amounts of wild-type PNPLA3 (PNPLA3(WT)) or mutant PNPLA3 (PNPLA3(I148M)) either in liver or adipose tissue. Overexpression of the transgenes in adipose tissue did not affect liver fat content. Expression of PNPLA3(I148M), but not PNPLA3(WT), in liver recapitulated the fatty liver phenotype as well as other metabolic features associated with this allele in humans. Metabolic studies provided evidence for 3 distinct alterations in hepatic TAG metabolism in PNPLA3(I148M) transgenic mice: increased formation of fatty acids and TAG, impaired hydrolysis of TAG, and relative depletion of TAG long-chain polyunsaturated fatty acids. These findings suggest that PNPLA3 plays a role in remodeling TAG in lipid droplets, as they accumulate in response to food intake, and that the increase in hepatic TAG levels associated with the I148M substitution results from multiple changes in hepatic TAG metabolism. The development of an animal model that recapitulates the metabolic phenotype of the allele in humans provides a new platform in which to elucidate the role of PNLPA3(I148M) in NAFLD.

Introduction/Background Two types of lower extremity exoskeleton rehabilitation training robots (named Aiwalk and Ailegs) had been the first powered gait orthosis (PGO) in China. The aim of this study conducted in patients with paraplegia was to verify the effectiveness and safety of the lower extremity exoskeleton rehabilitation training robot developed in China. Material and method Forty subjects (nine females and thirty-one males with the age of 38.1 ± 9.4 years, height of 169.8 ± 6.5 cm, weight of 62.2 ± 7.8 kg and the post-injury time of 67.8 ± 112.8 months) with T6–L2 spinal cord lesions participated in the study. The patients were characterized by a traumatic SCI with scale A (twenty-nine patients), B (four patients) and C (seven patients) according to American Spinal Injury Association score. All patients received a walking and standing program with the isocentric reciprocating gait orthosis (IRGO) and the PGOs. After 1 week of gait training, subjects walked using three types of orthosis in random order at their self-selected speed each half day and repeated ten times. A wireless single channel ECG recorder and a stop watch were used to measure the speed of walking and heart rate. Results Walking speed, the distance walked, the physiological cost index (PCI), orthosis wearing speed and rating of perceived exertion all improved with both PGOs as compared with the IRGO. Adverse events associated with PGO were calcaneus compression fracture (one patients) and mild skin pressure ulcer (two patients). Conclusion Patients with paraplegia (below T6 level) were able to ambulate efficiently using the powered exoskeleton for overground ambulation, providing potential use for functional gain and improved fitness. Fracture and skin damage may be the main risk focus in clinical application.

Obesity and insulin resistance are associated with deposition of triglycerides in tissues other than adipose tissue. Previously, we showed that a missense mutation (I148M) in PNPLA3 (patatin-like phospholipase domain-containing 3 protein) is associated with increased hepatic triglyceride content in humans. Here we examined the effect of the I148M substitution on the enzymatic activity and cellular location of PNPLA3. Structural modeling predicted that the substitution of methionine for isoleucine at residue 148 would restrict access of substrate to the catalytic serine at residue 47. In vitro assays using recombinant PNPLA3 partially purified from Sf9 cells confirmed that the wild type enzyme hydrolyzes emulsified triglyceride and that the I148M substitution abolishes this activity. Expression of PNPLA3-I148M, but not wild type PNPLA3, in cultured hepatocytes or in the livers of mice increased cellular triglyceride content. Cell fractionation studies revealed that approximately 90% of wild type PNPLA3 partitioned between membranes and lipid droplets; substitution of isoleucine for methionine at position 148 did not alter the subcellular distribution of the protein. These data are consistent with PNPLA3-I148M promoting triglyceride accumulation by limiting triglyceride hydrolysis.

OBJECTIVE—Our previous study suggests that Cidea, a member of Cide family proteins that share sequence homology with the DNA fragmentation factor and are expressed at high levels in brown adipose tissue, plays an important role in the development of obesity. Cideb, another member of Cide family protein, is highly expressed in the liver. We would like to understand the physiological role of Cideb in the regulation of energy expenditure and lipid metabolism. RESEARCH DESIGN AND METHODS—We generated Cideb-null mice by homolog recombination and then fed both wild-type and Cideb-null mice with high-fat diet (58% fat). We then characterized the animals’ adiposity index, food intake, whole-body metabolic rate, liver morphology, rate of fatty acid synthesis and oxidation, insulin sensitivity, and gene expression profile. RESULTS— Cideb-null mice had lower levels of plasma triglycerides and free fatty acids and were resistant to high-fat diet–induced obesity and live steatosis. In addition, Cideb mutant mice displayed significantly increased insulin sensitivity and enhanced rate of whole-body metabolism and hepatic fatty acid oxidation. More importantly, Cideb-null mice showed decreased lipogenesis and reduced expression levels of acetyl-CoA carboxylase, fatty acid synthase, and stearol-CoA desaturase. We further demonstrated that expression levels of sterol response element binding protein 1c was significantly decreased in Cideb-deficient mice. CONCLUSIONS—Our data demonstrate that Cideb is a novel important regulator in lipid metabolism in the liver. Cideb may represent a new therapeutic target for the treatment of obesity, diabetes, and liver steatosis.

Lipid droplets (LDs) are dynamic subcellular organelles whose growth is closely linked to obesity and hepatic steatosis. Cell death-inducing DNA fragmentation factor-α-like effector (CIDE) proteins, including Cidea, Cideb, and Cidec (also called Fsp27), play important roles in lipid metabolism. Cidea and Cidec are LD-associated proteins that promote atypical LD fusion in adipocytes. Here, we find that CIDE proteins are all localized to LD-LD contact sites (LDCSs) and promote lipid transfer, LD fusion, and growth in hepatocytes. We have identified two types of hepatocytes, one with small LDs (small LD-containing hepatocytes, SLHs) and one with large LDs (large LD-containing hepatocytes, LLHs) in the liver. Cideb is localized to LDCSs and promotes lipid exchange and LD fusion in both SLHs and LLHs, whereas Cidea and Cidec are specifically localized to the LDCSs and promote lipid exchange and LD fusion in LLHs. Cideb-deficient SLHs have reduced LD sizes and lower lipid exchange activities. Fasting dramatically induces the expression of Cidea/Cidec and increases the percentage of LLHs in the liver. The majority of the hepatocytes from the liver of obese mice are Cidea/Cidec-positive LLHs. Knocking down Cidea or Cidec significantly reduced lipid storage in the livers of obese animals. Our data reveal that CIDE proteins play differential roles in promoting LD fusion and lipid storage; Cideb promotes lipid storage under normal diet conditions, whereas Cidea and Cidec are responsible for liver steatosis under fasting and obese conditions.

A sequence polymorphism (rs738409, I148M) in patatin-like phospholipid domain containing protein 3 (PNPLA3) is strongly associated with nonalcoholic fatty liver disease (NAFLD), but the mechanistic basis for this association remains enigmatic. Neither ablation nor overexpression of wild-type PNPLA3 affects liver fat content in mice, whereas hepatic overexpression of the human 148M transgene causes steatosis. To determine whether the 148M allele causes fat accumulation in the liver when expressed at physiological levels, we introduced a methionine codon at position 148 of the mouse Pnpla3 gene. Knockin mice had normal levels of hepatic fat on a chow diet, but when challenged with a high-sucrose diet their liver fat levels increased 2 to 3-fold compared to wild-type littermates without any associated changes in glucose homeostasis. The increased liver fat in the knockin mice was accompanied by a 40-fold increase in PNPLA3 on hepatic lipid droplets, with no increase in hepatic PNPLA3 messenger RNA (mRNA). Similar results were obtained when the catalytic dyad of PNPLA3 was inactivated by substituting the catalytic serine with alanine (S47A). Conclusion: These data provide the first direct evidence that physiological expression of PNPLA3 148M variant causes NAFLD, and that the accumulation of catalytically inactive PNPLA3 on the surfaces of lipid droplets is associated with the accumulation of TG in the liver. (Hepatology 2015;61:108–118)

PNPLA3 (adiponutrin, calcium-independent phospholipase A(2) epsilon [iPLA(2)ε]) is an adipose-enriched, nutritionally regulated protein that belongs to the patatin-like phospholipase domain containing (PNPLA) family of lipid metabolizing proteins. Genetic variations in the human PNPLA3 gene (i.e., the rs738409 I148M allele) has been strongly and repeatedly associated with fatty liver disease. Although human PNPLA3 has triacylglycerol (TAG) hydrolase and transacylase activities in vitro, its in vivo function and physiological relevance remain controversial. The objective of this study was to determine the metabolic consequences of global targeted deletion of the Pnpla3 gene in mice. We found that Pnpla3 mRNA expression is altered in adipose tissue and liver in response to acute and chronic nutritional challenges. However, global targeted deletion of the Pnpla3 gene in mice did not affect TAG hydrolysis, nor did it influence energy/glucose/lipid homoeostasis or hepatic steatosis/injury. Experimental interventions designed to increase Pnpla3 expression (refeeding, high-sucrose diet, diet-induced obesity, and liver X receptor agonism) likewise failed to reveal differences in the above-mentioned metabolic phenotypes. Expression of the Pnpla3 paralog, Pnpla5, was increased in adipose tissue but not in liver of Pnpla3-deficient mice, but compensatory regulation of genes involved in TAG metabolism was not identified. Together these data argue against a role for Pnpla3 loss-of-function in fatty liver disease or metabolic syndrome in mice.

The upsurge in prevalence of obesity has spawned an epidemic of nonalcoholic fatty liver disease (NAFLD). Previously, we identified a sequence variant (I148M) in patatin-like phospholipase domain-containing protein 3 (PNPLA3) that confers susceptibility to both hepatic triglyceride (TG) deposition and liver injury. To glean insights into the biological role of PNPLA3, we examined the molecular mechanisms by which nutrient status controls hepatic expression of PNPLA3. PNPLA3 mRNA levels, which were low in fasting animals, increased ∼90-fold with carbohydrate feeding. The increase was mimicked by treatment with a liver X receptor (LXR) agonist and required the transcription factor SREBP-1c. The site of SREBP-1c binding was mapped to intron 1 of Pnpla3 using chromatin immunoprecipitation and electrophoretic mobility shift assays. SREBP-1c also promotes fatty acid synthesis by activating several genes encoding enzymes in the biosynthetic pathway. Addition of fatty acids (C16:0, C18:1, and C18:2) to the medium of cultured hepatocytes (HuH-7) increased PNPLA3 protein mass without altering mRNA levels. The posttranslational increase in PNPLA3 levels persisted after blocking TG synthesis with triascin C. Oleate (400 μM) treatment prolonged the half-life of PNPLA3 from 2.4 to 6.7 h. These findings are consistent with nutritional control of PNPLA3 being effected by a feed-forward loop; SREBP-1c promotes accumulation of PNPLA3 directly by activating Pnpla3 transcription and indirectly by inhibiting PNPLA3 degradation through the stimulation of fatty acid synthesis.

Cideb, a member of CIDE family proteins, has emerged as an important regulator in the development of obesity and diabetes by controlling fatty acid synthesis and VLDL secretion in hepatocytes. Here, we investigated the role of Cideb in cholesterol biosynthesis, uptake and storage in the liver by using Cideb-null mice as a model system. Cideb-null mice and wild-type mice were treated with normal diet (ND) or high cholesterol diet (HCD) for one month. The metabolic parameters of cholesterol metabolism and expression profiles of genes in cholesterol biosynthesis and storage were measured. Cideb-null mice had lower levels of plasma cholesterol and LDL when fed with both ND and HCD and increased rate of cholesterol absorption. Furthermore, the liver of Cideb-null mice has lower rates of cholesterol biosynthesis and reduced expression levels of sterol response element-binding protein (SREBP) cleavage-activation protein (SCAP), and lower levels of nuclear form of SREBP2 and its downstream target genes in cholesterol biosynthesis pathway under a normal diet treatment. On the contrary, hepatic cholesterol biosynthesis rate between wild-type and Cideb-null mice was similar after high cholesterol diet treatment. Interestingly, hepatic cholesterol storage in the liver of Cideb-null mice was significantly increased due to its increased LDL receptor (LDLR) and acyl-CoA cholesterol acyltransferase (ACAT) expression. Finally, we observed drastically reduced cholesterol levels in the heart of Cideb-null mice fed with a high cholesterol diet. Overall, our data suggest that Cideb is a novel regulator in controlling cholesterol homeostasis in the liver. Therefore, Cideb could serve as an important therapeutical target for the treatment of atherosclerosis and cardiovascular diseases.

We previously showed that Cidea(-/-) mice are resistant to diet-induced obesity through the upregulation of energy expenditure. The AMP-activated protein kinase (AMPK), consisting of catalytic alpha subunit and regulatory subunits beta and gamma, has a pivotal function in energy homoeostasis. We show here that AMPK protein levels and enzymatic activity were significantly increased in the brown adipose tissue of Cidea(-/-) mice. We also found that Cidea is colocalized with AMPK in the endoplasmic reticulum and forms a complex with AMPK in vivo through specific interaction with the beta subunit of AMPK, but not with the alpha or gamma subunit. When co-expressed with Cidea, the stability of AMPK-beta subunit was dramatically reduced due to increased ubiquitination-mediated degradation, which depends on a physical interaction between Cidea and AMPK. Furthermore, AMPK stability and enzymatic activity were increased in Cidea(-/-) adipocytes differentiated from mouse embryonic fibroblasts or preadipocytes. Our data strongly suggest that AMPK can be regulated by Cidea-mediated ubiquitin-dependent proteosome degradation, and provide a molecular explanation for the increased energy expenditure and lean phenotype in Cidea-null mice.

Obesity has become the most prevalent chronic disorder that affects large populations throughout the world. Obesity is due largely to the imbalance between energy intake and expenditure. Energy balance is regulated by interactions between various tissues such as adipose tissues, liver and skeletal muscle. Adipose tissues, which include brown adipose tissue (BAT) and white adipose tissue (WAT), play crucial roles in maintaining energy homeostasis. While WAT stores energy in the form of triglycerides; BAT increases energy expenditure through thermogenesis. Cide proteins including Cidea, Cideb and Fsp27, are expressed at high levels in BAT, liver and WAT, respectively. Cidea(-/-) mice exhibit increased lipolysis in BAT and are resistant to high fat diet-induced obesity and diabetes. Our recent data suggest that Cideb also plays an important role in the development of obesity by regulating various metabolic pathways in liver. The molecular mechanism of Cide protein regulation of metabolic networks and obesity is discussed.