Weiqin Chen

Patatin-like phospholipase domain-containing 3/adiponutrin deficiency in mice is not associated with fatty liver disease.

PNPLA3 (adiponutrin), a novel patatin‐like phospholipase domain‐containing enzyme, is expressed at high level in fat, but also in other tissues including liver. Polymorphisms in PNPLA3 have been linked to obesity and insulin sensitivity. Notably, a nonsynonymous variant rs738409(G) allele of the PNPLA3 gene was found to be strongly associated with both nonalcoholic and alcoholic fatty liver disease. We have generated Pnpla3−/− mice by gene targeting. Loss of Pnpla3 has no effect on body weight or composition, adipose mass, or development, whether the mice were fed regular chow or high‐fat diet or bred into the genetic obese Lepob/ob background. Plasma and liver triglyceride content and plasma aspartate aminotransferase and alanine aminotransferase levels were not different between Pnpla3+/+ and Pnpla3−/− mice while they were on regular chow, fed three different fatty liver‐inducing diets, or after they were bred into Lepob/ob background. Hepatic Pnpla5 messenger RNA (mRNA) levels were similar in wild‐type and Pnpla3−/− mice, although adipose Pnpla5 mRNA level was increased in Pnpla3−/− mice. A high‐sucrose lipogenic diet stimulated hepatic Pnpla3 and Pnpla5 mRNA levels to a similar degree, but it did not affect adipose or liver triglyceride lipase (ATGL, known also as Pnpla2) mRNA in Pnpla3+/+ and Pnpla3−/− mice. Finally, Pnpla3+/+ and Pnpla3−/− mice displayed similar glucose tolerance and insulin tolerance tests while on regular chow or three different fatty liver–inducing diets. Conclusion: Loss of Pnpla3 does not cause fatty liver, liver enzyme elevation, or insulin resistance in mice. (HEPATOLOGY 2010)

The Human Lipodystrophy Gene Product Berardinelli-Seip Congenital Lipodystrophy 2/Seipin Plays a Key Role in Adipocyte Differentiation

Mutations in the Berardinelli-Seip congenital lipodystrophy 2 gene (BSCL2) are the underlying defect in patients with congenital generalized lipodystrophy type 2. BSCL2 encodes a protein called seipin, whose function is largely unknown. In this study, we investigated the role of Bscl2 in the regulation of adipocyte differentiation. Bscl2 mRNA is highly up-regulated during standard hormone-induced adipogenesis in 3T3-L1 cells in vitro. However, this up-regulation does not occur during mesenchymal stem cell (C3H10T1/2 cells) commitment to the preadipocyte lineage. Knockdown of Bscl2 by short hairpin RNA in C3H10T1/2 cells has no effect on bone morphogenetic protein-4-induced preadipocyte commitment. However, knockdown in 3T3-L1 cells prevents adipogenesis induced by a standard hormone cocktail, but adipogenesis can be rescued by the addition of peroxisome proliferator-activated receptor-gamma agonist pioglitazone at an early stage of differentiation. Interestingly, pioglitazone-induced differentiation in the absence of standard hormone is not associated with up-regulated Bscl2 expression. On the other hand, short hairpin RNA-knockdown of Bscl2 largely blocks pioglitazone-induced adipose differentiation. These experiments suggest that Bscl2 may be essential for normal adipogenesis; it works upstream or at the level of peroxisome proliferator-activated receptor-gamma, enabling the latter to exert its full activity during adipogenesis. Loss of Bscl2 function thus interferes with the normal transcriptional cascade of adipogenesis during fat cell differentiation, resulting in near total loss of fat or lipodystrophy.

Berardinelli-Seip Congenital Lipodystrophy 2/Seipin Is a Cell-Autonomous Regulator of Lipolysis Essential for Adipocyte Differentiation

ABSTRACT Mutations in BSCL2 underlie human congenital generalized lipodystrophy. We inactivated Bscl2 in mice to examine the mechanisms whereby absence of Bscl2 leads to adipose tissue loss and metabolic disorders. Bscl2−/− mice develop severe lipodystrophy of white adipose tissue (WAT), dyslipidemia, insulin resistance, and hepatic steatosis. In vitro differentiation of both Bscl2−/− murine embryonic fibroblasts (MEFs) and stromal vascular cells (SVCs) reveals normal early-phase adipocyte differentiation but a striking failure in terminal differentiation due to unbridled cyclic AMP (cAMP)-dependent protein kinase A (PKA)-activated lipolysis, which leads to loss of lipid droplets and silencing of the expression of adipose tissue-specific transcription factors. Importantly, such defects in differentiation can be largely rescued by inhibitors of lipolysis but not by a gamma peroxisome proliferator-activated receptor (PPARγ) agonist. The residual epididymal WAT (EWAT) in Bscl2−/− mice displays enhanced lipolysis. It also assumes a “brown-like” phenotype with marked upregulation of UCP1 and other brown adipose tissue-specific markers. Together with decreased Pref1 but increased C/EBPβ levels, these changes highlight a possible increase in cAMP signaling that impairs terminal adipocyte differentiation in the EWAT of Bscl2−/− mice. Our study underscores the fundamental role of regulated cAMP/PKA-mediated lipolysis in adipose differentiation and identifies Bscl2 as a novel cell-autonomous determinant of activated lipolysis essential for terminal adipocyte differentiation.

Glucose-6-phosphate mediates activation of the carbohydrate responsive binding protein (ChREBP)

Carbohydrate response element binding protein (ChREBP) is a Mondo family transcription factor that activates a number of glycolytic and lipogenic genes in response to glucose stimulation. We have previously reported that high glucose can activate the transcriptional activity of ChREBP independent of the protein phosphatase 2A (PP2A)-mediated increase in nuclear entry and DNA binding. Here, we found that formation of glucose-6-phosphate (G-6-P) is essential for glucose activation of ChREBP. The glucose response of GAL4-ChREBP is attenuated by D-mannoheptulose, a potent hexokinase inhibitor, as well as over-expression of glucose-6-phosphatase (G6Pase); kinetics of activation of GAL4-ChREBP can be modified by exogenously expressed GCK. Further metabolism of G-6-P through the two major glucose metabolic pathways, glycolysis and pentose-phosphate pathway, is not required for activation of ChREBP; over-expression of glucose-6-phosphate dehydrogenase (G6PD) diminishes, whereas RNAi knockdown of the enzyme enhances, the glucose response of GAL4-ChREBP, respectively. Moreover, the glucose analogue 2-deoxyglucose (2-DG), which is phosphorylated by hexokinase, but not further metabolized, effectively upregulates the transcription activity of ChREBP. In addition, over-expression of phosphofructokinase (PFK) 1 and 2, synergistically diminishes the glucose response of GAL4-ChREBP. These multiple lines of evidence support the conclusion that G-6-P mediates the activation of ChREBP.

Remodeling of Retinal Fatty Acids in an Animal Model of Diabetes: A Decrease in Long-Chain Polyunsaturated Fatty Acids Is Associated With a Decrease in Fatty Acid Elongases Elovl2 and Elovl4

OBJECTIVE The results of the Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications cohort study revealed a strong association between dyslipidemia and the development of diabetic retinopathy. However, there are no experimental data on retinal fatty acid metabolism in diabetes. This study determined retinal-specific fatty acid metabolism in control and diabetic animals. RESEARCH DESIGN AND METHODS Tissue gene and protein expression profiles were determined by quantitative RT-PCR and Western blot in control and streptozotocin-induced diabetic rats at 3–6 weeks of diabetes. Fatty acid profiles were assessed by reverse-phase high-performance liquid chromatography, and phospholipid analysis was performed by nano-electrospray ionization tandem mass spectrometry. RESULTS We found a dramatic difference between retinal and liver elongase and desaturase profiles with high elongase and low desaturase gene expression in the retina compared with liver. Elovl4, an elongase expressed in the retina but not in the liver, showed the greatest expression level among retinal elongases, followed by Elovl2, Elovl1, and Elovl6. Importantly, early-stage diabetes induced a marked decrease in retinal expression levels of Elovl4, Elovl2, and Elovl6. Diabetes-induced downregulation of retinal elongases translated into a significant decrease in total retinal docosahexaenoic acid, as well as decreased incorporation of very-long-chain polyunsaturated fatty acids (PUFAs), particularly 32:6n3, into retinal phosphatidylcholine. This decrease in n3 PUFAs was coupled with inflammatory status in diabetic retina, reflected by an increase in gene expression of proinflammatory markers interleukin-6, vascular endothelial growth factor, and intercellular adhesion molecule-1. CONCLUSIONS This is the first comprehensive study demonstrating diabetes-induced changes in retinal fatty acid metabolism. Normalization of retinal fatty acid levels by dietary means or/and modulating expression of elongases could represent a potential therapeutic target for diabetes-induced retinal inflammation.

Aquaporin 7 Is a β-Cell Protein and Regulator of Intraislet Glycerol Content and Glycerol Kinase Activity, β-Cell Mass, and Insulin Production and Secretion

ABSTRACT To investigate if intracellular glycerol content plays a role in the regulation of insulin secretion in pancreatic β cells, we studied the expression of the glycerol channels, or aquaglyceroporins, encoded by the aquaporin 3 (Aqp3), Aqp7, and Aqp9 genes in mouse islets. We found expression of Aqp7 only, not that of Aqp3 or Aqp9, in the endocrine pancreas at both the mRNA (by reverse transcription-PCR) and protein (by immunohistochemistry) levels. Immunohistochemistry revealed a complete overlap between insulin and Aqp7 immunostaining in the pancreatic islet. Inactivation of Aqp7 by gene targeting produced viable and healthy mice. Aqp7−/− mice harbored an increased intraislet glycerol concentration with a concomitant increase of the glycerol kinase transcript level and enzyme activity. The islet triglyceride content in the Aqp7−/− mice was also increased compared to that in the Aqp7+/+ mice. Interestingly, Aqp7−/− mice displayed reduced β-cell mass and insulin content but increased insulin-1 and insulin-2 mRNAs. The reduction of β-cell mass in Aqp7−/− mice can be explained at least in part by a reduction in cell proliferation through protein kinase C and the c-myc cascade, with a reduction in the transcript levels of these two genes. Concomitantly, there was a decreased rate of apoptosis, as reflected by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling and caspase 3 and Bax expression in Aqp7−/− mice. Compared with Aqp7+/+ islets, islets isolated from Aqp7−/− mice secreted insulin at a higher rate under basal low-glucose conditions and on exposure to a high (450 mg/dl) glucose concentration. Aqp7−/− mice exhibited normal fasting blood glucose levels but elevated blood insulin levels. Their plasma glucose response to an intraperitoneal (i.p.) glucose tolerance test was normal, but their plasma insulin concentrations were higher than those of wild-type mice during the 2-h test. An i.p. insulin tolerance test showed similar plasma glucose lowering in Aqp7−/− and Aqp7+/+ mice, with no evidence of insulin resistance. In conclusion, we found that pancreatic β cells express AQP7, which appears to be a key regulator of intraislet glycerol content as well as insulin production and secretion.

Glucose-Mediated Transactivation of Carbohydrate Response Element-Binding Protein Requires Cooperative Actions from Mondo Conserved Regions and Essential Trans-Acting Factor 14-3-3

Carbohydrate response element-binding protein (ChREBP) is a basic helix-loop-helix/leucine zipper transcription factor that binds to the carbohydrate response element in the promoter of certain lipogenic and glycolytic genes. High glucose can activate ChREBP by releasing an intramolecular inhibition within the glucose-sensing module (GSM) that occurs in low glucose. We report here that the glucose response of GSM is mediated by cooperation between five conserved submodules known as Mondo conserved regions (MCRs) I through V within GSM. Deletion of individual MCRs leads to complete (for MCR II, III, and IV) or partial (MCR I) loss of glucose response of ChREBP. MCR IV is necessary and sufficient for inhibiting the transcriptional activity of ChREBP under low glucose. The roles of MCR II and III in glucose response of ChREBP are independent of and distinct from their function in controlling subcellular localization. We further demonstrate that, instead of inhibiting ChREBP activity as would be predicted from its cytoplasmic retentive function, 14-3-3 binding with MCR III is essential for the glucose responsiveness of ChREBP. The interaction between 14-3-3 and ChREBP is constitutive, indicating a permissive role of 14-3-3 in the glucose response of ChREBP. We further uncovered an unconventional 14-3-3 binding motif (residues 116-135) lacking phosphor-serine/threonine within MCR III, a predicted alpha-helix highly conserved in all Mondo proteins. We conclude that individual subdomains in the GSM (MCR I through V) play diverse but crucial roles in cooperation with essential trans-acting cofactors such as 14-3-3 proteins to mediate the glucose response of ChREBP.

Inactivation of Plin4 downregulates Plin5 and reduces cardiac lipid accumulation in mice

Plin4 is a lipid droplet protein (LDP) found predominantly in white adipose tissue (WAT). The Plin4 gene is immediately downstream of the Plin5 gene; the two genes exhibit distinct though overlapping tissue expression patterns. Plin4 is absent in brown adipose tissue (BAT) and liver and expressed at low levels in heart and skeletal muscle, whereas Plin5 is highly expressed in these oxidative tissues but at a low level in WAT. The physiological role of Plin4 remains unclear. We have generated Plin4(-/-) mice by gene targeting. Loss of Plin4 has no effect on body weight or composition or on adipose mass or development. However, the triacylglycerol (TAG) content in heart, but not other oxidative tissues such as BAT, soleus muscle, and liver, is markedly reduced in Plin4(-/-) mice. The heart of Plin4(-/-) mice displays reduced Plin5 mRNA and protein levels (by ~38 and 87%, respectively, vs. wild-type) but unchanged mRNA levels of other perilipin family genes (Plin2 and Plin3) or genes involved in glucose and lipid metabolism. Despite reduced cardiac TAG level, both young and aged Plin4(-/-) mice maintain normal heart function as wild-type mice, as measured by echocardiography. Interestingly, Plin4 deficiency prevents the lipid accumulation in the heart that normally occurs after a prolonged (48-h) fast. It also protects the heart from cardiac steatosis induced by high-fat diet or when Plin4(-/-) mice are bred into Lep(-/-) obese background. In conclusion, inactivation of Plin4 downregulates Plin5 and reduces cardiac lipid accumulation in mice.

Molecular Mechanisms Underlying Fasting Modulated Liver Insulin Sensitivity and Metabolism in Male Lipodystrophic Bscl2/Seipin-Deficient Mice

Bscl2(-/-) mice recapitulate many of the major metabolic manifestations in Berardinelli-Seip congenital lipodystrophy type 2 (BSCL2) individuals, including lipodystrophy, hepatomegly, hepatic steatosis, and insulin resistance. The mechanisms that underlie hepatic steatosis and insulin resistance in Bscl2(-/-) mice are poorly understood. To address this issue, we performed hyperinsulinemic-euglycemic clamp on Bscl2(-/-) and wild-type mice after an overnight (16-h) fast, and found that Bscl2(-/-) actually displayed increased hepatic insulin sensitivity. Interestingly, liver in Bscl2(-/-) mice after a short term (4-h) fast had impaired acute insulin signaling, a defect that disappeared after a 16-hour fast. Notably, fasting-dependent hepatic insulin signaling in Bscl2(-/-) mice was not associated with liver diacylglyceride and ceramide contents, but could be attributable in part to the expression of hepatic insulin signaling receptor and substrates. Meanwhile, increased de novo lipogenesis and decreased β-oxidation led to severe hepatic steatosis in fed or short-fasted Bscl2(-/-) mice whereas liver lipid accumulation and metabolism in Bscl2(-/-) mice was markedly affected by prolonged fasting. Furthermore, mice with liver-specific inactivation of Bscl2 manifested no hepatic steatosis even under high-fat diet, suggesting Bscl2 does not play a cell autonomous role in regulating liver lipid homeostasis. Overall, our results offered new insights into the metabolic adaptations of liver in response to fasting and uncovered a novel fasting-dependent regulation of hepatic insulin signaling in a mouse model of human BSCL2.

Altered Lipid Metabolism in Residual White Adipose Tissues of Bscl2 Deficient Mice

Mutations in BSCL2 underlie human congenital generalized lipodystrophy type 2 disease. We previously reported that Bscl2 −/− mice develop lipodystrophy of white adipose tissue (WAT) due to unbridled lipolysis. The residual epididymal WAT (EWAT) displays a browning phenotype with much smaller lipid droplets (LD) and higher expression of brown adipose tissue marker proteins. Here we used targeted lipidomics and gene expression profiling to analyze lipid profiles as well as genes involved in lipid metabolism in WAT of wild-type and Bscl2−/− mice. Analysis of total saponified fatty acids revealed that the residual EWAT of Bscl2−/− mice contained a much higher proportion of oleic18:1n9 acid concomitant with a lower proportion of palmitic16:0 acid, as well as increased n3- polyunsaturated fatty acids (PUFA) remodeling. The acyl chains in major species of triacylglyceride (TG) and diacylglyceride (DG) in the residual EWAT of Bscl2−/− mice were also enriched with dietary fatty acids. These changes could be reflected by upregulation of several fatty acid elongases and desaturases. Meanwhile, Bscl2−/− adipocytes from EWAT had increased gene expression in lipid uptake and TG synthesis but not de novo lipogenesis. Both mitochondria and peroxisomal β-oxidation genes were also markedly increased in Bscl2−/− adipocytes, highlighting that these machineries were accelerated to shunt the lipolysis liberated fatty acids through uncoupling to dissipate energy. The residual subcutaneous white adipose tissue (ScWAT) was not browning but displays similar changes in lipid metabolism. Overall, our data emphasize that, other than being essential for adipocyte differentiation, Bscl2 is also important in fatty acid remodeling and energy homeostasis.