Accumulation Of Fat Droplets Research Articles

Dietary constituents reduce lipid accumulation in murine C3H10 T1/2 adipocytes: A novel fluorescent method to quantify fat droplets

BackgroundAdipocyte volume (fat accumulation) and cell number (adipogenesis) is increased in obese individuals. Our objective was the identification of dietary constituents with inhibitory effects on triglyceride formation during adipogenesis. Therefore an in vitro adipose cell assay in murine C3H10 T1/2 cells was developed, which enabled rapid quantification of intracellular fat droplet accumulation during adipocyte differentiation. Results were corroborated by expression levels of several specific adipogenic and lipogenic genes which are known to regulate triglyceride accumulation.MethodsC3H10 T1/2 adipocyte differentiation was conducted with rosiglitazone in the presence of test compounds for 7 days. Accumulation of intracellular lipid droplets was measured using the Cellomics® ArrayScan® VTI HCS reader and SpotDetector® BioApplication from ThermoFisher. Fluorescent images were automatically acquired and analysed employing the fluorescent dyes BODIPY® 493/503 and Hoechst 33342, for staining neutral lipids and localisation of nuclei, respectively. The expression levels of adipogenic and lipogenic genes, such as PPARα and PPARγ, C/EBPα, aP2, adiponectin, LPL and HSL, CPT-1β, ACC1, Glut4 and FAS, were determined by quantitative RT-PCR. Dietary ingredients including PUFAs, carotenoids, polyphenols and catechins were tested for their effect on lipid accumulation.ResultsThe ω-3 PUFAs docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), the carotenoid β-carotene and hydroxytyrosol exhibited the strongest inhibitory effects on the rosiglitazone-stimulated lipid formation. (all-E)-lycopene and epigallocatechin gallate (EGCG) showed a moderate inhibition, whereas resveratrol did not reduce fat droplet formation. Additionally, it was demonstrated that adipogenic and lipogenic gene expression was attenuated. DHA, β-carotene and hydroxytyrosol inhibited the gene expression of PPARγ, C/EBPα, aP2 and CPT-1β.ConclusionThis in vitro assay in differentiating adipocytes enables automated detection and quantification of changes in lipid droplet number, size and intensity. The observed inhibitory effects of identified dietary constituents such as ω-3 PUFAs and β-carotene correlate with the modulation of genes involved in adipocyte differentiation.

Constitutively active Stat5A and Stat5B promote adipogenesis

The metabolic syndrome is an important social problem affecting many people in developed countries. Obesity is a leading cause of this syndrome, hence understanding molecular mechanisms underlying obesity is of prime importance for preventive medicine to develop novel methods to alleviate the corresponding social cost as well as for pharmaceutical companies to develop antimetabolic drugs. Since adipocytes play an important role in obesity, we explored the signaling pathways leading to differentiation of adipocytes. We used a preadipocyte cell line to monitor the differentiation of adipocytes, and virus-mediated gene transfer to assess the role of the transcription factor Stat5 in adipogenesis. Adipocyte differentiation was assessed by Northern blot and Western blot analyses as well as accumulation of fat droplets in cells. Promoter activity of the proadipogenic transcription factor peroxisome proliferator-activated receptor-gamma (PPARγ) was evaluated by luciferase assay. Virus-mediated gene transfer of the constitutively active form of both Stat5A and Stat5B resulted in enhanced adipocyte differentiation in the absence of fetal bovine serum (FBS) as judged by expression of proadipogenic factors as well as accumulation of fat droplets in cells. Such a proadipogenic effect of Stat5 is, in part, mediated by its ability to enhance transcription of PPARγ, a master transcriptional regulator in adipogenesis. The constitutively active form of Stat5A and Stat5B promoted adipocyte differentiation in the absence of FBS via induction of PPARγ.

황기홍국소스를 처리한 계육이 고지방 및 고콜레스테롤 식이 흰쥐의 체중과 혈청 및 간 조직의 지질 함량에 미치는 영향

Dietary effects of chicken treated with Hwangki-Beni Koji sauces (HBS) on serum and hepatic lipid profiles in rats fed high a fat and high cholesterol diet were investigated. Experimental plots (5 rats per each group) were divided into normal control diet group (NC), high fat and high cholesterol diet group (HFC), HFC plus 15% base sauce treated chicken supplemented diet group (HFC-BS), HFC plus 15% HBS-treated chicken supplemented diet group (HFC-HBS), and fed for 5 weeks. Feed intakes in HFC-HBS group were higher than those of HFC and HFC-BS groups, whereas body weight gain and feed efficiency ratio of the HFC-HBS group were lower than those of the HFC and HFC-BS groups. Serum alanine aminotransferase and aspartate aminotransferase activities of HFC-HBS group were significantly lower than those of HFC and HFC-BS groups. Level of serum triglyceride, total cholesterol and LDL-cholesterol in HFC-HBS group were 34.06%, 6.28% and 31.00% lower than those of HFC-BS group, respectively, whereas HDL-cholesterol level and atherogenic index in HFC-HBS group were 1.72-fold higher and 2.52-fold lower, respectively, than those of HFC-BS group. Level of hepatic total lipid, triglyceride and total cholesterol in HFC-HBS group were 9.53%, 5.25% and 22.20% lower than those of HFC-BS group, respectively. In the morphological results of liver, the hepatocytes of central vein lesion and intact hepatic cell plate are preserved well from steatosis in HFC-HBS group, whereas the hepatocytes in HFC and HFC-BS groups are distended by accumulation of multiple fat droplets, and sinusoids are occluded. In the above results, we expected that chicken treated with Hwangki-Beni Koji sauce may have functionalities of anti-obesity, anti-cholesterol and also, lowering anti-atherogenic index in high fat and high cholesterol diets.

Hepatocellular ballooning in NASH

Hepatocellular ballooning is a key finding in nonalcoholic steatohepatitis (NASH). It is conventionally defined by hemotoxylin and eosin (H&E) staining showing enlarged cells with rarefied cytoplasm and recently by changes in the cytoskeleton. Fat droplets are emerging as important organelles in cell metabolism. To address a possible relation between fat droplets and ballooning, we studied fat staining, H&E, and keratin 18 staining in human NASH. Sequential staining and high resolution imaging were used to study freshly prepared cryo-sections from 10 patients with histologically confirmed steatohepatitis using oil red O for fat droplet identification, H&E to identify ballooning, and anti-K18 to confirm cytoskeletal changes. High resolution images were captured at each stage using the Aperio Scanscope. To provide ultrastructural correlation, glutaraldehyde-fixed specimens were studied using transmission electron microscopy (TEM) with serial sectioning for localization of ballooned cells by light microscopy and TEM in identical specimens. Serial staining consistently demonstrated that hepatocellular ballooning is associated with fat droplet accumulation evident by oil red O positivity and depletion of cytoplasmic keratin 18 with K-18 positive Mallory-Denk bodies (MDB). TEM confirmed the association between osmium stained fat droplets, MDB formation, and cellular enlargement and suggested droplet-associated dilation of the endoplasmic reticulum. These results indicate a relationship between cellular ballooning, fat droplet accumulation, and cytoskeletal injury in NASH. We speculate that injury to multiple, organelles including fat droplets and endoplasmic reticulum, contribute to this characteristic finding.

JAM‐A regulates the progression of Non‐alcoholic Fatty Liver Disease (NAFLD)

Non‐alcoholic fatty liver disease (NAFLD) is characterized by an abnormal amount of fat accumulation in the liver, specifically more than 5% fat by weight. Little is known about how the fat accumulates in the liver, but it has been found that intestinal permeability due to leaky tight junctions may be a contributing factor. Our lab studies junctional adhesion molecule‐A (JAM‐A), a protein located at the tight junctions of epithelial and endothelial cells. Through its ability to homodimerize at the apical part of the lateral membrane, JAM‐A helps regulate the permeability and stability of the junction.This study's aim was to find what effect JAM‐A has on the development of NAFLD. To analyze this relationship, groups of Jam‐A (+/+) and Jam‐A (−/−) mice were put on either a high‐fat or low‐fat diet for 20 weeks. During this time the mice were weighed every two weeks and blood samples were taken every four weeks. At the end of the 20 weeks, the mice were sacrificed and the livers and fat pads were removed.We found that the variation in weight between the Jam‐A (−/−) groups was greater than that between the Jam‐A (+/+) groups. Also, the average area of the adipocytes in the high‐fat Jam‐A (−/−) group was greater than the average area in the high‐fat Jam‐A (+/+) group. Since the liver sends fat to tissues in the form of cholesterol, we compared the levels in the plasma. The high‐fat Jam‐A (−/−) mice had significantly higher LDL‐cholesterol and total cholesterol levels in the plasma than the other groups as well. After examining the stained sections of the livers, it was found that the livers of the high‐fat Jam‐A (−/−) mice showed more fat droplet accumulation than the high‐fat Jam‐A (+/+) mice. This suggests that ultimately the absence of JAM‐A from the tight junctions promotes the development of NAFLD. This project was funded by the Arnold and Mabel Beckman Foundation.

Current concepts of the pathogenesis of non-alcoholic fatty liver disease

Non-alcoholic fatty liver disease (NAFLD) - common chronic liver disease, characterized by pathological accumulation of fat droplets, not associatedwith alcohol. NAFLD is often a component of other diseases such as metabolic syndrome, diabetes, obesity, and contributes to the prevalence of CVDamong the population.Asymptomatic disease, the difficulty of diagnosis, the lack of a unified concept of treatment NAFLD - topical issues that require more in-depth studyand worthy of attention a wide range of specialists.

Characterization of a novel murine preadipocyte line, AP‐18, isolated from subcutaneous tissue: Analysis of adipocyte‐related gene expressions

Adipocyte lines are a useful tool for adipocyte research. Recently, a new preadipocyte line designated AP-18 was established from subcutaneous tissue of the C3H/He mouse. In this study, we further characterized AP-18 cells. Adipocyte differentiation was assessed by accumulation of fat droplets stained by Oil Red O. The expression of the preadipocyte- or adipocyte-specific genes and adipocytokine genes was analysed qualitatively by RT-PCR and quantitatively by real-time PCR in comparison with the LM cell, a murine fibroblast line, and the 3T3-L1 cell, respectively. AP-18 cells were fibroblastoid in maintenance culture. After the confluence, fat droplets were accumulated in 50-60% of the cells cultured in the medium alone and in 70-90% of the cells cultured with insulin within 2 to 3 weeks. The fat accumulation was not promoted by the addition of dexamethazone, IBMX (3-isobutyl-1-methylxanthine) or troglitazone in combination with insulin, which were obligatory for differentiation of the 3T3-L1 cell, a murine preadipocyte line. Throughout the differentiation, AP-18 cells expressed Pref-1, LPL, C/EBP beta, C/EBP delta, RXR alpha, C/EBP alpha, PPAR gamma, RXR gamma, aP2, GLUT4, SCD1, UCP2, UCP3, TNFalpha, resistin, leptin, adiponectin and PAI-1 genes, but not the UCP1 gene, indicating that the cell is derived from WAT (white adipose tissue). The time course of these gene expressions was similar to that of 3T3-L1 cells, although the expressions were slower and lower in AP-18 cells. These data indicate that AP-18 cells are preadipocytes originated from WAT and differentiate into adipocytes under more physiological conditions than 3T3-L1 cells. AP-18 may be useful in adipocyte research.

Osteogenic and adipogenic differentiation potential of an immortalized fibroblast-like cell line derived from porcine peripheral blood

Primary fibroblast-like cells isolated from the peripheral blood of a healthy pig were immortalized by transduction of cells with a replication-defective retrovirus vector expressing the E6/E7 proteins of human papillomavirus type 16 (pLXSN-16E6E7). The immortalized cells grow rapidly in cell culture and exhibit a distinct cell surface phenotype that was positive for CD90, CD44, collagen I, and vimentin and negative for CD14 and MHC II. Additionally, these immortalized blood derived-fibroblast-like cells had the potential to differentiate into osteoblasts and adipocytes in vitro as evidenced by the deposition of calcium, increased alkaline phosphatase activity, upregulated osteogenic and adipogenic marker gene expression, and accumulation of fat droplets in cells when osteogenic (dexamethasone, ascorbic acid, and β-glycerophosphate) or adipogenic supplements (dexamethasone, 3-isobutyl-1-methylxanthine, indomethacin, and insulin) were added to the culture. Overall, the results suggest that the immortalized blood-derived fibroblast-like cells exhibit some of the features of mesenchymal precursor cells, which may have implications in tissue repair and remodeling process.

Effect of ShanZha, a Chinese herbal product, on obesity and dyslipidemia in hamsters receiving high-fat diet

Aim The present study is designed to investigate the effect of ShanZha ( Crataegus pinnatifida) on obesity or dyslipidemia induced by high-fat diet in hamsters and characterize the role of PPARα in this action of ShanZha. Materials and methods We induced dyslipidemia and obesity in hamsters using high-fat diet and treated hamsters with ShanZha or vehicle for 7 days. We measured the body weight, adipose tissue weights, and food intake of hamsters. Plasma total cholesterol (TC), triglyceride (TG), LDL-cholesterol (LDL-C) and HDL-cholesterol (HDL-C) were determined at the beginning and end of this treatment. Effect of ShanZha on adipogenesis was examined in vitro and change of PPARα was analyzed using Western blot. Results The food intake, body weights, and weights of both brown and white adipose tissues were markedly reduced in hamsters receiving ShanZha as compared with the vehicle-treated control. Plasma levels of TC, TG and LDL-C were decreased by this ShanZha treatment while HDL-C was elevated. The effects of ShanZha were reversed by the combined treatment with PPARα antagonist, MK886. ShanZha inhibited the fat droplet accumulation in 3T3-L1 adipocytes in a dose-dependent manner and this effect was abolished by MK886. Western blot results showed activation of PPARα by ShanZha in hamster adipose tissue. Conclusion We suggest that ShanZha could activate PPARα to improve dyslipidemia or obesity.

Atypical presentation of VLCAD deficiency associated with a novel ACADVL splicing mutation

Very long chain acyl-CoA dehydrogenase (VLCAD) deficiency is an autosomal recessive inborn error of metabolism characterized by impaired mitochondrial beta-oxidation of fatty acids with a chain length between 14 and 18 carbons. While expansion of newborn screening has improved our ability to detect VLCAD deficiency in early childhood, the late-onset form of the disease still presents a significant diagnostic challenge. We report a 20-year-old female with VLCAD deficiency who first presented in infancy with hypoketotic hypoglycemia. In childhood the patient developed complex partial seizures that were aggravated by Lamotrigine treatment. The clinical course in early adulthood was complicated by recurrent, often unprovoked, episodes of rhabdomyolysis and myoglobinuria. In addition, she suffered from chronic myalgia, muscle weakness, and diffuse abdominal tenderness. A muscle biopsy revealed accumulation of fat droplets. Her acylcarnitine profile showed significantly elevated C14, C14:1, C16, and C18-carnitines. Sequence analysis of ACADVL revealed a heterozygous recurrent mutation c.848T>C (p.V283A) and a heterozygous novel splice mutation c.879-8T>A that results in the inclusion of six nucleotides from intron 9 into the transcript sequence. The molecular characterization of this novel mutation and its correlation with the clinical phenotype are discussed.

Pioglitazone Reduces ER Stress in the Liver: Direct Monitoring of in vivo ER Stress Using ER Stress-activated Indicator Transgenic Mice

It is known that endoplasmic reticulum (ER) stress is provoked under diabetic conditions and is possibly involved in the development of insulin resistance. In this study, using ER stress-activated indicator (ERAI) transgenic mice which express green fluorescent protein under ER stress conditions, we directly evaluated the effects of a diabetic agent pioglitazone on in vivo ER stress under diabetic conditions. In high fat and high sucrose diet-induced diabetic ERAI transgenic mice, 8 weeks of pioglitazone treatment reduced the accumulation of fat droplets in the liver and attenuated the development of insulin resistance. In the liver of the ERAI transgenic mice, ERAI fluorescence activity was clearly reduced as early as after 4 weeks of pioglitazone treatment, preceding the improvement of insulin resistance. In addition, after the pioglitazone treatment, serum free fatty acid and triglyceride levels were decreased, and serum adiponectin levels were increased. These data indicate that pioglitazone treatment suppresses ER stress in the liver which may explain, at least in part, the pharmacological effects of pioglitazone to reduce insulin resistance.

Effect of Transgenic Extrahepatic Expression of Betaine‐Homocysteine Methyltransferase on Alcohol or Homocysteine‐Induced Fatty Liver

Chronic alcohol feeding induces hyperhomocysteinemia (HHcy). Previously, we reported a protective role of betaine-homocysteine methyltransferase (BHMT) in homocysteine-induced injury in cultured hepatocytes. In this study, we investigated the direct role of BHMT in alcohol or homocysteine-induced liver injury. Betaine-homocysteine methyltransferase transgenic (Tg) mice were generated. Comparisons were made between the Tg and wild type (WT) mice in their response to intragastric alcohol infusion or to oral feeding of a high methionine low folate diet (HMLF). Expression of the Tg BHMT was increased in organs peripheral to the liver. The alcohol infusion for 4 weeks increased: plasma ALT by 5-fold in WT mice and 2.7-fold in Tg mice; plasma homocysteine by 7-fold in WT mice and 2-fold in Tg mice; liver triglycerides by 4-fold in WT mice and 2.5-fold in Tg mice. The alcohol-induced fatty liver was more severe in WT than in Tg mice based on H&E staining. The HMLF feeding for 4 weeks increased plasma ALT by 2-fold in WT mice and 1-fold in Tg mice; plasma homocysteine by 21-fold in WT mice and 3.3-fold in Tg mice; liver triglycerides by 2.5-fold in WT mice and 1.5-fold in Tg mice. HMLF induced accumulation of macro fat droplets in WT but not Tg mice. Betaine supplementation decreased partially the alcohol or HMLF-induced increase of ALT, homocysteine and liver lipids in WT mice. However, Tg mice were normal when fed both HMLF and betaine. In WT mice, both alcohol and HMLF induced moderate increase of sterol regulatory element binding protein 1 (SREBP1) protein which was partially reduced by betaine supplementation. In Tg mice, alcohol but not HMLF increased SREBP1. Carbohydrate responsive element-binding protein was increased by alcohol in either WT or Tg mice which was not affected by betaine supplementation. Ratio of S-adenosylmethionine (SAM) to S-adenosylhomocysteine (SAH) was reduced by 50% in WT and by 20% in Tg mice fed alcohol. Ratio of phosphatidylcholine (PC) to phosphatidylethanolamine (PE) was reduced in WT but not Tg mice fed alcohol. Changes in PE methyltransferase activities were not detected in response to alcohol or HMLF feeding but were increased by betaine. The BHMT Tg mice are resistant to alcohol or HMLF-induced HHcy and liver steatosis indicating that peripheral metabolism of homocysteine protected the liver without a direct effect of BHMT in the liver. Multiple mechanisms are involved in protection by betaine including increased SAM/SAH and PC/PE ratios.

Comparison of hypolipidemic activity of synthetic gallic acid–linoleic acid ester with mixture of gallic acid and linoleic acid, gallic acid, and linoleic acid on high-fat diet induced obesity in C57BL/6 Cr Slc mice

Hyperlipidemia is the major risk factors of heart disease such as atherosclerosis, stroke, and death. In the present study, we studied the effect of gallic acid (GA), linoleic acid (LA), mixture of GA and LA (MGL), and chemically synthesized gallic acid–linoleic acid ester (octadeca-9,12-dienyl-3,4,5-trihydroxybenzoate, GLE) on the ability to ameliorate hyperlipidemia in C57BL/6 mice fed a high-fat diet (HFD). GLE, GA, LA, and MGL were mixed with HFD and the composition of the test compounds were 1% of the diet for 7 weeks. After 7 weeks, the average body weight of ND and GLE groups was lower than that of HFD group ( P < 0.05). The liver weight of mice decreased ( P < 0.05) in all treatment groups relative to HFD fed group. The plasma lipids such as triglyceride and LDL-cholesterol were found to be decreased ( P < 0.05) in GLE, GA, LA, and MGL fed mice when compared to that of HFD fed mice. But high-density lipoprotein (HDL) cholesterol increased ( P < 0.05) in HFD and GLE fed mice when compared to that of ND fed mice. The hepatic accumulation of fat droplets of GA, LA, GLE, and MGL group showed considerably lower than that of HFD group. Adipose histology showed that GLE supplementation was found to be more effective in decreasing the size of adipocyte relative to those of other treatment groups. In conclusion, the supplementation of synthetic GLE from gallic acid and linoleic acid ester may have a potential hypolipidemic effect on mice fed high-fat diet. Further studies are required to prove GLE as a hypolipidemic agent.

Effect of High Fat Diet on the Volume of Liver and Quantitative Feature of Kupffer Cells in the Female Rat: A Stereological and Ultrastructural Study

The role of Kupffer cells (KCs) in nonalcoholic steatohepatitis (NASH) which is regarded as a major cause of cryptogenic cirrhosis of the liver was investigated using stereological methods and electron microscopy in the rat model. To our knowledge, there is no stereological study on the volume of liver, total number, numerical density, and nuclear height of KCs of liver in the female rat fed with a high fat diet (HFD) in the literature. 16 female Sprague Dawley rats were randomized into HFD and control group, with HFD and standard diet for 12 weeks, respectively. In this study, two basic research methods were used to analyze the samples. One was histopathological observation at both light and electron microscopic level. The other was stereological methods that consist of Cavalieri principle for liver volume estimation and physical disector method for estimation of numerical density and total number of KCs in the liver. Liver volume, both mean numerical density and total number of KCs, were statistically increased in HFD rats. Ultrastructurally, a significant decrease in the mean nuclear height of KCs in HFD rats was also found. In the control group, no abnormal change was observed, but in the HFD group, some changes such as diffuse steatosis, mononuclear cell infiltration, necrosis, fibrosis, accumulation of fat droplets and intra-cytoplasmic vacuoles, and swollen mitochondria with irregular membranes were observed in the hepatocytes. The number and activity of KCs are increased significantly in NASH induced by HFD, and KCs might be involved in the pathogenesis of steatohepatitis as previously attributed as a major cause of cryptogenic cirrhosis of the liver.

2-Cyano-lup-1-en-3-oxo-20-oic acid, a cyano derivative of betulinic acid, activates peroxisome proliferator-activated receptor in colon and pancreatic cancer cells

Betulinic acid (BA) is a phytochemical triterpenoid acid from bark extracts and is cytotoxic to cancer cells and tumors. We modified the A-ring of BA to give a 2-cyano-1-en-3-one moiety and the effects of the 2-cyano-lup-1-en-3-oxo-20-oic acid (CN-BA), 2-cyano derivative of BA, and its methyl ester (CN-BA-Me) were investigated in colon and pancreatic cancer cells. Both CN-BA and CN-BA-Me were highly cytotoxic to Panc-28 pancreatic and SW480 colon cancer cells. CN-BA and CN-BA-Me also induced differentiation in 3T3-L1 adipocytes, which exhibited a characteristic fat droplet accumulation induced by peroxisome proliferator-activated receptor gamma (PPARgamma) agonists. Based on these results, we investigated the activities of CN-BA and CN-BA-Me as PPARgamma agonists using several receptor-mediated responses including activation of transfected PPARgamma-responsive constructs, induction of p21 in Panc-28 cells and induction of caveolin-1 and Krüppel-like factor 4 in colon cancer cells. The results clearly demonstrated that both CN-BA and CN-BA-Me activated PPARgamma-dependent responses in colon (caveolin-1) and pancreatic (p21) cancer cells, whereas induction of KLF4 by these compounds in colon cancer cells was PPARgamma independent and also dependent on cell context. The PPARgamma agonist activities of CN-BA and CN-BA-Me were structure-, response/gene- and cell context-dependent suggesting that these compounds are a novel class of selective PPARgamma modulators with potential for clinical treatment of colon and pancreatic cancer.

Suppression of canonical Wnt/ -catenin signaling by nuclear plakoglobin recapitulates phenotype of arrhythmogenic right ventricular cardiomyopathy

Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVC) is a genetic disease caused by mutations in desmosomal proteins. The phenotypic hallmark of ARVC is fibroadipocytic replacement of cardiac myocytes, which is a unique phenotype with a yet-to-be-defined molecular mechanism. We established atrial myocyte cell lines expressing siRNA against desmoplakin (DP), responsible for human ARVC. We show suppression of DP expression leads to nuclear localization of the desmosomal protein plakoglobin and a 2-fold reduction in canonical Wnt/beta-catenin signaling through Tcf/Lef1 transcription factors. The ensuing phenotype is increased expression of adipogenic and fibrogenic genes and accumulation of fat droplets. We further show that cardiac-restricted deletion of Dsp, encoding DP, impairs cardiac morphogenesis and leads to high embryonic lethality in the homozygous state. Heterozygous DP-deficient mice exhibited excess adipocytes and fibrosis in the myocardium, increased myocyte apoptosis, cardiac dysfunction, and ventricular arrhythmias, thus recapitulating the phenotype of human ARVC. We believe our results provide for a novel molecular mechanism for the pathogenesis of ARVC and establish cardiac-restricted DP-deficient mice as a model for human ARVC. These findings could provide for the opportunity to identify new diagnostic markers and therapeutic targets in patients with ARVC.

Insulin and fatty acids regulate the expression of the fat droplet-associated protein adipophilin in primary human trophoblasts

This study was undertaken to test the hypothesis that insulin and fatty acids regulate adipophilin expression in cultured human trophoblasts. Cytotrophoblasts isolated from term human placentas were cultured in the absence or presence of insulin (10 nmol/L), and a mix of oleic and linoleic acid in serum-free medium. The expression of adipophilin as well as the fatty acid transport proteins (FATP) 2, 3, 4 and 6 was examined. Fat accumulation was quantified by BODIPY staining and fat uptake determined using [3H]-oleic acid. A combination of insulin and fatty acids enhanced the expression of adipophilin (2.3-fold, P < .05). In contrast, the expression of FATPs was unchanged. Furthermore, insulin and fatty acids increased the accumulation of fat droplets in trophoblasts by 4- to 5-fold (P < .05), but had no effect on oleic acid uptake. Insulin and fatty acids enhance the expression of adipophilin and the formation of fatty acid droplets in term human trophoblasts.

Mitochondrial cytopathy combined with Fanconi’s syndrome

Severe muscle weakness in Fanconi’s syndrome is rarely the result of mitochondrial cytopathy. We describe a rare case of a 9-year-old male with early onset of Fanconi’s syndrome. He developed severe proximal muscle weakness exacerbated by hypokalemia and hypophosphatemia in childhood. The muscle biopsy revealed increased accumulation of abnormal mitochondria and fat droplets in histochemical stains and electron microscopy. Mitochondrial cytopathy cannot be excluded in Fanconi’s syndrome with late onset of muscular impairment. Long-term follow-up of his clinical course is suggested to understand the natural history of this unusual case.

Knockout of the abetalipoproteinemia gene in mice: reduced lipoprotein secretion in heterozygotes and embryonic lethality in homozygotes.

Abetalipoproteinemia, an inherited human disease characterized by a near-complete absence of the apolipoprotein (apo) B-containing lipoproteins in the plasma, is caused by mutations in the gene for microsomal triglyceride transfer protein (MTP). We used gene targeting to knock out the mouse MTP gene (Mttp). In heterozygous knockout mice (Mttp+/- ), the MTP mRNA, protein, and activity levels were reduced by 50%, in both liver and intestine. Compared with control mice (Mttp+/+), chow-fed Mttp+/- mice had reduced plasma levels of low-density lipoprotein cholesterol and had a 28% reduction in plasma apoB100 levels. On a high-fat diet, the Mttp+/- mice exhibited a marked reduction in total plasma cholesterol levels, compared with those in Mttp+/+ mice. Both the livers of adult Mttp+/- mice and the visceral endoderm of the yolk sacs from Mttp+/- embryos manifested an accumulation of cytosolic fat. All homozygous embryos (Mttp-/-) died during embryonic development. In the visceral endoderm of Mttp-/- yolk sacs, lipoprotein synthesis was virtually absent, and there was a marked accumulation of cytosolic fat droplets. In summary, half-normal MTP levels do not support normal levels of lipoprotein synthesis and secretion, and a complete deficiency of MTP causes lethal developmental abnormalities, perhaps because of an impaired capacity of the yolk sac to export lipids to the developing embryo.

Histological changes induced by dietary phospholipids in intestine and liver of common carp ( Cyprinus carpio L.) larvae

Histological observations were made in common carp larvae in order to understand the origin of the phospholipid (PL) requirement of fish during their young stages. Larvae were fed for 6 or 8 days after start-feeding on semi-purified diets containing peanut oil and supplemented with or without different PL fractions enriched in phosphatidylcholine (PC) or phosphatidylinositol (PI). A group of larvae was also fed Artemia nauplii. Dietary PL deficiency was associated with an accumulation of fat droplets in the enterocytes of the anterior intestine, an increase in height of mucosal epithelium, a reduced total liver volume and mean hepatocyte volume. In contrast, diet supplementation with PC from hen egg yolk or from soybean prevented the intestinal steatosis and resulted in larger liver volume and larger hepatocyte volume. Larvae fed Artemia showed the same features as larvae fed PC-enriched diets. Hydrogenated PC also prevented the intestinal steatosis but resulted in small liver and hepatocyte size. Larvae fed PI-enriched diets exhibited intestinal steatosis, although less systematically than larvae fed PL-deficient diets, and reduced liver and hepatocyte size. Results support previous opinions that PL are needed for the absorption of neutral lipids although their beneficial effect appears to be independent of their emulsifying properties. The limiting stage seems to be the synthesis and exportation of chylomicrons or very low density lipoprotein (VLDL) by the enterocytes and not the intraluminal formation of micelles. PC appears to have a specific role for chylomicrons and VLDL synthesis and secretion, as in mammals. The reduced liver and hepatocyte size in the absence of PC could result from a decreased energy supply due to poor lipid absorption.