Differential association of adipophilin and TIP47 proteins with cytoplasmic lipid droplets in mouse enterocytes during dietary fat absorption

Recently, we found that enterocytes dynamically store triglycerides (TGs) in cytoplasmic lipid droplets (CLDs) during dietary fat absorption. A dynamic pool of TG in the form of CLDs which expands and depletes relative to time post dietary fat challenge is present in the absorptive cells of the small intestine, enterocytes. To identify cellular factors which may play a role in the regulation of this dynamic process we investigated the expression and localization of a lipid droplet associated protein family, PAT proteins, in enterocytes of mice chronically and acutely challenged by dietary fat. We found that adipophilin and Tip47 are the only PAT genes present in mouse intestinal mucosa and both genes are present at higher levels after high-fat challenges. We found TIP47 protein present in the intestine from chow and high-fat challenged mice; however, adipophilin protein was only present after high-fat challenges. In addition, TIP47 protein level was higher after an acute than a chronic high-fat challenge whereas adipophilin protein level was higher after a chronic than an acute high-fat challenge. We co-imaged TG in CLDs using CARS microscopy and TIP47 or adipophilin using immunocytochemistry in isolated enterocytes from mice challenged chronically and acutely by high levels of dietary fat. TIP47, but not adipophilin, coats CLDs in enterocytes after an acute high-fat challenge suggesting that TIP47 plays a role in the synthesis of CLDs from newly synthesized TG at the beginning of the process of dietary fat absorption in enterocytes. Adipophilin, on the other hand, coats CLDs only in enterocytes of chronic high-fat fed mice suggesting that adipophilin may play a role in the stabilization of TG stored in CLDs in longer term. These results suggest distinct roles for TIP47 and adipophilin in dietary fat absorption.     

Pancreatic triglyceride lipase deficiency minimally affects dietary fat absorption but dramatically decreases dietary cholesterol absorption in mice

This study generated pancreatic triglyceride lipase (PTL)-null mice to test the hypothesis that PTL-mediated hydrolysis of dietary triglyceride is necessary for efficient dietary cholesterol absorption. The PTL-/- mice grew normally and displayed similar body weight as their PTL+/+ littermates. Plasma lipid levels between animals of various PTL genotypes were similar when they were maintained on either a basal low fat diet or a western-type high fat/high cholesterol diet. Although the lack of a functional PTL delayed fat absorption during the initial hour of feeding a bolus load of olive oil containing [3H]triolein and [14C]cholesterol, the rate of [3H]triolein absorption was similar between PTL+/+ and PTL-/- mice after the initial 1-h period. Importantly, comparison of fecal fat content revealed similar overall fat absorption efficiency between PTL+/+ and PTL-/- mice. In contrast, the PTL-/- mice displayed significant decrease in both the rate and the amount of cholesterol absorbed after a single meal. The plasma appearance of [14C]cholesterol was found to be 75% lower (p < 0.0005) in PTL-/- mice compared with PTL+/+ mice after 4 h. The total amount of [14C]cholesterol excreted in the feces was 45% higher (p < 0.0004) in PTL-/- mice compared with PTL+/+ mice over a 24-h period. These results indicate that the delayed fat digestion due to PTL deficiency results in a significant reduction in cholesterol absorption, although other enzymes in the digestive tract may compensate for the lack of PTL in PTL-/- mice in fat digestion and absorption.     

Acylation of acylglycerols by acyl coenzyme A:diacylglycerol acyltransferase 1 (DGAT1). Functional importance of DGAT1 in the intestinal fat absorption

Acyl coenzyme A:diacylglycerol acyltransferase 1 (DGAT1) is one of the four intestinal membrane bound acyltransferases implicated in dietary fat absorption. Recently, it was found that, in addition to acylating diacylglycerol (DAG), DGAT1 also possesses robust enzymatic activity for acylating monoacylglycerol (MAG) (Yen, C. L., Monetti, M., Burri, B. J., and Farese, R. V., Jr. (2005) J. Lipid Res. 46, 1502-1511). In the current paper, we have conducted a detailed characterization of this reaction in test tube, intact cell culture, and animal models. Enzymatically, we found that triacylglycerol (TAG) synthesis from MAG by DGAT1 does not behave according to classic Michaelis-Menten kinetics. At low concentrations of 2-MAG (<50 microm), the major acylation product by DGAT1 was TAG; however, increased concentrations of 2-MAG (50-200 microm) resulted in decreased TAG formation. This unique product/substrate relationship is similar to MGAT3 but distinct from DGAT2 and MGAT2. We have also found that XP620 is an inhibitor that selectively inhibits the acylation of MAG by DGAT1 (IC(50) of human DGAT1: 16.6+/-4.0 nM (MAG as substrate) and 1499+/-318 nM (DAG as substrate); IC(50) values of human DGAT2, MGAT2, and MGAT3 are >30,000 nM). Using this pharmacological tool, we have shown that approximately 76 and approximately 89% of the in vitro TAG synthesis initiated from MAG is mediated by DGAT1 in Caco-2 cell and rat intestinal mucosal membranes, respectively. When applied to intact cultured cells, XP620 substantially decreased but did not abolish apoB secretion in differentiated Caco-2 cells. It also decreased TAG and DAG syntheses in primary enterocytes. Last, when delivered orally to rats, XP620 decreased absorption of orally administered lipids by approximately 50%. Based on these data, we conclude that the acylation of acylglycerols by DGAT1 is important for dietary fat absorption in the intestine.     

12R-lipoxygenase deficiency disrupts epidermal barrier function

12R-lipoxygenase (12R-LOX) and the epidermal LOX-3 (eLOX-3) constitute a novel LOX pathway involved in terminal differentiation in skin. This view is supported by recent studies showing that inactivating mutations in 12R-LOX and eLOX-3 are linked to the development of autosomal recessive congenital ichthyosis. We show that 12R-LOX deficiency in mice results in a severe impairment of skin barrier function. Loss of barrier function occurs without alterations in proliferation and stratified organization of the keratinocytes, but is associated with ultrastructural anomalies in the upper granular layer, suggesting perturbance of the assembly/extrusion of lamellar bodies. Cornified envelopes from skin of 12R-LOX-deficient mice show increased fragility. Lipid analysis demonstrates a disordered composition of ceramides, in particular a decrease of ester-bound ceramide species. Moreover, processing of profilaggrin to monomeric filaggrin is impaired. This study indicates that the 12R-LOX-eLOX-3 pathway plays a key role in the process of epidermal barrier acquisition by affecting lipid metabolism, as well as protein processing.     

Intact lysosome transport and phagosome function despite kinectin deficiency

The mechanism of cargo coupling to kinesin motor proteins is a fundamental issue in organelle transport along microtubules. Kinectin has been postulated to function as a membrane anchor protein that attaches various organelles to the prototype motor protein kinesin. To verify the biological relevance of kinectin in vivo, the murine kinectin gene was disrupted by homologous recombination. Unexpectedly, kinectin-deficient mice were viable and fertile, and no gross abnormalities were observed up to 1 year of age. The assembly of the endoplasmic reticulum was essentially unaffected in kinectin-deficient cells. Mitochondria appeared to be correctly distributed throughout the cytoplasm along the microtubules. Furthermore, the stationary distribution and the bidirectional movement of lysosomes did not depend on kinectin. Kinectin-deficient phagocytes internalized and cleared bacteria, indicating that phagosome trafficking and maturation are functional without kinectin. Thus, these data unequivocally indicate that kinectin is not essential for trafficking of lysosomes, phagosomes, and mitochondria in vivo.     

Ectopic overexpression of porcine DGAT1 increases intramuscular fat content in mouse skeletal muscle

The microsomal enzyme 1, 2-acyl CoA: diacylglyceroltransferase-1 (DGAT1) plays an important role in triglyceride storage in adipose tissue and expresses in skeletal muscle as well. The primary goal of the present study was to investigate the effect of porcine DGAT1 on intramuscular fat (IMF) content of transgenic mice produced by pronuclear microinjection with muscle specific promoter of porcine muscle creatine kinase (MCK). In normal chow-fed diet, 4 month-old male transgenic mice expressed more DGAT1, ACC1, UCP1, and FABP4 mRNAs and proteins in skeletal muscle than control mice by real-time PCR and western blot. No significant changes were detected for ACC2, CD36, ADRP, PPAR gamma and LPL. Triacylglycerol assay and soleus muscle sections showed overexpression of porcine DGAT1 in skeletal muscle increased intramyocellular triglyceride and percent of the total cell surface covered by lipid droplets. Thus, upregulation of porcine DGAT1 in skeletal muscle increases IMF content. The present study may further serve to develop transgenic pigs with higher IMF content and improved meat quality.     

Canalicular system of enterocytes at rest and its changes during lipid absorption

A study was made of the structural changes in different organoids of enterocytes of the rat small intestine at "rest" and during lipid absorption using ultrathin sections, impregnation with Ur-Pb-Cu, and continuous impregnation with OsO4. With the latter technique a specific canalicular system was found in the enterocytes, which we failed to observe on ultrathin sections. During lipid absorption the canalicular system underwent fragmentation. The vesicules on the trans-surface of the Golgi apparatus were enlarged, and the number of flattened cisterns was significantly lower. The changes in cellular organoids and their interrelations observed in the present study support the earlier hypothesis of the resynthesis of triglycerides within the enterocytes, the formation of chylomicrons and their transfer into the intercellular space.     

DGAT1 is not essential for intestinal triacylglycerol absorption or chylomicron synthesis

Dietary triacylglycerols are a major source of energy for animals. The absorption of dietary triacylglycerols involves their hydrolysis to free fatty acids and monoacylglycerols in the intestinal lumen, the uptake of these products into enterocytes, the resynthesis of triacylgylcerols, and the incorporation of newly synthesized triacylglycerols into nascent chylomicrons for secretion. In enterocytes, the final step in triacylglycerol synthesis is believed to be catalyzed primarily through the actions of acyl-CoA:diacylglycerol acyltransferase (DGAT) enzymes. In this study, we analyzed intestinal triacylglycerol absorption and chylomicron synthesis and secretion in DGAT1-deficient (Dgat1(-/-)) mice. Surprisingly, DGAT1 was not essential for quantitative dietary triacylglycerol absorption, even in mice fed a high fat diet, or for the synthesis of chylomicrons. However, Dgat1(-/-) mice had reduced postabsorptive chylomicronemia (1 h after a high fat challenge) and accumulated neutral-lipid droplets in the cytoplasm of enterocytes when chronically fed a high fat diet. These results suggest a reduced rate of triacylglycerol absorption in Dgat1(-/-) mice. Analysis of intestine from Dgat1(-/-) mice revealed activity for two other enzymes, DGAT2 and diacylglycerol transacylase, that catalyze triacylglycerol synthesis and apparently help to compensate for the absence of DGAT1. Our findings indicate that multiple mechanisms for triacylglycerol synthesis in the intestine facilitate triacylglycerol absorption.     

Effect of apolipoprotein A-IV genotype and dietary fat on cholesterol absorption in humans

We investigated the effect of the A-IV-2 allele, which encodes a Q360H substitution in apolipoprotein (apo) A-IV, and dietary fat on cholesterol absorption in humans. In three separate studies we compared fractional intestinal cholesterol absorption between groups of subjects heterozygous for the A-IV-2 allele (1/2) and homozygous for the common allele (1/1) receiving high cholesterol ( approximately 800 mg/day) diets with different fatty acid compositions. All subjects had the apoE 3/3 genotype. There was no difference in cholesterol absorption between the two genotype groups receiving a high saturated fat diet (33% of total energy as fat; 18% saturated, 3% polyunsaturated, 12% monounsaturated) or a low fat diet (22% of total energy as fat; 7% saturated, 7% polyunsaturated, 8% monounsaturated) diet. However, on a high polyunsaturated fat diet (32% of total energy as fat; 7% saturated, 13% polyunsaturated, 12% monounsaturated) mean fractional cholesterol absorption was 56. 7% +/- 1.9 in 1/1 subjects versus 47.5% +/- 2.1 in 1/2 subjects (P = 0.004). A post hoc analysis of the effect of the apoA-IV T347S polymorphism across all diets revealed a Q360H x T347S interaction on cholesterol absorption, and suggested that the A-IV-2 allele lowers cholesterol only in subjects with the 347 T/T genotype.We conclude that a complex interaction between apoA-IV genotype and dietary fatty acid composition modulates fractional intestinal cholesterol absorption in humans.     

Lgl1 deficiency disrupts hippocampal development and impairs cognitive performance in mice

Cellular polarity is crucial for brain development and morphogenesis. Lethal giant larvae 1 (Lgl1) plays a crucial role in the establishment of cell polarity from Drosophila to mammalian cells. Previous studies have found the importance of Lgl1 in the development of cerebellar, olfactory bulb, and cerebral cortex. However, the role of Lgl1 in hippocampal development during the embryonic stage and function in adult mice is still unknown. In our study, we created Lgl1‐deficient hippocampus mice by using Emx1‐Cre mice. Histological analysis showed that the Emx1‐Lgl1^?/? mice exhibited reduced size of the hippocampus with severe malformations of hippocampal cytoarchitecture. These defects mainly originated from the disrupted hippocampal neuroepithelium, including increased cell proliferation, abnormal interkinetic nuclear migration, reduced differentiation, increased apoptosis, gradual disruption of adherens junctions, and abnormal neuronal migration. The radial glial scaffold was disorganized in the Lgl1‐deficient hippocampus. Thus, Lgl1 plays a distinct role in hippocampal neurogenesis. In addition, the Emx1‐Lgl1^?/? mice displayed impaired behavioral performance in the Morris water maze and fear conditioning test.     

v-Abl signaling disrupts SOCS-1 function in transformed pre-B cells

The v-Abl oncogene activates Jak-Stat signaling during transformation of pre-B cells in mice. Disrupting Jak activation by deleting the Jak binding domain of v-Abl or by expressing a dominant-negative Jak1 decreases v-Abl transformation efficiency. As SOCS-1 is a known potent inhibitor of Jak kinases, the mechanism by which v-Abl bypasses SOCS-1 regulation to constitutively activate Jak kinases was investigated. SOCS-1 is expressed in v-Abl-transformed cells but is unable to inhibit v-Abl-mediated Jak-Stat signaling. In v-Abl transformants, SOCS-1 can inhibit cytokine signals, but it is more efficient at doing so when the cells are treated with STI571, an Abl kinase inhibitor. Downstream effects of v-Abl signaling include phosphorylation of SOCS-1 on nontyrosine residues, disruption of the interaction between SOCS-1 and the Elongin BC complex, and inhibition of SOCS-1-mediated proteasomal targeting of activated Jaks. These findings reveal a mechanism by which Jak-dependent oncogenes may bypass SOCS-1 inhibition.     

DGAT1, a new positional and functional candidate gene for intramuscular fat deposition in cattle

Intramuscular fat content, also assessed as marbling of meat, represents an important beef quality trait. Recent work has mapped a quantitative trait locus (QTL) with an effect on marbling to the centromeric region of bovine chromosome 14, with the gene encoding thyroglobulin (TG) being proposed as a positional and functional candidate gene for this QTL. Recently, the gene encoding diacylglycerol O-acyltransferase (DGAT1), which also has been mapped within the region of the marbling QTL, has been demonstrated to affect the fat content of milk. In the present study, the effects of a 5'-polymorphism of TG and of a lysine/alanine polymorphism of DGAT1 on the fat content of musculus (m.) semitendinosus and m. longissimus dorsi in 55 bovine animals (28 German Holstein and 27 Charolais) has been investigated. Significant effects were found for both candidate genes in both the breeds. These effects seem to be independent of one another because the alleles of the two polymorphisms showed no statistically significant disequilibrium. The DGAT1 effect is mainly on the m. semitendinosus. The TG polymorphism only affects m. longissimus dorsi. However, both intramuscular fat enhancing effects seem to be recessive. The possibility of two linked loci, acting recessively on intramuscular fat content, will require special strategies when selecting for higher marbling scores.     

Increased very low density lipoprotein secretion and gonadal fat mass in mice overexpressing liver DGAT1

Acyl-CoA:diacylglycerol acyltransferases (DGATs) catalyze the last step in triglyceride (TG) synthesis. The genes for two DGAT enzymes, DGAT1 and DGAT2, have been identified. To examine the roles of liver DGAT1 and DGAT2 in TG synthesis and very low density lipoprotein (VLDL) secretion, liver DGAT1- and DGAT2-overexpressing mice were created by adenovirus-mediated gene transfection. DGAT1-overexpressing mice had markedly increased DGAT activity in the presence of the permeabilizing agent alamethicin. This suggests that DGAT1 possesses latent DGAT activity on the lumen of the endoplasmic reticulum. DGAT1-overexpressing mice showed increased VLDL secretion, resulting in increased gonadal (epididymal or parametrial) fat mass but not subcutaneous fat mass. The VLDL-mediated increase in gonadal fat mass might be due to the 4-fold greater expression of the VLDL receptor protein in gonadal fat than in subcutaneous fat. DGAT2-overexpressing mice had increased liver TG content, but VLDL secretion was not affected. These results indicate that DGAT1 but not DGAT2 has a role in VLDL synthesis and that increased plasma VLDL concentrations may promote obesity, whereas increased DGAT2 activity has a role in steatosis.     

Comparative analysis of SGLT1 and GLUT2 transporters distribution in rat small-intestine enterocytes and Caco-2 cells during hexose absorption

The distribution of SGLT1 and GLUT2 hexose transporters has been evaluated in enterocytes of an isolated loop of the small intestine and Caco-2 cell culture after absorption of hexoses at their high and low concentrations. The SGLT1 transporter was found to be located in enterocytes along the edge of the intestinal villus. The GLUT2 transporter after loading with high hexose concentrations is located in the apical part of enterocytes. In culture, Caco-2 cells form a characteristic of enterocytes microvilli and the cell junction complex. During the incubation of the culture in solutions of glucose and galactose, the absorption of these sugars from the incubation medium was observed. The SGLT1 transporter in the Caco-2 cells is located in the apical and perinuclear enterocyte parts and is organized in globules. After loading with hexoses at low concentrations, the GLUT2 transporter is in the basal cell area. The Caco-2 cell culture can serve a model for studying the transport of sugar in the intestinal epithelium.     

Effects of DGAT1 deficiency on energy and glucose metabolism are independent of adiponectin

Mice lacking acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1), an enzyme that catalyzes the terminal step in triacylglycerol synthesis, have enhanced insulin sensitivity and are protected from obesity, a result of increased energy expenditure. In these mice, factors derived from white adipose tissue (WAT) contribute to the systemic changes in metabolism. One such factor, adiponectin, increases fatty acid oxidation and enhances insulin sensitivity. To test the hypothesis that adiponectin is required for the altered energy and glucose metabolism in DGAT1-deficient mice, we generated adiponectin-deficient mice and introduced adiponectin deficiency into DGAT1-deficient mice by genetic crosses. Although adiponectin-deficient mice fed a high-fat diet were heavier, exhibited worse glucose tolerance, and had more hepatic triacylglycerol accumulation than wild-type controls, mice lacking both DGAT1 and adiponectin, like DGAT1-deficient mice, were protected from diet-induced obesity, glucose intolerance, and hepatic steatosis. These findings indicate that adiponectin is required for normal energy, glucose, and lipid metabolism but that the metabolic changes induced by DGAT1-deficient WAT are independent of adiponectin and are likely due to other WAT-derived factors. Our findings also suggest that the pharmacological inhibition of DGAT1 may be useful for treating human obesity and insulin resistance associated with low circulating adiponectin levels.     

Manganese absorption and retention in rats is affected by the type of dietary fat

There is evidence that manganese (Mn) metabolism may be altered by the form and amount of dietary fat. Also, iron (Fe) absorption is greater with saturated fats, as compared to polyunsaturated fatty acids (PUFAs). The absorption of Fe and Mn are interrelated in many aspects; therefore, the form of dietary fat may indirectly alter Mn absorption. The reported studies were conducted to determine whether saturated fat, as compared to unsaturated fat, affected Mn absorption, retention, and metabolism. In experiment I, adult rats were fed diets containing either 0.7 or 100.4 μg/g Mn with the fat source as high-linoleic safflower oil or stearic acid. After 2 wk of equilibration, the animals were fed a test meal of ⁵⁴Mn followed by whole-body counting for 10 d. Manganese absorption was significantly (p<0.05) lower in the stearic acid group (0.9–4.8%) than in the safflower oil group (20–33.8%); however, the biological half-life was shorter in the safflower oil group. Retention of ⁵⁴Mn and total Mn was always significantly (p<0.05) greater in the safflower oil group when dietary Mn was low, but it was the same when dietary Mn was high. In experiment II, weanling rats were fed 1.3, 39.3, or 174.6 μg Mn/g and either stearate, high-oleic safflower oil or high-linoleic safflower oil for 8 wk. Long-term feeding of the stearate and low Mn-containing diet resulted in a significant (p<0.0001) reduction in heart superoxide dismutase activity and kidney and liver Mn concentrations compared to the other diets. These data show that stearic acid inhibitits Mn absorption, but it may not inhibit Mn retention when dietary Mn is high. The U.S. Department of Agriculture, Agricultural Research Service, Northern Plains Area, is an equal opportunity/affirmative action employer and all agency services are available without discrimination. Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable.     

Effect of adrenaline on kidney lysosome function in rats during prolonged soft tissue crushing

The total and unsedimentable activity of acid DNase, RNase, phosphatase and arylsulfatases A and B was examined in the rat kidneys during long-term compression of soft tissues in the presence of high excitability of the sympathoadrenal system. Injection of adrenalin to rats with trauma reduced the total activity of DNase, acid phosphatase and arylsulfatases A and B, particularly at the late periods of soft tissue compression, whereas the total activity of acid RNase slightly increased as compared with control. Compression of soft tissues after adrenalin preinjection was accompanied by a substantial rise of unsedimentable activity of the lysosomal enzymes under study in the kidneys. The activity of the enzymes in cytosol progressively ascended as the time of soft tissue injury increased.