Progress 10/01/14 to 09/30/19
Outputs
Target Audience:The outcomes this period are of interest to research scientists and pharmaceutical agencies Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Three doctoral students have obtained training and professional development in the course of their involvement in this research. Ms. Heli Xu has worked on the muscle of the FABP knockouts, focusing on the LFABP null mouse; she received her Ph.D. in January 2019. Mr. Atreju Lackey has focused on the intestine of the knockouts, particularly the IFABP null mouse. Mr. Lackey also bred and began studying the intestine-specific LFABP null mouse. Ms. Hiba Tawfeeq is breeding and studying the liver-specific LFABP null mouse. She is also using cultured cells to examine the effects of a common human polymorphism in LFABP on the oxidative status, as previous literature suggests that LFABP plays a role in regulating cell oxidation state. As noted in previous reports, the students are examining metabolomics data sets from plasma, liver, muscle, and intestine metabolites. They are developing enzyme assays, histology protocols in collaboration with Dr. Laurie Joseph, and other analytical procedures, as well as in vivo physiological studies, including gut motility, exercise performance, FGF21 sensitivity of liver, adipose, and muscle, Oxymax calorimetry analysis, and Seahorse analysis of mitochondrial oxygen consumption. All the students receive training in grant and manuscript writing, and in scientific seminar presentation. Students also attend scientific conferences, present posters at meetings, and meet regularly with seminar speakers who visit our campus. How have the results been disseminated to communities of interest?Seminar presentations, posters at conferences, publication of journal articles. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Our ongoing studies of intestinal lipid-binding proteins are leading to a paradigmatic shift in thinking about the functions of these proteins. We demonstrate that individual lipid-binding proteins, co-expressed in the intestinal absorptive cell, have unique functions and are important in sensing dietary lipid and signaling nutrient status to the body, in addition to their long appreciated role in intestinal lipid assimilation. For example, we have now shown that Liver fatty acid-binding protein (LFABP) signals dietary fat status to the muscle, impacting muscle energy storage and metabolism. Although LFABP is thought to function in intracellular lipid trafficking, studies of LFABP-null (LFABP-/-) mice have also indicated a role in regulating systemic energy homeostasis. We and others have reported that LFABP-/- mice become more obese than wild-type (WT) mice upon high-fat feeding. In the present studies we show that despite increased body weight and fat mass, LFABP-/- mice are protected from a high-fat feeding-induced decline in exercise capacity, displaying an approximate doubling of running distance compared with WT mice. To understand this surprising exercise phenotype, we focused on metabolic alterations in the skeletal muscle due to LFABP ablation. Compared with WT mice, resting skeletal muscle of LFABP-/- mice had higher glycogen and intramuscular triglyceride levels, as well as an increased fatty acid oxidation rate and greater mitochondrial enzyme activities, suggesting higher substrate availability and substrate utilization capacity. Dynamic changes in the respiratory exchange ratio during exercise indicated that LFABP-/- mice use more carbohydrate in the beginning of an exercise period and then switch to using lipids preferentially in the later stage. Consistently, LFABP-/- mice exhibited a greater decrease in muscle glycogen stores during exercise and elevated circulating free fatty acid levels post-exercise. We conclude that, since LFABP is not expressed in muscle, its ablation appears to promote interorgan signaling that alters muscle substrate levels and metabolism, thereby contributing to the prevention of high-fat feeding-induced skeletal muscle impairment. Another enterocyte FABP, intestinal FABP (IFABP), has now been shown to be critical for intestinal morphology and motility, in addition to a role in lipid absorption. IFABP is abundantly present in the cytosol of the small intestinal (SI) enterocyte. High fat (HF) feeding of IFABP-/- mice resulted in reduced weight gain and fat mass relative to wild-type (WT) mice. In the present studies we examined intestinal properties that may underlie the observed lean phenotype of high fat-fed IFABP-/- mice. No alterations in fecal lipid content were found, suggesting that the IFABP-/- mice are not malabsorbing dietary fat. However, the total excreted fecal mass for the IFABP-/- mice was increased relative to WT mice. Moreover, we found reduced intestinal transit time in the IFABP-/- mice. IFABP-/- mice were observed to have a shortened average villus length, thinner muscularis layer, reduced goblet cell density, and reduced Paneth cell abundance. The number of proliferating cells in the crypts of IFABP-/- mice did not differ from that of WT mice, suggesting that the blunt villi phenotype is not due to alterations in proliferation. IFABP-/- mice were observed to have altered expression of genes related to intestinal structure, while immunohistochemical analyses revealed increased staining for markers of inflammation. Taken together, the ablation of IFABP leads to changes in gut motility and morphology which likely contribute to the relatively leaner phenotype occurring at the whole-body level. Thus, these results suggest that IFABP is likely involved in dietary lipid sensing and signaling, influencing intestinal motility, intestinal structure, and nutrient absorption, thereby impacting systemic energy metabolism. In previous work from this project, we showed that LFABP binds not only fatty acids but also the other product of dietary triacylglycerol digestion, namely monoacylglycerols. Several MGs are ligands for cannabinoid receptors, and are thus termed endocannabinoids. It is known that endocannabinoids have a wide range of systemic actions including regulation of food intake, metabolism, and pain sensitivity. We have now found that another intestinal lipid-binding protein, cellular retinol binding protein 2 (RBP2), previously thought to bind only retinoids, also binds monoacylglycerols/endocannabinoids. Expressed solely in the adult small intestine, RBP2 is thought to facilitate dietary retinoid absorption. We now find that Rbp2-deficient (Rbp2-/-) male mice fed a chow diet exhibit by 6-7 months-of-age higher body weights, impaired glucose metabolism, and lower fasting blood, but greater hepatic triglyceride levels compared to matched controls. These metabolic phenotypes are also observed when young Rbp2-/- mice are fed a high fat diet. Our data do not support the inference that retinoid actions account for the phenotypes. We show that human RBP2 is a novel monoacylglycerol (MAG)-binding protein, interacting with the canonical endocannabinoid, 2-arachidonoylglycerol (2-AG) and the endocannabinoid-like 2-lineoylglycerol (2-LG), 2-oleoylglycerol (2-OG), and 1-arachidonoylglycerol (1-AG) with affinities comparable to retinol. X-ray crystallographic studies show that the MAGs bind in the identical binding pocket as retinol. When challenged with an acute oil gavage, Rbp2-/- mice show elevated mucosal levels of 2-MAGs but not all-trans-retinoic acid. This response is accompanied by significantly elevated blood levels of the gut hormone GIP (glucose-dependent insulinotropic polypeptide). These data indicate that RBP2, in addition to facilitating dietary retinoid absorption, modulates metabolism and enteroendocrine signaling acting through its capacity to bind MAGs. We propose that RBP2, acting via modulation of MAG metabolism and likely signaling, plays a heretofore unknown role in systemic energy balance and metabolism. Overall, the studies in this project have provided entirely new insights into the complex process of dietary lipid assimilation by the intestine. In particular, we show that the intestinal lipid-binding proteins LFABP, IFABP, and RBP2, are important not only for fatty acid and retinol absorption, as previously thought, but are also critically involved in sensing nutrient status and signaling information to the rest of the body, thereby regulating energy balance and metabolism.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Xu H, Gajda AM, Zhou YX, Panetta C, Sifnakis Z, Fatima A, Henderson GC, Storch J. Muscle metabolic reprogrammingunderlies the resistance of liver fatty acid-binding proteinnull mice to high-fat feedinginduced decline in exercisecapacity. J Biol Chem 2019 Aug 26. pii: jbc.RA118.006684. doi: 10.1074/jbc.RA118.006684
|
Progress 10/01/17 to 09/30/18
Outputs
Target Audience:The outcomes this period are of interest to research scientists and pharmaceutical agencies Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Three doctoral students are obtaining training and professional development in the course of their involvement in this research. Ms. Heli Xu has worked on the muscle of the FABP knockouts, focusing on the LFABP null mouse;Mr. Atreju Lackey has focused on the intestine of the knockouts, particularly the IFABP null mouse. Mr. Lackey is also breeding and studying the intestine-specific LFABP null mouse. Ms. Hiba Tawfeeq is breeding and studying the liver-specific LFABP null mouse. She is also using cultured cells to examine the effects of a common human polymorphism in LFABP on the oxidative status, as previous literature suggests that LFABP plays a role in regulating cell oxidation state. As noted in previous reports, the students are examining metabolomics data sets from plasma, liver, muscle, and intestine metabolites. They are developing enzyme assays, histology protocols in collaboration with Dr. Laurie Joseph, and other analytical procedures, as well as in vivo physiological studies, including gut motility, exercise performance, FGF21 sensitivity of liver, adipose, and muscle, and Oxymax calorimetry analysis. All the students receive training in grant and manuscript writing, and in scientific seminar presentation. As noted last year, Mr. Lackey successfully obtained an NIH F31 individual predoctoral fellowship to support the last two years of his Ph.D. work. Students also attend scientific conferences, present posters at meetings, and meet regularly with seminar speakers who visit our campus. How have the results been disseminated to communities of interest?Seminar presentations, posters at conferences, publication of journal articles. What do you plan to do during the next reporting period to accomplish the goals?Continue breeding the tissue specific enterocyte FABP knockouts and perform whole body phenotyping, which includes body weight, fat mass, indirect calorimetry, exercise performance, plasma metabolic profile, oral glucose, insulin, and fat tolerance tests, etc. Final completion of the tertiary structure of the IFABP-anandamide protein-ligand complex with our collaborator Dr. Ruth Stark. Finish experiments with the primary myocyte cultures now established in the lab,examine effects of fatty acids, endocannabinoids, growth factors, etc., on myocyte metabolic capacity using cells from WT and knockout mice. We are also about to commence a methodical collection of fecal mterial from all our mouse models in order to charcterize the gut microbime profiles of the animals. The studies will further our goal of understanding the unique roles for each of the FABPs found in the small intestine, to intestinal and whole body energy homeostasis.
Impacts
What was accomplished under these goals?
Liver fatty acid-binding protein (LFABP, FABP1) binds long chain fatty acids with high affinity, and is abundantly expressed in the liver and small intestine. Although LFABP is thought to function in intracellular lipid trafficking, studies of LFABP null (LFABP-/-) mice have also demonstrated a role in regulating systemic energy homeostasis. We reported that LFABP-/- mice become more obese than wild-type (WT) mice upon high fat feeding. Recently we found that despite increased body weight and fat mass, however, LFABP-/- mice were protected from a high fat feeding-induced decline in exercise capacity, displaying an approximate doubling of running distance compared with WT mice. To understand this surprising exercise phenotype, we focused on metabolic alterations in the skeletal muscle secondary to LFABP ablation. Compared to WT mice, resting skeletal muscle of LFABP-/- mice had higher glycogen and intramuscular triglyceride (IMTG) levels, as well as an increased fatty acid oxidation rate and greater mitochondrial enzyme activities, suggesting higher substrate availability and substrate utilization capacity. Based on the dynamic changes in respiratory exchange ratio during exercise, LFABP-/- mice utilized more carbohydrate in the beginning of an exercise period, and then switched to using lipids preferentially in the later stage. Consistently, LFABP-/- mice exhibited a greater decrease in muscle glycogen stores during exercise, and elevated circulating free fatty acid levels post-exercise. Thus, since it is not expressed in muscle, LFABP ablation appears to promote interorgan signaling which alters muscle substrate levels and metabolism, thereby contributing to the prevention of high fat feeding-induced skeletal muscle impairment. In contrast to the obese phenotype of LFABP null mice, we previously reported that high fat (HF) feeding of IFABP-/- mice resulted in lower weight gain and reduced fat mass relative to WT mice. This indicatesthat ablation of this intestine-specific protein leads to dramatic whole-body phenotypic alterations. To understand the mechanisms leading to these differences in systemic energy homeostasis, we examined intestinal properties that may underlie the observed lean phenotype. Fecal lipid analysis demonstrated that IFABP-/- mice did not differ in their fecal lipid content, suggesting they are not malabsorbing dietary fat. However, the total excreted fecal mass for the IFABP-/- mice was significantly increased relative to WT mice. Moreover we found reduced intestinal transit time in the IFABP-/- mice, which underlies the increased fecal excretion. During tissue collection it was noted that the small intestine of IFABP-/- mice appeared to be more fragile, thus we questioned whether they may have alterations in intestinal structure. Histological analysis demonstrated that IFABP-/- mice have a shortened average villus length, reduced goblet cell density, and a reduced Paneth cell abundance relative to WT mice. BrdU proliferation assays demonstrated that the amount of proliferating cells in the crypts of IFABP-/- mice does not differ from that of WT mice, suggesting that the blunt villi phenotype is likely not due to alterations in proliferation. IHC analyses revealed increased staining for markers of inflammation, which is often observed in animals that have increased intestinal permeability. Taken together, while the effects of IFABP ablation within the enterocyte may be relatively modest in terms of lipid, there are clear changes in gut motility and morphology which likely contribute to the dramatically different phenotypes occurring at the whole-body level. Thus, these results suggest that IFABP is likely involved in dietary lipid sensing and signaling, influencing intestinal motility, nutrient absorption, and intestinal structure, thereby impacting systemic energy metabolism. In further studies we have isolated mytocytes from WT and LFABP null muscle; this took a great deal of experimental 'tinkering' so as to reduce the numbers of fibroblasts and enrich for myocytes. Our initial results shows that muscle from high fat fed LFABP null mice have increased oxygen consumption rates; this supports our observations of increased exercise performance in the LFABP-/- mouse. Generation of tissue-specific LFABP-/- mice: We have now successfully bred both the liver-specific and the intestine-specific knockouts, with expression of Cre recombinase driven by the albumin promoter or the villin promoter, respectively. We have genotyped founders and, importantly, haveshown by Western blotting that the LFABP-int-/- expressed LFABP in liver but none in intestine, and that the LFABPliv-/- expresses LFABP in intestine but not in liver. Mice are breeding and at 8 weeks of age they are switched onto the 45% high saturated fat diet for phenotyping.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
DaSilva G, Boller M, Medina I, Storch J. Relative levels of dietary EPA and DHA impact gastric oxidation and essential fatty acid uptake. J Nutr Biochem 55:68-75, 2018
|
Progress 10/01/16 to 09/30/17
Outputs
Target Audience:Fellow nutritional science faculty and biochemists Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Three doctoral students are obtaining training and professional development in the course of their involvement in this research. Ms. Heli Xu is characterizing the muscle of the FABP knockouts and is now also examining plasma metabolites and liver metabolism, in order to understand how deletion of LFABP impacts the muscle. She continues to develop enzyme assays and other analytical procedures, and has begun some in vivo physiological studies, injecting the growth factor FGF21, which she found to be altered in the liver of the LFABP-/- mice, into the circulation to monitor effects in liver, muscle, and adipose tissue. She has begun to establish the procedures for generating primary myocytes from the LFABP knockout that will not only further characterize the metabolic changes at the cellular level, such as substrate oxidation rates, but will enable examination of how external stimuli impact the WT and LFABP null myocytes. Mr. Atreju Lackey has extended his histological analysis of the intestinal mucosa in the FABP null mice, with the training in histology done in the lab of our collaborator, Dr. Laurie Joseph. He has learned to do PAS staining for polysaccharides, and has discovered that the IFABP-/- mice fewer goblet cells, in addition to shorter intestinal villi, compared with WT mice. He is establishing gene expression assays for markers of intestinal structure and inflammation, to further understand the changes in intestinal morphology. The next phase of Mr. Lackey's thesis project will establish tissue-specific knockout mice, to elucidate the contributions of LFABP gene deletion in the intestine to the whole body phenotype. He has now successfully generated "floxed" LFABP mice, and is currently breeding these mice with Villin-Cre mice to obtain an intestine-specific LFABP knockout. Ms. Hiba Tawfeeq is breeding the "floxed" LFABP mice with Albumin-Cre mice to obtain a liver-specific LFABP knockout. She is also planning to breed the "floxed" LFABP mice with a Sox2-Cre, in order to obtain a whole body knockout, to compare to each of the tissue-specific nulls. The lab has experience in generating transgenic mice and in colony breeding and maintenance, and both Ms. Tawfeeq and Mr. Lackey are being trained in these procedures. Additional training: Ms. Xu took a writing course, since English is not her first language, and she has done outstanding work in that regard. All the students receive training in grant and manuscript writing, and in scientific seminar presentation. Indeed, Mr. Lackey obtained an NIH F31 individual predoctoral fellowship to support the last two years of his Ph.D. work. How have the results been disseminated to communities of interest?Publication of this and related work in peer reviewed journals. What do you plan to do during the next reporting period to accomplish the goals?Finish breeding the tissue specific enterocyte FABP knockouts and begin whole body phenotyping, which involves body weight, fat mass, indirect calorimetry, exercise performance, plasma metabolic profile, oral glucose, insulin, and fat tolerance tests, etc. Finishing the tertiary structure of the IFABP-anandamide protein-ligand complex with our collaborator Dr. Ruth Stark. Establish primary myocyte cultures in the lab, and examine effects of fatty acids, growth factors, etc., on myocyte metabolic capacity using cells from WT and knockout mice. Fatty acid profiles, obtained by ADIFABP analysis, will be used to guide the fatty acid incubation conditions. Overall, the studies will further our goal of understanding the unique roles for each of the FABPs found in the small intestine, to intestinal biology and also to whole body energy homeostasis.
Impacts
What was accomplished under these goals?
We have made substantial progress understanding the improved exercise capacity of LFABP-/- mice compared with WT: as reported previously, in contrast to the drastic decrease in exercise capacity in WT mice in response to high fat feeding, LFABP-/- mice were protected from high fat feeding-induced decline in exercise capacity, with an approximate doubling in running distance in a treadmill test. In the resting state, compared with WT mice, high fat-fed LFABP-/- mice had higher glycogen and triglyceride content in skeletal muscle, suggesting increased substrate availability for energy production. A marked increase in citrate synthase activity and a trend toward higher succinate dehydrogenase activity suggest greater mitochondrial function to support substrate utilization. Further, in the resting state, LFABP-/- mice had a higher rate of 14CO2 production from 14C-oleic acid in the skeletal muscle than WT mice, suggesting greater capacity for complete fatty acid oxidation, whereas the incomplete fatty acid oxidation levels, reflected by the production rate of acid soluble metabolite (ASM), were similar. qPCR analysis showed a decreased GLUT1 mRNA level in resting skeletal muscle, whereas the FA transporter CD36 was trending higher, perhaps suggesting a greater FA uptake over glucose. We also found increased levels of the transcription factors PGC1α and PPARα, in LFABP-/- muscle in the resting state, suggesting an increase in fatty acid oxidation and a possible induction of mitochondrial biogenesis. Compared with the WT mice, LFABP-/- mice had a similar level of mitochondrial DNA copies in gastrocnemius, soleus and quadriceps muscles (Fig. 5). Thus, the observed increases in mitochondrial function may be related to increased substrate levels and a better efficiency in substrate oxidation rather than an increased mitochondrial number. We also examined substrate utilization during exercise: During low-intensity exercise in the fasting state, LFABP-/- mice and WT mice seemed to have similar RER at all time points, but the average RER of the first and second half of exercise showed different pattern. In the first 9 min, LFABP-/- mice had a higher average RER than WT mice, suggesting more energy production from carbohydrate utilization; in the second half of exercise, LFABP-/- mice showed a lower average RER compared with WT mice, suggesting a preference in lipid oxidation to provide energy. In the skeletal muscle, the decrease in glycogen content during exercise was significantly higher in LFABP-/- mice than WT, supporting the higher carbohydrate utilization in the first half of exercise, where the muscle glycogen was the major fuel source. Post-exercise glucose in plasma were not different between WT and LFABP-/- mice, suggesting a similar carbohydrate source from circulation. In terms of lipid utilization, although the decrease in IMTG during exercise was trending lower in LFABP-/- mice, circulating FFA was significantly higher in post-exercise LFABP-/- mice, suggesting a greater FA supply in the plasma (possibly from lipolysis in adipose tissue) may contribute to better muscle energy production in LFABP-/- mice during the low-intensity exercise. We have made progress in understanding the lean phenotype of high fat fed IFABP-/- mice: As shown previously, high fat (HF) feeding of IFABP-/- mice resulted in lower weight gain and reduced fat mass relative to WT mice, revealing that ablation of this intestine-specific protein leads to dramatic whole-body phenotypic alterations. To understand the underlying mechanisms leading to these differences in systemic energy homeostasis, we examined properties of the intestine that may impact the observed lean phenotype. Fecal lipid analysis demonstrated that mice lacking IFABP did not differ in their fecal lipid content, suggesting they are not malabsorbing dietary fat. However, we found that the total excreted fecal mass varied significantly, with IFABP-/- mice having increased total fecal excretion relative to WT mice. This was likely secondary to reduced intestinal transit time, leading to increased excretion rates, in the IFABP-/- mice. These alterations suggest decreased net nutrient uptake and assimilation, explaining, at least in part, the body weight and body composition differences observed in HF fed IFABP-/- mice compared to WT. Interestingly, during tissue collection, it was observed that the small intestine of IFABP-/- mice appeared to be more fragile. Thus, we questioned whether the IFABP-/- mice may have alterations in intestinal structure that might underlie this apparent fragility. Gene expression analysis of SI mucosa showed that IFABP-/- mice have increased expression of claudin 2 and decreased expression of claudin 5, changes that are typically associated with decreased SI integrity. Additionally, histological analysis demonstrated that IFABP-/- mice have shortened villi and a reduced goblet cell density relative to WT mice, which may indicate a decreased contact area for structural junctions to form and hence, reduced structural support. Shorter villus height also suggests a reduced capacity for nutrient absorption. Taken together, while the effects of IFABP ablation within the enterocyte may be relatively modest in terms of lipid metabolism, there are clear changes in gut motility and morphology which likely contribute to the dramatically different phenotypes occurring at the whole-body level. Thus, these results suggest that IFABP is likely involved in dietary lipid sensing and signaling, influencing intestinal motility, nutrient absorption, and intestinal structure, thereby impacting systemic energy metabolism. In other studies, we examined effects of LFABP knockdown in cultured Caco-2 enterocytes: We generated an FABP1 knockdown model in Caco-2 cell line by stable antisense cDNA transfection (FABP1as). In these cells FABP1 expression was reduced up to 87%. No compensatory increase in FABP2 was observed, strengthening the idea of differential functions of both isoforms. In differentiated FABP1as cells, apical administration of oleate showed a decrease in its initial uptake rate and in long term incorporation compared with control cells. FABP1 depletion also reduced basolateral oleate secretion. The secreted oleate distribution showed an increase in FA/triacylglyceride ratio compared to control cells, probably due to FABP1's role in chylomicron assembly. Interestingly, FABP1as cells exhibited a dramatic decrease in proliferation rate. A reduction in oleate uptake as well as a decrease in its incorporation into the PL fraction was observed in proliferating cells. Overall, these studies indicate that FABP1 is essential for proper lipid metabolism in differentiated enterocytes, particularly concerning FA uptake and its basolateral secretion. Moreover, we show that FABP1 is required for enterocyte proliferation, suggesting that it may contribute to intestinal homeostasis. Finally, since much of our work involves the use of high fat diets, we undertook a study looking at effects of HF feeding on the gut microbial communities along the length of the entire gastrointestinal tract. Bacterial communities in the mouse cecum and feces are known to be altered by changes in dietary fat. The microbiota of the mouse small intestine, by contrast, has not been extensively profiled and it is unclear whether small intestinal bacterial communities shift with dietary fat levels. We compared the microbiota in the small intestine, cecum and colon in mice fed a low or high fat diet using 16S rRNA gene sequencing. Several genera were uniquely detected in the small intestine and included the aerotolerant anaerobe, Lactobacillus spp. A high fat diet was associated with significant weight gain and adiposity and with changes in the bacterial communities throughout the intestine, with changes in the small intestine differing from those in the cecum and colon.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Onishi JC, Campbell C, Moreau M, Patel F, Brooks AI, Zhou YX, Haggblom MM and Storch J. Bacterial communities in the small intestine respond differently to those in the cecum and colon in mice fed low and high fat diets. �Microbiology 163:1189-1197, 2017
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Wang Q, Rizk S, Bernard C, Kam D, Storch J, Stark RE. Protocols and Pitfalls in Obtaining Fatty Acid-Binding Proteins for Biophysical Studies of Ligand-Protein and Protein-Protein Interactions. Biochem Biophys Rep 10:318-324, 2017
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Rodriguez Sawicki L, Bottasso Arias NM, Scaglia N, Falomir Lockhard LJ, Franchini GR, Storch J, Corsico B. FABP1 knockdown in human enterocytes impairs proliferation and alters lipid metabolism. Biochim Biophys Acta 1862:1587-1594, 2017
|
Progress 10/01/15 to 09/30/16
Outputs
Target Audience:Fellow nutritional science faculty and biochemists Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?In the course of these studies, three doctoral students are obtaining training and professional development. Ms. Heli Xu is characterizing the muscle of the FABP knockouts. She has developed many enzyme assays and other analytical procedures that were new to the lab. She is also examining the plasma fatty acid profile, and next year will be beginning studies in primary myocytes from the LFABP knockout that will not only further characterize the metabolic changes at the cellular level (including for example Seahorse measurements of oxidation status), but will enable examination of how external stimuli impact the WT and LFABP null myocytes; these studies are aimed at elucidating the mechanism by which alterations in the liver and intestine impact the muscle. Mr. Atreju Lackey has focused on examining the intestinal mucosa in the FABP null mice, doing tissue histology (lipid staining and now beginning immunohistochemistry as noted above); the training in histology is in the lab of our collaborator, Dr. Laurie Joseph. Mr. Lackey's thesis project will be to elucidate the tissue-specific contributions of LFABP gene deletion to the whole body phenotype. As such he is also working on establishing the gene targeting methods for generating the tissue-specific LFABP knockout strains. Ms. Hiba Tawfeeq joined the lab very recently, and she will be focusing on the role of the endocannabinoid system in generating the alterations in food intake observed in the enterocyte FABP null mice. The lab has experience in generating transgenic mice and in colony breeding and maintenance, and both Ms. Xu and Mr. Lackey are being trained in these procedures. Individual Development Plans have been developed for these doctoral students; indeed the graduate program is instituting the use of these progress reports for all the students. The IDPs contain information on course requirements, research goals and progress, and other training modules including ethical conduct of research and appropriate care of vertebrate animals. Ms. Xu took a graduate writing course in Spring 2016. Although English is not her first language, she does very well in coursework which includes extensive written work, and it is clear that her English abilities have greatly improved. Mr. Lackey, was supported by an Underrepresented Minority supplement to this R01, is now a Graduate Assistant on this grant, and is submitting a revised individual F31 grant application that will, if successful, support the final two years of his Ph.D. work. Mr. Lackey's writing skills are excellent, and he did a terrific job developing the proposal, in consultation with the PI. There is no question that the process of developing the grant proposal and responding to the initial critiques have been tremendous learning experiences for him. Overall, training for the graduate students involves one on one mentoring by the PI and two senior technicians in the lab. Training also includes involvement of the students in biweekly lab meetings, monthly meetings of the Rutgers Center for Lipid Research, and attendance at departmental seminar series and other talks around the RU campus, as appropriate. It is also worth noting that this R01 enables the training of numerous undergraduates; each semester, there are three or four undergraduates who work alongside the technicians and graduate students, learning both specific bench-level methods as well as mouse husbandry and phenotyping. A number of these students stay with the lab for one or more years, completing honors projects for their degrees How have the results been disseminated to communities of interest?Publication of this and related work in peer reviewed journals. What do you plan to do during the next reporting period to accomplish the goals?Determination of the full structure of the IFABP-AEA protein-ligand complex, as well as obtaining an NMR structure of the LFABP-AEA complex. Despite the initial setback, we are now in a good position for generating the 'floxed' LFABP mouse, which will be followed by breeding with the albumin-Cre mouse to generate liver-specific LFABP null strain, and with the villin-Cre mouse to generate the intestine-specific LFABP null strain. Studies of the muscle tissue of the LFABP null mouse model will also continue, including histology to examine mitochondrial morphology and the localization of intramyocellular lipid stores. To examine further the role of the endocannabinoid system in mediating the effects of LFABP and IFABP deletion on organismal energy metabolism, WT and the two single KO mice are being used for tissue collection (mucosa, liver, brain, and plasma) following 12 weeks of high fat feeding. Analyses of endocannabinoids, expression of synthetic and hydrolytic enzymes and transporters, will be done. Food intake studies using the home-cage BioDaq system, which provides continuous recording of mouse eating, will continue, focusing on apparent differences in eating behavior during the light phase for the LFABP null and the double knockout mice that has been repeatedly observed. Since we find light phase hyperphagia in the LFABP nulls, studies of Clock machinery components will begin, analyzing the peripheral tissues intestine and liver, as well as the brain. We also plan to quantitate the unbound fatty acid profile in the plasma of our four genotypes--using ADIFAB fluorescence methodology developed by Kleinfeld; since a basic finding is that organismal metabolism is altered secondary to FABP ablation in intestine and liver, it is possible that resultant alterations in fatty acids in the bloodstream are playing a role in signaling other tissues such as muscle, for example, leading to changes in tissues that do not express these FABPs.
Impacts
What was accomplished under these goals?
Chemical shift perturbation analysis shows that the endocannabinoid anandamide (AEA) binds to iFABP. Examination of the perturbed residues indicates binding-induced alterations in the helix-turn-helix domain; determination of the full structure of the protein-ligand complex is ongoing. As for the other major endocannabinoid, 2-arachidonoylglycerol (2-AG), we previously reported LFABP binding of all long chain monoacylglycerol species examined, including 2-AG. Stable isotope-labeled LFABP has also been purified, and an NMR structure of the LFABP-AEA complex is being undertaken. The LFABP-2-AG complex is also planned, but of somewhat lower priority since we already obtained the solution structure for LFABP with monoolein. We have essentially completed lipid assimilation studies with the DKO (LFABP/IFABP double knockout) mice. We have now firmly established the surprising finding that DKO mice, as well as each single knockout) show no increase in fecal fat on a 45% kcal fat diet or even a 60% kcal fat diet, relative to WT. Thus at the level of fecal fat content (ug lipid/gm feces), FABP ablation has no effect, indicating no gross lipid malabsorption. The lipid composition of feces was also unchanged in the single and double knockouts. This supports our the idea that the enterocyte FABPs may not be essential for bulk lipid processing, as has been assumed for decades, but rather behave as lipid 'sensors' and/or chaperones, serving to transport ligands to signaling systems and/or transcription factors that then initiate subsequent changes in systemic energy metabolism. Nevertheless, to further interrogate the effects of the enterocyte FABPs on lipid absorption, we are currently examining the fecal output in these mice--fecal collections from WT, each single KO, and the DKO are being made on mice fed the 45% kcal fat diet. The goal is to determine whether the IFABP mice, which gain less weight than WT, and the LFABP mice, which gain more weight, are showing corresponding changes in total feces, despite the fact that the fecal content of their feces is unchanged. Our LFABP null mouse becomes obese upon feeding of a 45% kcal fat diet, but it nevertheless remains normoglycemic and in fact, displays both increased spontaneous activity levels, and markedly enhanced endurance exercise performance. We are therefore suggesting that the LFABP null mouse is a model of the 'metabolically healthy but obese" (MHO) state. Owing to the dramatic exercise phenotype, in-depth studies of the muscle of the LFABP-/- mice are being undertaken. We found increased myocellular triacylglycerol content, which could be providing fuel for enhanced exercise, as well as increased lipid oxidation enzyme activities. Recently, we also examined glycogen content of the skeletal muscle, and like the TG content, the glycogen content in resting muscle is significantly higher in the LFABP null mice than in WT. Calorimetry measurements performed during exercise suggest that there is no difference in fuel utilization during exercise, thus our working hypothesis is that the greater substrate availability (both TG and glycogen) in the LFABP muscle is responsible for the improved exercise endurance capacity in these mice. We are also examining upstream regulators of mitochondrial lipid oxidation, such as mitochondrial mass and mitochondrial fuel selection and metabolism. We are testing the hypothesis that the increased energy substrate levels in the muscle may be related to altered insulin sensitivity, initially by examining levels of phosphorylated IRS-1 and phospho-AMPK levels. To generate intestine-specific and liver-specific LFABP knockout models to understand the origin of the 'obese but healthy' phenotype of the global knockout, we decided to use CRISPR/Cas9 technology, but this has been more challenging than expected. Despite two attempts at simultaneous CRISPR/Cas-9 insertion of LoxP sites, we have not yet obtained a fully 'floxed' (LFABPf/f) mouse. However, we do have mice with a loxP site downstream of the fabp1 gene, and a fortuitous upstream deletion on the same allele. Zygotes from these mice are now being used to add the upstream loxP site using a newly designed single guide RNA (sgRNA) to specifically target the altered upstream site, thereby generating the LFABPf/+ mouse, which will then be bred to generate the LFABPf/f mouse. To examine further the role of the endocannabinoid system is mediating the effects of LFABP and IFABP deletion on organismal energy metabolism, WT and the two single KO mice are being used for tissue collection (mucosa, liver, brain, and plasma) following 12 weeks of high fat feeding. (Analyses of endocannabinoids, and expression of synthetic and hydrolytic enzymes and transporters, will commence once sufficient samples have been collected). The mouse model we are studying (LFABP knockout) becomes very obese but nevertheless does not become insulin resistant or hyperglycemic, and in fact it displays improved exercise performance relative to control (wild type) mice. This mouse may be useful as a model of humans who are considered "Metabolically Healthy Obese (MHO)." It is possible that by studying this mouse model, we can understand why some people become obese yet stay healthy, while most who become obese develop serious comorbidities such as diabetes and cardiovascular disease.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Davidson C, Fishman Y, Puskas I, Szeman J, Sohajda T, McCauliff LA, Sikora J, Storch J, Vanier MT, Szente L, Walkley SU and Dobrenis K. Efficacy and ototoxicity of different cyclodextrins in Niemann-Pick C disease. Ann Clin Transl Neurology 3:366-80, 2016.
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Progress 10/01/14 to 09/30/15
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?In the course of these studies, two doctoral students are obtaining training and professional development. Ms. Heli Xu has done the above-mentioned studies characterizing the muscle of the FABP knockouts. She has developed many enzyme assays, analytical procedures, and set up Western blots that were new to the lab (e.g. glycogen determination, phosphor-AMPK, cytochrome oxidase, etc). Mr. Atreju Lackey has focused on examining the intestinal mucosa in the FABP null mice, doing tissue histology (lipid staining and now beginning immunohistochemistry); the training in histology is in the lab of our ongoing collaborator, Dr. Laurie Joseph, with whom we published an examination of the effect of obesity and high fat feeding on the intestine of WT mice. Mr. Lackey is also working on establishing the gene targeting methods for generating the tissue-specific LFABP knockout strains. The lab has experience in generating transgenic mice and in colony breeding and maintenance, and both Ms. Xu and Mr. Lackey are being trained in these procedures. NIH-styleIndividual Development Plans have been developed for both of these doctoral students; indeed the graduate program is instituting the use of these progress reports for all the students. The IDPs contain information on course requirements, research goals and progress, and other training modules including ethical conduct of research and appropriate care of vertebrate animals. Ms. Xu will be taking a graduate writing course either in Spring 2016 or Fall 2016, depending on space availability, as English is not her first language (although she does very well in coursework which includes extensive written work); Mr. Lackey, who is currently being supported by an Underrepresented Minority supplement to an NIH R01 grant to the PI, just completed writing an individual F32 grant application (also to the NIH) that will, if successful, support the final three years of his Ph.D. work. Mr. Lackey's writing skills are excellent, and he did a terrific job developing the proposal, in consultation with the PI. There is no question that the process of searching and writing the grant proposal was a tremendous learning experience for him. Overall, training for both students involves one on one mentoring by the PI and two senior technicians in the lab. For the first six months of this funding period, the students were also mentored by a postdoctoral fellow, Dr. Angela Gajda. Training also includes involvement of the students in biweekly lab meetings, monthly meetings of the Rutgers Center for Lipid Research, and attendance at departmental seminar series. How have the results been disseminated to communities of interest?Publication of our work in peer reviewed journals What do you plan to do during the next reporting period to accomplish the goals?The generation of 'floxed' LFABP mouse strain should be completed, and breeding ongoing with the albumin-Cre mouse to generate liver-specific LFABP null strain, and with the villin-Cre mouse to generate the intestine-specific LFABP null strain. Studies of AEA binding to LFABP, and of 2-AG and AEA binding to IFABP, should be completed. NMR structural data for these complexes will be obtained, and analysis of data commenced. For the single and double knockout mice and the DKO, analyses of fecal lipid composition will be completed; these will augment the results obtained for total fecal fat. Studies of the muscle tissue of the mouse models will also continue, with additional analyses of enzyme mass and activities. Muscle histology will be undertaken to examine mitochondrial morphology. The localization of intramyocellular lipid is of particular interest, given the apparent increase in muscle TG recently found in the LFABP null mouse, in particular. Further studies of food intake, using the home-cage BioDaq system which provides continuous recording of mouse eating, will be undertaken, focusing on apparent differences in eating behavior during the light phase for the LFABP null and the double knockout mice, as recently observed. Expected confirmation of these results will support undertaking studies of Clock machinery components--in the peripheral tissues of enterocyte FABP expression i.e. intestine and liver, as well as in the brain. Initial studies will focus on qPCR determination of gene expression levels in WT and the three experimental lines (LFABP-/-, IFABP-/-, and DKO).
Impacts
What was accomplished under these goals?
We are examining whether LFABP binds the endocannabinoid anandamide (AEA); our previous work showed that LFABP binds 2-arachidonoylglycerol (2-AG), the other major endocannabinoid. As the initial studies using equilibrium binding and isothermal titration calorimetry indicate AEA binding, we are setting up to purify sufficient stable isotope-labeled LFABP to obtain an NMR structure of the LFABP-AEA complex, in collaboration with Dr. Ruth Stark's laboratory. In the IFABP null mouse, we found decreased food intake and altered endocannabinoid levels in intestinal mucosa. We are currently examining whether IFABP binds AEA; it does not appear to bind 2-AG, although we have yet to rule this out completely. We have almost completed our initial round of studies with the DKO (LFABP/IFABP double knockout) mice. We explored the surprising finding that DKO mice showed no dietary lipid malabsorption, even on a 45% kcal fat diet, by using a supraphysiological 60% kcal fat diet. The results show no differences in fecal fat between either single knockout or the DKO, from WT mice fed the 60% fat diet. This experiment lends strong support to our emerging idea that the enterocyte FABPs are not acting to perform bulk lipid processing, as has been assumed for decades. Rather, they are behaving as lipid 'sensors' and perhaps as chaperones, serving to transport ligands to signaling systems and/or transcription factors that then initiate subsequent changes in systemic energy metabolism. We found that the LFABP null mouse became very obese upon feeding of a 45% kcal fat diet, but that it nevertheless remained normoglycemic and in fact, displayed both increased spontaneous activity levels, and markedly enhanced endurance exercise performance. We are tentatively considering the LFABP null mouse to be a model of the 'metabolically healthy but obese" (MHO) state. Owing to the dramatic exercise phenotype, we have begun in-depth studies of the muscle of the LFABP-/- mice. The results to date show increased myocellular triacylglycerol content, which could be providing fuel for enhanced exercise. Further, initial studies show increased lipid oxidation enzyme activities. We are also examining upstream regulators of mitochondrial lipid oxidation, in particular total and phospho-AMPK levels, as well as mitochondrial mass, glycogen stores and metabolism, and muscle mitochondrial fuel selection and metabolism. The fabp1 gene, which encodes for LFABP, is highly expressed in both the liver and intestine. Thus we proposed to generate intestine-specific and liver-specific knockout models to understand the origin of the 'obese but healthy' phenotype of the global knockout. The generation of tissue=specific conditional knockouts is in process, with reagents being made for introducing LoxP sites in the fabp1 gene. We have elected to use CRISPR/Cas9 technology for this, and hope to soon have the 'floxed' mice (LFABPf/f mice) to begin breeding with albumin-Cre and villin-Cre expressing mice, to obtain the liver-specific and small intestine-specific LFABP null mice, respectively.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
McCauliff LA, Xu Z, Li R, Kodukula S, Ko DC, Scott MP, Kahn PC and Storch J. Multiple surface regions on the Niemann-Pick C2 protein facilitate intracellular cholesterol transport. J Biol Chem. 290:27321-31, 2015.
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Douglass JD, Zhou YX, Wu A, Zadrogra JA, Gajda AM, Lackey AI, Lang W, Chevalier KM, Sutton SW, Zhang SP, Flores CM, Connelly MA, and Storch J. Global deletion of monoacylglycerol lipase in mice delays lipid absorption and alters energy homeostasis and diet-induced obesity. J Lipid Res 56:1153-71, 2015.
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Gajda AM and Storch J. Enterocyte Fatty Acid Binding Proteins (FABPs): Different Functions of Liver- and Intestinal- FABPs in the Intestine. Prostaglandins, Leukotrienes & Essential Fatty Acids, 93:9-16, 2015.
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