Since the dawn of agriculture man has been genetically modifying crop plants to increase yield, quality and utility. In addition to selective breeding and hybridization we can utilize mutant populations and biotechnology to have greater control over crop plant modification than ever before. Increasing the production of plant oils such as soybean oil as a renewable resource for food and fuel is valuable. Successful breeding for higher oil levels in soybean, however, usually results in reduced protein, a second valuable seed component. We show that by manipulating a highly active acyl-CoA: diacylglycerol acyltransferase (DGAT) the hydrocarbon flux to oil in oilseeds can be increased without reducing the protein component. Compared to other plant DGATs, a DGAT from Vernonia galamensis (VgDGAT1A) produces much higher oil synthesis and accumulation activity in yeast, insect cells and soybean. Soybean lines expressing VgDGAT1A show a 4% increase in oil content without reductions in seed protein contents or yield per unit land area. Furthermore, we have screened a soybean fast neutrino population derived from M92-220 variety and found three high oil mutants that do not have reduced levels of protein. From the F2 plant populations we quantitatively pooled the high oil and low oil plants and performed comparative genomics hybridization (CGH). From the data it appears that two families have a 0.3 kb aberration in chromosome 14. We are performing further analysis to study this aberration and develop markers for molecular breeding. Mutagenic techniques are also useful for developing other traits such as early flowering varieties and adapting new high oil crops to a new region. Chia (Salvia hispanica) is an ancient crop that has experienced an agricultural resurgence in recent decades due to the high omega 3 fatty acid (ω-3) content of the seeds and good production potential. The area of cultivation has been expanded to Kentucky using mutagenized populations and the composition traits are similar to that of the original regions of cultivation in Central and South America.

New soybean lines have been developed with significantly higher oil, protein + oil and higher meal protein. These soybeans contain a VgD1 gene (highly active acyl-CoA:diacylglycerol acyltransferase, DGAT from Vernonia galamensis (VgDGAT1A) produces much higher oil synthesis and accumulation activity in soybean. Soybean with active DGAT from Vernonia galamensis (VgDGAT1A) has active TAG biosynthesis relative to other DGATs including from soybeans and Arabidopsis. DGATs catalyze the final step of TAG synthesis: DAG (diacylglycerol) + acyl-CoA → TAG + CoASH (Coenzyme A is notable for its role in the synthesis and oxidation of fatty acids, and the oxidation of pyruvate in the citric acid cycle). A thorough analysis of the major components in VgD1 lines, especially those of nutritional or anti-nutritional value including what else changed (decreased); and what remained at normal levels was conducted. A field study was conducted in Spindletop and Princeton KY, reviled no reduction in yield nor protein, and about 4 % (DW) more oil was obtained in Princeton and 2% (DW) in Spindeltop. No consistent reduction in the other seed composition.VgDGAT1A soybean lines indicated noticeably early maturation compared to the parental line. This is associated with higher expression of the flowering genes FT2 (FLOWERING LOCUS T2) and FT5 (FLOWERING LOCUS T5), for the high oil lines. A single recessive mutation in soybean (MIPS) myo-inositol 1-phosphate synthase, confers a seed phenotype of increase inorganic phosphate (Pi) crossed with high oil lines expressing a DGAT from Vernonia galamensis (VgDGAT1A) (VgD). The oil and protein were maintained compart to VgD. VgD X MIPS (VM), had 21.2, and 22 % oil in 2015, and 23.3 and 24.0 oil in 2016, and protein 46, 49 in 2015, and 37 and 39 % in 2016. Phosphate results suggesting the cross MV is still segregating for MIPS and more selection and planting are needed. Measurement of seed phosphate levels is an established technique for screening for low phytate mutants but to date, it has not been performed non-destructively from single soybean seeds. A protocol was developed greatly reducing the sample size thereby reducing the cost and time and saving a generation in the selection of low phytate mutant seeds based on the high Pi phenotype. Genotyping single seeds are useful in breeding and genetics while maintaining high germination rates. Nondestructive single-seed genomic DNA extraction protocols using 12 mg cotyledon tissue with a modified cetyl trimethyl ammonium bromide (CTAB) technique and a commercial seed DNA extraction kit using 1 mg cotyledon tissue were developed for dry soybean seeds and cross-verified with leaf DNA analysis.

A growing body of evidence implicates the importance of the endogenous cannabinoid 2-arachidonyl glycerol (2-AG) in memory regulation. The biosynthesis of 2-AG occurs primarily through the diacylglycerol lipases (DAGL-α and -β), with 2-AG serving as a bioactive lipid to both activate cannabinoid receptors and as a rate limiting precursor for the production of arachidonic acid and subsequent pro-inflammatory mediators. Gene deletion of DAGL-α shows decrements in synaptic plasticity and hippocampal neurogenesis suggesting this biosynthetic enzyme may be important for processes of normal spatial memory. Additionally, 2-AG is elevated in response to pathogenic events such as traumatic brain injury (TBI), suggesting its regulatory role may extend to conditions of neuropathology. As such, this dissertation investigates the in vivo role of DAGL-α and -β to regulate spatial learning and memory in the healthy brain and following neuropathology (TBI). The first part of this dissertation developed a mouse model of learning and memory impairment following TBI, using hippocampal-dependent tasks of the Morris water maze (MWM). We found modest, but distinct differences in MWM performance between left and right unilateral TBI despite similar motor deficits, histological damage, and glial reactivity. These findings suggest that laterality in mouse MWM deficit might be an important consideration when modeling TBI-induced functional consequences. The second part of this dissertation work evaluated DAGL-β as a target to protect against TBI-induced learning and memory deficit given its selective expression on microglia and the role of 2-AG as a precursor for eicosanoid production. The gene deletion of DAGL-β did not protect against TBI-induced MWM or motor deficits, but unexpectedly produced a survival protective phenotype. These findings suggest that while DAGL-β does not contribute to injury-induced memory deficit, it may contribute to TBI-induced mortality. The third and final set of experiments investigated the role of DAGL-α in mouse spatial learning and memory under physiological conditions (given the predominantly neuronal expression of DAGL-α). Complementary pharmacological and genetic manipulations produced task specific impaired MWM performance, as well as impaired long-term potentiation and alterations to endocannabinoid lipid levels. These results suggest that DAGL-α may play a selective role in the integration of new spatial information in the normal mouse brain. Overall, these data point to DAGL-α, but not DAGL-β, as an important contributor to hippocampal-dependent learning and memory. In contrast, DAGL-β may contribute to TBI-induced mortality.

Neutrophil granulocytes play an important role in the host defence against invading microorganisms and constitute the frontline of defence within the innate immune system and are among the first cells to arrive at the site of inflammation. Effective phagocytosis and killing of invading pathogens by neutrophils is of significant importance for successful resistance to infectious diseases. An important complication in diabetes mellitus is an increased sensitivity to infections and increased tissue damage, leading to many secondary diseases. This may in part be explained by an impaired function of neutrophil granulocytes. Since the exact mechanisms underlying defective neutrophil function in diabetes mellitus are not fully understood, the aim of the present study was to investigate the effects of elevated glucose and insulin concentrations on phagocytosis of opsonized yeast and on production of reactive oxygen metabolites (ROS) in normal human neutrophils. Elevated D-glucose concentrations (15-25 mM) inhibited the phagocytosis of C3bi- or IgG-opsonized yeast particles, which was neither an osmotic effect nor an effect due to reduced binding of opsonized yeast particles to the neutrophils. Inhibition of protein kinase C (PKC) by GF109203X or Go6976 could completely reverse the inhibitory effect of 25 mM D-glucose on phagocytosis. Diacylglycerol (DAG) dose-dependently inhibited phagocytosis and suboptimal inhibitory concentrations of DAG and glucose showed an additive inhibitory effect. Elevated concentrations of insulin (80-160 μU/ml) also inhibited neutrophil phagocytosis, an effect shown in part to be due to a delayed phagocytosis process. Insulin was found to increase the accumulation of cortical F-actin, without affecting the total cellular F-actin content. The PKCalpha/beta inhibitor, Go6976, abolished the insulin-mediated increase in cortical F-actin content and both Go6976 and the PKCalpha/beta/delta/epsilon-specific inhibitor GF109203X reversed the inhibitory effects of insulin on phagocytosis. The inhibition of phagocytosis by either glucose or insulin resulted in an expected reduction of intracellular respiratory burst. However, the extracellular release of ROS during phagocytosis was increased by insulin, but inhibited by glucose. The ability of insulin to enhance ROS production was found to be F-actin dependent. Data suggests that glucose inhibited intracellular respiratory burst activation by interfering with intracellular signaling downstream of PKC activation, whereas extracellular release of ROS was inhibited by glucose upstream of PKC signaling. Taken together these results suggest that both hyperglycemia and hyperinsulinemia inhibit complement receptor and Fc receptor-mediated phagocytosis in human neutrophils. Insulin, but not glucose, also induced an enhanced extracellular release of ROS during phagocytosis. The combination of reduced phagocytosis and alterations in ROS production may possibly explain both the increased sensitivity to infections and tissue damage seen in type 2 diabetes.

The neuropeptides orexin-A and orexin-B were discovered in 1998 and were first described as regulators of feeding behaviour. Later research has shown that they have an important role in the regulation of sleep. Two G protein-coupled receptors, OX1 and OX2 orexin receptors, mediate the cellular responses to orexins. The overall aim of this thesis was to investigate the OX1 orexin receptors signalling to phospholipases. Previous investigations have determined that orexin receptors induce Ca2+ elevations through both receptor-operated Ca2+ channels (ROCs) and store-operated Ca2+ channels (SOCs). In this thesis we investigated the importance of these influxpathways on orexin-mediated phospholipase (PLC) activation. The results demonstrate that ROC influx is enough to fully support orexin-stimulated PLC activation but that SOC influx has a further amplifying role. We also investigated the metabolites generated after PLC activation, inositolphosphates and diacylglycerol (DAG). The results indicate involvement of two different PLC activities with different substrate specificities one of them leading to DAG production without co-occurring IP3 production at low orexin receptor stimulation. The results also suggest that at even lower orexin receptor stimulation DAG is produced via the activation of phospholipase D. In this thesis we also investigated if the ubiquitous phospholipase A2 (PLA2) signalling system is involved in orexin receptor signalling. The results demonstrate that stimulation of the OX1 orexin receptors leads to arachidonic acid (AA) release. This release is fully dependent on Ca2+ influx, probably through ROC, and at the same time the studies demonstrate that ROC influx is partly dependent on PLA2 activation. At low orexin receptor activation the AA release seemed to in part rely on extracellular signal-regulated kinase. We also devised two methods to aid in these investigations. The first method enabled studies of the receptor-operated Ca2+ influx without interference of the co-occurring store-operated Ca2+ influx. This was done by the expression of IP3-metabolising enzymes IP3-3-kinase-A and IP3-5-phosphatase-I. The second method enables quantification of DAG and IP3 signalling in fixed cells using GFP-fused indicators, leading to a semi-quantitative but easily applicable pharmacological assay.

Obesity is the most common clinical disorder and is associated with various medical conditions in dogs. Appropriate dietary management potentially provides weight loss in a safe, healthy, and efficacious manner. In order to elucidate whether dietary fiber, carnitine, diacylglycerol (DAG), and low glycemic index (LGI) act on such dietary components, a series of studies was conducted: 1) the combination of dietary fiber/carnitine effect on short term (3 and 7 h) satiety and long term (6 weeks) canine weight loss, 2) the combination of dietary LGI/high glycemic index (HGI) starches and DAG/triacylglycerol (TAG) effect during a 9 week canine weight loss period, and 3) the DAG effect on triglyceride rich lipoprotein (TRL) metabolism isolated from canine plasma 3-4 h postprandially. The combination of dietary fiber/carnitine supplementation decreased both food and energy intake at 3 h post-feeding, suggesting that this combination diet provided 3 h post-meal satiety. This combination supplement also increased postprandial plasma B- hydroxybutyrate (BHB) at d 42 and body fat and weight loss at d 42 from baseline. This combination supplement did not alter plasma vitamin A distributions or concentrations although it contained high vitamin A as B-carotene. In the second study, the LGI diets resulted in a more pronounced body weight loss than the HGI diets due to lower diet digestibilities. These data are consistent with LGI diets decreasing metabolizable energy and consequently consuming less energy compared to the HGI diets. The DAG diets lowered postprandial plasma TAG at weeks 1 and 8 in and increased plasma BHB at week 8, suggesting an increase in fat oxidation. The combination of DAG/LGI decreased postprandial total cholesterol at week 8. Lipoprotein concentrations were not altered by diet types. Fasting lipoprotein lipase (LPL) and hepatic lipase (HL) activities were not affected by diets. In the final study, DAG ingestion decreased TRL and plasma TAG concentrations vs. TAG ingestion. The DAG enriched meal increased non-esterified fatty acid, monoacylglycerol, and 1,3-DAG and decreased TAG in TRLs which may be attributed to larger TRL particle size compared to the TAG meal. Consequently, the DAG derived TRLs showed increased affinity of core TAG for LPL and HL in vitro. Moreover, the intravenous injection of the DAG derived canine TRLs into mice underwent more rapid blood clearance associated with the greater hepatic uptake compared to the TAG derived TRL injection. In conclusion, the combination of dietary fiber/carnitine and DAG/LGI preferably reduced body weight and stimulated fat oxidation, which promotes overall weight loss. The postprandial plasma TAG lowering effect of DAG is the result, at least partially, from the efficient clearance of TRLs from blood circulation and their ability to act as a more efficient substrate for plasma lipolytic enzymes.

Master of Science / Biochemistry and Molecular Biophysics Interdepartmental Program / Timothy P. Durrett / Many Euonymus species express an acetyltransferase enzyme in their seeds which catalyzes the transfer of an acetyl group from acetyl-CoA to the sn-3 position of diacylglycerol (DAG) producing unusual acetyl-1,2-diacyl-sn-glycerols (acetyl-TAG). The presence of the sn-3 acetate group gives acetyl-TAG with unique physical properties over regular triacylglycerol (TAG) found in vegetable oils. The useful characteristics of acetyl-TAG oil offer advantages for its use as emulsifiers, lubricants, and 'drop-in' biofuels. One enzyme, Euonymus alatus diacylglycerol acetyltransferase (EaDAcT), responsible for acetyl-TAG synthesis in nature was previously isolated from the seeds of Euonymus alatus (burning bush) and expressed in the oilseed crop Camelina sativa. Expression of EaDAcT successfully led to production of high levels of acetyl-TAG in camelina seeds. To further increase acetyl-TAG accumulation in transgenic camelina seeds, multiple strategies were examined in this study. Expression of a new acetyltransferase enzyme (EfDAcT) isolated from the seeds of Euonymus fortunei, which was previously shown to possess higher in vitro activity and in vivo acetyl-TAG levels compared to EaDAcT, increased acetyl-TAG accumulation by 20 mol%. Suppression of the endogenous competing enzyme DGAT1 further enhanced acetyl-TAG accumulation to 90 mol% in selected transgenic line. Studying the regulation of EfDAcT transcript, protein, and acetyl-TAG levels during seed development further provided new insights on the factors limiting acetyl-TAG accumulation.

BACKGROUND: Lymphangioleiomyomatosis (LAM) is a rare disease characterized by cystic destruction of the lung. It occurs in 80% of people with Tuberous Sclerosis Complex disorder (TSC), a multisystem, autosomal dominant disorder caused by mutations in tumor suppressor genes TSC1 and TSC2. Spontaneous biallelic mutations in these genes can give rise to sporadic LAM. Mammalian target of rapamycin complex I (mTORC1), a master regulator of cellular anabolic metabolism is hyperactivated in LAM cells. Upregulation of protein synthesis and downregulation of autophagy creates a state of starvation stress that upregulates pathways of extracellular nutrient acquisition. Macropinocytosis, a form of clathrin-independent endocytosis, is upregulated in TSC2-deficient cells. We performed a high-throughput compound screen utilizing a repurposing drug library. We identified that ritanserin, a diacylglycerol kinase alpha (DGKA) inhibitor, synergizes with Chloroquine (CQ) to selectively inhibit proliferation of TSC2-deficient mouse embryonic fibroblasts (MEFs) compared to TSC2+/+ MEFs. OBJECTIVE: We hypothesized that TSC2-deficient cells rely on macropinocytosis to support their growth during the periods of stress and starvation and that ritanserin synergizes with CQ to inhibit proliferation in TSC2-deficient cells by inhibiting macropinocytosis. METHODS: Crystal violet-based proliferation assays were used to monitor the effect of pharmacological and genetic inhibition of DGKA on cell proliferation. Immunoblotting was used to measure the expression levels of TSC2, tS6R, pS6R, Cleaved PARP, Cleaved Caspase 3 and Actin. siRNA induced Htr2a knockdown and shRNA induced DGKA knockdown cell culture models were used to define the dual functions of ritanserin and observe their effects on macropinocytosis and cell proliferation. LC/MS was used to measure cell lipid content and how it changes in response to ritanserin. Fluorophore-labeled BSA and 70-kDa Dextran were used to measure macropinocytosis. Lysotracker was used to measure the number of lysosomes, while DQ-BSA was used to measure lysosomal functionality. RESULTS: TSC2-deficient cells express higher levels and show upregulated activity of DGKA. Genetic and pharmacologic inhibition of DGKA prevents TSC2-deficient cells from acquiring nutrients via macropinocytosis. Phospholipid metabolism is altered in TSC2-deficient cells, marked by the accumulation of phosphatidic acid and ceramides. Treatment with ritanserin leads to the accumulation of diacylglycerol and phospholipids, as well as a reduction in phosphatidic acid. CONCLUSIONS: TSC2-deficient cells rely on macropinocytosis to meet their metabolic needs. Diacylglycerol kinase alpha (DGKA) is required for macropinocytic nutrient uptake. Pharmacologic or genetic inhibition of DGKA creates metabolic stress in TSC2-deficient cells, which ultimately leads to increased apoptotic response to treatment with CQ. This project identifies a novel connection between mTOR signaling, lysosome metabolism and macropinocytosis, and a vulnerability that allows the selective targeting of LAM cells. / 2021-06-07T00:00:00Z

Chronic surgical denervation of the rat vas deferens leads to an enhanced contractile response of the tissue to norepinephrine in vitro. Norepinephrine produces a higher rate of protein kinase C translocation to the particulate fraction of denervated tissues as compared with the paired, control vas deferens. Diacylglycerol generation in response to norepinephrine and contractile responses to phorbol diacetate were not altered by chronic denervation of the vas deferens. However, the contractile response to norepinephrine in these tissues was less susceptible to the inhibitory effects of the calcium channel blocker nifedipine. A potential role of protein kinase C in sensitizing the contractile apparatus to mobilized calcium in denervation supersensitivity is discussed.

A variety of plants synthesize and store oil in the form of triacylglycerols (TAG) in their seed and nonseed tissues that are commonly used as vegetable oils. In seed tissues, an acyl CoA-dependent diacylglycerol (DAG) acyltransferase (DGAT) and/or -independent phospholipid:DGAT (PDAT) catalyze the conversion of DAG to TAG. In avocado fruit, which stores up to 70% oil by dry weight in mesocarp, it is hypothesized that both DGAT and PDAT are likely involved in TAG synthesis. To investigate, TAG content and composition and transcript levels for the acyltransferases in avocado fruit were quantified by gas chromatography and real-time polymerase chain reaction, respectively. Temporal, tissue-specific and phenotypic comparisons revealed that while DGAT1 gene expression was specifically associated with TAG accumulation, PDAT also correlated with higher levels of polyunsaturated fatty acid; DGAT2 was barely detectable. These studies suggest that TAG biosynthesis in nonseed tissues of avocado involves acyl CoA-dependent and -independent reactions.