Animal & Avian Sciences Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/2741
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Item COORDINATED TRAFFICKING OF HEME TRANSPORTERS BY CARGO SORTING COMPLEXES IS ESSENTIAL FOR ORGANISMAL HEME HOMEOSTASIS(2025) Dutt, Sohini; Hamza, Iqbal IH; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Heme, an iron-containing organic ring, is a vital cofactor responsible for diverse biologicalfunctions and is the major source of bioavailable iron in the human diet. As a hydrophobic and cytotoxic cofactor, heme must be transported in a highly controlled manner through membranes via specific intra- and inter-cellular pathways. However, the genes and pathways responsible for heme trafficking remain poorly understood. Unlike other metazoans, Caenorhabditis elegans cannot synthesize heme but requires heme for sustenance. Thus, C. elegans is an ideal animal model to identify heme trafficking pathways as it permits organismal heme homeostasis to be directly manipulated by controlling environmental heme. Heme is imported apically into the intestine by HRG-1-related permeases and exported basolaterally by MRP-5/ABCC5 to extra- intestinal tissues. Loss of mrp-5 causes embryonic lethality that can be suppressed by dietary heme supplementation raising the possibility that MRP-5-independent heme export pathways must exist. Here we show, by performing a forward genetic screen in mrp-5 null mutants, that loss of the vesicular cargo sorting Adaptor Protein complexes (AP-3) fully rescues mrp-5 lethality and restores heme homeostasis. Remarkably, intestinal heme accumulation due to mrp-5-deficiency causes a concomitant deficit in the lysosomal heme importer HRG-1 abundance and localization. Loss of both MRP-5 and AP-3 subunits resurrects HRG-1 levels and localization, thus underscoring the crucial role of HRG-1 in dictating mrp-5 mutant phenotypes. In the absence of MRP-5, heme is exported by SLC49A3 homolog, a previously uncharacterized transporter. Live- cell imaging reveals vesicular coalescence that facilitates heme transfer between the importers and exporters at the interface of lysosomal-related organelle. These results define a mechanistic model for metazoan heme trafficking and identifies SLC49A3 as a promising candidate for heme export in mammals.Item The study of hyperketonemia in the dairy cow.(2023) Barrientos-Blanco, Mario Alberto; Rico, Eduardo; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The metabolic phenomenon of ketosis in dairy cows has remained ambiguous, casting uncertainty over our understanding and its real implications. Ketosis, commonly defined as blood β-hydroxy-butyrate (BHB) ≥ 1.2 mM (i.e., hyperketonemia), has been observationally connected to the onset of peripartal metabolic disorders (e.g., infectious diseases, fatty liver), and reduced milk yield in dairy cows. Although BHB is currently used as standard biomarker for the prediction of negative health and performance outcomes during the peripartum, the nature of this relationship is ambiguous. In contraposition, recent discoveries in mammalian biology indicate BHB as therapeutic metabolite (e.g., alleviation of inflammation and oxidative stress). Our overreaching goal was to study the effects of BHB on dairy cow metabolism and health. In our first study, 6 multiparous (parity = 2.8 ± 0.9) Holstein mid-lactation dairy cows (128 ± 52 days in milk; DIM), were enrolled in a study to evaluate a ketogenic diet using calcium butyrate (—CaBu—; a ruminal ketone precursor) against an un-supplemented control (Control) in a crossover arrangement of treatments. The CaBu resulted in nutritional ketosis (P < 0.05) with blood BHB levels of 0.2 mM higher relative to Control. Although CaBu resulted in reduced dry matter intake (DMI; P < 0.05), milk production was not affected (P > 0.40), and feed efficiencies were improved (P < 0.05) relative to Control. No differences in glucose, NEFA, respiration rates, pain scores, or rectal temperatures were observed between treatments. In the second experiment, 8 multiparous Holstein (2.75 ± 0.89) mid-lactation dairy cows (140 ± 48 DIM), feed ad libitum, were enrolled in a in a crossover arrangement of treatments. The aim of the study was to evaluate the effect of ketones by intravenous infusion of either Na-BHB solution (2.5mM; EK) to sustain hyperketonemia —BHB > 1.2 mM and < 3.0 mM—, or NaCl as a control (2.5mM; Control) over a 72h period. A systemic lipopolysaccharide (LPS) challenge (E. coli 055:B5; 0,085 g/kg BW,) was intravenously administered at h 60 from infusion start. Cows sustained hyperketonemia throughout the 72h experimental period (1.4 BHB mM vs. 0.7 BHB mM in EK vs. Control, respectively). While DMI and milk production were not affected by the BHB infusion, the combination with the LPS challenge resulted in reductions of 20.8% (P < 0.05) and 40.1%, (P = 0.14) for both measurements in EK vs. Control, respectively. No differences were detected in the glucose and NEFA concentrations, but insulin was higher 46.6% (P < 0.05) in EK group. Among the immune markers, IL-1 was 30.8% higher (P < 0.05) in the EK group, and not differences were detected in TNF, IL-10, CRP, and caspase-1. As expected, the LPS challenge induced increased respiration rates, temperature, and pain scores over the time course of the evaluation (P < 0.001); however, respiration rates tended to be reduced in 8.4% (P < 0.1) and rectal temperature increased in 0.3% (P < 0.05) by the BHB treatment (P < 0.05). Our results are indicative that, in the absence of an immune challenge, hyperketonemia results in no negative impact on cow productivity and health. These data add support to our hypothesis that cofactors other than ketones may be necessary for the development of negative trajectories of health and performance of lactating dairy cows. Future studies will be required to confirm that BHB hyperketonemia metabolic effects could differ from ketosis disorder in dairy cows.Item The Importance of Transfer Receptor 1 in Adipose Tissue(2021) Mejia-Guevara, Yasmin; Kim, Byung-Eun; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Iron homeostasis is essential for maintaining cellular function in a variety of cell types. Transferrin receptor 1 (TfR1), which is expressed ubiquitously, facilitates cellular iron (Fe) uptake through receptor-mediated endocytosis of Fe-loaded transferrin. This study was undertaken to evaluate the importance of TfR1-mediated Fe import into adipose tissues for thermogenesis and systemic metabolism. We found that adipose-specific TfR1 knockout mice exhibited severe cold susceptibility upon acute cold exposure, leading to death of the mutant mice within hours. This phenotype was exacerbated by dietary Fe limitation and partially rescued by Fe administration. Knockout mice showed marked defects in oxidative phosphorylation components and lipid droplet homeostasis in adipose tissues. Furthermore, elevated levels of plasma glucose and insulin in the mutant’s hint at an unexpected connection between adipocyte Fe deficiency and diabetes. Altogether, our results suggest that TfR1-mediated Fe uptake is critical for multiple aspects of adipose function and systemic energy metabolism.Item MRP5 AND MRP9 PLAY A CONCERTED ROLE IN MALE REPRODUCTION AND MITOCHONDRIAL FUNCTION(2021) Chambers, Ian George; Hamza, Iqbal; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Heme is an essential iron-containing cofactor in proteins that perform diverse functions in biology. Free heme is not only hydrophobic but also generates cytotoxic peroxide radicals. In eukaryotes, heme synthesis occurs in the mitochondria but must be transported to different intracellular organelles to be utilized by hemoproteins, a process that remains poorly understood. In Caenorhabditis elegans, MRP5/ABCC5 is an essential heme exporter as mrp-5 knockout worms are unviable due to their inability to export nutritional heme from the intestine to extra-intestinal tissues. Heme supplementation restores viability of these mutants but fails to restore male reproductive deficits. By contrast, MRP5 in mammals regulates heme levels in the secretory pathway but shows no reproductive phenotypes. Phylogenetically, the closest homolog of MRP5 in vertebrates is MRP9/ABCC12, which is absent in C. elegans raising the possibility that MRP9 may genetically compensate for MRP5 lossin vertebrates. Here, we show that MRP5 and MRP9 double knockout (DKO) mice are viable but reveal significant male reproductive deficits, reminiscent of mrp-5 worms. Although MRP9 is highly expressed in sperm, MRP9 knockout mice show reproductive phenotypes only when MRP5 is absent. Unlike other ABCC transporters, these proteins localize to mitochondrial-associated membranes (MAMs), dynamic scaffolds that associate the mitochondria and endoplasmic reticulum. Consequently, combined loss of both transporters results in abnormal sperm mitochondria and reduced fertilization rates in DKO mice. Untargeted metabolomics show striking differences in metabolite profiles in the DKO testes, consistent with the localization of these transporters to MAMs where inter-organellar metabolite exchange occurs. RNA-seq results show significant alterations in genes related to mitochondria function and energy production, EIF2 signaling, and retinoic acid metabolism. Targeted functional metabolomics reveal retinoic acid levels are significantly lower in the DKO testes. These findings establish a model in which MRP5 and MRP9 play a concerted role in regulating normal male reproductive functions and mitochondrial sufficiency.