Project description:Age-related and/or high caloric intake driven changes in visceral adipose tissue contribute to the development of diabetes and cardiovascular disease. The lack of reliable, high-throughput methods to analyze the adipose tissue protein composition has limited our understanding of the protein networks responsible for metabolic regulation of various adipose depots. We have developed a proteomic approach using multiple-dimension liquid chromatography tandem mass spectrometry and multiplexed TMT labeling to analyze protein composition of epididymal adipose tissues isolated from mice fed either low or high fat diet for a short (8 weeks) or a long (18 weeks) term, and from mice that aged (26 weeks old) on low vs. high fat diets. Advancing age and high-fat diet feeding led to graded deterioration, with long-term high fat diet exposure being the worst of all measures of peripheral metabolic health such as body weight, adiposity, plasma fasting glucose, insulin, triglycerides, total cholesterol levels, and glucose and insulin tolerance tests. Epididymal adipose depot proteomic analysis identified >3300 proteins per sample. In response to short-term high fat diet, 43 proteins representing lipid metabolism (e.g., AACS, ACOX1, ACLY) and red-ox pathways (e.g., CPD2, CYP2E, SOD3) were significantly altered (FDR < 10%). Long-term high fat diet significantly altered 55 proteins associated with immune response (e.g., IGTB2, IFIT3, LGALS1) and rennin angiotensin system (e.g. ENPEP, CMA1, CPA3, ANPEP). Age-related changes on low fat diet significantly altered only 18 proteins representing mainly urea cycle (e.g., OTC, ARG1, CPS1), and amino acid biosynthesis (e.g., GMT, AKR1C6). Surprisingly, high fat diet driven age-related changes culminated with alterations in 155 proteins involving primarily the urea cycle (e.g., ARG1, CPS1), immune response/complement activation (e.g., C3, C4b, C8, C9, CFB, CFH, FGA), extracellular remodeling (e.g., EFEMP1, FBN1, FBN2, LTBP4, FERMT2, ECM1, EMILIN2, ITIH3) and apoptosis (e.g., YAP1, HIP1, NDRG1, PRKCD, MUL1) pathways. Our unbiased mass spectrometry method, tailored to adipose tissue, identified both age-related and high fat diet specific proteomic signatures. In addition to well-described immune and extracellular remodeling response to high fat feeding, we have uncovered the pronounced involvement of arginine metabolism in response to advancing age, and branched chain amino acid metabolism in early response to high fat feeding. We were able to uncouple age-related metabolic responses from those associated with caloric intake, a task otherwise difficult to achieve.

Project description:Preussin (Asperidine B) has recently shown its lipid-lowering effects by inhibiting intestinal absorption in human colorectal adenocarcinoma (Caco-2) cells and ex vivo intestinal loop in rats. While statins are widely prescribed as the primary line drug of choice for lowering hyperlipidemia, ezetimibe is the only drug that reduces intestinal cholesterol absorption, and is also optional for statin-nonresponsive individuals. To gain more understanding of the mechanisms of action and lead compounds, the derivatives of preussin: TP3, TP4, TP5, TP6, and TP7 have recently been synthesized, and the structures were confirmed using 1D and 2D nuclear magnetic resonance (NMR) spectroscopy. However, whether these derivatives exhibited inhibitory effects on cholesterol absorption is unknown. Thus, this study aims to investigate the most potent inhibitory effect of cholesterol absorption of preussin derivatives using fluorescent-micelle cholesterol transport in intestinal Caco-2 cells, confirmed by an in vivo cholesterol absorption assay. Possible targets of the lead compounds were identified using a protein binding assay and label-free quantification using proteomic analyses. Results showed that all tested preussin derivatives markedly inhibited cholesterol absorption in the intestinal Caco2 cells to a similar extent to preussin and ezetimibe. TP5 (preussin B) consistently displayed a significant reduction of plasma cholesterol, identical to preussin and ezetimibe, with the same potency in rats. In addition, the proteomic analysis found that substantial differentially expressed changes in six proteins are involved in the lipid metabolism pathway. Interestingly, GOT2, as one of these six proteins in the lipid transport pathway, consistently interacts with TP5. The molecular docking and dynamic prediction showed that TP5 coupled with GOT2 at a pocket comparable to its cofactor. These findings suggest that preussin B has therapeutic potential as a candidate for a cholesterol absorption inhibitor to treat hyperlipidemia.

Project description:Age-related and/or high caloric intake driven changes in visceral adipose tissue contribute to the development of diabetes and cardiovascular disease. The lack of reliable, high-throughput methods to analyze the adipose tissue protein composition has limited our understanding of the protein networks responsible for metabolic regulation of various adipose depots. We have developed a proteomic approach using multiple-dimension liquid chromatography tandem mass spectrometry and multiplexed TMT labeling to analyze protein composition of epididymal adipose tissues isolated from mice fed either low or high fat diet for a short (8 weeks) or a long (18 weeks) term, and from mice that aged (26 weeks old) on low vs. high fat diets. Advancing age and high-fat diet feeding led to graded deterioration, with long-term high fat diet exposure being the worst of all measures of peripheral metabolic health such as body weight, adiposity, plasma fasting glucose, insulin, triglycerides, total cholesterol levels, and glucose and insulin tolerance tests. Epididymal adipose depot proteomic analysis identified >3300 proteins per sample. In response to short-term high fat diet, 43 proteins representing lipid metabolism (e.g., AACS, ACOX1, ACLY) and red-ox pathways (e.g., CPD2, CYP2E, SOD3) were significantly altered (FDR < 10%). Long-term high fat diet significantly altered 55 proteins associated with immune response (e.g., IGTB2, IFIT3, LGALS1) and rennin angiotensin system (e.g. ENPEP, CMA1, CPA3, ANPEP). Age-related changes on low fat diet significantly altered only 18 proteins representing mainly urea cycle (e.g., OTC, ARG1, CPS1), and amino acid biosynthesis (e.g., GMT, AKR1C6). Surprisingly, high fat diet driven age-related changes culminated with alterations in 155 proteins involving primarily the urea cycle (e.g., ARG1, CPS1), immune response/complement activation (e.g., C3, C4b, C8, C9, CFB, CFH, FGA), extracellular remodeling (e.g., EFEMP1, FBN1, FBN2, LTBP4, FERMT2, ECM1, EMILIN2, ITIH3) and apoptosis (e.g., YAP1, HIP1, NDRG1, PRKCD, MUL1) pathways. Our unbiased mass spectrometry method, tailored to adipose tissue, identified both age-related and high fat diet specific proteomic signatures. In addition to well-described immune and extracellular remodeling response to high fat feeding, we have uncovered the pronounced involvement of arginine metabolism in response to advancing age, and branched chain amino acid metabolism in early response to high fat feeding. We were able to uncouple age-related metabolic responses from those associated with caloric intake, a task otherwise difficult to achieve.

			Action	DRS
	a_MTBLS12719_LC-MS_negative_reverse-phase_metabolite_profiling.txt	Txt
	a_MTBLS12719_LC-MS_positive_reverse-phase_metabolite_profiling.txt	Txt
	i_Investigation.txt	Txt
	m_MTBLS12719_LC-MS_negative_reverse-phase_metabolite_profiling_v2_maf.tsv	Tabular
	m_MTBLS12719_LC-MS_positive_reverse-phase_metabolite_profiling_v2_maf.tsv	Tabular

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Metabolic interactions of “catecholamine-cholesterol derivatives” as a potential mechanism for immunomodulation of chicken adipose tissue

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