Bryan A. Wilson

Diet-Gene Interactions and PUFA Metabolism: A Potential Contributor to Health Disparities and Human Diseases

The “modern western” diet (MWD) has increased the onset and progression of chronic human diseases as qualitatively and quantitatively maladaptive dietary components give rise to obesity and destructive gene-diet interactions. There has been a three-fold increase in dietary levels of the omega-6 (n-6) 18 carbon (C18), polyunsaturated fatty acid (PUFA) linoleic acid (LA; 18:2n-6), with the addition of cooking oils and processed foods to the MWD. Intense debate has emerged regarding the impact of this increase on human health. Recent studies have uncovered population-related genetic variation in the LCPUFA biosynthetic pathway (especially within the fatty acid desaturase gene (FADS) cluster) that is associated with levels of circulating and tissue PUFAs and several biomarkers and clinical endpoints of cardiovascular disease (CVD). Importantly, populations of African descent have higher frequencies of variants associated with elevated levels of arachidonic acid (ARA), CVD biomarkers and disease endpoints. Additionally, nutrigenomic interactions between dietary n-6 PUFAs and variants in genes that encode for enzymes that mobilize and metabolize ARA to eicosanoids have been identified. These observations raise important questions of whether gene-PUFA interactions are differentially driving the risk of cardiovascular and other diseases in diverse populations, and contributing to health disparities, especially in African American populations.

Evidence for a mitochondrial angiotensin-(1–7) system in the kidney

Evidence for an intracellular renin-angiotensin system (RAS) in various cell organelles now includes the endoplasmic reticulum, nucleus, and mitochondria (Mito). Indeed, angiotensin (ANG) AT1 and AT2 receptor subtypes were functionally linked to Mito respiration and nitric oxide production, respectively, in previous studies. We undertook a biochemical analysis of the Mito RAS from male and female sheep kidney cortex. Mito were isolated by differential centrifugation followed by a discontinuous Percoll gradient and were coenriched in Mito membrane markers VDAC and ATP synthase, but not β-actin or cathepsin B. Two distinct renin antibodies identified a 37-kDa protein band in Mito; angiotensinogen (Aogen) conversion was abolished by the inhibitor aliskiren. Mito Aogen was detected by an Aogen antibody to an internal sequence of the protein, but not with an antibody directed against the ANG I N terminus. ANG peptides were quantified by three direct RIAs; mitochondrial ANG II and ANG-(1-7) contents were higher compared with ANG I (23 ± 8 and 58 ± 17 vs. 2 ± 1 fmol/mg protein; P < 0.01, n = 3). 125I-ANG I metabolism primarily revealed the formation of 125I-ANG-(1-7) in Mito that reflects the endopeptidases neprilysin and thimet oligopeptidase. Last, immunoblot studies utilizing the ANG-(1-7)/Mas receptor antibody revealed the protein in isolated Mito from sheep renal cortex. Collectively, the current data demonstrate that Mito actively metabolize the RAS precursor protein Aogen, suggesting that ANG-(1-7) may be generated within Mito to establish an intramitochondrial RAS tone and contribute to renal mitochondrial function.

An angiotensin-(1–7) peptidase in the kidney cortex, proximal tubules, and human HK-2 epithelial cells that is distinct from insulin-degrading enzyme

Angiotensin 1-7 [ANG-(1-7)] is expressed within the kidney and exhibits renoprotective actions that antagonize the inflammatory, fibrotic, and pro-oxidant effects of ANG II. We previously identified an peptidase that preferentially metabolized ANG-(1-7) to ANG-(1-4) in the brain medulla and cerebrospinal fluid (CSF) of sheep (Marshall AC, Pirro NT, Rose JC, Diz DI, Chappell MC. J Neurochem 130: 313-323, 2014); thus the present study established the expression of the peptidase in the kidney. Utilizing a sensitive HPLC-based approach, we demonstrate a peptidase activity that hydrolyzed ANG-(1-7) to ANG-(1-4) in the sheep cortex, isolated tubules, and human HK-2 renal epithelial cells. The peptidase was markedly sensitive to the metallopeptidase inhibitor JMV-390; human HK-2 cells expressed subnanomolar sensitivity (IC50 = 0.5 nM) and the highest specific activity (123 ± 5 fmol·min(-1)·mg(-1)) compared with the tubules (96 ± 12 fmol·min(-1)·mg(-1)) and cortex (107 ± 9 fmol·min(-1)·mg(-1)). The peptidase was purified 41-fold from HK-2 cells; the activity was sensitive to JMV-390, the chelator o-phenanthroline, and the mercury-containing compound p-chloromercuribenzoic acid (PCMB), but not to selective inhibitors against neprilysin, neurolysin and thimet oligopeptidase. Both ANG-(1-7) and its endogenous analog [Ala(1)]-ANG-(1-7) (alamandine) were preferentially hydrolyzed by the peptidase compared with ANG II, [Asp(1)]-ANG II, ANG I, and ANG-(1-12). Although the ANG-(1-7) peptidase and insulin-degrading enzyme (IDE) share similar inhibitor characteristics of a metallothiolendopeptidase, we demonstrate marked differences in substrate specificity, which suggest these peptidases are distinct. We conclude that an ANG-(1-7) peptidase is expressed within the renal proximal tubule and may play a potential role in the renal renin-angiotensin system to regulate ANG-(1-7) tone.

The Ins and Outs of Angiotensin Processing within the Kidney

The kidney is a key target organ for bioactive components of the renin-angiotensin system (RAS); however, various renal cells such as the tubular epithelium contain an intrinsic RAS. The renal RAS can be functionally divided into ANG II-AT1 receptor and ANG-(1-7)-AT7/Mas receptor arms that functionally oppose one another. The current review considers both extracellular and intracellular pathways that potentially govern the formation and metabolism of angiotensin peptides within the renal proximal tubules.

Angiotensinogen Import in Isolated Proximal Tubules: Evidence for Mitochondrial Trafficking and Uptake

The renal proximal tubules are a key functional component of the kidney and express the angiotensin precursor angiotensinogen; however, it is unclear the extent that tubular angiotensinogen reflects local synthesis or internalization. Therefore, the current study established the extent to which angiotensinogen is internalized by proximal tubules and the intracellular distribution. Proximal tubules were isolated from the kidney cortex of male sheep by enzymatic digestion and a discontinuous Percoll gradient. Tubules were incubated with radiolabeled 125I-angiotensinogen for 2 h at 37°C in serum/phenol-free DMEM/F12 media. Approximately 10% of exogenous 125I-angiotensinogen was internalized by sheep tubules. Subcellular fractionation revealed that 21 ± 4% of the internalized 125I-angiotensinogen associated with the mitochondrial fraction with additional labeling evident in the nucleus (60 ± 7%), endoplasmic reticulum (4 ± 0.5%), and cytosol (15 ± 4%; n = 4). Subsequent studies determined whether mitochondria directly internalized 125I-angiotensinogen using isolated mitochondria from renal cortex and human HK-2 proximal tubule cells. Sheep cortical and HK-2 mitochondria internalized 125I-angiotensinogen at a comparable rate of (33 ± 9 vs. 21 ± 10 pmol·min-1·mg protein-1; n = 3). Lastly, unlabeled angiotensinogen (100 nM) competed for 125I-angiotensinogen uptake to a greater extent than human albumin in HK-2 mitochondria (60 ± 2 vs. 16 ± 13%; P < 0.05, n = 3). Collectively, our data demonstrate angiotensinogen import and subsequent trafficking to the mitochondria in proximal tubules. We conclude that this pathway may constitute a source of the angiotensinogen precursor for the mitochondrial expression of angiotensin peptides.

Identification of dipeptidyl peptidase 3 as the Angiotensin-(1-7) degrading peptidase in human HK-2 renal epithelial cells.

Angiotensin-(1-7) (Ang-(1-7)) is expressed within the kidney and exhibits renoprotective actions that antagonize the inflammatory, fibrotic and pro-oxidant effects of the Ang II-AT1 receptor axis. We previously identified a peptidase activity from sheep brain, proximal tubules and human HK-2 proximal tubule cells that metabolized Ang-(1-7); thus, the present study isolated and identified the Ang-(1-7) peptidase. Utilizing ion exchange and hydrophobic interaction chromatography, a single 80kDa protein band on SDS-PAGE was purified from HK-2 cells. The 80kDa band was excised, the tryptic digest peptides analyzed by LC-MS and a protein was identified as the enzyme dipeptidyl peptidase 3 (DPP 3, EC: 3.4.14.4). A human DPP 3 antibody identified a single 80kDa band in the purified enzyme preparation identical to recombinant human DPP 3. Both the purified Ang-(1-7) peptidase and DPP 3 exhibited an identical hydrolysis profile of Ang-(1-7) and both activities were abolished by the metallopeptidase inhibitor JMV-390. DPP 3 sequentially hydrolyzed Ang-(1-7) to Ang-(3-7) and rapidly converted Ang-(3-7) to Ang-(5-7). Kinetic analysis revealed that Ang-(3-7) was hydrolyzed at a greater rate than Ang-(1-7) [17.9 vs. 5.5 nmol/min/μg protein], and the Km for Ang-(3-7) was lower than Ang-(1-7) [3 vs. 12μM]. Finally, chronic treatment of the HK-2 cells with 20nM JMV-390 reduced intracellular DPP 3 activity and tended to augment the cellular levels of Ang-(1-7). We conclude that DPP 3 may influence the cellular expression of Ang-(1-7) and potentially reflect a therapeutic target to augment the actions of the peptide.

Sir Hans Adolf Krebs: Architect of Metabolic Cycles

Figure 2 Sir Hans Adolf Krebs (1900–1981) Sir Hans Adolf Krebs was born August 25, 1900, at the dawn of a new century in Hildesheim, Germany. He was the son of Dr. Georg Krebs, an ear, nose, and throat surgeon, and his wife Alma Davidson. As a young child, Krebs attended Lutheran schools, despite his Jewish heritage; his parents rarely even mentioned Judaism in the household. Hans Krebs went on to study medicine at the Universities of Gottingen, Freiburg-im-Breisgau, and Berlin. In 1925, he earned his MD degree at the University of Munich. Following his medical education, Dr. Krebs spent an additional year studying chemistry in Berlin. In 1926, he was appointed assistant to Professor Otto Warburg at the Kaiser Wilhelm Institute for Biology. He studied under Dr. Otto Warburg for 4 years before returning to clinical work. Later in his career, Dr. Krebs shared his sincere gratitude for Dr. Warburg’s mentoring and training in research. In Dr. Warburg’s laboratory, he learned manometry to measure oxygen consumption of tissue slices, which allowed the precise investigation of biochemical (metabolic) pathways from animal tissues. These techniques were essential tools Dr. Krebs later used to discover the citric acid cycle and other novel metabolic pathways. Between 1930 and 1933, Dr. Krebs practiced medicine at the Municipal Hospital at Altona under Professor L. Lichtwitz as well as Professor S. J. Thannhauser at the Medical Clinic of the University of Freiburg-im-Breisgau. In addition to his clinical duties, Dr. Krebs embarked on research in metabolism. One of the waste products the body must rid itself of is nitrogen, which it does by making and excreting urea. At the time it was known that urea production occurred in the liver, however the underlying pathways involved in urea metabolism were not defined. Without urea metabolism, the body does not have …

Assessment of the Renin–Angiotensin System in Cellular Organelle: New Arenas for Study in the Mitochondria

The renin-angiotensin system (RAS) is an important hormonal system composed of various protein and peptide components that contribute to blood pressure regulation. Although originally characterized as a circulating system, there is increasing evidence for the intracellular expression of RAS elements on the nucleus and mitochondria that may function in concert with or independent of the circulating system. The present chapter describes several experimental approaches to quantify the expression of RAS components in isolated mitochondria from the kidney. These approaches are intended to provide a framework to understand the mitochondrial RAS within a cell-free environment.

Peptidases and the Renin-Angiotensin System: The Alternative Angiotensin-(1-7) Cascade

The renin-angiotensin system (RAS) constitutes a key hormonal system in the physiological regulation of blood pressure via peripheral and central mechanisms. Dysregulation of the RAS is considered a major factor in the development of cardiovascular pathologies, and pharmacologic blockades of this system by the inhibition of angiotensin-converting enzyme (ACE) or antagonism of the angiotensin type 1 receptor (AT1R) are effective therapeutic regimens. The RAS is now defined as a system composed of different angiotensin peptides with diverse biological actions mediated by distinct receptor subtypes. The classic RAS comprises the ACE-Ang IIAT1R axis that promotes vasoconstriction, water intake, sodium retention and increased oxidative stress, fibrosis, cellular growth, and inflammation. The nonclassical or alternative RAS is composed primarily of the ACE2-Ang-(1-7)-AT7R pathway that opposes the Ang II-AT1R axis. In lieu of the complex aspects of this system, the current review assesses the enzymatic cascade of the alternative Ang-(1-7) axis of the RAS.

Percy Lavon Julian

Percy Lavon Julian (1899–1975) The contributions of Dr. Julian to the field of medicinal chemistry began humbly at the turn of the century in the segregated American South. Percy Lavon Julian was born on April 11, 1899, in Montgomery, AL. His formal public education was limited, while his secondary education ended at the eighth grade which was the norm for African Americans at that time. His father, who had been denied the opportunity to go to college, made it a priority to ensure that his children received this opportunity.14 Julian went on to complete two years of teacher training at the State Normal School for Negroes in Montgomery. In 1916, Julian became the first in his family to attend college when he was admitted to DePauw University in Green-castle, IN. During his first two years of college, Julian took remedial classes in addition to his college courses to make up for the academic deficiencies resulting from his limited primary education. Despite this, Julian was an excellent student in college and was elected as a member of both the Phi Beta Kappa and Sigma Xi honor societies. In 1920, Julian earned a bachelor’s degree in chemistry and was named valedictorian of his class. DePauw University circa 1916. A few African American students had been accepted to DePauw before Julian arrived; however, the culture was far from accepting. When he arrived, Julian was taken to off-campus housing because the dorms on campus were for “whites only.” Furthermore, it took a day and a half for Julian to find a diner that would serve him. This would be the beginning of many unwelcoming environments Julian would overcome and thrive in. Despite overcoming the barrier of obtaining a college education denied to his father, there were many more obstacles for Julian to overcome …