Consequences of NaCT/SLC13A5/mINDY deficiency: good versus evil, separated only by the blood-brain barrier.

NaCT/SLC13A5 is a Na+-coupled transporter for citrate in hepatocytes, neurons, and testes. It is also called mINDY (mammalian ortholog of 'I'm Not Dead Yet' in Drosophila). Deletion of Slc13a5 in mice leads to an advantageous phenotype, protecting against diet-induced obesity, and diabetes. In contrast, loss-of-function mutations in SLC13A5 in humans cause a severe disease, EIEE25/DEE25 (early infantile epileptic encephalopathy-25/developmental epileptic encephalopathy-25). The difference between mice and humans in the consequences of the transporter deficiency is intriguing but probably explainable by the species-specific differences in the functional features of the transporter. Mouse Slc13a5 is a low-capacity transporter, whereas human SLC13A5 is a high-capacity transporter, thus leading to quantitative differences in citrate entry into cells via the transporter. These findings raise doubts as to the utility of mouse models to evaluate NaCT biology in humans. NaCT-mediated citrate entry in the liver impacts fatty acid and cholesterol synthesis, fatty acid oxidation, glycolysis, and gluconeogenesis; in neurons, this process is essential for the synthesis of the neurotransmitters glutamate, GABA, and acetylcholine. Thus, SLC13A5 deficiency protects against obesity and diabetes based on what the transporter does in hepatocytes, but leads to severe brain deficits based on what the transporter does in neurons. These beneficial versus detrimental effects of SLC13A5 deficiency are separable only by the blood-brain barrier. Can we harness the beneficial effects of SLC13A5 deficiency without the detrimental effects? In theory, this should be feasible with selective inhibitors of NaCT, which work only in the liver and do not get across the blood-brain barrier.

Functional analysis of a species-specific inhibitor selective for human Na+-coupled citrate transporter (NaCT/SLC13A5/mINDY).

The Na+-coupled citrate transporter (NaCT/SLC13A5/mINDY) in the liver delivers citrate from the blood into hepatocytes. As citrate is a key metabolite and regulator of multiple biochemical pathways, deletion of Slc13a5 in mice protects against diet-induced obesity, diabetes, and metabolic syndrome. Silencing the transporter suppresses hepatocellular carcinoma. Therefore, selective blockers of NaCT hold the potential to treat various diseases. Here we report on the characteristics of one such inhibitor, BI01383298. It is known that BI01383298 is a high-affinity inhibitor selective for human NaCT with no effect on mouse NaCT. Here we show that this compound is an irreversible and non-competitive inhibitor of human NaCT, thus describing the first irreversible inhibitor for this transporter. The mouse NaCT is not affected by this compound. The inhibition of human NaCT by BI01383298 is evident for the constitutively expressed transporter in HepG2 cells and for the ectopically expressed human NaCT in HEK293 cells. The IC50 is ∼100 nM, representing the highest potency among the NaCT inhibitors known to date. Exposure of HepG2 cells to this inhibitor results in decreased cell proliferation. We performed molecular modeling of the 3D-structures of human and mouse NaCTs using the crystal structure of a humanized variant of VcINDY as the template, and docking studies to identify the amino acid residues involved in the binding of citrate and BI01383298. These studies provide insight into the probable bases for the differential effects of the inhibitor on human NaCT versus mouse NaCT as well as for the marked species-specific difference in citrate affinity.

Plasma Membrane Na⁺-Coupled Citrate Transporter (SLC13A5) and Neonatal Epileptic Encephalopathy.

SLC13A5 is a Na⁺-coupled transporter for citrate that is expressed in the plasma membrane of specific cell types in the liver, testis, and brain. It is an electrogenic transporter with a Na⁺:citrate3- stoichiometry of 4:1. In humans, the Michaelis constant for SLC13A5 to transport citrate is ~600 µM, which is physiologically relevant given that the normal concentration of citrate in plasma is in the range of 150-200 µM. Li⁺ stimulates the transport function of human SLC13A5 at concentrations that are in the therapeutic range in patients on lithium therapy. Human SLC13A5 differs from rodent Slc13a5 in two important aspects: the affinity of the human transporter for citrate is ~30-fold less than that of the rodent transporter, thus making human SLC13A5 a low-affinity/high-capacity transporter and the rodent Slc13a5 a high-affinity/low-capacity transporter. In the liver, SLC13A5 is expressed exclusively in the sinusoidal membrane of the hepatocytes, where it plays a role in the uptake of circulating citrate from the sinusoidal blood for metabolic use. In the testis, the transporter is expressed only in spermatozoa, which is also only in the mid piece where mitochondria are located; the likely function of the transporter in spermatozoa is to mediate the uptake of citrate present at high levels in the seminal fluid for subsequent metabolism in the sperm mitochondria to generate biological energy, thereby supporting sperm motility. In the brain, the transporter is expressed mostly in neurons. As astrocytes secrete citrate into extracellular medium, the potential function of SLC13A5 in neurons is to mediate the uptake of circulating citrate and astrocyte-released citrate for subsequent metabolism. Slc13a5-knockout mice have been generated; these mice do not have any overt phenotype but are resistant to experimentally induced metabolic syndrome. Recently however, loss-of-function mutations in human SLC13A5 have been found to cause severe epilepsy and encephalopathy early in life. Interestingly, there is no evidence of epilepsy or encephalopathy in Slc13a5-knockout mice, underlining the significant differences in clinical consequences of the loss of function of this transporter between humans and mice. The markedly different biochemical features of human SLC13A5 and mouse Slc13a5 likely contribute to these differences between humans and mice with regard to the metabolic consequences of the transporter deficiency. The exact molecular mechanisms by which the functional deficiency of the citrate transporter causes epilepsy and impairs neuronal development and function remain to be elucidated, but available literature implicate both dysfunction of GABA (γ-aminobutyrate) signaling and hyperfunction of NMDA (N-methyl-d-aspartate) receptor signaling. Plausible synaptic mechanisms linking loss-of-function mutations in SLC13A5 to epilepsy are discussed.

Ira Remsen, Osler, the Flexner Report, and the full-time plan.

A famous letter from Sir William Osler to Ira Remsen, dated September 1, 1911, concerns Osler's objections to the full-time plan, whereby clinical professors should focus on research and abstain from private practice. Previous accounts of this well-known episode make little or no mention of the recipient other than his being president of the Johns Hopkins University. Remsen, in retrospect, was uniquely positioned to champion ideas advanced by Abraham Flexner, the General Education Board of the Rockefeller Foundation, and some of Osler's former colleagues at Johns Hopkins, notably William H. Welch. Remsen had previously expressed the need for Hopkins to advance science-based medicine; he had introduced Abraham Flexner to the Carnegie Foundation (which led to the Flexner Report); and he appears to have been the first US-born person to possess both a medical degree and a doctorate in basic science. Caught in the middle of a faculty controversy, Remsen chose not to pursue the matter further for reasons that included a passive administrative style, concerns about his health, and friendship with Osler.

Sir William Osler: A forerunner of mindfulness in medical practice.

Increasing work hours, patient loads, and regulations have increased burnout among health care professionals, forcing many to neglect their own physical and emotional well-being. In response, several health care organizations are encouraging physicians to adopt mindfulness practices to reduce burnout and difficulties maintaining work-life balance. It is unclear whether mindfulness will improve health outcomes and patient satisfaction or will become another passing trend. With the stakes so high, this discussion should involve the founder of our modern education system, Sir William Osler.

An interview with Sir William Osler on resident work hours and physician responsibility in the 21st century.

Since 2003, duty hour regulations (DHRs) imposed by the Accreditation Council on Graduate Medical Education (ACGME) have been a controversial issue in the medical community. The conflict touches upon the ethical principles of beneficence, nonmaleficence, justice, and the activities of every physician. However, the controversy concerning DHR presents opportunities for reevaluating a physician's roles, responsibilities, and work-life balance in the 21st century. In this article, the DHR controversy is discussed through the thoughts and ideas of Sir William Osler using an interview format. An internal medicine residency program director adds comments to this discussion based on his understanding of current ACGME regulations.