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Transport Mechanisms for the Nutritional Supplement Β-Hydroxy-Β-Methylbutyrate (HMB) in Mammalian Cells

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Transport Mechanisms for the Nutritional Supplement Β-Hydroxy-Β-Methylbutyrate (HMB) in Mammalian Cells Pharm Res (2019) 36: 84 https://doi.org/10.1007/s11095-019-2626-3 RESEARCH PAPER Transport Mechanisms for the Nutritional Supplement β-Hydroxy-β-Methylbutyrate (HMB) in Mammalian Cells Jiro Ogura1 & Toshihiro Sato 1 & Kei Higuchi 1 & Yangzom D. Bhutia1 & Ellappan Babu 1 & Masayuki Masuda2 & Seiji Miyauchi2 & Ricardo Rueda3 & Suzette L. Pereira4 & Vadivel Ganapathy1 Received: 12 February 2019 /Accepted: 8 April 2019 /Published online: 17 April 2019 # Springer Science+Business Media, LLC, part of Springer Nature 2019 ABSTRACT transports HMB, but in a Na+-coupled manner. Other, yet Purpose β-Hydroxy-β-methylbutyrate (HMB), a nutritional unidentified, transporters mediate the major portion of HMB supplement, elicits anabolic activity in muscle. Here we inves- uptake in C2C12 and MCF7 cells. tigated the mechanism of HMB uptake in muscle cells. Methods Murine muscle cells (C2C12) and human mamma- KEY WORDS H+-coupled transport . MCT1 (SLC16A1) . ry epithelial cells (MCF7) were used for uptake. As HMB is a MCT4(SLC16A3) .Na+-coupledtransport .Skeletalmusclecell . monocarboxylate, focus was on monocarboxylate trans- SMCT1 (SLC5A8) porters, monitoring interaction of HMB with H+-coupled lac- tate uptake, and influence of H+ directly on HMB uptake. Involvement of MCT1–4 was studied using selective inhibi- ABBREVIATIONS tors and gene silencing. Involvement of human Na+/mono- AIDS Acquired immune deficiency syndrome carboxylate transporter SMCT1 was also assessed using HMB β-Hydroxy-β-methylbutyrate Xenopus oocytes. HPRT Hypoxanthine/guanine phosphoribosyl transferase + Results H -coupled lactate uptake was inhibited by HMB in KIC α-ketoisocaproate + both mammalian cells. HMB uptake was H -coupled and MCTs H+-Coupled monocarboxylate transporters inhibited by lactate. C2C12 cells expressed MCT1 and OATPs Organic anion transporting polypeptides MCT4; MCF7 cells expressed MCT1–4; undifferentiated OATs Organic anion transporters + C2C12 cells expressed SMCT1. SMCT1 mediated Na - PAH Para-aminohippurate coupled HMB transport. Inhibitors of MCT1/4, siRNA- SLC Solute carrier family mediated gene silencing, and expression pattern showed that SMCTs Na+-coupled monocarboxylate transporters MCT1–4 were responsible only for a small portion of HMB uptake in these cells. Conclusion HMB uptake in C2C12 and MCF7 cells is pri- INTRODUCTION marily H+-coupled and inhibited by lactate, but MCT1–4are − only partly responsible for HMB uptake. SMCT1 also β-Hydroxy-β-methylbutyrate [CH3C(OH)(CH3)CH2COO ]; HMB] is a natural metabolite of the branched chain amino * Vadivel Ganapathy acid leucine (1), produced in small amounts in the body by a [email protected] two-step process starting with the transamination of leucine to α-ketoisocaproate (KIC), followed by conversion of a fraction of 1 Department of Cell Biology and Biochemistry, Texas Tech University the produced KIC to HMB catalyzed by a KIC-dioxygenase Health Sciences Center, 3601 4th Street, Lubbock, TX 79430-6540, (2,3). Studies in animals and humans have demonstrated ben- USA efits of HMB in promoting skeletal muscle anabolism and at- 2 Department of Pharmaceutics, Toho University, Funabashi, tenuating muscle catabolism [Reviewed in Ref. 4–6]. Studies in Chiba 274-8510, Japan young exercising athletes have shown benefits of supplementing 3 Abbott Nutrition, Research & Development, 18004 Granada, Spain HMB with exercise to enhance muscle mass and strength 4 Abbott Nutrition, Research & Development, Columbus, OH 43219, [Reviewed in Ref. 7]. Several animal studies have reported USA on the beneficial effects of HMB in preserving muscle during 84 Page 2 of 14 Pharm Res (2019) 36: 84 catabolic conditions, including cancer cachexia (8), sepsis (9), Aldrich. The MCT1-specific inhibitor SR13800 (2,6-dihydro- immobilization (10), and response to steroids (11). These results 7-[(3-hydroxypropyl)thio]-2-methyl-4-(2-methylpropyl)-6-(1- have been supported by human studies that have demonstrated naphthalenylmethyl)-1H-pyrrolo[3,4-d]pyridazin-1-one) was ob- the benefit of HMB in preserving muscle mass during cancer tained from EMD Millipore Calbiochem (Billerica, MA, USA). cachexia (12), AIDS-related cachexia (13), extended immobili- Cell culture media were from Corning Cellgro (Manassas, VA, zation (14,15), and aging-related muscle loss, even in the ab- USA) and fetal bovine serum was from Atlanta Biologicals senceofexercise(16–18). (Norcross, GA, USA). L-[14C(U)]-Lactate (sodium salt) (specific HMB has a direct effect on muscle cells and modulates vari- radioactivity, 150 mCi/mmol) was purchased from Perkin Elmer ous metabolic pathways in the muscle. This has been demon- (Waltham, MA, USA). [1-14C]- β-Hydroxy-β-methylbutyrate strated in vitro using C2C12 myotubules, as well as in in vivo feed- (sodium salt) (American Radiolabeled Company, St. Louis, ing studies followed by analysis of muscle tissue. HMB has been MO, USA). MCT1 rabbit monoclonal antibody (ab179832) shown to downregulate pathways involved in muscle catabolism was obtained from Abcam (Cambridge, MA, USA). MCT1 rab- such as the ubiquitin-proteasomal degradation pathway (19)and bit polyclonal antibody (PA5–76687) was obtained from the autophagy pathway (20), and to upregulate pathways in- Thermo Fisher Scientific (Waltham, MA, USA). MCT4 rabbit volved in muscle anabolism via activation of mTOR (21,22) polyclonal antibody (LS-C406788) was obtained from LifeSpan and pathways involved in muscle regeneration and repair (23). BioSciences, Inc. (Seattle, WA, USA). β-Actin mouse antibody Upon intake of HMB as a supplement, plasma concentrations (C4) (sc-47,778) was obtained from Santa Cruz Biotechnology, have been shown to reach high micromolar levels in humans (7). Inc. (Dallas, TX, USA). Even though the exact site of action of HMB in muscle cells has not been identified, the plethora of biologic effects of HMB Analysis of SMCT1-Mediated HMB Transport in X. laevis coupled with its chemical nature as a monocarboxylate suggest at Oocytes least some involvement of intracellular actions of HMB in muscle cells. Despite all the existingdataontheeffectsofHMBon We expressed human SMCT1 (SLC5A8) heterologously in muscle, a big gap in our understanding of this molecule is the X. laevis oocytes by cRNA injection and used the oocytes for mechanism by which HMB enters the muscle cell to mediate its electrophysiology 4–5 days following the injection. For this, we effects. HMB, a monocarboxylate with a pKa value of 4.4, is used the oocyte expression plasmid pGH19 with human expected to exist almost entirely in deprotonated form. SLC5A8 cDNA as the insert. The structural features of the Therefore, facilitated transport rather than simple diffusion is cDNA insert are described in our previous publication (27). likely responsible for cellular entry of HMB. The goal of our The transport activity of SMCT1 was monitored in these oo- current study was to identify the transporters responsible for cytes using a two-microelectrode voltage-clamp technique (27). cellular uptake of HMB in muscle tissue. The membrane potential was clamped at −50 mV. Water- Because HMB is a monocarboxylate, we speculated involve- injected oocytes were used as negative controls. In SMCT1- ment of monocarboxylate transporters in the uptake of HMB expressing oocytes, the short-chain fatty acid butyrate was used into muscle cells. There are two well-known classes of trans- the positive control (27). The substrate-induced inward currents porters involved in the cellular uptake of various were used as a measure of SMCT1 transport activity. The monocarboxylates. These include H+-coupled monocarboxyl- ability of SMCT1 to transport HMB was monitored by exam- ate transporters (MCTs) and Na+-coupled monocarboxylate iningifexposureofSMCT1-expressingoocytestoHMBresult- transporters (SMCTs) (24–26). Here we examined the ability ed in an inward current. The relative affinities of SMCT1 for of the Na+-coupled, electrogenic, monocarboxylate transporter butyrate and HMB were determined using the substrate con- SMCT1 (SLC5A8) and the H+-coupled, electroneutral mono- centration versus the substrate-induced current relationship. carboxylate transporters MCT1 and MCT4 to transport HMB Michaelis-Menten equation describing a single saturable trans- in skeletal muscle cells. As MCT1 and MCT4 are known to be port system was used to calculate the kinetic constants for the expressed and play a critical role in tumor cells (24), we used the transport process. As the expression levels for SMCT1 varied human breast cancer cell line MCF7 as a control to evaluate the from oocyte to oocyte, the substrate-induced currents were nor- involvement of these two transporters in HMB uptake. malized by taking the maximum current induced by the sub- strate as 1 in each oocyte. The experiments were repeated at least with 5 different oocytes from at least two different animals. MATERIALS AND METHODS The composition of the oocyte-perfusion buffer for the analysis of substrate-induced currents was: 3 mM HEPES/3 mM Materials MES/3 mM Tris (pH 7.5), 100 mM NaCl, 2 mM KCl, + 1mMMgCl2,and1mMCaCl2.ForNa -free buffer, NaCl L-Lactate (sodium salt), butyrate (sodium salt), β-hydroxy-β- in the buffer was substituted iso-osmotically with N-methyl-D- methylbutyric acid (free acid), were purchased from Sigma glucamine chloride. Pharm Res (2019) 36: 84 Page 3 of 14 84 Cell Lines and Culture Conditions kit (Thermo Fisher Scientific) with bovine serum albumin as a standard. Fifteen micrograms of protein were subjected to Uptake studies were performed
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