Chemicals and Reagents.

Specific synthetic peptides and corresponding stable labeled peptides for OCT1, Na⁺/K⁺ ATPase, and calreticulin were purchased form New England Peptides (Boston, MA). Bicinchoninic acid assay kit, dithiothreitol, iodoacetamide, mass spectrometry–grade trypsin, pierce cell-surface protein isolation kit, High-performance liquid chromatography-grade acetonitrile, Dulbecco’s phosphate-buffered saline, and Dulbecco’s modified Eagle’s medium high-glucose medium were purchased form Thermo Fisher Scientific (Rockford, IL). Metformin (hydrochloride salt), thiamine (hydrochloride salt), quinidine, potassium chloride, magnesium sulfate, d-glucose, HEPES, sodium chloride, sodium bicarbonate, calcium chloride dipotassium phosphate, and formic acid were purchased from Sigma-Aldrich (St. Louis, MO). [¹⁴C]Metformin hydrochloride (114 mCi/mmol) was obtained from Moravek Biochemicals, Inc. (Brea, CA). Collagen-coated 24-well plates were purchased from Corning (Kennebunk, ME). Human hepatocyte thaw medium, INVITROGRO HI medium, INVITROGRO CP medium, and TORPEDO antibiotic mix, as well as cryopreserved human hepatocytes (PHH), were generously provided by BioIVT (Westbury, NY). OCT1-HEK293 cells were generously provided by SOLVO Biotechnology (Hungary).

Determination of Metformin Uptake CL_int into OCT1-HEK293 Cells and Human Hepatocytes (PHH).

OCT1-HEK293 cells were grown in T-75 cm² flasks with 20 ml of high-glucose Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 2 mM l-glutamine, 100 IU/ml penicillin, and 100 mg/ml streptomycin. Medium was changed daily, and cells were kept in a humidified incubator with 5% carbon dioxide at 37°C. For uptake assays, 0.5 million HEK293 cells per well were plated in a 24-well plate for 24 hours. For uptake assays using PHH, cryopreserved hepatocytes were thawed and plated 0.35 million per well in a 24-well collagen-coated plate with 0.5 ml/well TORPEDO containing INVITROGRO CP medium (1:9 v/v) for 5 hours.

To determine [¹⁴C]metformin uptake, HEK293 cells or PHH were washed three times with HEPES-buffered Krebs-Ringer bicarbonate buffer (HK buffer) (4.8 mM KCl, 1.2 mM K₂HPO₄, 1.2 mM MgSO₄, 5.6 mM glucose, 25 mM HEPES, 12 mM CaCl₂, 125 mM NaCl, 25 mM NaHCO₃) and incubated at 37°C. To initiate uptake, 500 μl of HK buffer containing 5 or 20 μM [¹⁴C]metformin (for HEK293 cells or PHH, respectively) with or without inhibitor (preincubated at 37°C) was added to the cells. After an appropriate time (over a period in which uptake was linear; 5 minutes for HEK293 cells and 15 minutes for PHH), [¹⁴C]metformin uptake into the cells was terminated by removing [¹⁴C]metformin-containing solution and immediately washing it three times with ice-cold HK buffer. Then, cells were lysed by adding 1 ml of 2% SDS solution to each well and incubating them on a shaker at 125 rpm for 1 hour at 37°C. After 1 hour, 700 μl of lysed solution from each well was used to measure radioactivity by Tri-Carb Liquid Scintillation Counters (PerkinElmer, Waltham, MA). In addition, 50 μl of lysed solution was used to measure protein content using the bicinchoninic acid assay method. [¹⁴C]Metformin uptake CL into the cells was calculated by the ratio of rate of [¹⁴C]metformin uptake and the [¹⁴C]metformin concentration in the media. Uptake CL_{int,in vitro} was calculated by dividing the slope of [¹⁴C]metformin accumulation over time by [¹⁴C]metformin concentration in media. CL_int calculated in absence and presence of inhibitor was considered as total CL (CL_int,total) and passive CL (CL_{int,in vitro,passive}), respectively. Active CL (CL_{int,in vitro,active}) was calculated by subtracting CL_{int,in vitro,passive} from CL_{int,in vitro,total.}

To determine the contribution of OCT1 or thiamine transporter 2 (THTR2) to [¹⁴C]metformin transport into PHH, [¹⁴C]metformin uptake assays were conducted in the presence or absence of 500 μM quinidine or 400 mM thiamine [a THTR2 substrate with Km of 3.4 mM (Liang et al., 2015)], respectively.

Quantification of Total and Percent Plasma Membrane Abundance of OCT1 in HEK293 Cells.

Data for the total and PMA of OCT1 in PHH were obtained from our previous publication (Kumar et al., 2019). OCT1 total and PMA in HEK293 cells was quantified using our previously published biotinylation approach (Kumar et al., 2017). HEK293 cells were grown in three independent experiments in a 75-cm² flask until 80% confluency and incubated at 37°C with 10 ml of 0.78 mg/ml sulfo-NHS-SS-biotin. After 1 hour, cells were lysed, and 50 μl of cell lysate was separated to quantify total abundance. From the rest of the total cell lysate, the biotinylated plasma membrane fragments (to quantify %PMA) were separated from the total cell lysate and intracellular membranes using the neutravidin resin columns. Briefly, 0.2 mg of total protein homogenate and biotinylated fraction were denatured by adding 45 μl of 100 mM ammonium bicarbonate buffer (pH 7.8), 25 μl of 3% sodium deoxycholate (w/v), 12.5 μl of 250 mM dithiothreitol, and 5 μl of 10 mg/ml human albumin and incubated at 95°C for 5 minutes. After incubation, samples were returned to room temperature, 25 μl of 200 mM iodoacetamide was added, and samples were incubated in the dark for 30 minutes. Then, 500 μl of ice-cold methanol, 200 μl of chloroform, and 450 μl of water were added. Samples were mixed and centrifuged at 12,000g for 10 minutes. Precipitated protein samples were redissolved in 45 μl of 100 mM ammonium bicarbonate buffer and digested with 20 μl (∼3.2 μg) of trypsin for 18 hours at 37°C in a shaker (300 rpm). Finally, the digestion was terminated by adding to the sample 20 μl of ice-cold acetonitrile containing the internal standard. Then, the sample was centrifuged at 5000g for 5 minutes at 4°C. The supernatant was collected and analyzed on AB Sciex 6500 triple-quadrupole mass spectrometer (Sciex, Framingham, MA) coupled to the Water Acquity Ultra-Performance Liquid Chromatography (UPLC) system (Waters Corporation, Milford, MA) operated in electrospray positive ionization mode using a previously described approach (Prasad et al., 2016). An Acquity UPLC HSS T3 Column (1.8 mm, 2.1 100 mm) with 0.2-mm inlet frits (Waters) was used for chromatographic separation and resolution. The flow rate of the mobile phase was 0.3 ml/min using a gradient ranging from 3% to 90% acetonitrile/water containing 0.1% formic acid. Signature peptides and product ions as well as their respective Stable Isotope Labelled (SIL) peptides used to quantify proteins were obtained from our previous report (Kumar et al., 2019). The ratio of abundance of protein in biotinylated fractions and total lysate was used to determine %PMA.

Estimation of In Vivo [¹¹C]Metformin CL_h,s,in by Compartmental Modeling of PET Imaging Data.

PET-imaged metformin hepatic radioactivity concentration-time data previously published by Gormsen et al. (2016) were reanalyzed using a blood input function and a one-tissue compartment model (Fig. 3A) with a fixed blood volume (SAAMII). This differed from the published analysis, in which radioactivity from the volume of blood within the liver tissue was not taken into account, plasma concentrations were used as input, and a two-compartment model was used (Gormsen et al., 2016).(1)(2)where A, C, B, L, , and represent amount, concentration, blood, liver, sinusoidal uptake CL, sinusoidal efflux CL, biliary efflux CL, and metabolic CL respectively. Since no biliary efflux was observed and the majority of metformin is excreted renally unchanged, biliary clearance and metabolic clearance were not included in eq. 1 (Hardie, 2007; Graham et al., 2011; Gormsen et al., 2016). The hepatic input blood concentration is a sum of the input via the portal vein (80%) and the hepatic artery (20%) (Schenk et al., 1962), where and are the observed [¹¹C]metformin arterial and portal vein concentrations, respectively (eq. 2). The volume of the liver was determined based on eq. 3 (Chan et al., 2006).(3)For male and female, sex = 1 and 0, respectively. The volume of blood in the liver was fixed to 10% of total blood volume, estimated based on body weight (75 ml/kg for males and 65 ml/kg for females) (Lautt, 1977; Stabin and Siegel, 2003; Pham and Shaz, 2013). Goodness of fit of the model to the data was assessed by the weighted residual plots, visual inspection of the predicted and observed data, and the confidence (CV%) in the estimates of the parameters.

Prediction of the In Vivo Metformin Blood Hepatic Uptake CL from PHH Using the MGPGL Approach.

Metformin in vivo blood intrinsic hepatic sinusoidal uptake CL () was predicted from PHH data using the MGPGL approach (eq. 4).(4)Liver weight was assumed to be 1500 g, B:P is the blood-to-plasma ratio (0.6), and MGPGL was 120 mg of total protein per gram of liver based on a previous report (1.2 × 10⁸ cells per gram of liver and 1 million cells ∼1 mg protein) (Jones et al., 2012; Gormsen et al., 2016; Kim et al., 2019).

Then, metformin sinusoidal uptake clearance was computed using the well stirred model (WSHM).(5)where fu is the fraction of metformin unbound in plasma (assumed to be 1), and Q is the hepatic blood flow (1500 ml/min) (Scheen, 1996).

Prediction of In Vivo Metformin Hepatic Uptake CL from OCT1- HEK293 Cells and PHH Using the REF Approach.

In vivo blood metformin hepatic intrinsic sinusoidal active uptake CL (CL_{int,s,active in}) was determined from in vitro metformin uptake CL in OCT1-HEK293 cells or PHH using the REF approach, corrected for OCT1 PMA in these cells or PHH (eq. 6). In doing so, the following assumptions were made: 1) OCT1 is the only transporter responsible for metformin hepatic uptake (see Results for justification) and 2) because it is impossible to use the biotinylation method for quantification of OCT1 PMA in liver tissue, the PMA abundance of OCT1 in liver tissue was assumed to be 100%.(6) is OCT1 abundance in HEK293 cells or PHH. is total OCT1 abundance in human liver tissue, and MGMPGL is milligrams of total membrane protein per gram of liver from individual control liver (n = 36) obtained from our previous report (Wang et al., 2016).

Also, the blood hepatic intrinsic sinusoidal passive uptake CL of metformin () was scaled based on liver weight and adjusted for the sinusoidal surface area (SSA) (37%; eq. 7) since the entire plasma membrane of hepatocytes is not exposed to blood (Esteller, 2008). The correction for SSA was not made for the active uptake of metformin into cells since OCT1 is thought to be present only on the sinusoidal membrane of the hepatocytes.(7)Finally, the total blood intrinsic hepatic sinusoidal uptake CL () was computed (eq. 8) to estimate the sinusoidal hepatic uptake clearance () using the WSHM (eq. 5).(8)Data are presented as means of REF-predicted based on 36 control livers, whereas the mean MGPGL-predicted was computed as a mean of four different lots of PHH.