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. 2024 Dec 18;10(51):eads5466. doi: 10.1126/sciadv.ads5466

Metformin targets mitochondrial complex I to lower blood glucose levels

Colleen R Reczek 1,*, Ram P Chakrabarty 1, Karis B D’Alessandro 1, Zachary L Sebo 1, Rogan A Grant 1, Peng Gao 2, Scott Budinger 1, Navdeep S Chandel 1,3,4,*
PMCID: PMC11654692  PMID: 39693440

Abstract

Metformin is among the most prescribed antidiabetic drugs, but the primary molecular mechanism by which metformin lowers blood glucose levels is unknown. Previous studies have proposed numerous mechanisms by which acute metformin lowers blood glucose, including the inhibition of mitochondrial complex I of the electron transport chain (ETC). Here, we used transgenic mice that globally express the Saccharomyces cerevisiae internal alternative NADH dehydrogenase (NDI1) protein to determine whether the glucose-lowering effect of acute oral administration of metformin requires inhibition of mitochondrial complex I of the ETC in vivo. NDI1 is a yeast NADH dehydrogenase enzyme that complements the loss of mammalian mitochondrial complex I electron transport function and is insensitive to pharmacologic mitochondrial complex I inhibitors including metformin. We demonstrate that NDI1 expression attenuates metformin’s ability to lower blood glucose levels under standard chow and high-fat diet conditions. Our results indicate that acute oral administration of metformin targets mitochondrial complex I to lower blood glucose.

Acute oral metformin administration lowers blood glucose levels by targeting mitochondrial complex I.

INTRODUCTION

Type 2 diabetes mellitus (T2DM) is a metabolic disorder characterized by high blood glucose and insulin resistance (1). The biguanide drug metformin has been a first-line treatment for individuals with T2DM and prediabetes for decades and is prescribed to more than 200 million people worldwide (2, 3). Metformin is thought to lower circulating blood glucose levels by inhibiting glucose production in the liver and augmenting glucose uptake by enterocytes in the intestine (4–6). In addition, metformin enhances the production of GDF15 in the intestines and kidneys (7–9), as well as the production of lactate-phenylalanine (Lac-Phe) in the intestines (10, 11), both of which are associated with weight loss. The molecular mechanisms by which metformin exerts its effects in these tissues is not fully understood (4). Investigators have attributed metformin’s ability to lower blood glucose levels to inhibition of mitochondrial complex I of the electron transport chain (ETC) (12), inhibition of mitochondrial glycerophosphate dehydrogenase (13, 14), and binding to the lysosomal PEN2 protein (15). These mechanistic discrepancies for metformin action may stem from the various means of metformin delivery and the differing metformin dosing amounts used across studies (5). Humans take a metformin pill orally, with the highest drug exposure to the intestines followed by the liver and kidney (16–18). In contrast, an intravenous injection of metformin bypasses the intestines and alters the pharmacokinetic properties of the drug (16, 19). A critical experiment still lacking in the field involves creating mice with mutations in the putative intracellular targets of metformin that allow these targets to function normally while being resistant to metformin’s inhibitory effects (20). Consequently, much of the current data rely on inferences about which specific targets metformin inhibits.

Metformin has been shown to reversibly inhibit mitochondrial complex I in vitro (12, 21–23). Recent structural data revealed potential metformin binding sites on mammalian mitochondrial complex I (24). Previously, our laboratory has found that expression of the Saccharomyces cerevisiae NDI1 protein can complement loss of the 45-subunit mammalian mitochondrial complex I electron transport function in cancer cells (21, 25), macrophages (26), neurons (27), and alveolar epithelial cells (28). NDI1 is resistant to mitochondrial complex I inhibitors like metformin, rotenone, IACS-010759, and piericidin A in vitro and in vivo (21, 29–32). Although inhibition of mitochondrial complex I has been proposed as a mechanism by which metformin lowers blood glucose levels, this hypothesis has not been assessed in vivo (4). Here, we tested the necessity of mitochondrial complex I inhibition by metformin to lower blood glucose levels by using mice that globally express NDI1.

RESULTS

Mice expressing NDI1 globally display normal glucose homeostasis

The S. cerevisiae internal alternative NADH dehydrogenase protein NDI1 is a homodimeric enzyme that catalyzes the oxidation of NADH in the mitochondrial matrix, passing those electrons to coenzyme Q similar to the 45-subunit mammalian mitochondrial complex I (Fig. 1A) (33, 34). Unlike mammalian mitochondrial complex I, NDI1 is insensitive to metformin and is unable to proton pump or generate reactive oxygen species (ROS). Therefore, to investigate whether metformin lowers blood glucose levels through inhibition of mitochondrial complex I in vivo, we generated a transgenic conditional whole-body NDI1-expressing mouse. We crossed homozygous β-actin-cre (BAC+/+) mice, which ubiquitously express Cre-recombinase under the control of the human beta actin gene promoter by the blastocyst stage of embryonic development, with mice that have the Lox-Stop-Lox (LSL)–NDI1 targeting construct inserted into the mouse Rosa26 locus (NDI1LSL/WT) (27). Littermate BAC+/− (hereafter referred to as control) mice and NDI1LSL/WT BAC+/− (hereafter referred to as NDI1) mice were born at the expected (50%) Mendelian ratio, and NDI1 mice were indistinguishable in size from their control littermates, indicating that whole-body expression of NDI1 does not affect embryonic development. It is important to note that endogenous mammalian mitochondrial complex I is present and functional in the NDI1 mice.

Fig. 1. NDI1 mice display normal glucose homeostasis.

Fig. 1.

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(A) Schematic representation of the mammalian mitochondrial electron transport chain containing the yeast NDI1 protein. Created with BioRender.com. (B) NDI1 mRNA expression in the livers of control and NDI1 mice. n = 13 control mice and n = 8 NDI1 mice (q = 3.7 × 10−39, Wald test). (C) MA plot of mRNA expression changes between control and NDI1 mice. n = 13 control mice and n = 8 NDI1 mice. Genes colored red are significantly up-regulated in NDI1 mice and genes colored blue are significantly down-regulated in NDI1 mice (q < 0.05, Wald test). (D and E) Multiple cohorts of control and NDI1 mice, 8 to 12 weeks of age and on a standard chow diet, were fasted overnight for 16 to 18 hours. Mice were then split into two groups, fasted and refed. Blood was taken either immediately (fasted) or 4 hours after standard chow diet food was provided (refed). Blood glucose (D) and plasma insulin (E) levels were assayed from these blood samples. n = 8 control fasted, n = 9 control refed, n = 6 NDI1 fasted, and n = 6 NDI1 refed in both (D) and (E). Results represent mean ± SEM. Two-way ANOVA (Bonferroni) shows no significance (n.s.) between genotypes within the fasted and refed groups. n.s.P > 0.9999 [(D); fasted], n.s.P = 0.5822 [(D); refed], n.s.P > 0.9999 [(E); fasted and refed]. (F) Glucose tolerance (2 g/kg, oral gavage) was assessed on multiple cohorts of 8- to 12-week-old standard chow diet–fed control and NDI1 mice fasted overnight for 16 to 18 hours. n = 17 control mice and n = 12 NDI1 mice. Results represent mean ± SEM. (G) Incremental area under the curve (iAUC) for the GTT in (F). Results represent mean + SEM. Statistical significance was determined using a two-tailed, unpaired t test and an α level of 0.05. n.s. means nonsignificant, n.s.P = 0.6853.

To confirm NDI1 expression in the NDI1 mice, we performed bulk RNA sequencing (RNA-seq) on the liver tissue from control and NDI1 mice. As expected, yeast NDI1 mRNA was only expressed in the liver tissue from NDI1 mice and not in the liver tissue from control mice (Fig. 1B). Of note, there was variability observed in the levels of NDI1 mRNA expression among the mice that express the NDI1 transgene. However, whole-body expression of NDI1 had only minimal transcriptomic impact in the liver compared to control mice (Fig. 1C). To determine whether NDI1 expression affects normal glucose homeostasis, we compared the blood glucose levels of control and NDI1 mice under fasted and refed conditions (4 hours ad libitum food access following an overnight fast). NDI1 mice displayed blood glucose levels similar to those of control mice in both the fasted and refed state (Fig. 1D). Blood glucose levels increased following refeeding as expected. Moreover, the NDI1 mice exhibited similar fasted and refed plasma insulin levels as control mice (Fig. 1E). Next, we performed a glucose tolerance test (GTT) on control and NDI1 mice. The mice were fasted overnight and then administered a bolus of glucose by oral gavage (2 g/kg body weight). Blood glucose levels were measured over a 2-hour period. NDI1 mice had a similar acute oral glucose tolerance compared to control mice (Fig. 1, F and G). Thus, at baseline, whole-body NDI1-expressing mice display normal glucose homeostasis and are indistinguishable from control mice.

NDI1 expression in mice attenuates metformin’s blood glucose–lowering effect

The transport of metformin into cells requires a family of plasma membrane organic cation transporters (OCTs) 1 to 3 and the plasma membrane monoamine transporter (PMAT) (19, 35, 36). To exclude the possibility that NDI1 expression alters metformin uptake into the liver and intestines, we measured metformin levels in liver and intestine homogenates after oral metformin administration. Control and NDI1 global-expressing mice had comparable levels of metformin in both the liver and intestines (Fig. 2, A and B, respectively). Thus, the global expression of NDI1 had no significant effect on the ability of the tissues to take up metformin.

Fig. 2. NDI1 expression attenuates the blood glucose–lowering effects of metformin in mice fed a standard chow diet.

Fig. 2.

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(A and B) Metformin levels were measured in the livers (A) and intestines (B) of 12- to 16-week-old standard chow diet–fed control and NDI1 mice fasted overnight for 16 to 18 hours and then administered metformin (200 mg/kg, oral gavage) or vehicle (water, oral gavage) 1 hour before tissue harvest. n = 5 control vehicle, n = 4 control metformin, n = 5 NDI1 vehicle, and n = 5 NDI1 metformin. Results represent mean ± SEM. Two-way ANOVA (Bonferroni) shows no significance (n.s.) between genotypes when treated with metformin, n.s.P > 0.9999 [(A) and (B)]. (C) Multiple cohorts of control and NDI1 mice, 8 to 12 weeks old and on a standard chow diet, were fasted overnight for 16 to 18 hours and then administered metformin (200 mg/kg, oral gavage) or vehicle (water, oral gavage) 30 min before a glucose (2 g/kg, oral gavage) tolerance test. n = 57 control vehicle, n = 64 control metformin, n = 26 NDI1 vehicle, and n = 79 NDI1 metformin. Results represent mean ± SEM. (D) Incremental area under the curve (iAUC) for the GTT in the presence or absence of metformin shown in (C). Results represent mean + SEM. Two-way ANOVA (Bonferroni) shows no significance (n.s.) between vehicle-treated control and vehicle-treated NDI1 mice, n.s.P = 0.4522, but shows statistical significance between metformin-treated control and metformin-treated NDI1 mice, *P < 0.0001.

To determine whether metformin mediates its blood glucose–lowering effects through inhibition of mitochondrial complex I, we performed a GTT in the presence or absence of metformin in control and NDI1 mice. Mice given the vehicle oral gavage, regardless of genotype, had a significant increase in their blood glucose levels following an oral glucose gavage (Fig. 2, C and D). Control mice administered metformin by oral gavage had a significantly lower blood glucose level compared to the vehicle-treated mice of either genotype (P < 0.05 for both comparisons). However, the ability of metformin to lower blood glucose in the control animals was significantly attenuated in the metformin-treated NDI1 mice (P < 0.05). Thus, the NDI1 mice have a blunted response to metformin’s blood glucose–lowering effect, indicating that acute oral administration of metformin lowers blood glucose levels by targeting mitochondrial complex I.

NDI1 expression attenuates metformin’s blood glucose–lowering effect in mice fed a high-fat diet

To assess their obesity-induced blood glucose levels, control and NDI1 mice (10 to 12 weeks of age) were placed on a high-fat diet (HFD) for at least 8 weeks. NDI1 mice on an HFD exhibited a similar weight gain to control mice (Fig. 3A). Moreover, control and NDI1 mice on an HFD had similar fasted and refed blood glucose (Fig. 3B) and plasma insulin (Fig. 3C) levels. Next, we performed a GTT on the HFD control and NDI1 mice in the presence or absence of metformin. The bolus of glucose increased the blood glucose levels similarly in HFD control and NDI1 mice treated with vehicle (Fig. 3, D and E). Metformin-treated HFD control mice had a significantly lower blood glucose level compared to the vehicle-treated mice of either genotype (P < 0.05 for both comparisons). However, the ability of metformin to lower blood glucose in the HFD control animals was significantly attenuated in the metformin-treated HFD NDI1 mice (P < 0.05). These results suggest that acute oral administration of metformin targets mitochondrial complex I to lower blood glucose levels in mice exposed to HFD.

Fig. 3. NDI1 expression attenuates the blood glucose–lowering effects of metformin in mice fed a high-fat diet.

Fig. 3.

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(A) Weight gain of control and NDI1 mice fed a 60 kcal% HFD ad libitum over an 8-week period. Mice were switched from a standard chow diet to an HFD at 10 to 12 weeks of age. n = 61 control mice and n = 57 NDI1 mice. Results represent mean ± SEM. (B and C) Multiple cohorts of control and NDI1 mice, on an HFD for 8 to 12 weeks, were fasted overnight for 16 to 18 hours. Mice were then split into two groups, fasted and refed. Blood was taken either immediately (fasted) or 4 hours after HFD food was provided (refed). Blood glucose (B) and plasma insulin (C) levels were assayed from these blood samples. n = 23 control fasted, n = 17 control refed, n = 17 NDI1 fasted, and n = 16 NDI1 refed in both (B) and (C). Results represent mean ± SEM. Two-way ANOVA (Bonferroni) shows no significance (n.s.) between genotypes within the fasted and refed groups. n.s.P > 0.9999 [(B); fasted and refed], n.s.P > 0.9999 [(C); fasted], n.s.P = 0.0946 [(C); refed]. (D) Multiple cohorts of control and NDI1 mice, on an HFD for 8 to 10 weeks, were fasted overnight for 16 to 18 hours and then administered metformin (200 mg/kg, oral gavage) or vehicle (water, oral gavage) 30 min before a glucose (2 g/kg, oral gavage) tolerance test. n = 31 control vehicle, n = 30 control metformin, n = 22 NDI1 vehicle, and n = 35 NDI1 metformin. Results represent mean ± SEM. (E) Incremental area under the curve (iAUC) for the GTT in the presence or absence of metformin shown in (D). Results represent mean + SEM. Two-way ANOVA (Bonferroni) shows no significance (n.s.) between vehicle-treated control and vehicle-treated NDI1 mice, n.s.P = 0.1808, but statistical significance between metformin-treated control and metformin-treated NDI1 mice, *P < 0.0001.

DISCUSSION

Metformin is a cost-effective oral medication generally administered twice daily to humans to reduce blood glucose levels. The molecular mechanisms by which acute metformin administration lowers blood glucose are not fully understood (4). Here, we generated whole-body NDI1-expressing mice to determine whether mitochondrial complex I inhibition by metformin is necessary for its blood glucose–lowering effect. We found that the blood glucose–lowering effect of acute metformin was diminished in mice expressing NDI1 compared to control mice after at least 8 weeks on a standard chow diet or HFD. The attenuation of the NDI1 mice to the blood glucose–lowering effects of metformin was not the result of altered basal glucose metabolism or impaired metformin uptake.

Our data illustrate the necessity of mitochondrial complex I inhibition for the blood glucose–lowering effects of metformin. Nevertheless, mice expressing NDI1 were not completely resistant to the glucose-lowering effects of metformin. This could be due, in part, to variable NDI1 expression within the NDI1 mouse line, the inability of NDI1 to proton pump or generate ROS, or metformin having multiple targets that fully mediate its ability to lower blood glucose levels. Future studies could potentially address this question by generating mice with a mutation in endogenous mitochondrial complex I that allows it to function normally while being resistant to metformin inhibition. Furthermore, tissue-specific expression of NDI1 could be used to dissect the cellular targets of metformin in the liver, intestine, or other tissues, and to elucidate the mechanism downstream of mitochondrial complex I inhibition responsible for glucose lowering in these cells.

Metformin has been shown to have beneficial effects beyond type 2 diabetes (4). Several retrospective studies have found a correlation between people taking metformin and a reduced risk of developing certain types of cancer (37, 38). The direct anticancer effect of metformin is mediated through inhibition of mitochondrial complex I (21, 39–42). A recent phase 3 trial on COVID-19 demonstrated that metformin could reduce hospitalizations and the incidence of Long Covid (43–45). Laboratory studies have suggested that metformin decreases inflammation by targeting mitochondrial complex I (46, 47). Moreover, metformin can reduce the risk of cardiovascular disease in patients without diabetes (48). Thus, metformin has been proposed as a drug that improves health span (49–51). In future studies, use of the NDI1 mice in these disease settings will be of interest to elucidate whether metformin mediates these pleiotropic effects through inhibition of mitochondrial complex I.

MATERIALS AND METHODS

Experimental design

The objective of this study was to use NDI1 in vivo to determine whether metformin mediates its blood glucose–lowering effect through inhibition of mitochondrial complex I.

Animals

β-Actin-cre mice were obtained from the Jackson Laboratory [B6N.FVB-Tmem163Tg(ACTB-cre)2Mrt/CjDswJ, stock no. 019099]. LSL-NDI1 mice have previously been described (27). Homozygous β-actin-cre-positive (BAC+/+) mice were crossed with heterozygous LSL-NDI1 (NDI1LSL/WT) mice to generate our control (BAC+/−) and NDI1 (NDI1LSL/WT BAC+/−) mice. The control and NDI1 animals used throughout this study were all male littermates and on a mixed C57BL6 (N/J) genetic background. Animal housing conditions and welfare were monitored in accordance with Northwestern University’s Institutional Animal Care and Use Committee’s (IACUC’s) policies. Animals were housed in a Northwestern Center for Comparative Medicine vivarium. This temperature- and humidity-controlled room (23°C with a 30 to 70% humidity range) had a standard 12-hour light and dark cycle. Control and NDI1 littermates were housed together in ventilated micro-insulator cages with free access to water and standard rodent chow (Envigo/Teklad LM-485) or a 60 kcal% HFD (Research Diets, D12492i) as described. Where noted, mice were fasted overnight for 16 to 18 hours. Food was removed from these mice at 5:00 to 7:00 p.m. local time (but free access to water was retained), and all experiments began by 12:00 p.m. (noon) local time the next day. After the procedure, live animals were immediately provided standard chow or HFD food. Weight measurements in mice were performed before the oral administration of glucose, metformin, or vehicle (water) to determine the volume of liquid to gavage. Mice on the 60 kcal% HFD were also weighed weekly for the first 8 weeks on the diet to assess weight gain. Mice were 8 to 16 weeks old for all procedures performed while on the standard chow diet (Figs. 1 and 2). For the HFD experiments (Fig. 3), all mice were placed on the HFD at 10 to 12 weeks of age, and they consumed 60 kcal% fat food daily for 8 to 12 weeks; therefore, the mice were 18 to 24 weeks old when the HFD experiments were performed. Disposable Cadence Science Malleable Stainless Steel Animal Feeding Needles (Thermo Fisher Scientific, no. 14-825-275) attached to BD Disposable Syringes with Luer-Lok Tips (Thermo Fisher Scientific, no. 14-823-30) were used throughout this study for the oral administration of glucose, metformin, or vehicle (water). All animal procedures conducted in this study were reviewed and approved by the IACUC at Northwestern University.

RNA sequencing

Standard chow diet–fed control and NDI1 mice, 12 to 16 weeks of age, were used for all sequencing experiments. Before harvest, mice were fasted overnight for 16 to 18 hours. On the day of harvest, mice were euthanized with isoflurane and whole liver tissue was placed in a cryovial and snap frozen in liquid nitrogen. The liver tissues were stored at −80°C until all samples were collected. RNA was extracted from liver tissues using a TissueRuptor II (QIAGEN) to homogenize the livers and the AllPrep DNA/RNA Micro Kit (QIAGEN, no. 80284) according to the manufacturer’s protocol.

RNA quality and quantity were assessed by Agilent 4200 TapeStation, using the RNA ScreenTape System (Agilent Technologies). RNA-seq libraries were prepared from 500 ng of total RNA using the NEBNext Ultra DNA Library Prep Kit for Illumina (NEB E7370L). Library quality control (QC) was then performed using TapeStation 4200 High Sensitivity DNA tapes (Agilent Technologies). Dual-indexed libraries were pooled and sequenced on a NextSeq2000 instrument (Illumina) for 100 cycles, single end, to an average sequencing depth of 20.2 million reads per sample. FASTQ files were generated using bcl-convert 4.0.3 using default parameters. To facilitate reproducible analysis, samples were processed using the publicly available nf-core/RNA-seq pipeline version 3.12.0 implemented in Nextflow 24.04.2.5914 using Singularity 3.8.1 with the minimal command nextflow run nf-core/rnaseq -r ‘3.12.0’ -profile nu_genomics --additional_fasta ‘transgenes.fa’ --star_index false --genome ‘GRCm38.’ Briefly, reads were trimmed using trimGalore! 0.6.7 and aligned to the hybrid genome described above using STAR 2.6.1d. Gene-level assignment was then performed using salmon 1.5.2.

All analysis was performed using custom scripts in R version 4.3.0 using the DESeq2 version 1.42.0 framework. A “local” model of gene dispersion was used as this better fit dispersion trends without obvious overfitting, and pairwise comparisons were performed using Wald tests on a single factor of genotype. α was set at 0.05 for all RNA-seq differential expression analysis (DEA). Otherwise, default settings were used. All code used in this analysis is available at https://doi.org/10.5281/zenodo.13923772 and at https://github.com/Chandel-Lab/Reczek_2024.

Metabolic phenotyping of fasted and refed mice

Multiple cohorts of control and NDI1 mice (10 to 15 animals each time) were fasted overnight for 16 to 18 hours and split into two groups the next day, fasted or refed. The fasted group of mice immediately had their blood drawn for metabolic phenotyping, while the refed group was provided standard chow or 60 kcal% HFD food ad libitum for 4 hours before having their blood drawn. Note that the refed group received the same type of food (standard chow or HFD) they had before their overnight fast. A small nick in the mouse’s tail vein was made and the first drop of blood was wiped away. Gentle massaging of the tail vein produced a second drop of blood, which was applied to the Contour Next blood glucose test strip inserted into the Contour Next blood glucose meter. Blood glucose levels were measured for each mouse and recorded.

Following the blood glucose measurement, additional blood from the mouse’s tail vein was collected to assess plasma insulin levels. Using a tail restraint and a heat lamp, 100 μl of blood was drawn from each animal’s tail vein using microhematocrit capillary tubes (Thermo Fisher Scientific, no. 22-362-566), which were then placed into a 1.5-ml microcentrifuge tube on ice. The blood samples were centrifuged at 14,000 rpm for 10 min at 4°C. Following centrifugation, the supernatant or plasma was collected and placed into a new tube. The plasma was stored at −80°C until analysis. Plasma insulin levels were determined using the Ultra-Sensitive Mouse Insulin ELISA kit (Crystal Chem, no. 90080) according to the manufacturer’s protocol.

Glucose tolerance test

All GTTs were performed on multiple cohorts of mice containing 10 to 15 animals. The tests were performed on different days and the cumulative data are shown. Animals were fasted overnight for 16 to 18 hours, and their fasting blood glucose levels were measured (time = 0 min) the next day using the Contour Next blood glucose test strips and meter. The second drop of blood from the animal’s tail vein was used for all blood glucose measurements. Immediately following the t = 0 measurement, each mouse received a bolus of glucose, 2 g/kg body weight of d-glucose dissolved in water, by oral gavage. Circulating glucose levels were measured and recorded from tail vein blood samples collected at t = 15, 30, 45, 60 and 120 min after glucose gavage. Baseline glucose tolerance of control and NDI1 mice was compared by examining the incremental area under the curve (iAUC), which is the area under the curve above the respective group’s t = 0 blood glucose measurement.

GTTs were also performed on mice in the presence or absence of metformin. Metformin (850 mg) tablets (Metformin Hydrochloride, Granules Pharmaceuticals Inc., NDC 70010-064-01) were crushed and dissolved in water. For these tolerance tests, mice were fasted overnight 16 to 18 hours and administered either vehicle (water) or metformin (200 mg/kg body weight) by oral gavage 30 min before the t = 0 fasting blood glucose measurement. Fasting blood glucose was assessed in each animal as above. Immediately following the t = 0 measurement, all mice, vehicle- and metformin-treated, received a bolus of glucose (2 g/kg body weight) by oral gavage. Blood glucose levels were again measured and recorded at t = 15, 30, 45, 60, and 120 min after glucose gavage. Glucose tolerance of control and NDI1 mice in the presence or absence of metformin was compared by examining the iAUC.

Metformin tissue concentration

Control and NDI1 mice fed standard chow were fasted overnight for 16 to 18 hours and were administered vehicle (water) or metformin (200 mg/kg body weight) by oral gavage the next day. One hour after administration, the mice were euthanized with isoflurane and their liver and intestine (i.e., jejunum) tissues were harvested (i.e., placed in a cryovial and snap frozen in liquid nitrogen). Liver and intestine tissues were then stored at −80°C until analysis. Soluble metabolites were extracted from the liver and intestine tissues using cold acetonitrile/water (80/20, v/v). For each milligram of liver tissue, 40 μl of cold acetonitrile/water (80/20, v/v) was added, while for each milligram of intestine tissue, 80 μl of the same solvent was used. The tissues were then disrupted using a rotor-stator homogenization device (QIAGEN TissueRuptor II). The homogenized samples underwent three cycles of freezing, thawing, and vortexing. Next, the samples were incubated at −20°C overnight to precipitate proteins. The next day, the samples were thawed on ice, vortexed, and centrifuged at 17,000g for 30 min at 4°C. The supernatants containing soluble metabolites, including metformin, were collected.

To make a standard curve, standard metformin (Metformin Hydrochloride, Cayman Chemical, no. 13118) concentrations ranging from 25 nM to 200 μM were used. Both the standards and the tissue samples were spiked with an equal amount of internal standards [2 μM Metformin-D6 (Cayman Chemical, no. 16921)]. Next, the standards, the liver tissue samples, and the intestine tissue samples were analyzed by high-performance liquid chromatography and triple quadrupole tandem mass spectrometry (HPLC-MS/MS). Specifically, the system consisted of a TSQ (Thermo) in line with an electrospray ion source (ESI) and a Vanquish (Thermo) UHPLC consisting of a binary pump, degasser, and auto-sampler outfitted with a XBridge C18 column (Waters, dimensions of 2.1 mm by 50 mm, 3.5 μM). Isocratic elution was performed with the mobile phase containing 0.1% formic acid in water/acetonitrile (35/65, v/v) at 0.15 ml/min. In positive mode, the capillary of ESI was set to 300°C, with sheath gas at 35 arbitrary units, auxiliary gas at 5 arbitrary units, and the spray voltage at 3.5 kV. A selective reaction monitoring (SRM) of the protonated precursor ion and the related product ions for metformin and metformin-D6 [mass/charge ratio (m/z) 130.15 → 71, 136.15→ 77, respectively] were monitored. The standard curve was derived from the peak area ratio of targets to internal standard with a linear regression R2 = 0.9991. Data acquisition and analysis were carried out by Xcalibur 4.1 software and TraceFinder 4.1 software, respectively (both from Thermo Fisher Scientific). Metformin concentrations in the tissue samples were determined by comparing the ratio of tissue metformin to the internal standard ratio to the standard curve.

Statistical analysis

Data are presented as mean + or ± SEM. The number of biological replicates (n) are indicated in the figure legends, and it refers to the number of individual animals per genotype group. Samples were not excluded from the analysis, randomization did not occur, and investigators were not blinded. A statistical method to predetermine sample size was not used. Statistical significance was determined using a two-tailed unpaired t test for single comparisons or a two-way analysis of variance (ANOVA) followed by post hoc tests using the Bonferroni method for multiple comparisons (GraphPad Prism software, version 10). Statistical significance (*) was defined as *P < 0.05. Each figure legend denotes the individual P values for the groups being compared.

All RNA-seq analyses were performed using custom scripts in R 4.3.0, all of which are publicly available on Zenodo and GitHub at https://doi.org/10.5281/zenodo.13923772 and at https://github.com/Chandel-Lab/Reczek_2024, respectively. Plotting was performed using ggplot2 3.4.4 unless otherwise noted. Statistics for these figures were added using ggsignif 0.6.4. In the box plots, box limits represent the interquartile range (IQR) with a center line at the median. Whiskers represent the largest point within 1.5 × IQR. All points were overlaid with horizontal jittering.

Acknowledgments

We thank the following core facilities at Northwestern University: Pulmonary NextGen Sequencing Core, the Robert H. Lurie Comprehensive Cancer Center Metabolomics Core, and the Transgenic and Targeted Mutagenesis Laboratory. We also would like to thank the staff of Northwestern University’s Center for Comparative Medicine. We are especially grateful to H. Abdala-Valencia (Northwestern University) for technical assistance with RNA-seq.

Funding: This work was supported by National Institutes of Health grant R35CA197532 (N.S.C.); National Institutes of Health grant P01HL154998-03 (N.S.C.); National Institutes of Health grant P01AG049665 (N.S.C.); National Heart, Lung, and Blood Institute grant T32HL076139-11 (C.R.R.); Northwestern University Pulmonary and Critical Care Division Cugell Predoctoral Fellowship (R.P.C.); Cellular and Molecular Basis of Disease grant T32GM008061 (K.B.D.); NRSA Training Program in Signal Transduction and Cancer grant T32CA070085 (Z.L.S.); Glenn Foundation for Medical Research Postdoctoral Fellowship in Aging Research (Z.L.S.); National Heart, Lung, and Blood Institute grant T32HL076139-21 (Z.L.S.); Schmidt Science Fellows, in partnership with the Rhodes Trust (R.A.G.); and the Simpson Querrey Fellowship in Data Science (R.A.G.).

Author contributions: Conceptualization: C.R.R., G.R.S.B., and N.S.C. Methodology: C.R.R., P.G., and N.S.C. Investigation: C.R.R., R.P.C., K.B.D., Z.L.S., R.A.G., P.G., and N.S.C. Validation: C.R.R. and N.S.C. Visualization: C.R.R., R.A.G., and N.S.C. Formal analysis: C.R.R., R.A.G., P.G., and N.S.C. Data curation: R.A.G. Software: R.A.G. Resources: P.G. and N.S.C. Supervision: G.R.S.B. and N.S.C. Funding acquisition: G.R.S.B. and N.S.C. Project administration: C.R.R., G.R.S.B., and N.S.C.. Writing—original draft: C.R.R. and R.A.G. Writing—review and editing: C.R.R., R.P.C., K.B.D., Z.L.S., R.A.G., and N.S.C.

Competing interests: The authors declare that they have no competing interests.

Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper. Raw FASTQ files, processed gene counts, and sample metadata for RNA-seq performed in this paper are available through GEO with accession number GSE278099. All code necessary to reproduce the RNA-seq analysis is available at https://doi.org/10.5281/zenodo.13923772 and at https://github.com/Chandel-Lab/Reczek_2024.

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