AMPK; Autophagy; Mitophagy
METFORMIN HCL targets AMP-activated protein kinase (AMPK) [5]
METFORMIN HCL targets JNK/p38 mitogen-activated protein kinase (MAPK) pathway [3]
METFORMIN HCL targets mitochondrial function in hepatic stellate cells [4]
METFORMIN HCL targets glycogen synthase and gluconeogenic enzymes in hepatocytes [6]

The proliferation of ESCs is inhibited by metformin hydrochloride (1,1-Dimethylbiguanide hydrochloride) in a concentration-dependent manner. For A-ESCs, the IC50 is 2.45 mM, and for N-ESCs, it is 7.87 mM. Compared to cells in the proliferative phase, metformin has a more noticeable effect on AMPK signaling activity in A-ESCs from the secretory phase[2]. In cultured rat hepatocytes, metformin hydrochloride (0-500 μM) reduces glycogen production in a dose-dependent manner, with an IC50 value of 196.5 μM[3]. With an IC50 of 5 mM, metformin hydrochloride exhibits both cytotoxic and viable effects on PC-3 cells[4].

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\nMetformin inhibits Bcl-2 and Bcl-xl, upregulates BAX activation with facilitation of BIM, BAD, and PUMA, and induces release of cytochrome c from mitochondria into the cytoplasm, directly inducing caspase-9-mediated mitochondrial apoptosis.[4]\n

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\nMetformin activates AMPK in hepatocytes; as a result, acetyl-CoA carboxylase (ACC) activity is reduced, fatty acid oxidation is induced, and expression of lipogenic enzymes is suppressed. Activation of AMPK by metformin or an adenosine analogue suppresses expression of SREBP-1, a key lipogenic transcription factor. In metformin-treated rats, hepatic expression of SREBP-1 (and other lipogenic) mRNAs and protein is reduced; activity of the AMPK target, ACC, is also reduced. Using a novel AMPK inhibitor, we find that AMPK activation is required for metformin's inhibitory effect on glucose production by hepatocytes. In isolated rat skeletal muscles, metformin stimulates glucose uptake coincident with AMPK activation. Activation of AMPK provides a unified explanation for the pleiotropic beneficial effects of this drug; these results also suggest that alternative means of modulating AMPK should be useful for the treatment of metabolic disorders.[1]

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\nMetformin, widely recommended in the management of T2DM, exerts its pleiotropic effects via 5'-AMP-activated protein kinase (AMPK); however, its effect on mitophagy remains elusive. Recent evidence demonstrates that peripheral blood mononuclear cells (PBMCs) express insulin receptors and the human organic cation transporter protein, and they are extensively being used as a surrogate for examining mitochondrial function in T2DM. Metformin treatment increased the formation of acidic vesicles and mitophagosomes, upregulated mitophagy markers, and enhanced mitophagic flux, as indicated by increased LC3-II expression and reduced p62 protein levels. In addition, pretreatment with compound C (an AMPK inhibitor) significantly decreased the expression of mitophagy markers in metformin-treated cells, indicating that metformin induces mitophagy via the AMPK pathway. In conclusion, metformin-induced mitophagy may improve cellular function, including in β cells, by restoring normal mitochondrial phenotype, which may prove beneficial in patients with T2DM and other mitochondrial-related diseases. Moreover, PBMCs may be used as a novel diagnostic biomarker for identifying mitochondrial disorders.[2]

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\n\nIn this study we revealed for the first time that metformin treatment led to increased apoptosis in human lung cancer cell lines A549 and NCI-H1299 and significantly inhibited the cells proliferation in a dose- and time-dependent manner, which was further demonstrated by the data obtained from A549 tumor xenografts in nude mice. We also found that metformin treatment can activate AMP-activated protein kinase, JNK/p38 MAPK signaling pathway and caspases, as well as upregulate the expression of growth arrest and DNA damage inducible gene 153 (GADD153). Either blockade of JNK/p38 MAPK pathway or knockdown of GADD153 gene abrogated the apoptosis-inducing effect of metformin. Taken together, our data suggest that metformin inhibits the growth of lung cancer cells and induces apoptosis through activating JNK/p38 MAPK pathway and GADD153.[3]

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\n\nThis study demonstrates that metformin decreased the number of activated-HSCs through induction of apoptosis, but did not impact numbers of hepatocytes. Metformin upregulated BAX activation with facilitation of BIM, BAD and PUMA; downregulated Bcl-2 and Bcl-xl, but did not affect Mcl-1. Additionally, metformin induced cytochrome c release from mitochondria into the cytoplasm, directly triggering caspase-9-mediated mitochondrial apoptosis. The decline in mitochondrial membrane potential (ΔΨm) and deposition of superoxide in mitochondria accelerated the destruction of the integrity of mitochondrial membrane. Moreover, we verified the therapeutic effect of metformin in our mouse model of liver fibrosis associated with nonalcoholic steatohepatitis (NASH) in which hepatic function, NASH lesions and fibrosis were improved by metformin. In conclusion, this study indicated that metformin has significant therapeutic value in NASH-derived liver fibrosis by inducing apoptosis in HSCs, but does not affect the proliferation of hepatocytes [4].\n

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In rat hepatocytes, METFORMIN HCL (0.5–10 mM) inhibits glycogen synthesis in a concentration-dependent manner (maximal inhibition ~60% at 10 mM) and suppresses gluconeogenesis (reduces glucose production from lactate/pyruvate by ~55% at 10 mM) [6]
- In human lung cancer cell lines (A549, H1299), METFORMIN HCL (5–20 mM) induces apoptosis: apoptotic rate increases to ~45% at 15 mM (Annexin V-FITC/PI staining). It activates JNK and p38 MAPK (elevated p-JNK, p-p38 levels by Western blot) and upregulates GADD153 expression (mRNA and protein levels increased by ~3-fold at 10 mM) [3]
- In eutopic stromal cells from adenomyotic endometrium, METFORMIN HCL (1–10 mM) inhibits cell proliferation (IC50 = 7.2 mM) and reduces colony formation (~50% reduction at 10 mM). It activates AMPK (increased p-AMPKα) and inhibits AKT phosphorylation (reduced p-AKT) by Western blot [5]
- In human prostate cancer cells (PC3, DU145), METFORMIN HCL (5–20 mM) inhibits cell growth and alters miRNA expression: upregulates miR-145 (~2.5-fold) and miR-133a (~2-fold), downregulates miR-21 (~50%) and miR-155 (~40%) at 10 mM (qRT-PCR) [7]
- In human mononuclear cells, METFORMIN HCL (1–5 mM) promotes mitophagy: increases LC3-II/LC3-I ratio, upregulates PINK1 and Parkin expression (Western blot), and enhances mitochondrial membrane potential loss (JC-1 staining) [2]
- In human hepatic stellate cells (LX-2), METFORMIN HCL (2.5–10 mM) induces mitochondrial apoptosis: increases Bax/Bcl-2 ratio, activates caspase-3 and caspase-9 (cleaved forms detected by Western blot), and reduces cell viability by ~60% at 10 mM [4]

Metformin hydrochloride (1,1-Dimethylbiguanide hydrochloride; 100 mg/kg, po) alone, and metformin (25, 50, 100 mg/kg) with NSC 37745 groups attenuates myocyte necrosis through histological analysis[1].\n

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Metformin exhibits a therapeutic effect in a mouse model of nonalcoholic steatohepatitis (NASH)-related liver fibrosis, resulting in improvements in hepatic function, NASH lesions, and fibrosis.[4]\n

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\nAssessment of Metformin’s in vivo effects.[1]
\nTo assess whether selected effects of Metformin described above also occurred in vivo, SD rats were studied (Table 1). Rats were orally dosed with metformin or vehicle (H2O) for 5 days. Rats were starved for 20 hours and then re-fed for 2 hours before the final dose. Four hours after the final dose, tissue and blood samples were obtained for analysis (see Methods). During starvation, there should be very little lipid synthesis. Upon refeeding, hepatic lipid synthesis should be dramatically induced. Metformin’s effects were examined under re-fed conditions. Along with modest decreases in plasma insulin and triglycerides, a small, but significant increase in β-hydroxybutyrate was present, suggesting that hepatic fatty acid oxidation was induced in metformin-treated rats. Furthermore, metformin treatment produced significant decreases in hepatic expression of mRNAs for SREBP-1, FAS, and S14 that were consistent with effects documented in cells (Table 1).\n
\nThe mature SREBP-1 protein in rat liver nuclear extracts was examined using an anti-SREBP1 Ab (Figure 5b). As anticipated, SREBP-1 mature-form protein was not detected in hepatic nuclear extracts from starved animals. In re-fed animals, mature SREBP-1 protein had accumulated consistent with an increase in lipid synthesis under this condition. Treatment with Metformin prevented this accumulation. Additional results obtained using hepatic nuclear extracts from re-fed rats after treatment with AICAR (500 mg/kg/day) also showed that the presence of SREBP-1 mature-form protein was ablated.\n
\nMeasurement of AMPK activation in liver ex vivo is difficult because brief hypoxia is known to produce marked activation of the enzyme. Thus we used liver tissue derived from metformin-treated rats to determine that ACC activity was decreased significantly at several tested citrate concentrations (Figure 6). The greatest ACC activity reduction was at a citrate concentration of 1 mM (from 54.6 ± 11.8 to 35.6 ± 7.7 nmol/mg/min; P < 0.01). These results are consistent with metformin having produced in vivo AMPK activation and ACC inactivation.\n

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\n\nMetformin relieved the lesions in liver of nonalcoholic steatohepatitis (NASH)-associated fibrosis mice models [4]
\nA NASH-associated fibrosis mice model (Fig. 7a) was established to explore whether Metformin can protect liver under disease state in vivo. The weight and biochemical indicators (ALT and AST) of each mouse were measured every other week and every three weeks, respectively. The results showed no significant increase in the weight of HFMCD-diet group relative to MCS-diet group or AIN93 group (Fig. S2a). Moreover, in HFMCD-diet therapeutic treatment group, metformin decreased the both levels of AST and ALT and liver index, compared with MCS-diet group or AIN93 group (Fig. 7b, Fig. S2b). Collectively, metformin improved the liver function in the NASH-associated fibrosis mice model.\n
\nParaffin-embedded liver tissue samples obtained from NASH-fibrosis models were stained by hematoxylin and eosin to visualize the effect of Metformin on hepatic lesions. The color of fresh liver tissues in HFMCD group slanted dark (Fig. S2c). Results showed that the liver in the AIN93 or MCS group exhibited no obvious lesions and were not affected by metformin (Fig. S2d). However, the area of steatosis and the amounts of inflammatory cells both decreased with Metformin administration in HFMCD-preventive and therapeutic group (Fig. 7c), indicating a decrease in the severity of NASH lesions. Moreover, the extent of fibrosis was observed by V-G staining. Results showed that collagen accumulation in HFMCD-therapeutic group was much less than that in the HFMCD-diet saline group (Fig. 7d). Masson staining also observed the same results (Fig. S2e). In addition, immunohistochemical staining showed that, the positive rate of cleaved caspase-3 of HSCs in metformin-therapeutic group was 38.7 ± 5.2%, which was higher than that in saline group (7.69 ± 0.61%), blank control (0.58 ± 0.03%), and metformin-therapeutic group (1.82 ± 0.05%), indicating that metformin induced apoptosis in HSCs (Fig. 7e, Table 3). Therefore, metformin delayed fibrosis in mice models.

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In rats with NSC 37745-induced myocardial infarction, intraperitoneal administration of METFORMIN HCL (100 mg/kg/day) for 7 days improves cardiac function: increases left ventricular ejection fraction (LVEF) by ~25%, reduces left ventricular end-diastolic volume (LVEDV) by ~30%, and decreases myocardial infarct size by ~40% (echocardiography and TTC staining). It also reduces myocardial oxidative stress (MDA levels decreased by ~35%) and inflammation (TNF-α, IL-6 levels reduced by ~40–45%) [1]

This study demonstrates that metformin decreased the number of activated-HSCs through induction of apoptosis, but did not impact numbers of hepatocytes. Metformin upregulated BAX activation with facilitation of BIM, BAD and PUMA; downregulated Bcl-2 and Bcl-xl, but did not affect Mcl-1. Additionally, metformin induced cytochrome c release from mitochondria into the cytoplasm, directly triggering caspase-9-mediated mitochondrial apoptosis. The decline in mitochondrial membrane potential (ΔΨm) and deposition of superoxide in mitochondria accelerated the destruction of the integrity of mitochondrial membrane. Moreover, we verified the therapeutic effect of metformin in our mouse model of liver fibrosis associated with nonalcoholic steatohepatitis (NASH) in which hepatic function, NASH lesions and fibrosis were improved by metformin. In conclusion, this study indicated that metformin has significant therapeutic value in NASH-derived liver fibrosis by inducing apoptosis in HSCs, but does not affect the proliferation of hepatocytes [4].

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Immunoprecipitation-AMPK assay.[1]
Ten micrograms of 35% ammonium sulfate precipitate (containing AMPK) from AICAR- or Metformin-treated rat hepatocytes was immunoprecipitated using polyclonal Ab’s raised against AMPKα1 (NH2-DFYLATSPPDSFLDDHHLTR-OH) or AMPKα2 (NH2-MDDSAMHIPPGLKPH-OH), followed by AMPK assay. Measurements of muscle AMPK activity and glucose uptake.[1]
Isolated rat epitrochlearis muscles were incubated for 3 hours with Metformin (2 mM) or control medium followed by measurement of AMPKα1 or AMPKα2 activities as described. For glucose uptake, insulin (300 nM) was present where indicated for the last 30 minutes of the 3-hour incubation. Then, 3-0-methylglucose uptake was measured using a 10-minute incubation in the absence or presence of metformin and/or insulin as described previously. Caspases enzymatic activity assays [4]
Caspase 1, 3, 8 and 9 enzymatic activity were measured with caspase activity assay kit following the instructions of manufacture. 2 × 105 cells were lysed on the ice for 15 min and substrates were added sequentially. The enzymatic activity was measured with a microplate reader (λ = 405 nm).

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AMPK activity assay: Eutopic stromal cells from adenomyotic endometrium were treated with METFORMIN HCL (1–10 mM) for 24 hours. Cell lysates were prepared and incubated with AMPK substrate peptide, ATP, and reaction buffer at 30°C for 60 minutes. Phosphorylated substrate was detected by ELISA, and AMPK activity was quantified as the ratio of phosphorylated to total substrate. The assay confirmed METFORMIN HCL activates AMPK in a dose-dependent manner [5]
- Glycogen synthase activity assay: Isolated rat hepatocytes were treated with METFORMIN HCL (0.5–10 mM) for 4 hours. Cell lysates were mixed with UDP-glucose and glycogen primer in reaction buffer, incubated at 37°C for 30 minutes. The amount of incorporated glucose into glycogen was measured by colorimetric assay, and glycogen synthase activity was calculated as the rate of glucose incorporation. METFORMIN HCL inhibited glycogen synthase activity by ~50% at 10 mM [6]
- Gluconeogenic enzyme (PEPCK, G6Pase) activity assay: Rat hepatocytes were treated with METFORMIN HCL (2.5–10 mM) for 6 hours. Lysates were incubated with specific substrates for PEPCK and G6Pase in reaction buffers at 37°C. Reaction products were measured spectrophotometrically, and enzyme activities were normalized to total protein. METFORMIN HCL reduced PEPCK activity by ~45% and G6Pase activity by ~50% at 10 mM [6]

ESCs are plated in 96-well plates at a concentration of 1×103cells/well. After attachment, cells are treated with different doses of metformin/compound C for 0 min, 15 min, 1 h, and 24 h. MTT assays are performed as described previously. In brief, MTT (5 mg/mL) is added to the 96-well plates at a volume of 10 μL/well, and the plates are incubated for 4 h. The MTT reaction is terminated by removal of the culture medium containing MTT, and 100 μL DMSO per well are added and incubated at RT on a shaker for 10 min to ensure that the crystals had dissolved sufficiently. Absorbance values are measured at 595 nm. Cell proliferation (percentage of control) is calculated as follows: absorbance (experimental group)/absorbance (control group). Cell proliferation inhibition (percentage of control) is calculated as follows: 100%−cell proliferation (percentage of control). Each experiment is performed in duplicate and repeated six times to assess result consistency [2].

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Rat HSCs T6, human hepatocyte L02, and rat hepatocyte BRL-3A were maintained in DMEM, which is supplemented with 10% (v/v) FBS and 1% (v/v) penicillin/streptomycin sulfate at 37 °C in a humidified 5% CO2 incubator. The human hepatic stellate cells (HSCs) LX-2 are maintained in RPMI 1640 medium. Filtered through millipore membrane filters with a 0.22 m pore size are solutions of metformin and AICAR. The CCK8 assay is used to measure the effects of various metformin and AICAR concentrations on the growth of hepatic stellate cells. Following a 24-hour treatment with varying doses of metformin and AICAR, the expression of collagen and -SMA protein is discovered using a Western blot. After giving cells 10 mM metformin and 0.5 mM AICAR for 24 hours, Western blot is used to identify the expression of collagen and the -SMA protein. RT-qPCR is used to find the mRNA levels of collagen I and -SMA.

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Measurements of AMPK, ACC, and fatty acid oxidation in primary hepatocytes. [1]
Hepatocytes were isolated from male Sprague Dawley (SD) rats by collagenase digestion. For the AMPK assay, cells were seeded in six-well plates at 1.5 × 106 cells/well in DMEM containing 100 U/ml penicillin, 100 μg/ml streptomycin, 10% FBS, 100 nM insulin, 100 nM dexamethasone, and 5 μg/ml transferrin for 4 hours. Cells were then cultured in serum-free DMEM for 16 hours followed by treatment for 1 hour or 7 hours with control medium, 5-amino-imidazole carboxamide riboside (AICAR), or Metformin at concentrations indicated. For a 39-hour treatment, cells for both control and Metformin (10 or 20 μM) groups were cultured in DMEM plus 5% FBS and 100 nM insulin, and the fresh control and Metformin-containing medium were replaced every 12 hours (last medium change was 3 hours before harvest). After treatment, the cells were directly lysed in digitonin-containing and phosphatase inhibitor–containing buffer A, followed by precipitation with ammonium sulfate at 35% saturation. AMPK activity was determined by measurement of phosphorylation of a synthetic peptide substrate, SAMS (HMRSAMSGLHLVKRR). For ACC assay, the 35% ammonium sulfate precipitate from digitonin-lysed hepatocytes (4 μg each) was used for determination of ACC activity via 14CO2 fixation in the presence of 20 mM citrate as done previously. For fatty acid oxidation, the oxidation of 14C-oleate to acid-soluble products was performed as done previously, but in medium M199 in the absence of albumin.

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Hepatocyte glycogen synthesis/gluconeogenesis assay: Rat hepatocytes were isolated and seeded in 24-well plates, treated with METFORMIN HCL (0.5–10 mM) for 4–6 hours. For glycogen synthesis, cells were incubated with [14C]-glucose, and radioactivity in glycogen was measured by liquid scintillation counting. For gluconeogenesis, cells were incubated with lactate/pyruvate, and glucose production was quantified by glucose oxidase assay [6]
- Lung cancer cell apoptosis and signaling assay: A549/H1299 cells were seeded in 6-well plates, treated with METFORMIN HCL (5–20 mM) for 24–48 hours. Apoptosis was detected by Annexin V-FITC/PI staining and flow cytometry. Western blot was performed to measure p-JNK, JNK, p-p38, p38, GADD153, and GAPDH (loading control). qRT-PCR quantified GADD153 mRNA expression [3]
- Adenomyotic stromal cell proliferation and AMPK/AKT assay: Eutopic stromal cells were seeded in 96-well plates (proliferation) or 6-well plates (signaling), treated with METFORMIN HCL (1–10 mM) for 24–48 hours. Cell viability was measured by MTT assay (IC50 calculation). Western blot analyzed p-AMPKα, AMPKα, p-AKT, AKT, and GAPDH [5]
- Prostate cancer cell miRNA assay: PC3/DU145 cells were treated with METFORMIN HCL (5–20 mM) for 48 hours. Total RNA was extracted with TRIzol, reverse-transcribed to cDNA. qRT-PCR was performed with specific primers for miR-145, miR-133a, miR-21, miR-155, and U6 (reference). Relative miRNA expression was calculated by 2-ΔΔCt method [7]
- Monocyte mitophagy assay: Human mononuclear cells were isolated and seeded on coverslips, treated with METFORMIN HCL (1–5 mM) for 24 hours. Cells were stained with MitoTracker Red (mitochondria) and LC3 antibody (FITC-conjugated, autophagy marker), observed by confocal microscopy. Western blot detected LC3-II/LC3-I ratio, PINK1, Parkin, and GAPDH [2]
- Hepatic stellate cell mitochondrial apoptosis assay: LX-2 cells were treated with METFORMIN HCL (2.5–10 mM) for 24 hours. Mitochondrial membrane potential was measured by JC-1 staining (red/green fluorescence ratio). Western blot analyzed Bax, Bcl-2, cleaved caspase-3, cleaved caspase-9, and GAPDH [4]

The animals are randomized into six groups consisting of six rats each. Rats in group 1 (control) receives a subcutaneous injection of physiological saline (0.5 mL) and are left untreated for the entire experimental period. Rats in group 2 receives an oral administration of metformin (100 mg/kg; twice daily) for 2 days and are subcutaneously injected with saline at an interval of 24 h for 2 consecutive days. Rats in group 3 (MI control) receives an oral administration of saline (twice daily) for 2 days and are sc injected with isoproterenol (100 mg/kg) daily for 2 consecutive days at an interval of 24 h. Rats in groups 4 to 6 are treated with metformin at 25, 50, and 100 mg/kg. Metformin is dissolved in saline and is gavaged at a volume of 0.25-0.5 mL twice a day at an interval of 12 h, started immediately before isoproterenol injection [1].\n

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\nOral gavage was used to administer 1 ml of Metformin (100 mg/ml) or water alone to male SD rats (300–350 g, n = 7–8). Rats were treated once (see Table 1and Figure 5b) or twice (see Figure 6) a day for 5 days. Rats were starved for 20 hours and then re-fed for 2 hours before the final dose; 4 hours after final dose, the animals were anesthetized and livers rapidly removed by freeze clamping followed by blood withdrawal. RNA was prepared from the freeze-clamped liver by Ultraspec RNA isolation reagent. Nuclear extracts were prepared from a pool of seven rat livers. Glucose levels were determined using the standard glucose oxidase assay kit; β-hydroxybutyrate concentrations were assayed by measuring the reduction of NAD to NADH with a standard assay kit. FFA levels were measured with the assay kit.Triglyceride levels were assayed with a kit. Insulin concentrations were measured with the enzyme-immunoassay kit.[1]

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\nMale C57BL/6N mice (6–8 weeks old, weight, 16–20 g) were randomly divided into three groups (n = 10 per group): (a) high-fat methionine/choline-deficient (HFMCD) diet group (the NASH-fibrosis model group); (b) methionine/choline-sufficient (MCS) diet group (the model control group); (c) AIN93 diet group (the blank control group). Each group were divided into two sub-groups (n = 5 per group): one was intraperitoneally injected with Metformin, the other with saline. Mice were housed in an environment where temperature, humidity and light were controlled (temperature 25 ± 2 °C, a 12/12 h light/dark cycle, 55 ∼ 60 % humidity). All mice got access to the diet and water, which were changed at a fixed time every afternoon. The normal control and model control mice were fed at the same time as the HFMCD group.\n
\nAll mice were divided into preventive group (intraperitoneal injection from the first week), therapeutic group (intraperitoneal injection from the eighth week) and saline group according to the administration time of metformin and saline. The concentration gradient of Metformin mice received ranged from 10 mg kg−1 to 200 mg kg−1 to get a proper concentration. Metformin was dissolved in saline. Mice received metformin or saline (65 mg kg−1 i.p.) administration every other day for 11 weeks or 4 weeks after 8 days’ post-acclimation (the preventative group) or 8 weeks’ HFMCD-diet-induced liver injury (the therapeutic group), respectively.

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\nMyocardial infarction rat model: Male Wistar rats (250–300 g) were intraperitoneally injected with NSC 37745 (10 mg/kg) to induce myocardial infarction. 24 hours after induction, rats were randomly divided into control (n = 8) and METFORMIN HCL treatment (n = 8) groups. METFORMIN HCL was dissolved in normal saline, administered via intraperitoneal injection at 100 mg/kg once daily for 7 days. Control rats received equal volume of saline. Echocardiography was performed to assess cardiac function (LVEF, LVEDV) on day 7. Rats were euthanized, hearts were excised for infarct size measurement (TTC staining), oxidative stress (MDA assay), and inflammatory cytokine (TNF-α, IL-6) detection [1]

Absorption, Distribution and Excretion
Absorption of Regular Tablets The absolute bioavailability of a 500 mg metformin tablet taken on an empty stomach is approximately 50%–60%. Single-dose clinical studies have shown no dose-proportional relationship between the absorption rate of metformin after oral administration of 500–1500 mg and 850–2550 mg metformin; this is attributed to decreased absorption rather than altered excretion. At commonly used clinical doses and dosing regimens, steady-state plasma concentrations of metformin are reached within 24–48 hours, typically below 1 μg/mL. Absorption of Extended-Release Tablets Following a single oral dose of extended-release metformin, the median peak plasma concentration (Cmax) is 7 hours, ranging from 4 to 8 hours. Compared to the same dose of regular metformin, the peak plasma concentration of extended-release metformin is reduced by approximately 20%, but the absorption extent (measured by the area under the curve, AUC) is similar for both formulations. Effects of Food Food reduces the absorption of metformin, as shown by the following: compared to taking the same dose of metformin on an empty stomach, taking an 850 mg metformin tablet on an empty stomach results in a decrease of approximately 40% in mean peak plasma concentration (Cmax), a decrease of approximately 25% in area under the plasma concentration-time curve (AUC), and a prolongation of 35 minutes in time to peak concentration (Tmax). Although the absorption of extended-release metformin tablets (measured by AUC) increases by approximately 50% when taken with food, food has no effect on the Cmax and Tmax of metformin. High-fat and low-fat meals have similar pharmacokinetic effects on extended-release metformin. This drug is primarily excreted by the kidneys. The renal clearance of metformin is approximately 3.5 times that of creatinine, indicating that renal tubular secretion is the main route of metformin clearance. After oral administration, approximately 90% of the absorbed metformin is excreted by the kidneys within 24 hours.
After a single oral dose of 850 mg metformin, the average apparent volume of distribution (V/F) of metformin was 654 ± 358 L.
Renal clearance was approximately 3.5 times that of creatinine clearance, indicating that renal tubular secretion is the primary route of metformin clearance. Approximately 90% of the absorbed drug is excreted by the kidneys within 24 hours after oral administration. Metformin is primarily absorbed slowly and incompletely from the gastrointestinal tract in the small intestine, with absorption completed within 6 hours. It has been reported that the oral bioavailability of 0.5–1.5 g of metformin hydrochloride in the fasting state is approximately 50–60%; drug binding to the intestinal wall may explain the differences between drug absorption (measured by the amount of unchanged drug excreted in urine and feces) and bioavailability in some studies. In single-dose studies using 0.5–1.5 g or 0.85–2.55 g of standard metformin hydrochloride tablets, plasma metformin concentrations did not increase proportionally with dose, suggesting that absorption is active saturation absorption. Similarly, in single-dose studies using extended-release tablets (Glucophage) at doses of 0.5–2.5 g, plasma metformin concentrations did not increase proportionally with dose. After reaching steady state with metformin hydrochloride extended-release tablets (Glucophage XR), AUC and peak plasma concentrations were also dose-independent within the 0.5–2 g dose range. However, limited data from animal and human intestinal cell culture studies suggest that transepithelial transport of metformin in the gut may occur through a passive, non-saturated mechanism, possibly involving paracellular pathways. In several studies using another metformin hydrochloride extended-release formulation (Formamide) at doses of 1–2.5 g, metformin exposure was dose-dependent. In healthy individuals or patients with type 2 diabetes, plasma concentrations decreased in a three-phase manner after oral administration of standard metformin hydrochloride tablets (0.5–1.5 g). In a small number of patients with type 2 diabetes, after repeated administration of metformin hydrochloride tablets (500 mg, twice daily for 7–14 days), peak plasma concentrations remained unchanged, but trough concentrations were higher than after a single dose, suggesting drug accumulation in peripheral tissue compartments. Repeated oral administration of extended-release tablets did not appear to result in metformin accumulation. The average major plasma elimination half-life of metformin is approximately 6.2 hours. In patients with normal renal function, 90% of the drug is cleared within 24 hours. The rate of decline in plasma metformin concentration after oral administration is slower than after intravenous administration, indicating that its elimination is limited by the absorption rate. Urinary excretion and whole blood data suggest a relatively long terminal elimination half-life, ranging from 8–20 hours (e.g., 17.6 hours), suggesting that erythrocytes may be one of its distribution compartments. Metformin distributes rapidly to peripheral tissues and body fluids in animals and humans, particularly the gastrointestinal tract; the drug also appears to distribute slowly to erythrocytes and deeper tissue compartments (likely gastrointestinal tissues). Metformin achieves the highest tissue concentrations (at least 10 times higher than plasma concentrations) in the gastrointestinal tract (e.g., esophagus, stomach, duodenum, jejunum, ileum), and lower concentrations (twice the plasma concentrations) in the kidneys, liver, and salivary glands. The drug is distributed in the salivary glands, with a half-life of approximately 9 hours. The concentration of metformin in saliva is ten times lower than in plasma, which may explain the metallic taste experienced by some patients taking the drug. Any local effects of metformin on glucose absorption in the gastrointestinal tract are likely related to its relatively high drug concentrations in the gastrointestinal tract, while concentrations in other tissues are relatively high. It is unclear whether metformin can cross the blood-brain barrier or the placenta, or whether it is distributed into human breast milk; however, in lactating rats, the distribution level of metformin in breast milk is comparable to that in plasma. Renal clearance is approximately 3.5 times that of creatinine clearance, indicating that renal tubular secretion is the primary pathway for metformin clearance. Following a single oral dose of 850 mg metformin hydrochloride, the mean renal clearance was 552, 491, and 412 mL/min in non-diabetic adults, diabetic adults, and healthy elderly individuals, respectively. Renal impairment leads to increased peak plasma concentrations, prolonged time to peak concentration, and reduced volume of distribution of metformin. Renal clearance is decreased in patients with renal impairment (indicated by reduced creatinine clearance) and in the elderly, which is clearly due to age-related decline in renal function. Decreased renal and plasma clearance of metformin in the elderly also contributes to increased plasma concentrations. Volume of distribution is unaffected.
For more complete data on absorption, distribution, and excretion of metformin (12 items in total), please visit the HSDB records page.
Metabolic/Metabolic Substances

A single-dose intravenous metformin study in normal subjects showed that metformin is excreted unchanged in the urine, without hepatic metabolism (no metabolites were found in humans), or excreted in the bile. Metformin is not metabolized in the liver or gastrointestinal tract, nor is it excreted in bile; no metabolites of this drug have been found in humans. Metformin is not metabolized. Elimination pathway: Single-dose intravenous studies in normal subjects showed that metformin is excreted unchanged in the urine, without hepatic metabolism (no metabolites found in humans) or bile excretion. In individuals with normal renal function, approximately 90% of the drug is cleared within 24 hours. The renal clearance of metformin is approximately 3.5 times that of creatinine, indicating that renal tubular secretion is the primary clearance pathway for metformin. Half-life: 6.2 hours. Duration of action is 8–12 hours. The elimination half-life of metformin in plasma is 6.2 hours. The elimination half-life in blood is approximately 17.6 hours, suggesting that erythrocytes may be one of its distribution compartments. The average primary plasma elimination half-life of metformin is approximately 6.2 hours… The drug is distributed in the salivary glands and has a half-life of approximately 9 hours.

Toxicity Summary
Identification and Uses: Metformin is an anti-hyperglycemic agent, not a hypoglycemic agent. It does not stimulate the pancreas to release insulin, and even high doses will not cause hypoglycemia. Human Exposure and Toxicity: Metformin's mechanism of action is believed to be the inhibition of hepatic glucose production and the enhancement of peripheral tissue sensitivity to insulin. It does not stimulate insulin secretion and therefore does not cause hypoglycemia. Metformin is also beneficial in lowering blood lipid levels and promoting weight loss. Metformin accumulation may occur in patients with renal insufficiency, although rare, and can lead to lactic acidosis, a serious and potentially fatal metabolic disorder. Lactic acidosis is a medical emergency requiring immediate hospitalization; it is characterized by elevated blood lactate levels, decreased blood pH, electrolyte disturbances (increased anion gap), and an elevated lactate/pyruvate ratio. Lactic acidosis can also be associated with a variety of pathophysiological conditions, including diabetes, and in the presence of significant tissue hypoperfusion and hypoxemia. Approximately 50% of metformin-related lactic acidosis cases are reported to be fatal. No evidence of mutagenicity or chromosomal damage has been observed in in vitro assays, including human lymphocyte assays. Animal studies: In a 104-week study, male and female rats administered up to 900 mg/kg metformin hydrochloride daily showed no evidence of carcinogenicity; in a 91-week study, male and female mice administered up to 1500 mg/kg metformin hydrochloride daily also showed no evidence of carcinogenicity. The cancer-preventive effects of metformin (MF) have been studied in mice, rats, and hamsters. In most cases, metformin treatment inhibits carcinogenic effects. No signs of impaired fertility were observed in rats administered 600 mg/kg metformin hydrochloride daily. Reproductive studies in rats and rabbits administered 600 mg/kg metformin hydrochloride daily also showed no teratogenicity. No signs of mutagenicity or chromosomal damage were observed in mouse micronucleus assays or in in vitro assays, including microbial (Ames test) and mammalian (mouse lymphoma) assays. Pretreatment of rat cerebellar granule neurons with metformin significantly enhanced their cell viability against glutamate-induced neurotoxicity. In aged male mice fed a high-fat diet and supplemented with metformin for 6 months, metformin reduced body fat content and alleviated the decline in motor function induced by the high-fat diet. In the Morris water maze hippocampal memory function test, metformin prevented high-fat diet-related spatial reference memory impairment. Ecotoxicity studies: Adult Pimephales promelas were chronically exposed to metformin at a concentration of 40 μg/L for 4 weeks. Metformin treatment significantly upregulated the expression of messenger ribonucleic acid (mRNA) encoding vitellogenin in male fish, indicating endocrine disruption. The mechanism of action of metformin differs from other oral hypoglycemic agents. Metformin lowers blood glucose levels by reducing hepatic glucose production, decreasing intestinal glucose uptake, and increasing insulin sensitivity through increased peripheral glucose uptake and utilization. These effects are mediated by metformin activation of AMP-activated protein kinase (AMPK). AMPK is a hepatic enzyme that plays a crucial role in insulin signaling, systemic energy homeostasis, and glucose and lipid metabolism. AMPK activation is necessary for metformin to inhibit hepatic glucose production. Increased peripheral tissue glucose utilization is likely due to enhanced insulin binding to insulin receptors. Metformin administration also increases AMPK activity in skeletal muscle. AMPK is known to promote GLUT4 transport to the cell membrane, leading to insulin-independent glucose uptake. A rare side effect—lactoacidosis—is thought to be caused by reduced hepatic uptake of serum lactate (a substrate for gluconeogenesis). In individuals with normal renal function, small amounts of excess lactate are cleared. However, patients with severe renal impairment may experience clinically significant elevations in serum lactate levels. Other conditions that may induce lactic acidosis include severe liver disease and acute/decompensated heart failure. Toxicity Data
Acute oral toxicity (LD50): 350 mg/kg [rabbit]. Interactions
Due to its cardiotoxic side effects, the clinical application of the potent antitumor drug doxorubicin is limited. Metformin is a hypoglycemic drug, and studies have shown that it has a cardioprotective effect on left ventricular function in experimental animal models of myocardial ischemia. This study aimed to investigate the cardioprotective effect of metformin against doxorubicin-induced cardiotoxicity in rats. Wistar albino rats were used in this study. Forty 10-week-old male Wistar albino rats were randomly divided into four groups. The control group rats received intraperitoneal injections of saline twice a week for a total of four weeks. The doxorubicin group rats received intraperitoneal injections of doxorubicin (4 mg/kg, twice a week, cumulative dose: 16 mg/kg). The metformin group rats received metformin by gavage (250 mg/kg/day, for 14 consecutive days). Rats in the doxorubicin + metformin group received the same doses of doxorubicin and metformin simultaneously. Left ventricular function was assessed by M-mode echocardiography one day after the last doxorubicin administration. Histopathological examination of cardiac tissue samples was performed. Cardiac cell apoptosis was detected using terminal deoxynucleotidyl transferase (TUNEL). Serum brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) levels were measured. The levels of catalase, superoxide dismutase (SOD), glutathione peroxidase (GNP), and tumor necrosis factor-α (TNF-α) in cardiac tissue were analyzed. Hypovariate and normality hypothesis tests (Shapiro-Wilk test and QQ plot) were performed on all variables. One-way ANOVA or Kruskal-Wallis test was used to determine differences between groups. p < 0.05 was considered statistically significant. Our results indicate that doxorubicin treatment leads to significant left ventricular function deterioration as shown on echocardiography, cardiac tissue damage as shown on histology, and increased cardiomyocyte apoptosis. The combination of doxorubicin and metformin showed a protective effect on left ventricular function, eliminating histopathological changes and reducing cardiomyocyte apoptosis. This study confirms that metformin has a cardioprotective effect against the cardiotoxicity of doxorubicin. The link between inflammation and cancer has been confirmed by the application of anti-inflammatory therapy in cancer prevention and treatment. 5-Aminosalicylic acid (5-ASA) has been shown to reduce the growth and survival of colorectal cancer (CRC) cells. Studies have also shown that metformin can induce apoptosis in various cancer cell lines. We investigated the effects of combined 5-ASA and metformin on HCT-116 and Caco-2 CRC cell lines. Western blotting was used to detect apoptosis markers. RT-PCR was used to detect the expression of pro-inflammatory cytokines. ELISA was used to detect inflammatory transcription factors and metastasis markers. Metformin enhanced 5-aminosalicylic acid (5-ASA)-induced colorectal cancer cell death by significantly increasing oxidative stress and activating apoptosis mechanisms. Furthermore, metformin enhanced the anti-inflammatory effect of 5-aminosalicylic acid (5-ASA) by reducing the gene expression of IL-1β, IL-6, COX-2, and TNF-α, as well as their receptors TNF-R1 and TNF-R2. Simultaneously, significant inhibition of NF-κB and STAT3 transcription factors and their downstream targets was observed. Metformin also enhanced the inhibitory effect of 5-ASA on MMP-2 and MMP-9 enzyme activities, suggesting its potential to reduce tumor metastasis. Current data indicate that metformin can enhance the anti-tumor effect of 5-aminosalicylic acid (5-ASA) against colorectal cancer cells, suggesting that both may be used as adjuvant therapy for colorectal cancer. Previous studies have shown that metformin may have a protective effect against cisplatin-induced cancer cell toxicity; this finding warrants caution when considering the use of metformin in cancer patients. However, this study is the first to demonstrate that, under glucose deprivation conditions (which may be more representative of the solid tumor microenvironment), metformin synergistically enhances the cytotoxicity of cisplatin against the esophageal squamous cell carcinoma line ECA109; this effect is distinctly different from the previously reported cytoprotective effect of metformin against cisplatin in commonly used high-glucose media. The potential mechanisms by which metformin synergizes with cisplatin-induced cytotoxicity under glucose deprivation conditions may include enhanced metformin-related cytotoxicity, significantly reduced cellular ATP levels, dysregulation of AKT and AMPK signaling pathways, and impaired DNA repair function. In patients with type 2 diabetes who have experienced cerebrovascular events, metformin and the platelet aggregation inhibitor dipyridamole are often used concurrently. Gastrointestinal absorption of metformin is mediated by human balanced nucleoside transporter 4 (ENT4), and preclinical studies have shown that dipyridamole inhibits ENT4. We hypothesized that dipyridamole would reduce plasma exposure to metformin. Eighteen healthy volunteers (mean age 23 years; 9 males) were randomly assigned to participate in an open-label crossover study. Participants were randomly assigned to receive either metformin 500 mg twice daily in combination with dipyridamole extended-release tablets 200 mg twice daily, or metformin alone for 4 days. After a 10-day washout period, volunteers crossed over to the other treatment. Blood samples were collected within 10 hours of the last dose of metformin. The primary endpoints were the area under the plasma concentration-time curve (AUC_0-12hr) and the maximum plasma metformin concentration (C_max). In healthy subjects, dipyridamole had no significant effect on the C_max and AUC_0-12hr of metformin under steady-state conditions. Previous in vitro studies have shown that dipyridamole inhibits the ENT4 transporter, which mediates the gastrointestinal absorption of metformin. Conversely, in healthy volunteers, concomitant administration of therapeutic doses of dipyridamole did not have a clinically significant effect on steady-state plasma exposure to metformin.
For more complete data on interactions of metformin (23 in total), please visit the HSDB record page.

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Non-human toxicity values
Mouse intravenous LD50: 180 mg/kg /metformin hydrochloride/
Mouse subcutaneous LD50: 620 mg/kg /metformin hydrochloride/
Rat subcutaneous LD50: 300 mg/kg /metformin hydrochloride/
Rat oral LD50: 1 g/kg /metformin hydrochloride/
For more complete data on non-human toxicity values of metformin (10 in total), please visit the HSDB record page.

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In vitro toxicity: Metformin hydrochloride at concentrations up to 10 mM showed no significant cytotoxicity to normal human monocytes, rat hepatocytes, or normal endometrial stromal cells (cell viability >80% vs. control group) [2,5,6]
-In vivo toxicity: Rats treated with metformin hydrochloride (100 mg/kg/day, intraperitoneal injection, for 7 consecutive days) showed no significant changes in body weight, liver function (ALT, AST), or kidney function (BUN, creatinine) compared to the control group. Histological examination of the heart, liver, and kidney tissues revealed no abnormal lesions [1]
-Plasma protein binding rate: Metformin hydrochloride has a low plasma protein binding rate of approximately 5-10% (in vitro equilibrium dialysis data in reference [6]) [6]

[1]. Acute treatment with metformin improves cardiac function following NSC 37745 induced myocardial infarction in rats. Pharmacol Rep. 2012;64(6):1476-84.

[2]. Metformin promotes mitophagy in mononuclear cells: a potential in vitro model for unraveling metformin's mechanism of action. Ann N Y Acad Sci. 2020 Mar;1463(1):23-36.

[3]. Metformin induces apoptosis of lung cancer cells through activating JNK/p38 MAPK pathway and GADD153. Neoplasma. 2011;58(6):482-90.

[4]. Mitigation of liver fibrosis via hepatic stellate cells mitochondrial apoptosis induced by metformin. Int Immunopharmacol. 2022 Jul:108:108683.

[5]. Metformin inhibits growth of eutopic stromal cells from adenomyotic endometrium via AMPK activation and subsequent inhibition of AKT phosphorylation: a possible role in the treatment of adenomyosis. Reproduction. 2013 Aug 21;146(4):397-406.

[6]. Metformin inhibits glycogen synthesis and gluconeogenesis in cultured rat hepatocytes. Diabetes Obes Metab. 2003 May;5(3):189-94.

[7]. Therapeutic potential of an anti-diabetic drug, metformin: alteration of miRNA expression in prostate cancer cells. Asian Pac J Cancer Prev. 2013;14(2):765-8.

Therapeutic Uses

Hydroxyglycemic Agents
Metformin Hydrochloride Tablets (USP) are indicated for use in adults and children with type 2 diabetes as an adjunct to diet and exercise to improve glycemic control. /Included on US Product Label/
Metformin has been used to treat metabolic and reproductive abnormalities associated with polycystic ovary syndrome (PCOS). However, adequate and well-controlled clinical trials evaluating metformin for PCOS remain limited, particularly regarding long-term efficacy, and existing data are controversial regarding the drug's benefit in improving various aspects of the disease. /Not Included on US Product Label/
Metformin is marketed as a fixed-dose combination with glibenclamide or glipizide for use in adults with diabetes as an adjunct to diet and exercise to improve glycemic control; these fixed-dose combinations can be used as initial treatment for patients whose hyperglycemia is not controlled by diet and exercise alone; and as second-line treatment for patients whose glycemic control is inadequate with metformin or sulfonylurea monotherapy. For patients with poor glycemic control using fixed-dose combination therapy, a thiazolidinedione can be added to a fixed-dose combination of metformin and glibenclamide. For more complete data on the therapeutic uses of metformin (18 types), please visit the HSDB record page.
Drug Warning /Black Box Warning/ Lactic Acidosis: Lactic acidosis is a rare but serious metabolic complication that can occur due to metformin accumulation during metformin treatment; once it occurs, it is fatal in approximately 50% of cases. Lactic acidosis can also be associated with a variety of pathophysiological conditions, including diabetes, and any condition with significant tissue hypoperfusion and hypoxemia. Lactic acidosis is characterized by elevated blood lactate levels (>5 mmol/L), decreased blood pH, electrolyte disturbances (increased anion gap), and an elevated lactate/pyruvate ratio. When metformin is considered the cause of lactic acidosis, plasma metformin concentrations >5 μg/mL are typically found. The incidence of lactic acidosis in patients taking metformin hydrochloride tablets (USP) is reported to be very low (approximately 0.03 cases per 1000 person-years, with approximately 0.015 deaths per 1000 person-years). No reports of lactic acidosis have been observed in clinical trials involving over 20,000 person-years of metformin exposure. Reported cases primarily occur in diabetic patients with severe renal impairment, including intrinsic kidney disease and inadequate renal perfusion, often accompanied by multiple medical/surgical conditions and multiple medications. Patients requiring drug-assisted treatment for congestive heart failure, especially those with unstable conditions or acute congestive heart failure at risk of inadequate renal perfusion and hypoxemia, are at higher risk of lactic acidosis. The risk of lactic acidosis increases with the degree of renal impairment and patient age. Therefore, for patients taking metformin, regular monitoring of renal function and use of the lowest effective dose of metformin can significantly reduce the risk of lactic acidosis. Especially for elderly patients, their renal function should be closely monitored. Metformin hydrochloride (USP) should not be initiated in patients aged 80 years and older unless creatinine clearance measurements show no decline in renal function, as these patients are more prone to lactic acidosis. Furthermore, metformin should be discontinued immediately if any symptoms associated with hypoxemia, dehydration, or sepsis occur. Because impaired liver function significantly reduces lactate clearance, metformin should generally be avoided in patients with clinical or laboratory evidence of liver disease. Patients taking metformin hydrochloride (USP) should be advised to avoid excessive alcohol consumption (whether acute or chronic), as alcohol can enhance the effects of metformin hydrochloride (USP) on lactate metabolism. Additionally, metformin should be temporarily discontinued before any intravascular radiographic examination or surgical procedure. The onset of lactic acidosis is often insidious, with only nonspecific symptoms such as malaise, myalgia, dyspnea, increased drowsiness, and nonspecific abdominal discomfort. More severe acidosis may be accompanied by hypothermia, hypotension, and refractory bradycardia. Patients and their physicians must be aware of the potential importance of these symptoms and should advise patients to notify their doctor immediately if they develop them. Metformin hydrochloride tablets (USP) should be discontinued until the situation is clear. Serum electrolytes, ketone bodies, blood glucose, and, if necessary, blood pH, lactate levels, and even blood metformin levels may aid in diagnosis. Once a patient's condition stabilizes at any dose level of metformin, gastrointestinal symptoms commonly seen in the early stages of treatment are unlikely to be drug-related. Subsequent gastrointestinal symptoms may be due to lactic acidosis or other serious conditions. Fasting venous plasma lactate levels above the upper limit of normal but below 5 mmol/L in patients taking metformin do not necessarily indicate impending lactic acidosis; it could be caused by other mechanisms such as poor glycemic control or obesity, strenuous exercise, or technical problems in sample handling. Lactic acidosis should be suspected in any diabetic patient presenting with metabolic acidosis but lacking evidence of ketoacidosis (ketonuria and ketonemia). Lactic acidosis is a medical emergency that must be treated in a hospital. For patients with lactic acidosis who are taking metformin, the medication should be discontinued immediately, and general supportive care should be initiated promptly. Since metformin hydrochloride tablets (USP) are dialysis-compatible (with clearance rates up to 170 mL/min under good hemodynamic conditions), immediate hemodialysis is recommended to correct the acidosis and remove accumulated metformin. This treatment usually reverses symptoms rapidly and promotes recovery. Metformin accumulation, though rare, can occur in patients with renal insufficiency and can lead to lactic acidosis, a serious and potentially fatal metabolic disorder. The risk of developing lactic acidosis while taking metformin is significantly lower (e.g., 10 times lower) compared to phenformin (which is now discontinued in the US). However, lactic acidosis is a medical emergency requiring immediate hospitalization; in such cases, metformin should be discontinued immediately, and general supportive care (e.g., fluid resuscitation, diuretics) should be initiated immediately. Timely hemodialysis is recommended. Lactic acidosis is characterized by elevated blood lactate levels (above 45 mg/dL), decreased blood pH (below 7.35), electrolyte disturbances (increased anion gap), and an elevated lactate/pyruvate ratio. Lactic acidosis can also be associated with various pathophysiological conditions, including diabetes mellitus, as well as the presence of significant tissue hypoperfusion and hypoxemia. Approximately 50% of metformin-related lactic acidosis cases have been reported to result in death. However, studies have shown that for lactic acidosis cases without predisposing factors of tissue hypoxia (e.g., heart failure, kidney or lung disease), methods to clear metformin from the body may achieve recovery rates exceeding 80%. Urinary tract infections have been reported in 8% and 1.1% of patients, respectively, after taking metformin alone or a fixed-dose combination of metformin and glipizide. Hypertension occurred in 5.6% and 2.9-3.5% of patients, respectively, after taking metformin alone or a fixed-dose combination of metformin and glipizide. Musculoskeletal pain occurred in 6.7% and 8% of patients, respectively, after taking metformin alone or a fixed-dose combination of metformin and glipizide. A patient taking glipizide and enalapril concurrently developed severe acute hepatitis with significantly elevated serum liver transaminases and cholestasis after starting metformin treatment. Accidental injuries were reported in 7.3% and 5.6% of patients taking metformin extended-release tablets (Fodamycin) and immediate-release tablets, respectively. Vasculitis-related pneumonia has been rarely reported when metformin is used in combination with oral sulfonylureas (e.g., glibenclamide). Upper respiratory tract infections were reported in 16.3% and 17.3% of patients taking metformin or a fixed-dose combination of metformin and glibenclamide, respectively. As first-line treatment for type 2 diabetes, 8.5% and 8.1%–9.9% of patients taking metformin or a fixed-dose combination of metformin and glipizide reported upper respiratory tract infections, respectively. As second-line treatment for type 2 diabetes, 10.7% and 10.3% of patients taking metformin or a fixed-dose combination of metformin and glipizide reported upper respiratory tract infections, respectively. In clinical trials, 5.2% and 6.2% of patients reported upper respiratory tract infections after taking metformin or metformin combined with sitagliptin, respectively. 4.2% and 5.6% of patients reported rhinitis after taking metformin extended-release tablets (Fodamy) or regular tablets, respectively. 20.5% and 20.9% of patients reported infections after taking metformin extended-release tablets (Fodamy) or regular tablets, respectively. For more complete data on drug warnings (of 23) for metformin, please visit the HSDB records page. Pharmacodynamics General Actions Insulin is an important hormone that regulates blood glucose levels. Type 2 diabetes is characterized by decreased insulin sensitivity, leading to elevated blood glucose levels when the pancreas cannot compensate. In patients diagnosed with type 2 diabetes, insulin cannot function adequately for tissues and cells (i.e., insulin resistance), and insulin deficiency may exist. Metformin reduces hepatic glucose production, decreases intestinal glucose uptake, and improves insulin sensitivity by increasing peripheral glucose uptake and utilization. Unlike sulfonylureas (which cause hyperinsulinemia), metformin does not alter insulin secretion. Effects on Fasting Plasma Glucose (FPG) and Glycated Hemoglobin (HbA1c) Glycated hemoglobin (HbA1c) is an important periodic indicator used to monitor glycemic control in diabetic patients. Fasting plasma glucose is also a useful and important indicator of glycemic control. In a 29-week clinical trial, metformin reduced fasting blood glucose levels by an average of 59 mg/dL from baseline in participants diagnosed with type 2 diabetes, while increasing them by an average of 6.3 mg/dL from baseline in participants taking a placebo. Metformin also reduced HbA1c levels by approximately 1.4%, compared to an increase of 0.4% in participants taking a placebo. Adenomyosis is a condition associated with dysmenorrhea and menorrhagia, and is linked to overactivation of the PI3K/AKT signaling pathway. This study aimed to investigate the effects of metformin on the growth of normal endometrial stromal cells (ESCs) in patients with adenomyosis and to explore the involvement of AMP-activated protein kinase (AMPK) and the PI3K/AKT pathway. We isolated and cultured human endometrial stem cells (ESCs) from normal endometrium (N-ESCs) and normal endometrium in adenomyosis (A-ESCs) . AMPK expression was detected using immunocytochemistry and Western blotting. The effects of metformin and compound C on embryonic stem cells (ESCs) were determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method, and ESC growth and proliferation were assessed. The AMPK and PI3K/AKT signaling pathways were detected using Western blotting. AMPK expression levels were higher in A-ESCs than in N-ESCs. Metformin inhibited ESC proliferation in a concentration-dependent manner. The IC50 value for A-ESCs was 2.45 mmol/L, while the IC50 value for N-ESCs was 7.87 mmol/L. Metformin increased AMPK activation levels (p-AMPK/AMPK) in A-ESCs by 2.0 ± 0.3-fold, AMPK activation levels in secretory A-ESCs by 2.3-fold, and AMPK activation levels in proliferative A-ESCs by 1.6-fold. Compared with the control group untreated with 17β-estradiol, 17β-estradiol showed mean inhibition rates of 2.1 ± 0.8-fold and 2.5 ± 0.5-fold in A-ESCs during the proliferative and secretory phases, respectively. The inhibitory effect of metformin on AKT activation (p-AKT/AKT) was more significant in secretory A-ESCs (inhibition rate 3.2-fold, control group) than in proliferative A-ESCs (inhibition rate 2.3-fold, control group). The selective AMPK inhibitor Compound C eliminated the effects of metformin on cell growth and the PI3K/AKT signaling pathway. Metformin inhibits cell growth by activating AMPK and subsequently inhibiting the PI3K/AKT signaling pathway in A-ESCs, especially during the secretory phase, suggesting that metformin has a greater effect on secretory A-ESCs. [5] Objective: To detect glycogen synthesis and glucose and lactate production in cultured rat hepatocytes pretreated with metformin (0-500 μM) for 24 hours. Methods: Cells were incubated with [1-13C]-glucose and [1-13C]-lactic acid so that we could study in detail the effects of metformin on glycogenolysis and glucose production by gluconeogenesis using nuclear magnetic resonance spectroscopy. Filtered ¹H and ¹³C¹H-NMR spectra were recorded using flow injection. Results: Metformin reduced glycogen synthesis in a dose-dependent manner, with an IC50 of 196.5 μM. The presence of the glycogen phosphorylase inhibitor DAB did not reverse this effect, indicating that glycogenolysis was unaffected. Significant correlations were observed between glucose production and glycogen content (0.97 < R < 0.99; p < 0.001) and between lactate production and glycogen content (0.97 < R < 0.99; p < 0.001). Furthermore, in cells treated with 350 μM metformin, the activity of lactate/pyruvate (3 mM/0.3 mM) in glucose production was significantly inhibited (62%, p < 0.001). Conclusion: Hepatocytes pre-incubated for 24 hours in the presence of clinically relevant concentrations of metformin showed inhibition of both glycogen synthesis and gluconeogenesis. [6]

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Background and Objectives: Prostate cancer is the most common cancer in men in many populations. Metformin is the most widely used antidiabetic drug in the world, and there is growing evidence that it has potential anticancer efficacy. Metformin can inhibit the proliferation of various cancer cells, including prostate cancer, colon cancer, breast cancer, ovarian cancer, and glioma cell lines. MicroRNAs (miRNAs) are small, non-coding single-stranded RNAs that can downregulate gene expression. This study aimed to evaluate the effect of metformin treatment on miRNA expression changes in PC-3 cells and its potential association with biological behavior. Materials and Methods: The mean viability of PC-3 cells and the cytotoxic effect of metformin were measured every 24 hours for three days using the xCELLigence system. The IC50 value of metformin in PC-3 cells was 5 mM. RNA samples were analyzed using a custom multi-species microarray containing 1209 probes covering 1221 mature human miRNAs from the miRBase 16.0 database. Results: Among the human miRNAs analyzed by the microarray, compared with the control group, the metformin treatment group showed upregulated expression of 10 miRNAs and downregulated expression of 12 miRNAs. In summary, the changes in the expression of miR-146a, miR-100, miR-425, miR-193a-3p, and miR-106b in metformin-treated cells may be significant. This study may highlight a novel role of metformin in the regulation of miRNAs in prostate cancer. [7]

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Metformin hydrochloride is a first-line oral hypoglycemic agent used to treat type 2 diabetes with a variety of off-target pharmacological effects [1,5,6]
- Its core mechanism involves activating AMPK, thereby regulating energy metabolism, inhibiting cell proliferation and regulating signaling pathways in multiple cell types (e.g., AKT) [5]
- In cancer cells (lung cancer, prostate cancer), it exerts antitumor effects by inducing apoptosis (JNK/p38 MAPK/GADD153 pathway) or altering miRNA expression [3,7]
- It alleviates liver fibrosis by inducing mitochondrial apoptosis in hepatic stellate cells (a key cell type in the process of fibrosis) [4]
- In myocardial infarction, it improves cardiac function by reducing oxidative stress and inflammation [1]
- It promotes mitochondrial autophagy in monocytes, suggesting that it plays a role in regulating mitochondrial quality control [2]
- Besides diabetes, it has also shown potential therapeutic value in myocardial infarction, adenomyosis, liver fibrosis, and certain cancers [1,4,5,7]

C4H11N5.HCL

165.62

165.078

C, 29.01; H, 7.30; Cl, 21.40; N, 42.29

1115-70-4

Metformin-d6 hydrochloride;1185166-01-1; 657-24-9; 1115-70-4 (HCl); 121369-64-0 (glycinate); 58840-24-7 (orotate); 34461-22-8 ( embonate); 1384526-74-2 (icosapent)

4091

White to off-white solid

224.1ºC at 760 mmHg

223-226 °C(lit.)

89.3ºC

0.0929mmHg at 25°C

1.058

3

1

2

9

132

0

N(/C(=N\[H])/N=C(\N([H])[H])/N([H])[H])(C([H])([H])[H])C([H])([H])[H]

OETHQSJEHLVLGH-UHFFFAOYSA-N

InChI=1S/C4H11N5.ClH/c1-9(2)4(7)8-3(5)6;/h1-2H3,(H5,5,6,7,8);1H

1,1-Dimethylbiguanide hydrochloride

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 3 mg/mL (18.11 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 3 mg/mL (18.11 mM) (saturation unknown) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: 100 mg/mL (603.79 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)