Low-dose Metformin Treatment Research Articles

Low-Dose Metformin Treatment Reduces In Vitro Growth of the LL/2 Non-small Cell Lung Cancer Cell Line.

Lung cancer maintains a relatively small survival rate (~19%) over a 5-year period and up to 80-85% of all lung cancer diagnoses are Non-Small Cell Lung Cancer (NSCLC). To determine whether metformin reduces non-small cell lung cancer (NSCLC) LL/2 cell growth, cells were grown in vitro and treated with metformin for 48 h. qPCR was used to assess genes related to cell cycle regulation and pro-apoptotic markers, namely Cyclin D, CDK4, p27, p21, and HES1. Treatment with 10 mM metformin significantly reduced HES1 expression (p = 0.011). Furthermore, 10 mM metformin treatment significantly decreased REDD1 (p = 0.0082) and increased p-mTOR Ser2448 (p = 0.003) protein expression. Control cells showed significant reductions in phosphorylated p53 protein expression (p = 0.0367), whereas metformin treated cells exhibited reduced total p53 protein expression (p = 0.0078). There were no significant reductions in AMPK, PKB/AKT, or STAT3. In addition, NSCLC cells were treated for 48 h. with 10 mM metformin, 4 µM gamma-secretase inhibitor (GSI), or the combination of metformin (10 mM) and GSI (4 µM) to determine the contribution of respective signaling pathways. Metformin treatment significantly reduced total nucleus expression of the proliferation maker Ki-67 with an above 65% reduction in Ki-67 expression between control and metformin-treated cells (p = 0.0021). GSI (4 µM) treatment significantly reduced Ki-67 expression by ~20% over 48 h (p = 0.0028). Combination treatment (10 mM metformin and 4 µM GSI) significantly reduced Ki-67 expression by more than 50% over 48 h (p = 0.0245). As such, direct administration of metformin (10 mM for 48 h) proved to be an effective pharmaceutical agent in reducing the proliferation of cultured non-small cell cancer cells. These intriguing in vitro results, therefore, support the further study of metformin in appropriate in vivo models as an anti-oncogenic agent and/or an adjunctive therapy.

P–288 Changes in gene and protein expression in human endometrial cancer cell lines after low dose metformin treatment over time

Abstract Study question Does metformin treatment lead to temporal expression changes of specific genes and proteins in endometrial cancer cell lines? Summary answer The expression of three different genes and proteins was investigated, of which all displayed changes over time. What is known already Endometrial cancer (EC) is one of the most common malignancies among postmenopausal women. A long-term estrogen effect on the endometrium often seen in women with obesity or the polycystic ovary syndrome (PCOS), as well as type II diabetes mellitus (T2DM) are well-known risk factors for the development and progression of EC. Metformin is a biguanide used in the treatment of T2DM patients and off label in women with PCOS. Moreover, metformin displays anti-tumor and anti-proliferative effects in various cancer types, including EC. In that regards BCL2L11, CDH1 and CDKN1A play an important role in apoptotic pathways, proliferation and invasion processes. Study design, size, duration The EC cells were cultured in normal (5.5 mmol/L) or high (17 mmol/L) glucose medium supplemented with 10 nmol/L ß-estradiol. The cells were treated with low dose metformin (1.0 mmol/L) for 2, 6, 24, 48 and 168 h (7 d). In addition, EC cells were treated with a combination of metformin and insulin (100 ng/mL) or remained untreated. Five independent experiments were fulfilled and untreated cells served as controls. Participants/materials, setting, methods The study was accomplished using two different human EC cell lines. HEC–1A represents an estrogen-independent EC, whereas Ishikawa represents the more common, estrogen-dependent EC. Proteins were extracted, quantified with a BCA assay, and the protein expression of BCL2L11, CDH1 and CDKN1A was analyzed by western blots. Furthermore, total RNA was extracted, transcribed to cDNA and Taqman real-time PCR was carried out to measure the expression of the associated genes, using fold change (FC) as parameter. Main results and the role of chance The expression of the selected genes, analyzed by RT-PCR, changed in both cell lines over time as follows: After 6 h, metformin induced a decrease in the expression of BCL2L11 (FC = 0.7) and CDH1 (FC = 0.75), whereas the expression of CDKN1A slightly increased (FC = 0.95–1.35). After 24 h, BCL2L11 expression increased in normal glucose groups (FC = 1.3, high glucose: FC = 0.93) and CDH1 expression decreased in combination with metformin and high glucose (FC = 0.7, normal glucose: FC = 1.1). CDKN1A expression was increased by metformin in both cell lines after 24 h (FC = 1.2–1.8). After 48 h of metformin treatment, expression for all three genes was only slightly changed (FC = 0.9–1.0). After 7 d it was observed that the combination of high glucose and metformin (i.e. like obese T2DM patients) led to an increased expression of BCL2L11, CDH1 and CDKN1A (FC = 1.4–2.9) in the presence and absence of insulin, whereas metformin induced a decreased expression of CDH1 and CDKN1A (FC = 0.5–0.75) in normal glucose medium. BCL2L11, CDH1 and CDKN1A expression was investigated at the protein level as well. Limitations, reasons for caution The results cannot be directly transferred to metformin treatment of patients, since the study was carried out in vitro. Additionally, further studies including more timepoints would indicate a more precisely gene and protein expression over time. Wider implications of the findings: This is the first in vitro study showing the temporal changes of BCL2L11, CDH1 and CDKN1A expression, genes related to tumorigenesis due to low dose metformin over time, suggesting differentially pathways in long term metformin treatment using physiologically achievable metformin levels. Trial registration number Not applicable

Renoprotective Effects of Low‐dose Metformin Treatment in a Mouse Model of Diabetic Kidney Disease

Background Diabetes mellitus (DM) affects more than 30 million people in the United States. About one-third of DM patients develop diabetic kidney disease (DKD). Metformin is a widely prescribed antidiabetic medication for patients with type 2 diabetes; however, its direct effects on renal function are poorly understood. Hence, our study aimed to investigate the effects of a non-glucose lowering, low-dose metformin treatment against the progression of DKD using a genetic mouse model of type 2 diabetes. Methods Our study utilized db/db mice, which spontaneously develop type 2 diabetes due to a mutation in leptin receptors. To aggravate the degree of renal injury, each mouse had its left kidney removed at ten weeks of age. Heterozygous, non-diabetic mice were used as the control group. The study had four treatment groups: 1) vehicle-treated control, 2) metformin-treated control, 3) diabetic, vehicle-treated, and 4) diabetic, metformin-treated. Mice were treated with either vehicle or metformin (100 mg/kg/day) for four weeks. Urine and kidney tissue samples were collected at the end of the study to measure the markers of renal dysfunction, such as elevations in urine albumin-creatinine ratio (UACR) and kidney injury molecule 1 (KIM-1), and the markers of renal fibrosis, such as transforming growth factor-beta (TGF-β) and alpha-smooth muscle actin (α-SMA). Results Our results indicate that low-dose metformin-treatment decreased UACR (117.53 ± 61.56 vs. 205.3 ± 112.5); however, it did not alter the elevated fasting blood glucose levels or plasma creatinine levels in diabetic mice. Kidneys from metformin-treated diabetic mice revealed reduced KIM-1 immunostaining (1.5 ± 0.5 vs. 2.3 ± 0.7) compared to vehicle-treated diabetic mice. Similarly, the metformin-treated diabetic mice kidneys showed decreased immunostaining for TGF-β (0.7 ± 0.2 vs. 1.3 ± 0.3). Moreover, the renal protein expression of α-SMA (assessed via western blotting) was significantly reduced in metformin-treated diabetic mice than the vehicle-treated counterparts. Conclusion Our findings suggest that low-dose metformin treatment ameliorates renal dysfunction and fibrosis associated with DKD. Future studies are warranted to ascertain the renoprotective effects of low-dose Metformin treatment against the progression of DKD.

Low-dose metformin treatment in the subacute phase improves the locomotor function of a mouse model of spinal cord injury.

Metformin, a first-line drug for type-2 diabetes, has been shown to improve locomotor recovery after spinal cord injury. However, there are studies reporting no beneficial effect. Recently, we found that high dose of metformin (200 mg/kg, intraperitoneal) and acute phase administration (immediately after injury) led to increased mortality and limited locomotor function recovery. Consequently, we used a lower dose (100 mg/kg, i.p.) metformin in mice, and compared the effect of immediate administration after spinal cord injury (acute phase) with that of administration at 3 days post-injury (subacute phase). Our data showed that metformin treatment starting at the subacute phase significantly improved mouse locomotor function evaluated by Basso Mouse Scale (BMS) scoring. Immunohistochemical studies also revealed significant inhibitions of microglia/macrophage activation and astrogliosis at the lesion site. Furthermore, metformin treatment at the subacute phase reduced neutrophil infiltration. These changes were in parallel with the increased survival rate of spinal neurons in animals treated with metformin. These findings suggest that low-dose metformin treatment for subacute spinal cord injury can effectively improve the functional recovery possibly through anti-inflammation and neuroprotection. This study was approved by the Institute Animal Care and Use Committee at the University of Texas Medical Branch (approval No. 1008041C) in 2010.

Low-Dose Metformin Reprograms the Tumor Immune Microenvironment in Human Esophageal Cancer: Results of a Phase II Clinical Trial

The tumor immune microenvironment (TIME) has an important impact on response to cancer immunotherapy using immune checkpoint inhibitors. Specifically, an "infiltrated-excluded"/"cold" TIME is predictive of poor response. The antidiabetic agent metformin may influence anticancer immunity in esophageal squamous cell carcinoma (ESCC). We analyzed matched pre- and posttreatment ESCC specimens in a phase II clinical trial of low-dose metformin treatment (250 mg/day) to evaluate direct anti-ESCC activity and TIME reprogramming. Follow-up correlative studies using a carcinogen-induced ESCC mouse model were performed with short-term (1 week) or long-term (12 weeks) low-dose metformin (50 mg/kg/day) treatment. In the clinical trial, low-dose metformin did not affect proliferation or apoptosis in ESCC tumors as assayed by Ki67 and cleaved caspase-3 immunostaining. However, metformin reprogrammed the TIME toward "infiltrated-inflamed" and increased the numbers of infiltrated CD8+ cytotoxic T lymphocyte and CD20+ B lymphocyte. Further, an increase in tumor-suppressive (CD11c+) and a decrease in tumor-promoting (CD163+) macrophages were observed. Metformin augmented macrophage-mediated phagocytosis of ESCC cells in vitro. In the ESCC mouse model, short-term metformin treatment reprogrammed the TIME in a similar fashion to humans, whereas long-term treatment further shifted the TIME toward an active state (e.g., reduction in CD4+ FoxP3+ regulatory T cells) and inhibited ESCC growth. In both humans and mice, metformin triggered AMPK activation and STAT3 inactivation, and altered the production of effector cytokines (i.e., TNFα, IFNγ, and IL10) in the immune cells. Low-dose metformin reprograms the TIME to an activated status and may be a suitable immune response modifier for further investigation in patients with ESCC.

Low‐Dose Metformin Treatment Ameliorates Renal Dysfunction and Fibrosis in a Mouse Model of Diabetic Nephropathy

BackgroundDiabetes mellitus is the leading cause of chronic kidney disease and end‐stage renal disease in the United States. About one‐third of patients with diabetes develop renal dysfunction, commonly referred to as diabetic nephropathy. Metformin is a widely prescribed antidiabetic medication for patients with type 2 diabetes; however, its direct effects on the renal function besides normalizing glucose levels in patients with diabetic nephropathy are poorly understood. Thus, the objective of our study was to investigate the effects of a non‐glucose lowering, low‐dose metformin treatment on the diabetic kidney using a genetic mouse model of type 2 diabetes.MethodsOur study utilized db/db mice (Leprdb), which harbor a mutation in their leptin receptors, and hence, show the signs and symptoms similar to those experienced in patients with type 2 diabetes. To accelerate the progression of nephropathy, each mouse had their left kidney removed at ten weeks of age. Heterozygous mice were used as a control group for the study. Our study had four treatment groups (n=8 per group): 1) Vehicle‐treated control (non‐diabetic), 2) Metformin‐treated control (non‐diabetic), 3) Diabetic, vehicle‐treated, and 4) Diabetic, metformin‐treated. Mice were treated with either vehicle or metformin (100 mg/kg/day) for four weeks. Fasting blood glucose and urine samples were collected at the beginning and end of the study. Plasma and kidney tissue samples were collected at the end of the study. Markers of renal dysfunction such as elevations in urine albumin‐creatinine ratio (UACR) and plasma creatinine were measured in the samples collected from the study. Alpha‐smooth muscle (α‐SMA), a marker of renal fibrosis, were measured in kidney samples via Western blotting.ResultsOur results showed that the fasting blood glucose (FBG) levels were elevated in the diabetic mice (470.29 ±73.35 mg/dL) at the beginning of the study and were gradually increased with disease progression (672.57 ±107.88 mg/dL). Our results also demonstrated that low‐dose metformin treatment did not lower FBG levels in diabetic mice (583.38 ±195.77 mg/dL). However, metformin treatment was able to prevent the increase in UACR in diabetic mice (205.3 ±112.5 vs. 117.53 ±61.56 μg albumin/mg creatinine). Results from the plasma creatinine levels show that diabetic mice had increased plasma creatinine levels (0.72 ±0.07 mg/dL) compared to the control mice (0.51 ±0.06 mg/dL). Intriguingly, low‐dose metformin treatment did not prevent the diabetes‐induced elevations in plasma creatinine levels. Western blot analysis for α‐SMA indicated that only the diabetic mice expressed α‐SMA in the kidney and low‐dose metformin treatment diminished renal expression of α‐SMA in diabetic mice.ConclusionOur findings suggest that low‐dose metformin treatment improves renal function, and ameliorates renal fibrosis observed with diabetic nephropathy. Further studies are required to investigate the mechanisms that underlie the nephroprotective effects of low‐dose metformin therapy in diabetic mice.Support or Funding InformationFundingThis study was supported by intramural funds received from Drake University through Harris Research Endowment, Kresge Endowment, and Provost fund.

AMPK activation protects from neuronal dysfunction and vulnerability across nematode, cellular and mouse models of Huntington's disease.

The adenosine monophosphate activated kinase protein (AMPK) is an evolutionary-conserved protein important for cell survival and organismal longevity through the modulation of energy homeostasis. Several studies suggested that AMPK activation may improve energy metabolism and protein clearance in the brains of patients with vascular injury or neurodegenerative disease. However, in Huntington's disease (HD), AMPK may be activated in the striatum of HD mice at a late, post-symptomatic phase of the disease, and high-dose regiments of the AMPK activator 5-aminoimidazole-4-carboxamide ribonucleotide may worsen neuropathological and behavioural phenotypes. Here, we revisited the role of AMPK in HD using models that recapitulate the early features of the disease, including Caenorhabditis elegans neuron dysfunction before cell death and mouse striatal cell vulnerability. Genetic and pharmacological manipulation of aak-2/AMPKα shows that AMPK activation protects C. elegans neurons from the dysfunction induced by human exon-1 huntingtin (Htt) expression, in a daf-16/forkhead box O-dependent manner. Similarly, AMPK activation using genetic manipulation and low-dose metformin treatment protects mouse striatal cells expressing full-length mutant Htt (mHtt), counteracting their vulnerability to stress, with reduction of soluble mHtt levels by metformin and compensation of cytotoxicity by AMPKα1. Furthermore, AMPK protection is active in the mouse brain as delivery of gain-of-function AMPK-γ1 to mouse striata slows down the neurodegenerative effects of mHtt. Collectively, these data highlight the importance of considering the dynamic of HD for assessing the therapeutic potential of stress-response targets in the disease. We postulate that AMPK activation is a compensatory response and valid approach for protecting dysfunctional and vulnerable neurons in HD.

Inhibition of Polo-like Kinase 1 (Plk1) Enhances the Antineoplastic Activity of Metformin in Prostate Cancer

The widely used anti-diabetic drug metformin has been shown to exert strong antineoplastic actions in numerous tumor types, including prostate cancer (PCa). In this study, we show that BI2536, a specific Plk1 inhibitor, acted synergistically with metformin in inhibiting PCa cell proliferation. Furthermore, we also provide evidence that Plk1 inhibition makes PCa cells carrying WT p53 much more sensitive to low-dose metformin treatment. Mechanistically, we found that co-treatment with BI2536 and metformin induced p53-dependent apoptosis and further activated the p53/Redd-1 pathway. Moreover, we also show that BI2536 treatment inhibited metformin-induced glycolysis and glutamine anaplerosis, both of which are survival responses of cells against mitochondrial poisons. Finally, we confirmed the cell-based observations using both cultured cell-derived and patient-derived xenograft studies. Collectively, our findings support another promising therapeutic strategy by combining two well tolerated drugs against PCa proliferation and the progression of androgen-dependent PCa to the castration-resistant stage.

Metformin suppresses gluconeogenesis by inhibiting mitochondrial glycerophosphate dehydrogenase.

Metformin is considered to be one of the most effective therapeutics for the treatment of type 2 diabetes (T2D) since it specifically reduces hepatic gluconeogenesis without increasing insulin secretion, inducing weight gain, or posing a risk of hypoglycemia1,2. For over half a century, this agent has been prescribed to T2D patients worldwide, yet the underlying mechanism by which metformin inhibits hepatic gluconeogenesis remains unknown. Here we show that metformin non-competitively inhibits the redox shuttle enzyme mitochondrial glycerophosphate dehydrogenase (mGPD), resulting in an altered hepatocellular redox state, reduced conversion of lactate and glycerol to glucose, and decreased hepatic gluconeogenesis. Acute and chronic low-dose metformin treatment effectively reduced endogenous glucose production (EGP), while increasing cytosolic redox and decreasing mitochondrial redox states. Antisense oligonucleotide (ASO) knockdown of hepatic mGPD in rats resulted in a phenotype akin to chronic metformin treatment, and abrogated metformin-mediated increases in cytosolic redox state, decrease in plasma glucose concentrations and inhibition of EGP. These findings were replicated in whole-body mGPD knockout mice. These results have significant implications for understanding the mechanism of metformin’s blood glucose lowering effects and provide a novel therapeutic target for T2D.

Effects of metformin on peripheral insulin sensitivity and intracellular lipid contents in muscle and liver of overweight Japanese subjects

Recent studies suggest that insulin resistance is associated with increased intrahepatic lipid (IHL) and intramyocellular lipid (IMCL) contents. While metformin improves insulin resistance mainly in liver, its effects on IHL and IMCL have not been clarified yet. The aim of this study was to investigate the effects of low-dose metformin (750 mg/day) on peripheral insulin sensitivity, IHL and IMCL. Before and 3 months after low-dose metformin therapy, eight overweight/obese Japanese subjects [body mass index (BMI) >25 kg/m2] were studied with blood sampling, measurement of IHL and IMCL by 1H magnetic resonance spectroscopy and glucose infusion rate (GIR) during euglycaemic-hyperinsulinaemic clamp as an index of peripheral insulin sensitivity. A 3-month low-dose metformin therapy did not alter body weight, total body fat, fat distribution or physical activity level but increased GIR by 31% (from 6.24 +/- 0.86 to 7.82 +/- 0.82 mg/kg/min, p < 0.01). Although metformin treatment did not alter IMCL (from 4.1 +/- 1.0 to 4.2 +/- 0.9, not significant), it decreased IHL by 21% (from 15.9 +/- 2.8 to 11.8 +/- 2.2%, p < 0.05). A 3-month low-dose metformin treatment improved peripheral insulin sensitivity and reduced IHL, without significantly changing BMI, adiposity or IMCL.

Acute Metformin Therapy Confers Cardioprotection Against Myocardial Infarction Via AMPK-eNOS–Mediated Signaling

Clinical studies have reported that metformin reduces cardiovascular end points of type 2 diabetic subjects by actions that cannot solely be attributed to glucose-lowering effects. The therapeutic effects of metformin have been reported to be mediated by its activation of AMP-activated protein kinase (AMPK), a metabolite sensing protein kinase whose activation following myocardial ischemia has been suggested to be an endogenous protective signaling mechanism. We investigated the potential cardioprotective effects of a single, low-dose metformin treatment (i.e., 286-fold less than the maximum antihyperglycemic dose) in a murine model of myocardial ischemia-reperfusion (I/R) injury. Nondiabetic and diabetic (db/db) mice were subjected to transient myocardial ischemia for a period of 30 min followed by reperfusion. Metformin (125 microg/kg) or vehicle (saline) was administered either before ischemia or at the time of reperfusion. Administration of metformin before ischemia or at reperfusion decreased myocardial injury in both nondiabetic and diabetic mice. Importantly, metformin did not alter blood glucose levels. During early reperfusion, treatment with metformin augmented I/R-induced AMPK activation and significantly increased endothelial nitric oxide (eNOS) phosphorylation at residue serine 1177. These findings provide important information that myocardial AMPK activation by metformin following I/R sets into motion events, including eNOS activation, which ultimately lead to cardioprotection.

Acute effect of low dose metformin treatment on insulin resistance and hyperandrogenemia in insulin resistant women with polycystic ovary syndrome (PCOS)

Acute effect of low dose metformin treatment on insulin resistance and hyperandrogenemia in insulin resistant women with polycystic ovary syndrome (PCOS)