Metformin-treated Animals Research Articles

Elucidating metformin action on the ovary and fertility in healthy mice.

The hypoglycemic drug metformin has shown reproductive effects in women, although its mechanism of action is not fully understood. In this study, we demonstrate the direct effects of metformin on the ovary of healthy mice, with no alterations in fertility. Metformin is a hypoglycemic drug widely used in type-2 diabetes (T2D) patients. In recent years, this drug has been suggested as a treatment for gestational diabetes and recommended to women with ovarian hyperstimulation syndrome (PCOS) to increase the chances of pregnancy or avoid early miscarriages. However, the exact effects of metformin on the female reproductive tract in general, and on the ovary in particular, are still not completely understood. In this study, we analyzed the effect of metformin on fertility and ovarian physiology in healthy female mice. We found that this drug altered the estrous cycle, early follicular development, serum estradiol and progesterone levels, and ovarian steroidogenic enzyme expression. Moreover, ovarian angiogenesis was lower in metformin-treated animals compared with untreated ones, whereas natural or gonadotropin-induced fertilization rates remained unchanged. However, offspring of metformin-treated animals displayed decreased body weight at birth. In this work, we unraveled the main effects of metformin on the ovary, isolated from other conditions such as hyperglycemia and hyperandrogenism, which is essential for a better understanding of metformin's mechanisms of action on reproduction and fertility.

Progression of established non-diabetic chronic kidney disease is halted by metformin treatment in rats

Current treatment strategies for chronic kidney disease (CKD) mainly focus on controlling risk factors. Metformin, a first-line drug for type 2 diabetes, exerts beneficial pleiotropic actions beyond its prescribed use and incipient data have revealed protective effects against the development of kidney impairment. This study evaluated the therapeutic efficacy of metformin and canagliflozin, a sodium-glucose cotransporter-2 (SGLT2) inhibitor recently approved by the United States Food and Drug Administration to treat diabetic nephropathy, in slowing the progression of established non-diabetic CKD. Rats with adenine-induced CKD were assigned to different treatment groups to receive either 200 mg/kg metformin, four or five weeks after the start of the adenine diet (established mild-moderate CKD), or 25 mg/kg canagliflozin four weeks after the start of the diet, by daily oral gavage administered during four weeks. Each treatment group was compared to a vehicle group. Chronic adenine dosing resulted in severe CKD in vehicle-treated rats as indicated by a marked rise in serum creatinine levels, a marked decrease in creatinine clearance, and a disturbed mineral metabolism. Metformin, but not canagliflozin, halted functional kidney decline. Additionally, kidneys of metformin-treated animals showed less interstitial area and inflammation as compared to the vehicle group. Proteomic analyses revealed that metformin's kidney-protective effect was associated with the activation of the Hippo signaling pathway, a highly conserved multiprotein kinase cascade that controls tissue development, organ size, cell proliferation, and apoptosis. Thus, metformin demonstrated therapeutic efficacy by halting the progression of established CKD in a rat model.

Kv1.3 Channel Blockade Improves Inflammatory Profile, Reduces Cardiac Electrical Remodeling, and Prevents Arrhythmia in Type 2 Diabetic Rats

PurposeKv1.3 channel regulates the activity of lymphocytes, macrophages, or adipose tissue and its blockade reduces inflammatory cytokine secretion and improves insulin sensitivity in animals with metabolic syndrome and in genetically obese mice. Thus, Kv1.3 blockade could be a strategy for the treatment of type 2 diabetes. Elevated circulating levels of TNFα and IL-1b mediate the higher susceptibility to cardiac arrhythmia in type 2 diabetic rats. We hypothesized that Kv1.3 channel blockade with the psoralen PAP1 could have immunomodulatory properties that prevent QTc prolongation and reduce the risk of arrhythmia in type 2 diabetic rats.MethodsType 2 diabetes was induced to Sprague-Dawley rats by high-fat diet and streptozotocin injection. Diabetic animals were untreated, treated with metformin, or treated with PAP1 for 4 weeks. Plasma glucose, insulin, cholesterol, triglycerides, and cytokine levels were measured using commercial kits. ECG were recorded weekly, and an arrhythmia-inducing protocol was performed at the end of the experimental period. Action potentials were recorded in isolated ventricular cardiomyocytes.ResultsIn diabetic animals, PAP1 normalized glycaemia, insulin resistance, adiposity, and lipid profile. In addition, PAP1 prevented the diabetes-induced repolarization defects through reducing the secretion of the inflammatory cytokines IL-10, IL-12p70, GM-CSF, IFNγ, and TNFα. Moreover, compared to diabetic untreated and metformin-treated animals, those treated with PAP1 had the lowest risk of developing the life-threatening arrhythmia Torsade de Pointes under cardiac challenge.ConclusionKv1.3 inhibition improves diabetes and diabetes-associated low-grade inflammation and cardiac electrical remodeling, resulting in more protection against cardiac arrhythmia compared to metformin.

Postnatal metformin treatment alters rat Sertoli cell proliferation and daily sperm production.

The direct correlation between Sertoli cell number and sperm production capacity highlights the importance of deciphering external factors that modify Sertoli cell proliferation. A growing body of evidence in vitro suggests that metformin, the main pharmacological agent for type 2 diabetes treatment in children, exerts anti-proliferative effects on Sertoli cells. The aims of this study were to investigate the effect of metformin administration during postnatal period on Sertoli cell proliferation and on cell cycle regulators expression and to analyze the impact of this treatment on the sperm production capacity in adulthood. Sprague Dawley rat pups were randomly divided into two groups: MET (receiving daily 200mg/kg metformin, from Pnd3 to Pnd7 inclusive) and control (receiving vehicle). BrdU incorporation was measured to assess proliferation. Gene expression analyses were performed in Sertoli cells isolated from animals of both groups. Daily sperm production and sperm parameters were measured in adult male rats (Pnd90) that received neonatal treatment. MET group exhibited a significant decrease in BrdU incorporation in Sertoli cells. Concordantly, MET group showed a reduction in cyclin D1 and E2 expression and an increase in p21 expression in Sertoli cells. In addition, metformin-treated animals displayed lower values of daily sperm production on Pnd90. These results suggest that metformin treatment may lead to a decrease in Sertoli cell proliferation, a concomitant altered expression of cell cycle regulators and ultimately, a reduction in daily sperm production in adult animals.

Metformin enhances protection in guinea pigs chronically infected with Mycobacterium tuberculosis

Tuberculosis (TB) is a chronic inflammatory disease that is often associated with alterations in systemic and cellular metabolism that resolves following successful antimicrobial drug treatment. We hypothesized that altered systemic glucose metabolism as a consequence of Mycobacterium tuberculosis (Mtb) infection, contributes to TB pathogenesis, and when normalized with anti-glycemic drugs would improve clinical outcomes. To test this hypothesis, guinea pigs were treated daily with the anti-diabetic drug metformin starting 4 weeks prior or concurrent with aerosol exposure to the H37Rv strain of Mtb. In the chronic stages of infection, Mtb infected metformin-treated animals had restored systemic insulin sensitivity but remained glucose intolerant as determined by oral glucose tolerance testing. Despite persistent glucose intolerance, metformin-treated guinea pigs had a 2.8-fold reduction in lung lesion burden and a 0.7 log decrease in CFUs. An alternative hypothesis that metformin treatment improved clinical disease by having a direct effect on immune cell energy metabolism was tested using extracellular flux analysis and flow cytometry. The proinflammatory immune response to Mtb infection in untreated guinea pigs was associated with a marked increase in energy metabolism (glycolysis and mitochondrial respiration) of peripheral blood mononuclear cells (PBMCs), which was normalized in metformin-treated guinea pigs. Moreover, both CD4+ and CD8+ T lymphocytes from Mtb infected, metformin treated animals maintained a more normal mitochondrial membrane potential while those isolated from untreated animals had persistent mitochondrial hyperpolarization. These data suggest that metformin promotes natural host resistance to Mtb infection by maintaining immune cell metabolic homeostasis and function during the chronic stages of active TB disease.

Metformin improves vascular and metabolic insulin action in insulin-resistant muscle.

Insulin stimulates glucose disposal in skeletal muscle in part by increasing microvascular blood flow, and this effect is blunted during insulin resistance. We aimed to determine whether metformin treatment improves insulin-mediated glucose disposal and vascular insulin responsiveness in skeletal muscle of insulin-resistant rats. Sprague-Dawley rats were fed a normal (ND) or high-fat (HFD) diet for 4 weeks. A separate HFD group was given metformin in drinking water (HFD + MF, 150 mg/kg/day) during the final 2 weeks. After the intervention, overnight-fasted (food and metformin removed) anaesthetised rats underwent a 2-h euglycaemic-hyperinsulinaemic clamp (10 mU/min/kg) or saline infusion. Femoral artery blood flow, hindleg muscle microvascular blood flow, muscle glucose disposal and muscle signalling (Ser473-AKT and Thr172-AMPK phosphorylation) were measured. HFD rats had elevated body weight, epididymal fat pad weight, fasting plasma insulin and free fatty acid levels when compared to ND. HFD-fed animals displayed whole-body and skeletal muscle insulin resistance and blunting of insulin-stimulated femoral artery blood flow, muscle microvascular blood flow and skeletal muscle insulin-stimulated Ser473-AKT phosphorylation. Metformin treatment of HFD rats reduced fasting insulin and free fatty acid concentrations and lowered body weight and adiposity. During euglycaemic-hyperinsulinaemic clamp, metformin-treated animals showed improved vascular responsiveness to insulin, improved insulin-stimulated muscle Ser473-AKT phosphorylation but only partially restored (60%) muscle glucose uptake. This occurred without any detectable levels of metformin in plasma or change in muscle Thr172-AMPK phosphorylation. We conclude that 2-week metformin treatment is effective at improving vascular and metabolic insulin responsiveness in muscle of HFD-induced insulin-resistant rats.

Metformin treatment reduces motor and neuropsychiatric phenotypes in the zQ175 mouse model of Huntington disease

Huntington disease is a neurodegenerative condition for which there is no cure to date. Activation of AMP-activated protein kinase has previously been shown to be beneficial in in vitro and in vivo models of Huntington’s disease. Moreover, a recent cross-sectional study demonstrated that treatment with metformin, a well-known activator of this enzyme, is associated with better cognitive scores in patients with this disease. We performed a preclinical study using metformin to treat phenotypes of the zQ175 mouse model of Huntington disease. We evaluated behavior (motor and neuropsychiatric function) and molecular phenotypes (aggregation of mutant huntingtin, levels of brain-derived neurotrophic factor, neuronal inflammation, etc.). We also used two models of polyglutamine toxicity in Caenorhabditis elegans to further explore potential mechanisms of metformin action. Our results provide strong evidence that metformin alleviates motor and neuropsychiatric phenotypes in zQ175 mice. Moreover, metformin intake reduces the number of nuclear aggregates of mutant huntingtin in the striatum. The expression of brain-derived neurotrophic factor, which is reduced in mutant animals, is partially restored in metformin-treated mice, and glial activation in mutant mice is reduced in metformin-treated animals. In addition, using worm models of polyglutamine toxicity, we demonstrate that metformin reduces polyglutamine aggregates and restores neuronal function through mechanisms involving AMP-activated protein kinase and lysosomal function. Our data indicate that metformin alleviates the progression of the disease and further supports AMP-activated protein kinase as a druggable target against Huntington’s disease.

Genistein enhances the secretion of glucagon-like peptide-1 (GLP-1) via downregulation of inflammatory responses

Glucagon-like peptide-1 (GLP-1) an incretin hormone, is known to regulate the glucose-mediated insulin secretion. However, reduction in the level of GLP-1 is considered to be a major cause for the reduction of GLP-1-dependent insulin secretory response. Genistein an isoflavone, is an important polyphenol and has wide range of therapeutic potentials, but its therapeutic effects alone and/or in combination with metformin on GLP-1 secretion have not been investigated yet. Hence, we aimed to investigate the stimulatory action of genistein in combination with metformin on GLP-1 via downregulation of inflammatory mediators, hyperlipidemia and hyperglycemia in alloxan-induced diabetic rats. Diabetes was induced in experimental rats by single administration of alloxan intraperitoneally. Metformin (50 mg/kg/day), genistein (20 mg/kg/day) and combination of genistein and metformin was administered in alloxan-induced diabetic rats. We found that genistein alone and/or in combination with metformin significantly increased the serum level (P < 0.01) and tissue content (P < 0.05) of GLP-1 in intestine when compared with that of metformin-treated animals. Similarly, genistein alone and/or in combination with metformin also resulted in normoglycemia (P < 0.001), glucose tolerance (P < 0.01), insulin sensitivity (P < 0.0001), hyperlipidemia (P < 0.01), liver and kidney function biomarkers (P < 0.01) as compared to that of metformin-treated experimental animals. Moreover, genistein alone and/or in combination with metformin also downregulated the inflammatory responses by decreasing the levels of interleuin-6, tumor necrosis factor-α and C-reactive protein in serum (P < 0.05) and intestine (P < 0.001) more efficiently as compared to that of metformin-treated experimental animals. The downregulation of inflammatory responses in intestine, was positively associated with increased secretion of GLP-1 from intestine. Histopathology of pancreas and intestine also showed that genistein significantly improved the deleterious effects of alloxan on pancreas and intestine. Hence, our work provides new insights on the synergistic effects of genistein and metformin on GLP-1 secretion. This may significantly improve the perception for proposing new GLP-1-based synergistic approaches for the treatment of diabetes mellitus.

Effect of Intracoronary Metformin on Myocardial Infarct Size in Swine.

Metformin has been demonstrated to decrease infarct size (IS) and prevent postinfarction left ventricular (LV) remodeling in rodents when given intravenously at the time of reperfusion. It remains unclear whether similar cardioprotection can be achieved in a large animal model. The objective of this study was to determine whether intravascular infusion of metformin at the time of reperfusion reduces myocardial IS in a porcine model of acute myocardial infarction. In a blinded and randomized preclinical study, closed-chest swine (n=20) were subjected to a 60-minute left anterior descending coronary artery occlusion to produce myocardial infarction. Contrast-enhanced computed tomography was performed during left anterior descending coronary artery occlusion to assess the ischemic area-at-risk. Animals were randomized to receive either metformin or vehicle as an initial intravenous bolus (5 mg/kg) 8 minutes before reperfusion, followed by a 15-minute left coronary artery infusion (1 mg/kg per minute) commencing with the onset of reperfusion. Echocardiography and computed tomographic imaging of LV function were performed 1 week later, at which time the heart was removed for postmortem pathological analysis of area-at-risk and IS (triphenyltetrazolium chloride). Baseline variables including hemodynamics and LV function were similar between groups. Peak circulating metformin concentrations of 374±35 µmol/L were achieved 15 minutes after reperfusion. There was no difference between the area-at-risk as a percent of LV mass by computed tomography (vehicle: 20.7%±1.1% versus metformin: 19.7%±1.3%; P=0.59) or postmortem pathology (22.4%±1.2% versus 20.2%±1.2%; P=0.21). IS relative to area-at-risk averaged 44.5%±5.0% in vehicle-treated versus 38.2%±6.8% in metformin-treated animals ( P=0.46). There was no difference in global function 7 days after myocardial infarction as assessed by echocardiography or computed tomographic ejection fraction (56.2%±2.6% versus 56.3%±2.4%; P=0.98). In contrast to rodent hearts, postconditioning with high-dose metformin administered immediately before reperfusion does not reduce IS or improve LV function 7 days after myocardial infarction in swine. These results reinforce the importance of rigorously testing therapies in large animal models to facilitate clinical translation of novel cardioprotective therapies.

Metformin inhibits stromal aromatase expression and tumor progression in a rodent model of postmenopausal breast cancer

BackgroundObesity and type II diabetes are linked to increased breast cancer risk in postmenopausal women. Patients treated with the antidiabetic drug metformin for diabetes or metabolic syndrome have reduced breast cancer risk, a greater pathologic complete response to neoadjuvant therapy, and improved breast cancer survival. We hypothesized that metformin may be especially effective when targeted to the menopausal transition, as this is a lifecycle window when weight gain and metabolic syndrome increase, and is also when the risk for obesity-related breast cancer increases.MethodsHere, we used an 1-methyl-1-nitrosourea (MNU)-induced mammary tumor rat model of estrogen receptor (ER)-positive postmenopausal breast cancer to evaluate the long-term effects of metformin administration on metabolic and tumor endpoints. In this model, ovariectomy (OVX) induces rapid weight gain, and an impaired whole-body response to excess calories contributes to increased tumor glucose uptake and increased tumor proliferation. Metformin treatment was initiated in tumor-bearing animals immediately prior to OVX and maintained for the duration of the study.ResultsMetformin decreased the size of existing mammary tumors and inhibited new tumor formation without changing body weight or adiposity. Decreased lipid accumulation in the livers of metformin-treated animals supports the ability of metformin to improve overall metabolic health. We also found a decrease in the number of aromatase-positive, CD68-positive macrophages within the tumor microenvironment, suggesting that metformin targets the immune microenvironment in addition to improving whole-body metabolism.ConclusionsThese findings suggest that peri-menopause/menopause represents a unique window of time during which metformin may be highly effective in women with established, or at high risk for developing, breast cancer.

Lack of Effects of Metformin and AICAR Chronic Infusion on the Development of Hypertension in Dahl Salt-Sensitive Rats.

In the kidney, reabsorption via the epithelial sodium channel (ENaC) is involved in long-term blood pressure control. Previously we demonstrated that ENaC hyperactivity is associated with development of salt-sensitive (SS) hypertension in Dahl SS rats. AMP-activated kinase (AMPK), playing a role in cellular energy homeostasis, has been shown to decrease ENaC activity. Here, we tested whether metformin and AICAR, two drugs that activate AMPK, affect the development of salt-induced hypertension. High salt diet significantly increased mean arterial pressure (MAP) in Dahl SS rats. Blood pressure elevation was accompanied by a short-term decline of heart rate and increased circadian arterial pressure dipping. Metformin and AICAR were delivered intravenously at doses of 200 and 20 mg/kg/day, respectively. However, both control and drug-treated groups had similar development of high blood pressure within 3 weeks of 8% NaCl dietary salt intake. In the metformin-treated animals MAP reached 164.9 ± 9.1 mmHg, which was not significantly different from the control group (171.8 ± 5.6 mmHg). Patch clamp analysis revealed that the metformin-treated rats had no difference in the activity of ENaC. AICAR treatment also did not affect the development of hypertension and kidney injury. MAP reached 182.8 ± 4.8 and 178.0 ± 2.8 mmHg in AICAR and vehicle treated groups, respectively. Of note, we found that high-salt diet activated AMPK in the Dahl SS rats, and treatment with these AMPK activators had no significant further effect on AMPK activity. We conclude that AMPK activators, at least under these conditions, do not affect development of hypertension during high-salt diet in the Dahl SS rat model.

Metformin ameliorates insulitis in STZ-induced diabetic mice.

Background & AimsMetformin is currently the most widely used first-line hypoglycemic agent for diabetes mellitus. Besides glucose-lowering action, there is increasingly interest in the potential anti-inflammatory action of this drug. In the present study, we investigated the actions of metformin on experimental insulitis using STZ-induced diabetic mice.MethodsMice with acute diabetes induced by STZ were administered metformin by gavage. Changes of blood glucose and body weight, and the daily amount of food and water intake were measured. Pancreatic tissues were collected for histologic analyses. Pathological assessment and immunohistochemistry analysis were used to determine the effect of metformin on insulitis. Inflammatory cytokines in the pancreas and insulin levels were measured through ELISA analysis.ResultsMetformin significantly reduced blood glucose levels and improved aberrant water intake behavior in experimental diabetic mice. No significant differences were observed in terms of body weight and food intake behavior in metformin-treated animals. In the STZ-induced model of diabetes, we found the appearance of pronounced insulitis. However, metformin administration reduced the severity of insulitis assessed by blind pathological scoring. In addition, metformin treatment improved insulin levels in experimental diabetic mice. ELISA assay revealed decreased levels of inflammatory response marker IL-1β and TNF-α in the pancreatic tissues following metformin treatment.ConclusionMetformin attenuated insulitis in the STZ-induced mice model of diabetes. This islet-protective effect might be partly correlated with the anti-inflammatory action of metformin.

The role of metformin in the prevention of diabetic nephropathy in experimental type 2 diabetes

Introduction and purpose. A number of landmark trials have demonstrated clear benefits of metformin therapy in the prevention of macrovascular outcomes. Nevertheless, there is a lack of robust evidence to suggest whether metformin therapy will have similar beneficial outcomes in one of the most serious type 2 diabetes-related renal microvascular complications known as diabetic nephropathy. The study aimed to evaluate the effects of ten-week metformin treatment on renal morphofunctional changes in rats with non-genetic type 2 diabetic nephropathy. Materials and methods. Starting at 3 weeks after unilateral nephrectomy, adult male Wistar rats were fed the high-fat diet for 5 weeks, and then successively received nicotinamide (230 mg/kg) and streptozotocin (65 mg/kg) intraperitoneally in 15-min interval. Results. Starting at 11 weeks after confirmation of diabetes, metformin treatment did not attenuate routine renal dysfunction markers such as creatinine, creatinine clearance and albuminuria compared to placebo-treated diabetic group, and glomerulosclerosis index and glomerular expression of type IV collagen didn't significantly change either. Nevertheless, level of urinary kidney injury molecule-1, considered to be the marker of tubular damage in diabetes, was significantly lower in metformin-treated animals. Moreover, reduction of tubulointerstitial lesion tended to be significant. Conclusions. Under conditions of diabetic nephropathy modeling, metformin has shown direct protective effects against diabetic tubular disturbance. To assess long-term renal outcomes of these findings, more pre-clinical studies and clinical trials are required.

Metformin Effect on Nontargeted Metabolite Profiles in Patients With Type 2 Diabetes and in Multiple Murine Tissues

Metformin is the first-line oral medication to increase insulin sensitivity in patients with type 2 diabetes (T2D). Our aim was to investigate the pleiotropic effect of metformin using a nontargeted metabolomics approach. We analyzed 353 metabolites in fasting serum samples of the population-based human KORA (Cooperative Health Research in the Region of Augsburg) follow-up survey 4 cohort. To compare T2D patients treated with metformin (mt-T2D, n = 74) and those without antidiabetes medication (ndt-T2D, n = 115), we used multivariable linear regression models in a cross-sectional study. We applied a generalized estimating equation to confirm the initial findings in longitudinal samples of 683 KORA participants. In a translational approach, we used murine plasma, liver, skeletal muscle, and epididymal adipose tissue samples from metformin-treated db/db mice to further corroborate our findings from the human study. We identified two metabolites significantly (P < 1.42E-04) associated with metformin treatment. Citrulline showed lower relative concentrations and an unknown metabolite X-21365 showed higher relative concentrations in human serum when comparing mt-T2D with ndt-T2D. Citrulline was confirmed to be significantly (P < 2.96E-04) decreased at 7-year follow-up in patients who started metformin treatment. In mice, we validated significantly (P < 4.52E-07) lower citrulline values in plasma, skeletal muscle, and adipose tissue of metformin-treated animals but not in their liver. The lowered values of citrulline we observed by using a nontargeted approach most likely resulted from the pleiotropic effect of metformin on the interlocked urea and nitric oxide cycle. The translational data derived from multiple murine tissues corroborated and complemented the findings from the human cohort.

Effects of metformin on bone healing around titanium implants inserted in non-diabetic rats.

To evaluate the effects of metformin on bone healing around titanium implants inserted in non-diabetic rats. Twenty Wistar rats were randomly assigned to one of the following groups: control group (n=10): rats without metformin treatment; MT group (n=10): rats treated with metformin (40mg/kg/day by gavage). At thirty days after implant placement, animals were euthanized. Histometric measurements of bone-to-implant contact (BIC) and bone area (BA), in addition to immunohistochemical analysis of the number of cells stained for RANKL and OPG, were assessed in the cortical and medullary areas around implants. The percentages of BIC and BA in the cortical bone were significantly lower in the MT group than in the control group (P<0.05). Furthermore, the medullary bone around the implants inserted in the metformin-treated animals exhibited an increased number of RANKL-stained cells than that around the implants inserted in the control animals (P<0.05). Metformin negatively affected osseointegration by reducing the percentages of BIC and BA and increasing the expression of RANKL around titanium implants inserted in non-diabetic rats.

In Vitro and In Vivo Effects of Metformin on Osteopontin Expression in Mice Adipose-Derived Multipotent Stromal Cells and Adipose Tissue.

Metformin is applied not only as antidiabetic drug, but also in the treatment of obesity or as antiaging drug. The first part of the research discussed the effect of metformin at concentrations of 1 mM, 5 mM, and 10 mM on the morphology, ultrastructure, and proliferation potential of mice adipose-derived multipotent mesenchymal stromal cells (ASCs) in vitro. Additionally, we determined the influence of metformin on mice adipose tissue metabolism. This study has shown for the first time that metformin inhibits the proliferative potential of ASCs in vitro in a dose- and time-dependent manner. In addition, we have found a significant correlation between the activity of ASCs and osteopontin at the mRNA and protein level. Furthermore, we have demonstrated that 5 mM and 10 mM metformin have cytotoxic effect on ASCs, causing severe morphological, ultrastructural, and apoptotic changes. The reduced level of OPN in the adipose tissue of metformin-treated animals strongly correlated with the lower expression of Ki67 and CD105 and increased caspase-3. The metformin influenced also circulating levels of OPN, which is what was found with systemic and local action of metformin. The results are a valuable source of information regarding the in vitro effect of metformin on adipose-derived stem cells.

The Effect of Metformin on the Myocardial Tolerance to Ischemia-Reperfusion Injury in the Rat Model of Diabetes Mellitus Type II

In recent years, evidence has been accumulated that metformin, an antidiabetic drug in the biguanide class, in addition to its well-recognized glucose-lowering effect, can also reduce cardiovascular mortality in the patients with type 2 diabetes mellitus (T2DM). Besides, there are a few experimental studies on the possibility of the direct anti-ischemic effect of the drug in both type 1 diabetes mellitus and T2DM. In our study, myocardial tolerance to ischemia in rats with neonatal streptozotocin T2DM was investigated using the model of global ischemia-reperfusion of the isolated perfused heart. Metformin was administered i.p. at a dose of 200 mg/kg/day for 3 days prior to isolated heart perfusion. The results showed that both the infarct size and postischemic recovery of left ventricular function were not different between controls and metformin-treated animals. At the same time, the infarct size in the T2DM animals was significantly lower than that in the controls (24.4 ± 7.6% versus 45.0 ± 10.4%, resp., P < .01), indicative of the metabolic preconditioning in T2DM. It follows that the protocol of metformin administration used in this study had not elicited cardioprotective effect in animals with T2DM so that the different mechanism(s) may underlie the beneficial effect of metformin on cardiovascular complications in patients with T2DM which, however, would need further investigation.

Androgen Receptor Antagonism and an Insulin Sensitizer Block the Advancement of Vaginal Opening by High-Fat Diet in Mice1

Reduced hypothalamic sensitivity to steroid negative feedback may contribute to the onset of puberty. In high fat-fed rodents, the timing of vaginal opening (VO) is advanced, suggesting that puberty begins earlier. Because obesity can increase androgens, which interfere with normal steroid feedback in adult females, we hypothesized that androgens reduce hypothalamic sensitivity to negative feedback during puberty and that blocking androgen action would prevent advanced VO in high fat-fed mice. Age at VO was examined in mice fed high-fat or low-fat diets from weaning and treated with the androgen receptor antagonist flutamide or vehicle (controls). VO was advanced in high-fat vs. low-fat controls, and flutamide blocked this advancement. VO was also delayed in low fat-fed flutamide-treated females, suggesting involvement of androgens in the timing of normal puberty. We next investigated if high-fat diet-induced insulin resistance contributes to early VO, as elevated insulin can stimulate androgen production. VO was examined in mice on either diet treated with the insulin sensitizer metformin. Metformin blocked high-fat advancement of VO but did not alter the timing of VO in low fat-fed mice. Insulin was elevated in high fat-fed females that had undergone VO compared with age-matched low fat-fed or metformin-treated animals on either diet that had not undergone VO. Together, these data suggest a model in which metabolic changes induced by high-fat diet, including transient increased circulating insulin, act in part by increasing androgen action to influence the timing of puberty in females.

Metabolic recovery of adipose tissue is associated with improvement in insulin resistance in a model of experimental diabetes

Obesity and insulin resistance are highly correlated with metabolic disturbances. Both the excess and lack of adipose tissue can lead to severe insulin resistance and diabetes. Adipose tissue plays an active role in energy homeostasis, hormone secretion, and other proteins that affect insulin sensitivity, appetite, energy balance, and lipid metabolism. Rats with streptozotocin-induced diabetes during the neonatal period develop the classic diabetic picture of hyperglycemia, hypoinsulinemia, and insulin resistance in adulthood. Low body weight and reduced epididymal (EP) fat mass were also seen in this model. The aim of this study was to investigate the glucose homeostasis and metabolic repercussions on the adipose tissue following chronic treatment with antidiabetic drugs in these animals. In the 4th week post birth, diabetic animals started an 8-week treatment with pioglitazone, metformin, or insulin. Animals were then killed, EP fat pads were excised, and blood samples were collected for biological and biochemical assays. Pioglitazone and insulin treatments, but not metformin, reduced hyperglycemia, polydipsia, and polyphagia. Although all antidiabetic therapies improved insulin sensitivity, this was particularly noteworthy in the pioglitazone-treated rats. Furthermore, a recovery of adipose mass and insulin levels were observed in pioglitazone- and insulin-, but not metformin-treated animals. Treatments with insulin or pioglitazone were able to correct significantly, but not completely, the metabolic abnormalities, parallel to full recovery of adipose mass, indicating that not only the low insulin levels but also the lack of adipose tissue might play a significant role on the pathophysiology of this particular diabetes model.

Effect of metformin on survival rate in experimental sepsis

Because "metformin-associated lactic acidosis" refers to metformin and concurrent pathologies as co-precipitating factors, the respective impact in the outcome of metformin therapy, metformin accumulation, and general diseases should be determined. We therefore constructed a model of sepsis in mice treated with metformin at a dose corresponding to clinical practice, or to accumulation. 460 mice were separated in 3 groups: no metformin therapy, a 7-day metformin therapy at 50 mg.kg(-1).day(-1) (MET50) or 500 mg.kg(-1).day(-1) (MET500). Blood was drawn on day 7 in 40 metformin-treated animals for determining metformin concentrations. The 420 other mice were divided in 14 subgroups according to the amount of an intra-peritoneal inoculum of E. coli ranging from 5.103 to 1010 CFU/ml in order to construct a lethal dose curve. The survival rate was assessed at 7, 13, 24, 36, 60 and 120 hours thereafter. Plasma metformin concentrations were 0.26 +/- 0.13 mg/l in MET50, and 4.63 +/- 1.92 mg/l in MET500. The comparative analysis of the survival rates at 120 hours showed no difference of mortality, always occurring for an inoculum amount > 10(8) CFU/ml. Comparing the survival rates from time 0 to 120 hours using Kaplan-Meyer curves and the Logrank test, there was no difference between the different groups. Metformin, even at a dose mimicking accumulation, does not aggravate the mortality rate in this model of sepsis. Consequently, metformin can not be considered as toxic in such a condition.