The incretin hormone glucagon-like peptide-1 enhances glucose-dependent insulin secretion and is therefore a most attractive therapeutic approach for the treatment of Type 2 Diabetes Mellitus. The anti-diabetic drug, metformin, has previously been shown to increase circulating levels of GLP-1, although its mechanism of action is currently unknown. Neither metformin nor AICAR (activators of AMPK) directly stimulated GLP-1 secretion from the L-cell in vitro. However, oral treatment of rats with metformin enhanced plasma levels of active and total GLP-1, independent of GLP-1 degradation. Furthermore, pre-treatment with the general muscarinic antagonist, atropine, or the M3 antagonist, 4-DAMP, decreased metformin–induced GLP-1 secretion, while M1 and M2 antagonists did not. Chronic bilateral subdiaphragmatic vagotomy had no effect, while the GRP antagonist, RC-3095, reduced metformin-induced GLP-1secretion. Therefore, I conclude that metformin-induced GLP-1 secretion occurs in part through the parasympathetic nervous system, the M3 and GRP receptors, but is independent of the vagus nerve.

The incretin hormone glucagon-like peptide-1 enhances glucose-dependent insulin secretion and is therefore a most attractive therapeutic approach for the treatment of Type 2 Diabetes Mellitus. The anti-diabetic drug, metformin, has previously been shown to increase circulating levels of GLP-1, although its mechanism of action is currently unknown. Neither metformin nor AICAR (activators of AMPK) directly stimulated GLP-1 secretion from the L-cell in vitro. However, oral treatment of rats with metformin enhanced plasma levels of active and total GLP-1, independent of GLP-1 degradation. Furthermore, pre-treatment with the general muscarinic antagonist, atropine, or the M3 antagonist, 4-DAMP, decreased metformin–induced GLP-1 secretion, while M1 and M2 antagonists did not. Chronic bilateral subdiaphragmatic vagotomy had no effect, while the GRP antagonist, RC-3095, reduced metformin-induced GLP-1secretion. Therefore, I conclude that metformin-induced GLP-1 secretion occurs in part through the parasympathetic nervous system, the M3 and GRP receptors, but is independent of the vagus nerve.

Ovarian cancers are the most lethal gynecological malignancies, responsible for more than 150,000 deaths around the globe annually. Among ovarian cancers, high-grade serous ovarian cancer has a 5-year survival rate of only 40%. This poor survival is due to a widespread lack of understanding of this disease, from suboptimal prevention and screening methods to failures in treatment. Moving towards novel prevention and treatment methods requires better models of ovarian cancer that phenotypically and genetically recapitulate the features of ovarian cancers that are seen clinically. This thesis highlights the characterization of a novel syngeneic model of high-grade serous ovarian cancer that exhibits the growth, expression profile, histology, and a tumor-initiating cell population that closely resembles human disease. We expand on our initial characterization of the STOSE model in a proof-of-principle study using deep learning of second-harmonic generation and two-photon-excited-fluorescence images to classify normal compared to cancerous tissues. The use of deep learning for image classification based on extracellular matrix and cellular structure could have robust application to complementing common histological examination of tissues and in treatment planning. Building on the changes in structure found in normal compared to cancerous ovarian tissue and recent research that showed age-associated fibrosis develops in murine ovaries, we assessed the non-hereditary ovarian cancer risk factors of age and ovulation number for their effects in altering ovarian tissue structure. This thesis concludes with the first evidence of ovarian fibrosis in non-pathological post-menopausal human ovaries. We show that ovarian fibrosis correlates with the development of a pre-metastatic (tumor-permissive) niche, revealing a novel avenue of research into ovarian cancer risk. Interestingly, age-associated fibrosis could be prevented or reversed by metformin use, revealing a possible mechanism for the previously identified ovarian cancer risk reduction seen with metformin use and further supporting the use of metformin for ovarian cancer prevention.

Obesity is the most common antenatal comorbidity, affecting one in five of the antenatal population in the UK. It is associated with adverse outcomes for mother and baby in both the short and long term. Increasing data suggest that maternal obesity may programme offspring later life obesity and premature mortality, with high birth weight being a marker for increased risk. The mechanism by which maternal obesity causes excessive neonatal birth weight is incompletely understood but considerable evidence implicates insulin resistance and/or hyperglycaemia. There are currently no effective interventions to mitigate the effects of obesity during pregnancy. In this thesis, we present the findings from a randomised, double blind, placebo controlled trial designed to examine the efficacy of metformin, an insulin-sensitising agent, in obese pregnant women. The aim of the trial was to determine whether giving metformin to obese pregnant women from between 12 and 16 weeks’ gestation until birth, would improve maternal and fetal outcomes. The primary outcome measure was birth weight of the baby, using this as a surrogate marker for the future life risk of the child developing obesity. Nested within this large clinical trial were a series of mechanistic sub-studies. To examine the effect of metformin on maternal insulin resistance at 36 weeks’ gestation, we used the hyperinsulinaemic euglycaemic clamp with concomitant use of stable isotope tracers. This enabled us to characterise in greater detail insulin sensitivity, endogenous glucose production and lipolysis. To determine the effect of metformin on maternal and fetal body composition we used magnetic resonance imaging and spectroscopy. This allowed us to quantify subcutaneous and intra-abdominal adipose tissue deposition and hepatic and skeletal muscle ectopic lipid deposition in the mother; and to measure subcutaneous adipose tissue deposition, hepatic lipid and hepatic volume in the fetus. To determine the effect of metformin on maternal endothelial function, we measured endothelium-dependent flow-mediated dilatation at the beginning and end of pregnancy. Change in diameter of the brachial artery in response to a flow stimulus created by arterial occlusion was measured using ultrasound imaging. We found no significant effect of metformin on birth weight. Mean birth weight was 3463 g (SD 660) in the placebo group and 3462 g (SD 548) in the metformin group (adjusted mean difference in z score –0·029, 95% CI –0·217 to 0·158; p=0·7597). Subjects taking metformin did demonstrate increased insulin sensitivity (M/I difference between means during high dose insulin of 0.02 [95% CI 0.001 to 0.03] milligrams per kilogram fat free mass per minute per pmol/L, p=0.04) but also enhanced endogenous glucose production (difference between means 0.54 [95% CI 0.08 to 1.00] milligrams per kilogram fat free mass per minute, p=0.02), compared with those taking placebo. We did not demonstrate any differences between treatment groups in maternal subcutaneous and intra-abdominal adipose tissue, or ectopic lipid deposition, or in fetal body fat distribution and liver volume. Participants in both treatment groups demonstrated a decline in endothelium-dependent flow-mediated dilatation between early and late pregnancy but there were no differences in the magnitude of that decline between the treatment groups. In conclusion, metformin, administered to obese, non-diabetic pregnant women, does not have any significant effect on birth weight of the baby. Our clamp studies demonstrated that subjects taking metformin were indeed more insulin-sensitive than those taking placebo, but the higher endogenous glucose production in this group suggests a reduced ability to suppress hepatic glucose production in response to insulin. This increased glucose flux may in part explain the lack of effect of metformin on fetal nutrition and growth. We can conclude that metformin, should not be used as an intervention in obese pregnant women to prevent excess birth weight. The global obesity epidemic is one of the greatest public health challenges we face and the cycle of disadvantage continues to be perpetuated to the next generation. The lack of any effective interventions for this high-risk group remains a significant concern and an important area for further research.

Das Biguanid Metformin besitzt in Prostatakarzinomzellen eine proliferationsinhibierende Wirkung unter anderem über die Aktivierung von p53, die eine Überexpression von microRNA-205 über einen direkten Induktionsmechanismus bewirkt. Somit konnte im Rahmen der vorliegenden Arbeit microRNA-205 als Effektor der proliferationsinhibierenden Wirkung von Metformin im Prostatakarzinom identifiziert werden. / The biguanide metformin has a proliferation-inhibiting effect in prostate carcinoma cells, among other things via the activation of p53, which causes the overexpression of microRNA-205 via a direct induction mechanism. Thus, within the scope of the present work, microRNA-205 could be identified as an effector of the proliferation-inhibiting effect of metformin in prostate carcinoma.

The generation of human induced pluripotent stem cells (hiPSCs) from human fibroblasts has revolutionized cell therapy by providing a source of autologous cells for transplantation. Several studies have demonstrated that transplantation of hiPSC-derived neural stem cells (hiPSC-NSCs) increases regeneration and recovery following stroke, supporting their therapeutic potential. However, major concerns for translating hiPSC transplantation therapy to the clinic are efficacy and safety. Therefore, there is demand to develop an optimal strategy to enhance the engraftment and regenerative capacity of transplanted hiPSC-NSCs. The recent published work shows that metformin, an FDA approved drug, is an optimal neuroregenerative agent that not only promotes the proliferation of neural stem cells but also enhances their neuronal differentiation. In this regard, we hypothesize that preconditioning of hiPSC-NSCs with metformin before transplantation into the stroke-damaged brain will improve engraftment and regenerative capabilities of hiPSC-NSCs, further enhancing cell-mediated functional recovery. Here we show that treatment of hiPSC-NSCs with metformin enhances the proliferation and differentiation of hiPSC-NSCs in culture even after withdrawal of metformin treatment, showing its promise as a novel preconditioning strategy. Furthermore, transplantation of preconditioned hiPSC-NSCs into a rat endothelin-1 ischemic stroke model showed an improved engraftment capability 1-week post-transplant. In addition, metformin preconditioned grafts survived longer compared to naïve grafts and were detectable at 8 weeks post-stroke. However, cell transplantation did not result in improve functional recovery when compared to sham group in this model. These studies represent a vital step in the optimization of hiPSC-NSC based transplantation to promote post-stroke recovery.

Background Preclinical data suggests that metformin may have anti-cancer effects to reduce breast cancer incidence and improve cancer prognosis. However, the current evidence in observational studies is inconclusive. A systematic review and meta-analysis was conducted to assess the effect of metformin on the incidence and mortality of breast cancer in diabetic patients. Methods A comprehensive literature search was performed on Medline (Pubmed), EMBASE, and the Cochrane library from inception to November 2016 with no language restrictions. Outcomes were incidence of breast cancer and all-cause mortality. Risk of bias and overall quality of evidence was assessed using the Newcastle Ottawa Scale and GRADE respectively. A meta-analysis was performed using the most adjusted odds ratios (ORs) or hazard ratios (HRs) and 95% confidence intervals (95% CI) as effect measures. Results A total of 12 observational studies were included for breast cancer incidence and 11 studies for all-cause mortality. No significant association was found between metformin exposure and incidence of breast cancer (OR: 0.93, 95% CI: 0.85-1.03, I2 = 35%). A 45% risk reduction was observed for all-cause mortality (HR: 0.55, 95%CI: 0.44-0.70, I2=81%). Presence of publication bias is strongly suspected for both outcomes. Conclusion The use of metformin in standard cancer therapy may improve overall survival of diabetic patients with breast cancer. No effect of metformin on the incidence of breast cancer was observed. Interpretation of results is limited by the observational nature of the studies and methodological biases. Clinical trials are warranted to determine the role of metformin in breast cancer risk reduction and prognosis. / Thesis / Master of Health Sciences (MSc)

>Magister Scientiae - MSc / Diabetes, a worldwide disease, is classified into two types, type 1 or insulin-dependent and type 2 or noninsulin-dependent. Based on reports published by the International Diabetes Federation, the total number of those suffering from diabetes is growing every year. Statistics predict that type 2 diabetes, currently affecting about 8% of the adult population, would spread at such a pace that by 2030, more than 40 million cases of diabetes would be found throughout the world. On the other hand, studies revealed that patients with type 2 diabetes mellitus (T2DM) have a lower incidence of tumour development than healthy controls and that patients diagnosed with cancer have a lower risk of mortality when treated with metformin. However, the frequent use of metformin with low oral bioavailability ranging between 40-60% in the intestinal environment leads to large accumulation on the enterocytes. / 2024-02-24

The antihyperglycaemic drug metformin has become the most widely prescribed drug treatment for the management of type 2 diabetes mellitus. Despite being prescribed for over 50 years, the precise molecular mechanisms underlying metformin’s therapeutic effects remain poorly understood. Newly recognised health benefits of metformin, irrespective of diabetes status, have led to proposals of ‘re-purposing’ metformin for treatment of cancer, cardiovascular disease and ageing; conditions regularly associated with impaired regulation of the mammalian/mechanistic target of rapamycin complex 1 (mTORC1) but not safely treatable with its inhibitor, rapamycin. Here we report that in the liver, the primary target tissue of metformin, metformin regulates mTORC1 signalling by inhibiting its activation by amino acids in an AMPK-independent manner. Furthermore, we present evidence to suggest that this occurs through a reversible mechanism ‘upstream’ of the amino acid sensor involving inhibition of hepatic uptake of leucine, a potent stimulator of mTORC1 activity. Using gene expression studies, we identified a role for metformin in decoupling uptake of small and large neutral amino acids, such as glutamine and leucine, from a favourable sodium gradient, involving significant reduction in mRNA expression of SNAT2. Consistent with impaired hepatic uptake and removal from the plasma, elevations in plasma concentrations of branched chain amino acids (BCAAs) and glutamine were observed in non-diabetic humans with chronic heart failure (CHF) receiving metformin. Furthermore, elevated plasma concentrations of leucine were significantly associated with improved plasma glucose and fasting insulin resistance index parameter (FIRI). Taken together, these results suggest a role for metformin in controlling mTORC1 via amino acid transport, akin to hepatic protein restriction. This study highlights the potential for ‘re-purposing’ metformin for use as a protein restriction mimetic in treatment of age-related diseases including cardiovascular disease, cancer and diabetes.

Ulm, Univ., Diss., 2008.