Is non-insulin dependent glucose uptake a therapeutic alternative? Part 1: physiology, mechanisms and role of non insulin-dependent glucose uptake in type 2 diabetes.

Several decades of research for treating type 2 diabetes have yielded new drugs but the actual experience with the available oral antidiabetic compounds clearly shows that therapeutic needs are not matched. This highlights the urgent need for exploring other pathways. All cell types have the capacity to take up glucose independently of insulin, whereby basal but also hyperglycaemia-promoted glucose supply is ensured. Although poorly explored, insulin-independent glucose uptake might nevertheless represent a therapeutic target, as an alternative to the clear limits of actual drug treatments. This review not only critically examines some major pathways not requiring insulin (although they may be influenced by the hormone) but importantly, this analysis extends to the clinical applicability of these potential therapeutic principles by also considering their predictable tolerability for long-term therapy. In particular vascular safety (the ultimate problem linked with diabetes) will be envisaged because of the ubiquitous distribution of glucose transporters and some linked mechanisms. Several mechanisms can be identified which do not require insulin for their functioning. The first part of this review deals with the description, the regulation and the limits of some mechanisms representing potential pharmacological targets capable of having a highly significant impact on glucose uptake. These selected topics are: a) unmasking and/or activation of glucose transporters in cell plasma membranes, b) insulin mimetics acting at postreceptor level, c) activation of AMPK, d) increasing nitric oxide and e) increasing glucose-6P and glycogen stores.

Is non-insulin dependent glucose uptake a therapeutic alternative? Part 2: Do such mechanisms fulfil the required combination of power and tolerability?

The worldwide burden of diabetes, the unavoidable worsening which is observed in long-term clinical trials despite treatment and the close link between glycaemia and microangiopathy appeal for much stronger treatment strategies. This, in turn, either requires polypharmacy (with new risks) or new, more powerful drugs to be invented. The first part of this review dealt with a thorough analysis of pros and cons for some selected pathways which could potentially increase glucose uptake without necessitating insulin. The choice of such targets for developing completely new drugs, however, requires a favourable background from existing tentatives with either drugs or cell biology approaches. Moreover, because vascular complications are what must ultimately be avoided when treating diabetic patients, we must be sure that increasing glucose uptake in a fashion which is no more controlled by normal physiology is compatible with the physiology of vascular cells (long-term tolerance). The aspect of drug side-effects must therefore be considered systematically. For reasons which are individually developed, it appears that each of the potential pathways analyzed either lacks sufficient power and/or is likely to induce side effects which are not acceptable for long-term application. The fact that GLUT-1 transporters are ubiquitously distributed even extends this cardinal question to the general principle of increasing glucose uptake. In conclusion a precise evaluation suggests that, although non-insulin dependent glucose uptake represents (3/4) of whole body glucose transport, it is difficult to consider such mechanisms able to generate a new treatment fulfilling the unavoidable request of combined efficacy and tolerability.

The antihyperglycaemic effect of metformin: therapeutic and cellular mechanisms.

Metformin is regarded as an antihyperglycaemic agent because it lowers blood glucose concentrations in type 2 (non-insulin-dependent) diabetes without causing overt hypoglycaemia. Its clinical efficacy requires the presence of insulin and involves several therapeutic effects. Of these effects, some are mediated via increased insulin action, and some are not directly insulin dependent. Metformin acts on the liver to suppress gluconeogenesis mainly by potentiating the effect of insulin, reducing hepatic extraction of certain substrates (e.g. lactate) and opposing the effects of glucagon. In addition, metformin can reduce the overall rate of glycogenolysis and decrease the activity of hepatic glucose-6-phosphatase. Insulin-stimulated glucose uptake into skeletal muscle is enhanced by metformin. This has been attributed in part to increased movement of insulin-sensitive glucose transporters into the cell membrane. Metformin also appears to increase the functional properties of insulin- and glucose-sensitive transporters. The increased cellular uptake of glucose is associated with increased glycogen synthase activity and glycogen storage. Other effects involved in the blood glucose-lowering effect of metformin include an insulin-independent suppression of fatty acid oxidation and a reduction in hypertriglyceridaemia. These effects reduce the energy supply for gluconeogenesis and serve to balance the glucose-fatty acid (Randle) cycle. Increased glucose turnover, particularly in the splanchnic bed, may also contribute to the blood glucose-lowering capability of metformin. Metformin improves insulin sensitivity by increasing insulin-mediated insulin receptor tyrosine kinase activity, which activates post-receptor insulin signalling pathways. Some other effects of metformin may result from changes in membrane fluidity in hyperglycaemic states. Metformin therefore improves hepatic and peripheral sensitivity to insulin, with both direct and indirect effects on liver and muscle. It also exerts effects that are independent of insulin but cannot substitute for this hormone. These effects collectively reduce insulin resistance and glucotoxicity in type 2 diabetes.

Oxidative stress as a therapeutic target in diabetes: revisiting the controversy.

Oxidative stress has been repetitively shown to be a hallmark of many diseases linked with metabolic or vascular disorders. Therefore diabetes represents an ideal candidate for studying the consequences of oxidative stress and its treatment. Indeed diabetes constitutes a multiple source of free radicals, starting very early in the disease process and worsening over the course of disease. In view of the typical characteristics of diabetes, oxidative stress is expected to have a double impact, on both metabolic and vascular functions. It is therefore particularly disappointing to note the dramatic failure of clinical trials with antioxidants, although it must be pointed out that such studies have not been performed with only diabetic patients. This review describes the many different aspects of oxidative stress in diabetes and proposes possible explanations for the apparent lack of efficacy of antioxidant treatments in patients. Some verifications seem warranted before a definitive conclusion can be drawn about the validity of this therapeutic concept.

Membrane physiology as a basis for the cellular effects of metformin in insulin resistance and diabetes.

Many recent data provide new, original insights into the mechanisms of action of the antidiabetic Metformin. Careful selection of most relevant data in terms of dosage prompted this original review, largely devoted to the drug action at the cell level and whose hypotheses/conclusions are tentatively interpreted according to corresponding basic scientific knowledge. Metformin interferes with several processes linked to HGP (gluconeogenesis, glycogenolysis and their regulatory mechanisms), lowering glucose production and resensitizing the liver to insulin. The hepatic drug effect is largely favoured by prevailing glycemia. In peripheral tissues, metformin potentiates the effects of both hyperglycemia and hyperinsulinemia. Increase in glucose-mediated glucose transport is mainly mediated by an improvement in the glucose transporter's intrinsic activity. Potentiation of the hormone effect relates to an increase in insulin receptor tyrosine kinase activity. Both mechanisms (insulin signalling and glucose transport) result in the activation of glycogen synthase, a limiting enzyme in the causal defects of NIDDM. Exciting findings show that, conversely, priming cells with very low insulin concentrations also leads to full expression of metformin's antidiabetic activity. Specific investigations confirm a working hypothesis defining the site of action as the cell membrane level. Indeed metformin corrects membrane fluidity and protein configuration disturbed by the diabetic state and which interfere with normal protein-protein or protein-lipid interactions required for proper functioning of the processes regulating glucose transport/metabolism. It is proposed that membrane changes largely represent a common denominator explaining metformin effects on various systems involved in receptor signalling and related functions.

Microcirculation in insulin resistance and diabetes: more than just a complication.

The microvascular bed is an anatomical entity which is governed by specific, highly regulated mechanisms which are closely adapted to the specific function of each vascular segment. Among those, small arteriolar vasomotion and capacity of small vessels to constrict in response to physical and humoral stimuli play a major role. Other processes of importance for the adequacy of nutritive perfusion are haemorheological properties of whole blood and red cells, adhesiveness of leukocytes and capillary permeability. This review provides some description of these phenomena, how they impact on organ function and how they appear in diabetes. Metformin, as a unique example among the drug arsenal, exerts various effects preferentially at the level of smallest vessels (arterioles, capillaries, venules). This review summarises our actual knowledge and includes several new data showing its high potential for reducing microvascular dysfunction. Most of these unique properties have also been demonstrated in non-diabetic animals or humans, suggesting they are intrinsic to the drug and not secondary to diabetic metabolic improvement. A particular focus is put on the relevance of metformin's capacity to stimulate slow wave arteriolar vasomotion and improve functional capillary density, whereby nutritive flow can be re-established. Finally, the implication of microcirculation in other aspects of insulin resistance and diabetes, such as macroangiopathy and metabolic control, is discussed and strengthens the concept of a broad involvement of microvascular dysfunction in these diseases as well as the potential interest of introducing adapted treatment early in the history of a patient's diabetes.

Antiatherogenic properties of metformin: the experimental evidence.

Cardiovascular disease (CVD) is the major determining factor of morbidity and mortality in type 2 diabetic patients. The established relationship between type 2 diabetes and atherosclerosis has fueled suggestions that anti-diabetic drugs with beneficial effects on CV risk factors may help attenuate the atherosclerotic process in diabetic patients. Metformin is a hypoglycaemic agent widely used in the management of type 2 diabetes. In addition to its insulin-sensitising action, this drug has favourable effects on various CV risk factors and reduces macrovascular complications in obese type 2 diabetic patients. This review summarises in vivo and in vitro experimental evidence on the antiatherogenic properties of metformin.

Mitochondrial metabolism and type-2 diabetes: a specific target of metformin.

Several links relate mitochondrial metabolism and type 2 diabetes or chronic hyperglycaemia. Among them, ATP synthesis by oxidative phosphorylation and cellular energy metabolism (ATP/ADP ratio), redox status and reactive oxygen species (ROS) production, membrane potential and substrate transport across the mitochondrial membrane are involved at various steps of the very complex network of glucose metabolism. Recently, the following findings (1) mitochondrial ROS production is central in the signalling pathway of harmful effects of hyperglycaemia, (2) AMPK activation is a major regulator of both glucose and lipid metabolism connected with cellular energy status, (3) hyperglycaemia by inhibiting glucose-6-phosphate dehydrogenase (G6PDH) by a cAMP mechanism plays a crucial role in NADPH/NADP ratio and thus in the pro-oxidant/anti-oxidant cellular status, have deeply changed our view of diabetes and related complications. It has been reported that metformin has many different cellular effects according to the experimental models and/or conditions. However, recent important findings may explain its unique efficacy in the treatment of hyperglycaemia- or insulin-resistance related complications. Metformin is a mild inhibitor of respiratory chain complex 1; it activates AMPK in several models, apparently independently of changes in the AMP-to-ATP ratio; it activates G6PDH in a model of high-fat related insulin resistance; and it has antioxidant properties by a mechanism (s), which is (are) not completely elucidated as yet. Although it is clear that metformin has non-mitochondrial effects, since it affects erythrocyte metabolism, the mitochondrial effects of metformin are probably crucial in explaining the various properties of this drug.

Oxidative stress: the special case of diabetes.

The implication of oxidative stress (OS) in diabetes is a major concern for the development of therapeutics aimed at improving the metabolic and/or vascular dysfunctions of this burdening disease. Ample evidence is available suggesting that OS is present in essentially all tissues and can even be observed in prediabetic states. This raises the question of the origin of OS and suggests that, although hyperglycemia is largely linked with free radical production, its role may mainly be the aggravation of a preexisting state. Indeed other factors are also causally linked to OS, such as hormones and lipids. The main debate is about the pertinence of antioxidant therapy since the large scale clinical trials performed recently have essentially failed to show any significant improvement in metabolic or vascular disturbances of diabetic patients. However this conclusion must be tempered by the fact that they have mainly been using vitamin E +/-C; indeed many arguments suggest that either the choice or the application modalities of these substances may have been inadequate. Potential reasons for the actual failure of antioxidant therapy in diabetes are discussed; the indisputable involvement of OS in this disease still leaves hope for alternative therapeutic approaches.

In defense of microvascular constriction in diabetes.

In sharp contrast to vasodilatation, vasoconstriction is not subjected to great consideration in vascular physiological and pharmacological investigations in diabetes. However, vasoconstriction is a main regulatory process in smaller vessels, in particular in the small arterioles. Here it is linked to maintenance of local blood pressure and avoids capillary hyperperfusion/hypertension. This highlights the importance of constrictor processes in the microvascular bed as opposed to larger vessels. It is manifested by a series of phenomena such as precapillary vasomotion, venoarteriolar reflex and myogenic response. Animal and human studies indeed reported defects in small vessel constrictor reactivity in diabetes but also evidence for disturbances already present in nondiabetic, insulin resistant states. This abnormality is present whether humoral, neuronal or physical stimuli are used for test. This article overviews the adequate literature and discusses both vascular and metabolic implications induced by defective vasoconstriction.

Generation of reactive oxygen species by endothelial and smooth muscle cells: influence of hyperglycemia and metformin.

There is evidence that reactive oxygen intermediates (ROI) play an important role in the pathogenesis of vascular complications in diabetes. On the other hand, metformin, one of the most often used antidiabetic compounds has not only been shown to reduce the risk for vascular complications, but in addition these protective effects are largely independent of its well-known antihyperglycemic action. Therefore, to explain the vasculoprotective effects of metformin, a direct antioxidative action of this compound has been suggested. We show here that human endothelial cells (HUVEC) generate ROI not only in response to high glucose (30 mmol/l glucose), but also in response to palmitic acid, and advanced glycation end-products (carboxymethyllysine and S100 proteins). Metformin inhibited the production of ROI in response to all these stimuli. By double staining-dichlorofluorescein as marker of ROI and Mitotracker CMH-Ros for mitochondria-the mechanism of ROI generation was analyzed in more detail in smooth muscle cells. Our data suggest that ROI are generated by uncoupling of the mitochondrial respiratory chain as well as by activation of the cytosolic NADPH-oxidase. A complete inhibition of ROI generation is only achieved by simultaneous inhibition of the mitochondrial electron flux (theonyltrifluoroacetone) and NADPH-oxidase (apocynin). Our data suggest that the various processes contributing to generation of ROI are closely linked. Activation of AMP kinase may represent an important mechanism to understand the antioxidative effects of metformin on the mitochondrial and cytosolic generation of ROI.

Serotonin, 5-HT2 receptors, and their blockade by naftidrofuryl: a targeted therapy of vascular diseases.

The importance and the various effects of serotonin (5-HT) in cardiovascular diseases are reviewed, with particular emphasis on the involvement of 5-HT2 receptors as mediators of the biological responses of vessels and blood platelets to 5-HT. The importance of 5-HT in peripheral and cerebral ischemia is shown by the key role it plays in inducing vasoconstriction, platelet aggregation, vascular permeability, and cell proliferation. Of particular importance is the 5-HT-selective hypersensitivity developing in vessels/platelets shortly after acute ischemia or early in the development of chronic vascular diseases. The mechanisms of action of naftidrofuryl are described, showing that this drug offers a particularly interesting profile of having both metabolic and vascular effects. Naftidrofuryl improves glucose aerobic metabolism by an action on succinodehydrogenase and improves the blood supply and the ischemic damage of the vessel wall by blocking specifically 5-HT2 receptors. The latter property permits an inhibition of the deleterious, multiple effects of 5-HT at sites of vascular injury, without influencing the general circulatory bed. Therefore, naftidrofuryl appears to be an anticonstrictor and not, as previously thought, a vasodilator. As a consequence, naftidrofuryl has a targeted impact without vasodilator-linked side effects such as hypotension or the steal phenomenon.

Effects of metformin on bile salt transport by monolayers of human intestinal Caco-2 cells.

The antidiabetic biguanide metformin has been shown to increase faecal excretion of bile salts in type 2 diabetes. Cultured human intestinal Caco-2 cell monolayers provide a model of human enterocytes. These monolayers are used here to determine the effect of metformin on the secondary-active, sodium-linked transfer of 14C-glycocholate from the apical (brush border) to the basolateral (serosal) surface. During 24-h incubations, 10-2 mol/l metformin significantly reduced 14C-glycocholate transfer. This could not be attributed to alterations of monolayer integrity or Na+-K+ ATPase pump activity. For example, the secondary-active transport of glucose and proline was not interrupted, and the inhibitory effect of metformin on bile salt transport was additive to the inhibitory effect of ouabain. The results suggest that metformin can act directly on intestinal enterocytes to reduce the active transfer of bile salts by a mechanism that is independent of Na+-K+ ATPase activity.

Differential effects of metformin on bile salt absorption from the jejunum and ileum.

AIMS: The antidiabetic drug metformin is often associated with a small reduction in total circulating cholesterol, but the mechanism responsible is unknown. As bile salts contribute significantly to cholesterol homeostasis, this study has investigated the effect of metformin on the absorption of bile salts by the jejunum and ileum, and their transfer into bile. METHODS: Sodium-[1-14C]-glycocholate was administered into the jejunum or ileum of anaesthetized rats with and without metformin (250 mg/kg). Appearance of 14C-glycocholate in plasma and bile was followed for 150 min. RESULTS: Absorption of 14C-glycocholate from the ileum, which is a high-capacity active process, was 10-fold greater than absorption from the jejunum, which is mainly a passive process. Metformin increased threefold the absorption of 14C-glycocholate from the jejunum. Metformin similarly increased the appearance of jejunal 14C-glycocholate in plasma and bile. In contrast to the jejunum, absorption of 14C-glycocholate from the ileum was suppressed by more than half with metformin. This was associated with corresponding reductions of 14C-glycocholate in plasma and bile. DISCUSSION: Thus, metformin induced a large suppression of active bile salt absorption from the ileum compared with a small increase in passive absorption from the jejunum. This suggests that the ileal effect of metformin to reduce overall bile salt absorption could contribute to the modest cholesterol-lowering effect of this drug.

Demonstration of defective glucose uptake and storage in erythrocytes from non-insulin dependent diabetic patients and effects of metformin.

1. Red blood cells can store glucose and may thus participate in blood glucose homeostasis. We investigated if a defect in this process exists in non-insulin dependent diabetes (NIDD). 2. Blood was obtained in fasting conditions from 10 normal and 10 newly diagnosed NIDD patients (before and after 4 weeks Metformin therapy). Washed erythrocytes were resuspended in media containing various glucose concentrations (4.4, 6.6, 8.8 and 13.2 mmol/L). Total glucose uptake was calculated as the sum of the measurements of lactate as well as free glucose, the latter being determined before and after addition of amyloglucosidase to the pellet. 3. Cells from diabetics showed a pronounced reduction in glucose uptake, particularly in their capacity to store glucose as glycogen (reactive to amyloglucosidase). Metformin treatment almost normalized glycogen levels, whereas lactate declined concomitantly in the pellet. 4. Our data demonstrate that a defect in glucose uptake exists in erythrocytes from NIDD patients, affecting both free and stored glucose, and that this defect is reversed by Metformin treatment, indicating that this drug can increase glycogen levels even in insulin-insensitive cells. 5. Thus, in view of their total mass, erythrocytes may be important in the impaired glucose homeostasis in NIDD, in particular in marked hyperglycaemia such as after a meal.