Microvascular diseases: is a new era coming?

The microvascular bed is an anatomical entity which comprises myriads of small arterioles, capillaries and venules. Microvessels and surrounding tissue metabolism are tightly coupled; consequently they are equipped with many, very specific and fine-tuned mechanisms allowing permanent, precise regulation of nutrient delivery. The review thoroughly describes the structure and physiology of arterioles and capillaries as well as the specialized means to investigate them. Microcirculation has been largely neglected for decades, mainly because of lack of technical possibilities for visualization and quantitation. However the past years have completely renewed the scientific interest, due to the combination of the availability of new techniques in human research and the recognition that the microcirculation is autonomically and causally involved in diseases previously thought to be essentially a question of macrocirculation. Today we start to see that microangiopathy is not only a consequence of large vessel diseases but can be the source of many pathologies in both cardiovascular and metabolic disorders, the best example -developed here- being the cardiometabolic syndrome or prediabetes. With very few exceptions, pentoxifylline and the antidiabetic metformin, no specific treatments have been developed for treating disorders at the microcirculatory level. Metformin has unique, intrinsic actions specifically at the level of terminal arterioles, which are completely independent of its antidiabetic effect. Other drugs are shortly described which have revealed a potential interest in this field. Our review aims at showing that microcirculation is entering a new era, starting with rapidly increasing knowledge of its intimate functioning and worth specific pharmacological developments.

Microcirculation and the metabolic syndrome.

The present review, to the authors' knowledge, is the first to specifically address the relationships between microcirculation and metabolic syndrome, a cluster of metabolic and cardiovascular modifications highly prevalent in the general population. Its close link to overweight and insulin resistance makes it the main cause of the worldwide burden of type 2 diabetes. However, metabolic syndrome is also observed in many other diseases, particularly, but not exclusively, those where insulin resistance is a main feature. Analysis of the literature reveals that this clinical situation is invariably linked to microvascular disturbances, such as abnormalities in arteriolar reactivity, capillary recruitment, permeability, and hemorheology. A particularly interesting observation is that these defects in small vessel structure and function are seen very early in life or disease. Very importantly, they further suggest that microcirculatory abnormalities may be not only secondary but also causal to the development and/or aggravation of insulin resistance and metabolic syndrome. Mechanisms responsible for these modifications remain largely unknown, but insulin's vascular effects in the microvascular network, detailed in this review, are at least one example of such connections. The existing data point to a clear, at least bidirectional, relationship between microcirculation and metabolic syndrome. Additional studies should determine the level of reciprocal causality.

Metformin improves skin capillary reactivity in normoglycaemic subjects with the metabolic syndrome.

AIMS: Insulin resistance and a parental history of diabetes mellitus are independently associated with endothelial dysfunction. Oxidative stress has a pivotal role in the pathophysiology of vascular injury. Metformin, in addition to its glucose-lowering properties, has vasculoprotective effects. We investigated whether metformin has beneficial effects on the nutritive skin capillary circulation and deceases oxidative stress in a group at high risk for Type 2 diabetes mellitus (T2DM) and cardiovascular disease. METHODS: Thirty normoglycaemic subjects with the metabolic syndrome (MS),who had first-degree relatives with T2DM, participated. The mean age was 39.1 +/- 8.4 years and body mass index (BMI) 35.7 +/- 4.8 kg/m2 (mean +/- SD). SUBJECTS: were randomized 1 : 1 to receive placebo (n=14) or metformin (n=16; 1700 mg/day) in a double-blind study. At baseline and post treatment, blood and urine samples were collected for biochemical and 8-epi-prostaglandin F2alpha (8-epi-PGF2alpha) analysis, respectively. Microcirculation was assessed by nailfold videocapillaroscopy, analysing afferent (AF), efferent (EF) and apical (AP) diameters of capillary loops, functional capillary density (FCD), red blood cell velocity at rest (RBCV), after 1 min arterial occlusion (RBCVmax) and time (TRBCVmax)taken to reach it. RESULTS: Groups did not differ significantly in anthropometric, clinical, laboratory or microvascular measurements at baseline. In the metformin group, weight,BMI, systolic blood pressure and fasting plasma glucose fell, and lipid profile and microcirculatory parameters FCD, AF, EF, AP, RBCVmaxand TRBCVmax improved (all P<0.01). No relationship between clinico-laboratory parameters and microvascular reactivity was observed, except for changes in total and low density lipoprotein-cholesterol and RBCVmax* 8-epi-PGF2alpha did not change significantly in either group. CONCLUSIONS: Metformin improved skin capillary reactivity in normoglycaemic MS subjects independently of significant changes in 8-epi-PGF2alpha levels.

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.

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.

Albumin antioxidant capacity is modified by methylglyoxal.

OBJECTIVE: Oxidative stress seems to play a major role in diabetic vascular complication development. Plasma albumin, via its thiol groups, is the main extracellular antioxidant molecule. Methylglyoxal (MG) is a very reactive dicarbonyl compound increased in diabetes which strongly modifies proteins by non-enzymatic glycosylation. The aim of this work was to study if MG could modify albumin antioxidant capacity. METHODS: Bovine serum albumin was incubated with 1 mM MG at 37 degrees C for 7 days (MG-BSA). Albumin physico-chemical changes were evaluated by tryptophan autofluorescence measurement in the presence or in the absence of a quencher (acrylamide). Albumin antioxidant capacity was determined by thiol measurement using Ellman's reagent as well as in a cellular system (HeLa cells stressed by H2O2). RESULTS: MG-BSA exhibited important modifications as shown by conformational changes, decreased tryptophan autofluorescence (30%) and significant thiol loss (40%). MG-BSA led to important modifications resulting in oxidation and loss of albumin antioxidant capacity. MG-BSA modifications were close to the one observed in albumin isolated from diabetic patients. CONCLUSION: Our results suggest that deleterious effects induced by carbonyl stress in diabetes could also originate from a loss of albumin antioxidant capacity by dicarbonyl compound attack. The biological consequences of these findings have now to be investigated.

Noninvasive orthogonal polarization spectral imaging as applied to microvascular studies in mice.

In vivo observations of the mouse microcirculation can hardly be performed due to technical difficulties, limiting the knowledge that could be obtained from gene manipulated mice models. The aim of the present study was to check the applicability of a novel optical system, the orthogonal polarization spectral technology, to study the mouse microcirculation. In anaesthetized mice, the spinotrapezius muscle microcirculation was observed in situ. The diameter of precapillary arterioles was measured before and after a pharmacological or hormonal stimulation. High-contrast images of the muscle microcirculation were obtained and significant vasodilatation of arterioles was observed after topical applications of acetylcholine, sodium nitroprusside, and insulin. As compared to conventional techniques, orthogonal polarization spectral imaging makes it possible to assess and study microvascular beds in mice, which were inaccessible until now, allowing the use of gene manipulated mice to investigate, for example, the mechanisms involved in the development of diabetic microangiopathy.

Effect of glycated LDL on microvascular tone in mice: a comparative study with LDL modified in vitro or isolated from diabetic patients.

AIMS/HYPOTHESIS: In vitro studies have suggested that glycation of LDL might be implicated in diabetic microangiopathy. We therefore investigated the in vivo effects of LDL glycated in vitro on the mouse skeletal muscle arteriolar tone. Since glycation naturally occurs during diabetes, we also tested the effects of LDL isolated from diabetic patients. METHODS: In anaesthetized mice, the spinotrapezius muscle microcirculation was observed, in situ, using the orthogonal polarization spectral imaging technology. The diameter of terminal (<20 microm) and small arterioles (20-40 microm) was measured before and after a bolus intravenous injection of glycated LDL followed by a continuous perfusion (115 micro g/kg/min). RESULTS: A slight decrease of terminal and small arterioles diameter (<10%) was observed with native LDL and LDL isolated from healthy subjects. In contrast, mildly glycated LDL induced a clear vasoconstriction of arterioles (>15%), which was further increased when highly glycated LDL was perfused (>22%). LDL isolated from diabetic patients mimicked the vasoconstriction obtained with in vitro mildly glycated LDL, which underwent similar glycation as those isolated from diabetic patients. CONCLUSION/INTERPRETATION: Our results show in vivo that acute perfusion of both types of glycated LDL (artificially or naturally modified), cause major microvascular modification by enhancing arteriolar tone in skeletal muscle. These findings highlight a new role of glycated LDL at the level of microvessels. We suggest that glycation of LDL could contribute to the impaired vascular reactivity observed in 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.

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.

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.

Mild acute renal failure potentiates metformin accumulation in the diabetic rat kidney without further impairment of renal function.

OBJECTIVES: To analyze, in acute renal failure (ARF) in diabetic rats, how moderate functional ARF would modify metformin (MET) pharmacokinetics and if plasma and renal tissue MET accumulation could aggravate renal insufficiency and/or elicit plasma lactate accumulation. METHODS: Streptozotocin-induced diabetic rats were allocated to four groups: control, MET, ARF, ARF-MET (6-7 rats per group). MET (100 mg/kg/day) was given per os for two weeks before ARF was induced by drinking restriction and enalapril treatment. The effects of MET and/or ARF were examined in vivo on renal function in conscious rats (metabolic cages) and ex vivo on renal vascular reactivity (isolated kidney). RESULTS: MET treatment (plasma level: 5.3 +/- 1.4 microg/ml, mean+/-SEM), resulted in biguanide accumulation in cortex and medulla (53 +/- 17 and 80 +/- 40 microg/g respectively). MET was devoid of any effect on creatinine clearance, mean blood pressure or renal vascular resistance, but moderately increased plasma lactate (3.8 +/- 0.5 vs 3.2 +/- 0.2 mM, P<0.05) and decreased angiotensin II-induced renal vasoconstriction. ARF, although mild, decreased renal MET clearance (0.29 +/- 0.05 vs 1.01 +/- 0.31 ml/min/100 g, P<0.05) and increased plasma and renal tissue MET levels (x 2-4). MET however did not worsen the fall in glomerular filtration rate, nor modify renal vascular reactivity. ARF did not change the MET-elicited moderate increase in plasma lactate. CONCLUSION: Despite the increase in MET plasma and renal tissue levels subsequent to moderate ARF, no harmful metabolic effect on plasma lactate and no further impairment of renal function was observed in MET-treated diabetic rats subjected to ARF.

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.

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.

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.

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.

Increased intraabdominal adipose tissue mass in fructose fed rats: correction by metformin.

Summary. The aim of the present study was to investigate the effect of metformin on insulin sensitivity, adipose tissue mass and sympathetic nervous system (SNS) activity in fructose fed rats. Male Sprague-Dawley rats were fed for six weeks either on a standard diet (C group) or on a high-fructose diet (F group, 10% in drinking water). In each group, half of the animals received metformin in drinking water for the last 4 weeks (500 mg/kg x day, C+M and F+M). Hyperinsulinemic-euglycemic clamps (6 mU insulin/kg.min) were performed on awake unrestrained rats to test insulin resistance. Six-week fructose diet induced a reproducible insulin resistance (31.1 +/- 1.9 C vs 22.5 +/- 3.2 mg glucose/kg.min F, p<0.05). Metformin treatment prevented insulin resistance (31.1 +/- 1.9 C vs 30,2 +/- 1.8 mg glucose/kg x min F+M, ns). To measure SNS activity, rats received, ten minutes before sacrifice, an i.p. injection of NSD (m-hydroxybenzylhydrazine, inhibitor of DOPA decarboxylase, 100 mg/kg). DOPA accumulation was used as an index of SNS activity and measured in superior cervical, coeliac ganglias, retroperitoneal and epidydimal adipose tissues. SNS activity was increased in F group only in coeliac ganglia (16.8 +/- 1.1 C vs 22.6 +/- 2.2 ng DOPA/ganglia, F group, p<0.05) and not in superior cervical ganglia (8.4 +/- 0.7 C vs 8.6 +/- 0.7 ng DOPA/ganglia, F group, ns). Metformin had no effect on SNS activity in coeliac ganglia of control animals (15.9 +/- 1.7 C+M vs 16.8 +/- 1.1 ng DOPA/coeliac ganglia C, ns) but prevented the increase in SNS activity in fructose fed animals (22.6 +/- 2.2 F vs 16.3 +/- 2.8 ng DOPA/coeliac ganglia F + M). In fructose fed rats, metformin significantly increased sympathetic activity in retroperitoneal white adipose tissue (RPWAT) resulting in a marked decrease in depot mass but had no effect on epidydimal WAT. In conclusion, our results demonstrate that fructose diet caused a selective increase of SNS activity in coeliac ganglia. Metformin increased SNS activity in RPWAT resulting in a significant reduction in RPWAT mass, lowered SNS activity in coeliac ganglia to control values and restore whole body insulin sensitivity.

In vivo kinetics of 123 iodine-labelled insulin in skeletal muscle of patients with type 2 diabetes. Effect of metformin.

BACKGROUND: The capillary filtration of albumin (CFA) is often increased in diabetic patients. However, the transcapillary transfer of insulin is considered to be a key stage in insulin action. The aim was to study the in vivo kinetics of 123I-labelled human insulin in the skeletal muscle of type 2 diabetic patients with an increase in CFA and to evaluate the effect of metformin, using a noninvasive method. METHODS: Ten type 2 diabetic patients and 6 healthy control subjects were investigated. After an i.v injection of 123I-labelled insulin, venous samples were drawn during 75 minutes and radioactivity was counted externally by a gammacamera on the contralateral forearm before, during and after venous compression. The changes in the serum percentages of bound and free 123I were followed during the entire test and the forearm iodine bound to insulin was estimated by multiplying the forearm counted radioactivity by the serum percentage of bound iodine at the same time. RESULTS: In the diabetic patients the maximal increase in the forearm iodine bound to insulin during venous compression was lower (p=0.06), and 10 minutes after removal of venous compression the forearm retention of labelled insulin was significantly lower (p<0.0005) than in controls. After one month of metformin treatment, retention of labelled insulin significantly increased (p<0.001) but was still significantly lower than in the controls (p<0.001). CONCLUSION: The in vivo kinetics of (123) I-labelled insulin procedure allows the study of skeletal muscle metabolism provided venous compression is exerted. In type 2 diabetic patients a reduction of insulin transfer from capillary to tissue despite an increase in CFA, and a reduction of the time spent by insulin in the tissues contribute to insulin resistance. The latter disorder may be improved by metformin.