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The Antihyperglycaemic Effect of Metformin Therapeutic and Cellular Mechanisms

Nicolas F. Wiernsperger1 and Clifford J. Bailey2

1 Lipha SA, an affiliate of Merck KGaA, Darmstadt, Germany 2 Department of Pharmaceutical Sciences, Aston University, Birmingham, England

Abstract 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 potentiat- ing 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 glu- cose-lowering effect of metformin include an insulin-independent suppression of fatty acid oxidation and a reduction in hypertriglyceridaemia. These effects re- duce 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.

REVIEW ARTICLE Drugs 1999; 58 Suppl. 1: 31-390012-6667/99/0001-0031/$04.50/0 © Adis International Limited. All rights reserved.

Since its introduction in 1957, metformin has become an established treatment for type 2 (non– insulin-dependent) diabetes mellitus.[1,2] It is used as monotherapy and in combination with other

types of oral antidiabetic agent or insulin, thereby offering a unique profile of therapeutic effects. The blood glucose-lowering effect of metformin is complemented by potentially beneficial effects on

blood lipid profiles and improvements in various micro- and macrovascular parameters. Metformin does not cause weight gain, and tends to reduce hyperinsulinaemia, which serves to counter insulin resistance and its clinical sequelae.

Typically, metformin reduces basal hypergly- caemia by 1 to 3 mmol/L and decreases haemo- globin A1c (HbA1c) by 1 to 2%.[3-5] However, metformin alone does not precipitate overt hypoglycaemia; hence its designation as an anti- hyperglycaemic agent. Metformin appears to re- quire the presence of insulin to lower blood glucose levels, although the drug does not stimulate insulin secretion. Metformin exerts a variety of insulin-de- pendent and insulin-independent actions, although the insulin-independent effects are not a substitute for insulin. Moreover, metformin has different ac- tions in different tissues, which collectively ac- count for the blood glucose-lowering effect.[6,7] This review considers the sites and mechanisms of action responsible for the antihyperglycaemic ef- fect of metformin.

1. Antihyperglycaemic Effects

The main gluco-regulatory effects of metformin involve suppression of hepatic glucose output, in- creased peripheral glucose utilisation, reduced fatty acid utilisation and increased glucose turn- over, particularly in the splanchnic bed (table I). In addition, metformin alters glucose handling by erythrocytes and reduces hypertriglyceridaemia. Thus, metformin can affect a variety of gluco-reg- ulatory processes, both directly and indirectly via an improved metabolic environment.

The variety of effects exerted by metformin ap- pears to be related, at least in part, to the markedly different concentrations of the drug in different tis- sues seen at different times after administration.

Metformin is rapidly absorbed and rapidly ex- creted unchanged in the urine.[8] In clinical use an oral dose of 500 to 1000mg results in a maximum concentration of 1 to 3 mg/L (about 1 to 2 × 10–5 mol/L) in venous plasma after about 2 hours.

When 50 mg/kg metformin was given orally to normal and streptozotocin diabetic mice (equiva- lent to 3000mg in a 60kg person on a weight-for- weight basis), the maximum concentration of metformin was about 3 × 10–5 mol/L in peripheral venous plasma and 5 to 6 × 10–5 mol/L in the he- patic portal vein.[9] Extremely high concentrations of metformin accumulated in the walls of the jeju- num and ileum (up to 3 × 10–3 mol/L) compared with the liver (up to 3 × 10–4 mol/L) or muscle and fat (up to approximately 5 × 10–5 mol/L).

1.1 Hepatic Glucose Output

There is substantial evidence that metformin re- duces hepatic gluconeogenesis. Studies in isolated perfused livers and hepatocytes from animals have shown that metformin acts directly on the liver to reduce gluconeogenesis from a range of substrates including lactate, pyruvate, alanine, glutamine and glycerol.[10-12] In the absence of added insulin there is little effect until the metformin concentration is above that normally found within the hepatic portal vein. However, in the presence of insulin, therapeu- tic concentrations of metformin have been reported to suppress gluconeogenesis, thus showing a syn- ergistic effect with insulin (fig. 1).[11,12] This effect was increased in the presence of raised glucose concentrations,[13] and metformin also suppressed the gluconeogenic effect of glucagon.[13,14] While low concentrations of metformin promote the anti- gluconeogenic action of insulin, higher concentra- tions can exert several non–insulin-dependent effects that contribute to a reduction in hepatic glucose production. These include decreased he- patic uptake of gluconeogenic precursors such as lactate and possibly amino acids.[15,16] High con- centrations of metformin might also reduce the mito- chondrial NAD to NADH ratio,[11] causing a slight lowering of cellular ATP that is sufficient to en- hance the flux through pyruvate kinase.[17]

Table I. Major blood glucose-lowering effects of metformin

Parameter Effect

Hepatic glucose output Decreased

Peripheral glucose utilisation Increased

Fatty acid oxidation Decreased

Glucose turnover Increased

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Therapeutic and supratherapeutic concentra- tions of metformin have been consistently shown to cause only partial suppression of gluconeogen- esis.[1,7] This is consistent with clinical experience to show that metformin is rarely associated with hypoglycaemia to any degree, and that the drug does not cause serious or fatal hypoglycaemia, even after overdosage.[1]

In addition to partial suppression of gluconeo- genesis, metformin appears also to reduce hepatic glucose output by decreasing the overall rate of glycogenolysis.[7] No consistent effect of metformin has been demonstrated on total hepatic glycogen content, but there are reports that metformin can increase both glycogenesis and gly- cogenolysis.[7] In the livers of diabetic mice, metformin increased levels of the active forms of both glycogen synthase and glycogen phosphoryl- ase, indicating increased glycogen turnover.[18] Pa- tients with type 2 diabetes treated with metformin showed a reduction in overall glycogenolysis that was associated with decreased cycling of glucose- 6-phosphate.[19]

Reductions in both gluconeogenesis and glyco- genolysis by metformin probably reflect in part the drug’s suppressive effect on hepatic glucagon ac- tivity.[13,14] Glucagon activates hepatic adenylate

cyclase, and the ability of insulin to inhibit adenyl- ate cyclase is reduced in insulin-resistant diabetic states. Metformin restores the capacity of insulin to inhibit hepatic adenylate cyclase, thus providing an explanation for the improvement in hepatic in- sulin action.[20-22] Metformin also decreases the ac- tivity of glucose-6-phosphatase in the livers of di- abetic animals and insulin-resistant liver cells, which indicates a further means of reducing he- patic glucose output.[7,21,23-25]

1.2 Peripheral Glucose Utilisation

Although metformin exerts little glucose-low- ering activity in normoglycaemic individuals with normal insulin sensitivity, it increases insulin- stimulated glucose utilisation under conditions of hyperglycaemia and/or insulin resistance.[6,7] For example, metformin increased glucose utilisation during a hyperglycaemic-hyperinsulinaemic infu- sion in normal rats and during hyperinsulinaemic glucose clamp studies in mildly hyperglycaemic diabetic rats.[26,27] The bulk of insulin-stimulated peripheral glucose utilisation takes place in skele- tal muscle, and several studies have shown that ad- ministration of metformin enhances glucose up- take and glycogen synthesis in muscle of diabetic animals[18,26-28] (fig. 2) and patients with type 2

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G lu

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Fig. 1. Interaction of insulin and metformin to suppress gluconeogenesis from lactate (10 mmol/L) and pyruvate (1 mmol/L) by isolated hepatocytes from 48-hour starved rats after incubation for 1 hour.[12] Values are mean ± standard error; n = 6. * p < 0.05, ** p < 0.01, *** p < 0.001 vs control; † p < 0.05, †† p < 0.01 vs insulin alone.

Antihyperglycaemic Mechanisms of Metformin 33

© Adis International Limited. All rights reserved. Drugs 1999; 58 Suppl. 1

diabetes.[29] In some studies, metformin treatment also produced a small increase in glucose oxidation but no increase in lactate production by skeletal muscle.[6,7]

Addition of metformin in vitro enhanced insu- lin-stimulated glucose uptake by skeletal muscle biopsies from diabetic and nondiabetic insulin-re- sistant individuals.[30,31] However, short term in vitro studies in normal muscle have been inconclu- sive, suggesting that a direct effect of metformin on insulin-stimulated glucose uptake is usually ev- ident only in insulin-resistant states.[7] It is appre- ciated that metformin will also increase insulin sensitivity indirectly through a general improve- ment in the metabolic environment that results from the drug’s other antihyperglycaemic mecha- nisms.[6,7] There is also a possibility that metformin might enhance the mass action effect of glucose, in which hyperglycaemia itself enhances glucose dis- posal.[32,33]

Metformin has been reported to increase insu- lin-stimulated glucose uptake by adipose tissue in

most, but not all, studies with this tissue.[7] How- ever, other actions of metformin that increase glu- cose turnover and utilisation usually cause the ex- penditure of sufficient energy for adipose depots not to be increased. Indeed weight stabilisation or slight weight loss is commonly observed during metformin therapy.[1]

The mechanism through which metformin en- hances insulin-stimulated glucose uptake in insu- lin-resistant states has been attributed mainly to in- creased translocation to and activity of glucose transporters in the plasma membrane (discussed below).

1.3 Fatty Acid Oxidation

In vitro studies in muscle and liver have shown that biguanides can reduce the rate of fatty acid oxidation.[34] The oxidation of palmitate by rat di- aphragm homogenates was reduced by 30% with metformin 10–4 mol/L, with no direct effect on glu- cose oxidation[35] (S. Muntoni, personal communi- cation). This provides a means whereby metformin can correct any imbalance of the glucose-fatty acid (Randle) cycle in the diabetic state.

In indirect calorimetric studies in patients with type 2 diabetes, lipid oxidation was decreased by metformin, whereas glucose oxidation was slightly increased.[36,37] Suppression of fatty acid oxidation (about 15 to 25%) correlated approximately with suppression of hepatic glucose production, sug- gesting that reduced hepatic energy supply from fatty acid oxidation contributed to reduced gluco- neogenesis.[37] The decrease in fatty acid oxidation occurred in the basal state and was not related to prevailing insulin levels. Metformin can decrease fatty acid turnover and reduce circulating free fatty acid (FFA) levels, probably via increased re-ester- ification rather than decreased lipolysis.[37] How- ever, decreased FFA levels appear to be insufficient to account for the reduction in fatty acid oxidation, which indicates a direct action of metformin that is independent of insulin levels.[37]

Metformin often reduces circulating triglycer- ide levels in hypertriglyceridaemic patients; this is associated with decreased synthesis and in-

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Fig. 2. Muscle glycogen level and muscle glycogenic index (3H- 3-glucose counts/gram of tissue × plasma 3H-glucose specific activity × 100) of hind-limb muscle from n2 streptozotocin dia- betic rats after a euglycaemic insulin clamp procedure.[26] Com- pared with that in nondiabetic (control) rats, glycogen accumulation was impaired in the diabetic state and normalised by metformin treatment (100 mg/kg/day for 5 weeks). Values are mean ± standard error; n = 8. * p < 0.01 vs control.

34 Wiernsperger & Bailey

© Adis International Limited. All rights reserved. Drugs 1999; 58 Suppl. 1

creased clearance of very low density lipopro- teins (VLDL).[1,7,38,39] Reductions in triglyceride levels reduce insulin resistance[40,41] and provide an indirect route through which metformin can im- prove the metabolic environment and reduce hyperglycaemia.

1.4 Glucose Turnover

Administration of metformin to insulin-resis- tant obese Zucker rats increased rates of glucose turnover and glucose recycling, which suggests po- tential mechanisms for increased glucose expendi- ture and a contribution to the lowering of blood glucose levels.[42] This appears to be achieved, at least in part, by high concentrations of metformin in the intestinal wall that stimulate anaerobic me- tabolism of glucose and cause the release of lactate into the portal vein.[43] Indeed, metformin causes a greater stimulation of glucose utilisation (per unit weight of tissue) by the intestine than the skeletal muscle.[27,42,43] The lactate is metabolised by the liver and peripheral tissues.[43]

Erythrocytes of patients with type 2 diabetes show impaired uptake of glucose and storage of glycogen under conditions of hyperglycaemia.[44] Interestingly, metformin corrects this by increas- ing glucose uptake and glycogen levels in these

cells (fig. 3).[44] This substantiates the view that metformin can promote certain non−insulin- dependent (as well as insulin-dependent) glucose transport mechanisms.

2. Cellular Mechanisms

Metformin counters insulin resistance predom- inantly by enhancing insulin action. It also exerts effects on glucose transport that are not acutely sensitive to insulin, and it appears to reduce defects in cell membrane fluidity that are caused by glycosylation.

2.1 Increased Glucose Transport

Metformin increased translocation of the insu- lin-sensitive glucose transporter isoform GLUT4 into the plasma membrane in several in vitro stud- ies in adipocytes.[45,46] Increased translocation of GLUT1 was also noted in some studies in ad- ipocytes and L6 muscle cells, but there was no change in the total pool of transporters, which im- plied a lack of effect on transporter synthesis.[45,47] Although these studies involved chiefly supra- therapeutic concentrations of metformin, adminis- tration of the drug to insulin-resistant fatty rats in- creased insulin-stimulated translocation of GLUT4 and GLUT1 into the plasma membrane of ad-

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Controls Diabetes Metformin-treated

†

†

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Glucose concentration (mmol/L) = 6.6 = 8.8 = 13.2

Fig. 3. Glucose uptake by erythrocytes from nondiabetic individuals (controls) and patients with type 2 diabetes before and after metformin treatment (850mg 3 times daily for 4 weeks). Erythrocytes were incubated at different glucose concentrations. Values are mean ± standard error; n = 10. * p < 0.05, ** p < 0.001 vs control; † p < 0.001 vs before treatment.

Antihyperglycaemic Mechanisms of Metformin 35

© Adis International Limited. All rights reserved. Drugs 1999; 58 Suppl. 1

ipocytes without causing de novo transporter syn- thesis.[48] To account for the accompanying in- crease in glucose transport, it was suggested that metformin also increased the functional activity of glucose transporters.[48] This is corroborated by studies in skeletal muscle, vascular smooth muscle cells and erythrocytes, which suggests that metformin can enhance the glucose transport ca- pacity of GLUT4 and (probably to a greater extent) GLUT1 transporters that are present in the plasma membrane.[48-51] Interestingly, metformin in- creased insulin-stimulated glucose transport by Xenopus laevis oocytes, which are devoid of GLUT4, but no such effect was seen in the absence of insulin.[52] Glucose transport by GLUT3 into monocytes of patients with type 1 (insulin-depend- ent) diabetes was increased by metformin,[53] which further supports the view that metformin can enhance some insulin-insensitive glucose transport mechanisms as well as those that are acutely insulin sensitive.[7] Indeed, metformin enhances the mass action effect of glucose on glucose uptake (e.g. by erythrocytes) in patients with type 2 diabe- tes.[32,44,51]

Thus, metformin appears to improve the trans- membrane transfer capacity of glucose transporters and to increase the translocation of transporters into the cell membrane. These effects require the presence of insulin or high levels of glucose, which suggests that metformin enhances the function of transporters preactivated by their natural stimu- lants.

2.2 Increased Insulin Action

Since the therapeutic efficacy of metformin is dependent on the presence of insulin (albeit not necessarily at high levels), the relationship be- tween metformin and insulin has been the subject of much recent research.[7,54] Although metformin has been shown to increase the number of insulin receptors in various diabetic states, this does not appear to be related to its blood glucose-lowering effect.[1,54] Nevertheless, metformin increases most biological functions of insulin (at least in in- sulin-resistant states), including glucose transport,

and glycogen and lipid synthesis.[7] This suggests that metformin might influence insulin receptor signalling or an early event in the postreceptor sig- nalling process that is common to the diversity of insulin-mediated events known to be augmented by metformin. Particular attention has therefore fo- cused upon insulin receptor phosphorylation, tyro- sine kinase activity and the activation of insulin receptor substrate (IRS) proteins. In vitro studies in Xenopus oocytes and studies in erythrocytes from metformin-treated patients with type 2 diabetes showed that metformin increased insulin receptor tyrosine kinase activity (fig. 4).[55,56] Other studies have also indicated that metformin enhances tyro- sine kinase pathways.[57-60] In Xenopus oocytes, metformin stimulated insulin receptor tyrosine ki- nase[55] and glycogen synthase[52] activity (fig. 5) in a concentration-dependent manner within the therapeutic range. Further studies in oocytes have indicated that increased insulin receptor tyrosine kinase activity in the presence of metformin is as- sociated with the activation of phospholipase C,

0 0 0.1 1.0 10 100

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Before After

Fig. 4. Insulin-stimulated insulin receptor tyrosine kinase activity of insulin receptors solubilised from erythrocytes of patients with type 2 diabetes before and after metformin treatment (850mg twice daily for 10 weeks).[56] Values are mean ± standard error; n = 14. * p < 0.0001 vs before treatment.

36 Wiernsperger & Bailey

© Adis International Limited. All rights reserved. Drugs 1999; 58 Suppl. 1

elevated levels of inositol-1,4,5-trisphosphate (IP3) and raised intracellular levels of free Ca++.[55]

From these observations, metformin appears to bring about an increase in tyrosine kinase activity of the β subunit of the insulin receptor. This in turn facilitates the phosphorylation of receptor-associ- ated IRS proteins.[57,58] Thereafter, key compo- nents of the postreceptor signalling cascades will be stimulated, e.g. phosphatidylinositol-3-kinase (PI3-kinase), a known intermediate in the insulin signalling pathway that leads to increased glucose uptake.[59,61] The mechanism through which metformin enhances insulin-mediated insulin re- ceptor tyrosine kinase activity is not established. However, there is evidence that metformin alters the physicochemical properties of cell membranes, in particular reversing the detrimental effects on membrane fluidity that occur with non-enzymatic glycosylation in the presence of raised glucose levels.[62]

3. Conclusion

Metformin is an effective antihyperglycaemic agent with a unique portfolio of therapeutic effects

and a variety of cellular actions. It lowers blood glucose levels predominantly by enhancing the ac- tion of insulin, but it also exerts some effects under hyperglycaemic conditions that are not acutely de- pendent on insulin. However, these latter effects cannot substitute for the action of insulin. Thus, metformin improves insulin sensitivity in insulin- resistant and hyperglycaemic states. The main therapeutic effects responsible for reducing blood glucose levels are reduced hepatic glucose output and increased peripheral glucose utilisation. In ad- dition, metformin decreases fatty acid oxidation and increases glucose turnover. At the cellular level, metformin influences membrane events af- fecting tyrosine kinase activity and glucose trans- port processes. In particular, metformin increases insulin-mediated insulin receptor tyrosine kinase activity, which leads to the augmentation of a range of insulin signals. Metformin also increases the functional activity of glucose transporters, possi- bly via effects on the physicochemical properties of cell membranes.

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Correspondence and reprints: Dr C.J. Bailey, Department of Pharmaceutical Sciences, Aston University, Aston Triangle, Birmingham B4 7ET, England.

Antihyperglycaemic Mechanisms of Metformin 39

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