European Journal of Clinical Investigation (1994) 24, Suppl. 3,3-10

Pharmacology of a-glucosidase inhibition

H. BISCHOFF Bayer AG, Institute for Cardiovascular and Arteriosclerosis Research, Wuppertal, Germany

Abstract. The development of a-amylase and brush- border a-glucosidase inhibitors is reviewed. The mode of action as well as pharmacological and pharmaco- dynamic properties of selected inhibitors with special regard to the most thoroughly investigated Q-

glucosidase inhibitor acarbose are discussed. Inhibi- tion of intestinal a-glucosidases delays the digestion of starch and sucrose, flattens the postprandial blood glucose excursions, and thus mimics the effects of dieting on hyperglycaemia, hyperinsulinaemia and hypertriglyceridaemia. Therefore, the mechanism of a-glucosidase inhibition represents the pharmacologi- cal optimization of the dietary principle of delayed carbohydrate absorption.

In pre-clinical studies using diabetic animals the oral administration of acarbose improved the meta- bolic state and reduced the blood glucose area under the curve. As a consequence, the process of non- enzymatic glycation of proteins was retarded as indicated by reduced glycated haemoglobin, glomer- ular basement membranes or advanced glycation end- products (AGES) in collagen. These improved bio- chemical parameters correlated with beneficial effects against the development of diabetic nephropathy and neuropathy. Thus, the treatment of diabetic animals with acarbose does not only improve the metabolic state but has also the potential to delay, or possibly prevent, the development of diabetic complications.

Keywords. Acarbose, a-glucosidase inhibitors, dia- betic animals, diabetic nephropathy, diabetic neuro- pathy, miglitol.

Introduction The aim of anti-diabetic therapy of IDDM and NIDDM patients is to reach normoglycaemia and to reduce insulin resistance in! NIDDM thereby improv- ing metabolic control with the intention to prevent diabetic late complications. The manifestations of nephropathy, neuropathy, retinopathy and micro- angiopathy in diabetics is highly correlated with the long-term quality of metabolic control and blood glucose concentration [l]. Diet therapy plays an important role, not only in obese patients in order

Correspondence: H. Bischoff, Bayer AG, Institute for Cardio- vascular and Arteriosclerosis Research, Bayer AG, D-5600 Wuppertal 1, Germany.

to reduce overweight, but primarily as the basis of diabetes treatment.

Diet regimen and the control of nutrient entry, with the aim of avoiding glucose and anabolic hormone peaks, is broadly accepted as basic treat- ment of diabetes mellitus [2]. Diabetic patients should, therefore, divide their daily food intake into as many small portions as possible, spread over the day. They should particularly reduce, or better still avoid, the consumption of fast digestible carbohy- drates such as refined starch products or sucrose, to avoid excessive postprandial hyperglycaemia and hypertriglyceridaemia.

Carbohydrate digestion Carbohydrates are the major constituents of the human diet, particularly those recommended for diabetic patients by the national and international diabetes associations [3,4]. As mono-, di- and poly- saccharides they represent not only the major part quantitatively but also the main energy supply. How- ever, only the relatively infrequent monosaccharides (glucose, fructose, etc.) can be absorbed and are readily taken up from the small intestine [5 ] .

The most important dietary carbohydrate compo- nents, starch and sucrose, have to be broken down enzymatically to monosaccharides by a-glucosidases before they can be absorbed (Fig. 1). With the refined carbohydrates of our Western diet this usually takes place very rapidly in the upper parts of the small intestine. Consequently, due to lack of substrate, the lower parts (ileum) are normally not involved, although all enzyme activities required for the carbo- hydrate digestion are also present in this part of the digestive tract [6]. As a consequence of the high digestion rate in the upper part of the small intestine a postprandial rise in blood glucose is generally rapid and high following a carbohydrate load.

Based on these ideas, Puls et al. [7] looked for a new pharmacological approach to improve the metabolic control of diabetics as the postprandial hyperglycaemia was not adequately treated by the standard pharma- cotherapy with sulfonylureas or insulin.

Assuming that inhibition of a-glucosidases should delay the digestion of starch and sucrose and thus mimic the effects of dieting on hyperglycaemia, hyper- insulinaemia and hypertriglyceridaemia, they started

3

4 H. BISCHOFF

Starch Sucrose

4 I

Oligosaccharides

Glucose + Fructose 1

Glucose

r, Absorption

Figure 1. Schematic diagram of enzymatic degradation of p l y - and oligosaccharides and sucrose by intestinal a-glucosidases.

searching for inhibitors of carbohydrate digestive enzymes in the late 1960s. The aim was to optimize pharmacologically the therapeutic principle of delaying carbohydrate absorption.

Inhibitors of intestinal a-glucosidases

a-amylase inhibitors The first enzyme inhibitors Puls and colleagues found inhibited the pancreatic a-amylase. A protein, BAY D 7791, isolated from wheat flour, inhibited in vitro and in vivo the digestion of raw starch. However, the inhibitory effect was weak against cooked starch and the effect against a mixed carbo- hydrate meal including sucrose was disappointing [8]. This was also true for the more potent a-amylase inhibitors BAY E 4609, tendamistat and trestatin because they did not inhibit the most important step, the cleavage of oligo- and disaccharides by the key- enzymes-the membrane-bound a-glucosidases glu- coamylase, dextrinase, isomaltase, maltase and sucrase [9]. Inhibitors which retard digestion both of sucrose and starch are, therefore, regarded as super- ior to a-amylase inhibitors [71.

<OH

Figure 2. Structural formula of acarbose.

A

1 -deoxynojirirnycin-derivatives

cw-D--8--D--p*c

MDL-25,637

Erniglitate Castanospermine

MDL 73945

Valiolarnine derivative

I CHZOH

Voglibose

Acarviosine derivative

1

D L

Al-5662

Figure 3. Structural formula of various cr-glucosidase inhibitors.

Inhibitors of brush-border a-glucosidases Acarbose, a pseudo-tetrasaccharide of microbial origin (Actinoplanes) that potently inhibits in vitro and in vivo the brush-border enzymes glucoamylase, dextrinase, maltase and sucrase as well as the pancreatic a-amylase was the first compound that fulfilled these demands [7,9,10] (Fig. 2). In experimen- tal animals and human subjects, inhibition of carbo- hydrate digestive enzymes by acarbose resulted in a significant decrease in the postprandial rise in blood glucose after a mixed carbohydrate load [9,11,12].

Over the past I5 years, other inhibitors of intestinal brush-border a-glucosidases showing a broad spec- trum of activity have been described and are partially under clinical development [9J 3.141. These more recent inhibitors (Fig. 3) include miglitol, emiglitate,

PHARMACOLOGY OF a-GLUCOSIDASE INHIBITION 5

\m\ from starch

CW;W %on Acarbose

on on

Miaovillus

Microvilli

Enlerocyte I Figure 4. Schematic diagram of enzymatic hydrolysis of oligosac- charides and competitive inhibition of intestinal brush-border a- glucosidases by acarbose. Adapted from H. Bischoff, Act Endokr Stoffw 1991;12:25-32.

voglibose, AI-5662, castanospermine, MDL-25637 and MDL-73945. Among these a-glucosidase inhibitors, acarbose remains the most thoroughly investigated.

Mode of action Acarbose is structurally similar to a typical oligosac- charide derived from starch digestion (Fig. 4). Due to the presence of the intramolecular nitrogen, acarbose attaches to the carbohydrate binding site of the a- glucosidase enzyme (e.g. sucrase) with an affinity exceeding that of the normal substrate (e.g. sucrose) by a factor of 104-105 [9]. The enzymatic reaction stops because the C-N linkage in the acarviosine unit of acarbose cannot be cleaved. As long as acarbose remains bound to the a-glucosidase enzyme, ingested carbohydrates cannot be digested and glucose cannot be released for absorption. However, in spite of its high affinity for a-glucosidases, acarbose is reversibly bound and its inhibition kinetic is competitive. Due to this mode of action, carbohydrates not digested in the upper part of the small intestine are transported to the ileum. This allows the distal ileum to take part in the carbohydrate digestive process [ 1 11. Acarbose thus delays carbohydrate digestion, prolongs digestion time and reduces the rate of glucose absorption. As a result, the postprandial rise in blood glucose is decreased. However, acarbose does not interact with the intestinal Na+-dependent glucose transporter and, thus, does not influence the absorption of orally administered glucose!

Pharmacological and pharmacodynamic effects of a-glucosidase inhibitors Acarbose dose-dependently reduces postprandial increments of blood glucose, exhibiting EDSO-values between 1.0 and 1.5 mg kg-I of body weight either in rats or men administered oral loads of sucrose or starch [12]. As a consequence of this attentuation and flatten- ing of postprandial glycaemia, glucose-stimulated insulin secretion is diminished. This effect on insulin secretion is frequently more pronounced than the reduction in postprandial glycaemia, particularly in hyperinsulinaemic animals. This has also been demonstrated for other inhibitors (Fig. 5 ) e.g. migli- to1 (BAY M 1099) or emiglitate (BAY 0 1248) [13]. Although structurally different from acarbose both the deoxynojirimycin derivates, miglitol and emiglitate, follow similar enzyme kinetics [9]. As was shown by Lembcke et al. [15] the inhibitory mechanism is competitive and the binding to the enzymes is rever- sible. Compared to acarbose the in vitro data reveal some higher inhibitory potency for miglitol and a pronounced higher activity for emiglitate (Table 1). In contrast to acarbose both deoxynojirimycin deri- vatives do not affect the pancreatic a-amylase.

However, there are crucial pharmacokinetical differ- ences between acarbose and both the deoxynojirimycin derivatives. Acarbose, as a pseudo-tetrasaccharide is only minimally absorbed (1 -2%) and will be trans- ported through the intestinal tract to the colon. Both miglitol and emiglitate structurally related to mono- saccharides will be absorbed in the jejunum and only minor amounts reach the ileum and colon [16]. In carbohydrate-loading tests with rats miglitol is some- what higher according to the markedly lower Ki- values in vitro (Table l), emiglitate is at least 5-fold more potent compared with acarbose. However, there is another new quality-emiglitate-which is differ- ent from all other inhibitors and shows a long-lasting inhibitory effect in rats [13]. The postprandial blood glucose and insulin rises after a carbohydrate load administered 4 h and even up to I7 h after giving the substance to rats were still reduced (Fig. 6).

Since a-glucosidase inhibitors are only effective in combination with digestible carbohydrates, they will be ineffective when administered with a carbohydrate- free meal. However, the ingestion of carbohydrates not

Table 1. Inhibition of intestial a-glucosidases: Ki-values (mol I-') of acarbose, miglitol and emiglitate (a-glucosidases from porcine

intestinal mucosa) [13]

Sucrase Maltase Isomaltase

Acarbose. 1.3 x 10-71 1.1 x 10-61 very weak Miglitol 1.4 x 10-7 3.5 x 10-7 5.1 x lo-* Emiglitate 3.9 x lo-* 1.9 x 10-7 3.2 x 10-7

Personal communication from Dr Wingender. Bayer AG, Wuppertal, Germany.

6 H-BISCHOFF

Blood glucose rnrnol I-’

AUC in ?h of CHO

control

7

* 0-0 control, CHO 100 0 P

0.1 mg BAY M 1099 50%

A 0.3 mg BAY M 1099 35% ./

3i 0 f , I I 1

0 15 30 45 min

p u IRI mt-’ 1 000 -

cooked starch + sucrose

AUC inob of CHO

control control, CHO 1 00

500 -

O A , I I 1 0 15 30 45 min

Figure 5. Mean blood glucose and serum insulin concentrations in female, obese (fa, fa) Zucker-rats IS, 30 and 45 min after a mixed carbohydrate load ( 1 g cooked starch +2 g sucrose/kg) fmiglitol (BAY M 1099) p.0. or saline alone. The percentages refer to the area under curve values (CHO-control = 100%); n = 6 rats/group.

digested by a-glucosidases (e.g. lactose, D-glucosidic linkage) will also not contribute to drug efficacy. Thus, an acute effect of a-glucosidase inhibitors is observed only when they are administered together with carbohydrates that are digestible by a- glucosidases. However, it is necessary, and this applies to all a-glucosidase inhibitors, that they have to be administered in doses that allow all carbohydrates, which are digestible by a-glucosidases, to be digested and absorbed completely within the small intestine. Otherwise, undigested carbohydrates will enter the colon and, as a consequence of bacterial fermenta- tion, give rise to side-effects such as flatulence, meteorism and possibly diarrhoea. As anti-diabetic agents, a-glucosidase inhibitors are definitely not designed to promote loss of calories through malab- sorption. Indeed, their intended mode of action is to delay carbohydrate digestion and absorption without promoting the overspill of nutrients into the colon. For this reason, it is important to base the dosage on carbohydrate digestive capacity and dietary regimen and to avoid overdosage.

It is a common observation in patients that gastro- intestinal symptoms possibly occurring in the begin-

ning of the treatment become more infrequent after a few weeks on acarbose and finally almost disappear. This phenomenon is not due to reduced efficacy of acarbose but to an adaptation of the carbohydrates’ digestive enzyme capacity. Due to lack of substrate, in normal fed animals, a-glucosidase activity is much lower in the distal part of the small intestine of rats compared to proximal segments [17,18]. It was shown for sucrase activity in mice by Lee et al. [19] that this decline was reversed by acarbose treatment. We obtained similar alterations in 21 d feeding experi- ments with rats. Because acarbose is a very strong inhibitor of sucrase, we selected the isomaltase due to the very weak isomaltase inhibitory activity of acar- bose [9]. The small intestine was divided into three segments of equal length and the isomaltase activity was determined in the upper, middle and distal segments. Figure 7 verifies a proximal to distal declin- ing gradient also for total isomaltase activity. Acar- bose treatment increased the isomaltase activity in the middle and more pronounced in the distal segments (6-7-fold) after feeding a standard diet 1121. Equiva- lent effects were observed when raw oatmeal was mixed with the standard diet (50%) without the

PHARMACOLOGY OF a-GLUCOSIDASE INHIBITION 7

80 1

70 - 60 - 50 - 40 - 30 - 20 - 10-

0 -

Blood glucose area under curve

A CHO BAY 0 1248/kg

o 0.10 mg

-A 1-00 mg

BAY 0 1248/kg

BAY 0 1248/kg

100 9 7 '/o

60% 46%

0 10 20 30 min

Serum insulin area under curve 1400 - 1200 - 1000 - 800 - 600 - 400 - 200 - 0 2

0.03 mg BAY 0 1248/kg 11 9% 1 B

100

0.1 0 rng BAY 0 1248/kg 44'10

A 1.00 rng BAY 0 1248/kg 13%

mg BAY 0 1248/kg 44%

r I I 1 0 10 20 30 min

Figure 6. Effects of emiglitate (BAY 0 1248) on postprandial area under curve values of blood glucose (A) and serum insulin concentrations (B). Emiglitate was administered orally 4 h before a second sucrose load (2g kg-I); n = 6 rats/group.

inhibitor. From these results we can conclude that alimentary carbohydrates that reach the lower half of the small intestine due to rich fibre content also promote the synthesis of a-glucosidase enzymes. This clearly shows that new synthesis of a-glucosi- dases by acarbose treatment in the distal small intes- tine is a very physiological process due to the supply with undigested carbohydrates. Consequently, because of higher carbohydrates digestive capacity, in the second half of the small intestine the tolerability of acarbose is improved after a few weeks of treat- ment.

From the primary pharmacological effects, it can be concluded that a-glucosidase inhibitors, such as acarbose, act to reduce postprandial hyperglycaemia without stimulating insulin secretion. Since insulin also acts as a lipogenic hormone this represents a beneficial new approach to the treatment of diabetes, especially NIDDM, compared to currently available

agents such as insulin and sulfonylureas. Because the antihyperglycaemic effect of sulfonylureas is mediated primarily through the stimulation of insulin secretion, use of these agents can promote undesirable conse- quences such as body weight gain and hyperlipidaemia.

This difference between a-glucosidase inhibitors and sulfonylureas was illustrated in an experiment that measured triglyceride uptake into adipose tissue after rats were given a mixed meal containing radio- labelled triolein (Fig. 8). With sulfonylurea adminis- tration, the postprandial storage of triglyceride in adipose tissue was clearly increased, after 1 h 3-fold compared to controls. In contrast, acarbose adminis- tration significantly reduced triglyceride uptake due to decreased insulin secretion. Furthermore, after com- bined administration of sulfonylurea and acarbose, the sulfonylurea-induced increase in triglyceride uptake was completely abolished. As a result of its effect on reducing postprandial insulin secretion,

8 H. BISCHOFF

4

U/rat = standard diet EZZZZi 50% oatmeal (control = 100%)

0 40 mg acarbosef 7 100gdiet .

.*

middle dista

Figure 7. Isomaltase activity of the upper, middle or distal part of the small intestine of rats 21 d after feeding a standard diet with or without 50 g% raw oatmeal or 40mg acarbose IOOg-' diet; n = 5-6 rats per segment and group. *P < 0.01 vs. standard diet; **I' < 0.001 vs. standard diet.

acarbose also significantly reduced carbohydrate- driven hepatic lipogenesis. This was shown by feed- ing a high sucrose diet to normal Wistar rats [20] and to genetically obese Zucker-rats [21]. The serum elevations of insulin and triglycerides were dose- dependently reduced by acarbose (10, 20 and 40mg loo-' g diet) and, at the higher dose, abolished.

These studies thus show that acarbose improves not only postprandial hyperglycaemia and hyperinsulin- aemia, but may also have some impact on lipid metabolism.

dpm x 1000 g-' tissue

200 - + control, mixed meal

glisoxepid + acarbose

glisoxepid A acarbose

150-

100 -

I I I I 0 1.0 2.0 4.0 h

Figure 8. Mean radioactivity from orally given ["c] triolein in perirenal adipose tissue at various times after loading fasted rats with a meal containing protein, olive oil and carbohydrates with and without a sulfonylurea (glisoxepid, 006mg rat-') or acarbose (1.2mg rat-') or glisoxepid in combination with acarbose. n = 6 rats/group.

Effects on hyperglycaemia and non-enzymatic glycation in diabetic animals Each antidiabetic pharmacotherapy has the aim to delay or avoid the development of diabetic late com- plications. The relationship between glycated proteins, advanced glycation endproducts (AGEs) and diabetic late complications has not been elucidated so far [22]. However, the development of diabetic late complica- tions correlates with the height of long-term blood glucose concentrations [l] which can be controlled by the measurement of glycated haemoglobin. Because the glycation is a non-enzymatic process the degree of protein glycation follows the law of mass action and depends predominantly on the free glucose concentra- tion. Therefore, a consistently lowered amount of glucose in the blood should lead directly to fewer glycation reactions and development of AGEs.

Acarbose, however, did not only show a reduction of the glucose area under the curve in hyperinsulinae- mic or NIDDM-type animal models [23]; Sima & Chakrabarti [24] demonstrated such an effect also for insulin-dependent BB/W-rats. As shown in Fig. 9, by measuring the glucose concentration very tightly controlled in 2 h intervals for 24 h the area under Curve was reduced by 40%.

Improved blood glucose control in diabetic ani- mals under acarbose treatment does not only lead to lower glycated haemoglobin concentrations but also to a prevention of the glycation process of glomeru- lar basement membranes in streptozotocin-diabetic rats as was shown by Cohen et ul. [25]. Further- more, in an equivalent study it was shown that the development of advanced glycation endproducts (AGEs) in the connective tissue was reduced con- cerning the skin collagen and prevented concerning the tendon collagen compared with the untreated diabetic group [26].

30 1 20 -

15-

10 -

5 - INSULIN I

PHARMACOLOGY OF a-GLUCOSIDASE INHIBITION 9

Effects on diabetic late complications

Nephroparhy In accordance with the reduced glycation of glomer- ular basement membrane by a-glucosidase inhibition in streptozotocin-diabetic rats [25], acarbose (40 mg 100 g-' diet) completely prevented the diabetic renal hypertrophy in these rats that was increased by 35% in the untreated rats (Fig. 10). Using genetically diabetic mice, Lee [27] could demonstrate that after 10 weeks on acarbose (20 and 40mg 100 g-' feed) the kidneys revealed diminished deposits of immuno- globulin (IgG, IgM and IgA) in the glomerular mesangium in a dose-dependent manner, and that thickening of the glomerular mesangium was signifi- cantly reduced. In a different NIDDM-type animal model Cohen & Rosenmann [28] showed that acar- bose (40mg lOOg-I diet) significantly lowered the incidence and severity of glomerulosclerosis in the Cohen-diabetic rat. Furthermore, the longevity of acarbose-treated diabetic animals was longer com- pared with untreated animals. Only three out of 15 untreated control animals survived the 7 month study period. However, after acarbose treatment 12 out of 15 diabetic animals were still alive after that period of time.

Neuroparhy Neuropathy is one of the most frequent complications of diabetes and is not restricted to a single type of diabetes but occurs both in type I and type I1 diabetes. In animal experiments, aldose-reductase inhibitors have shown that they delay the development of diabetic neuropathy. Interestingly, acarbose which acts by a completely different mechanism was like- wise capable to retard the development of diabetic polyneuropathies in the BB/W-rat as was shown by

kidney weight/ body weight * * * P < 0.05 against diabetic control

P < 005 against control

*

Control Diabetic Diabetic rats rats rats

Figure 10. Effect of acarbose (40mg 100g-I diet) on kidney hypertrophy in streptozotocin-diabetic rats after 8 weeks of treat- ment; n = 4 ratslgroup. Adapted from M. Cohen er al. Gen Pharmacol22, 1991 (see ref. 25).

+ Acarbose

R-BAR values

0 control rats

0 BB-rat + 20 mg- diabetic BB-rat

acarbose 100 g I diet

30

20

10

0 2 months 4 months 0

Figure 11. Effect of acarbose on diabetic polyneuropathy in type I- diabetic BB/W-rats. Determination of the autonomic respiratory function (R-BAR values) in non-diabetic control, diabetic BB/W and diabetic BB/W-rats treated with acarbose (20mg 1OOg-1 diet) for 2 and 4 months; n = 6 rats/group. X f SEM. P < 0.001. Adapted from A. Sima et a/. Diabetologia 35, 1992 (see ref. 24).

Sima & Chakrabarti [24]. The vagal autonomic neuropathy in the diabetic BB/W-rat is characterized by a progressive decrease in R-BAR values (a measure of respiration-related variations of heart rates), which precedes and accompanies the development of struc- tural abnormalities [29]. Because this impairment in autonomic polyneuropathy is an age-dependent process [30], the decline in R-BAR values also occurred in healthy, non-diabetic control rats; this process, however, is accelerated in the diabetic state (Fig. 11). The reduction of glucose area in diabetic BB/W-rats over a 4 month period of treatment with acarbose (20 mg 100 g-1 diet) resulted in a complete prevention of the decrease in R-BAR values (Fig. 1 l), suggesting that acarbose had a protective effect on the development of autonomic neuropathy. These func- tional improvements by acarbose treatment were accompanied by the complete prevention of nodal neuroanatomical abnormalities consisting of para- nodal swelling, paranodal demyelination and remye- linated nodes which were all significantly increased in non-treated diabetic rats. Only the frequency of axo- glial dysjunction was not completely, but partially (-40%), decreased by acarbose treatment.

Conclusion By the mechanism of a-glucosidase inhibition, the digestion and absorption of carbohydrates are delayed. The a-glucosidase inhibitor acarbose dose- dependently reduces the postprandial blood glucose and serum insulin peaks after carbohydrate loads, due to these primary and secondary effects:

(1) glucosuria in diabetic animals is markedly lowered;

(2) the hyperinsulinaemia and concordantly the impaired glucose tolerance of insulin resistant and genetically diabetic rats are improved;

(3) the blood glucose area and the glycation of proteins are reduced;

10 H. BISCHOFF

(4) the carbohydrate-driven and insulin-stimulated hepatic synthesis and secretion of triglycerides (VLDL) in rats are lowered resulting in a reduced hypertriglyceridaemia.

Finally, in preclinical studies using diabetic animals acarbose treatment demonstrated that this new therapeutic principle exhibited beneficial effects on diabetic nephropathy and neuropathy. Due to the improvement of the metabolic state, a-glucosidase inhibitors may have the potential to delay or, possi- bly, to prevent the development of diabetic late complications.

References 1 Pirart J. Diabetes mellitus and its degenerative complications: a

prospective study of 4,400 patients observed between 1947 and 1973. Diabetes Care 1978;1:168-88.

2 Creutzfeldt W, Folsch UR, eds. Delaying absorption as a therapeutic principle in metabolic diseases. Stuttgart: G. Thieme Verlag, 1983.

3 Diabetes and Nutrition Study Group of the European Associa- tion for the Study of Diabetes. Statement. Nutritional recom- mendations for individuals with diabetes mellitus. Diabetes Nutr Metab 1988;1:145-9.

4 American Diabetes Association. Nutritional recommendations and principles for individuals with diabetes mellitus 1986. Diabetes Care 198710: 126-32.

5 Elsenhans B, Caspary WF. Absorption of carbohydrates. In: Caspary EF, ed. Structure and Function of the Small Intestine. Amsterdam: Excerpta Medica, 1987:139-59.

6 Newcomer AD, McGill DB. Distribution of disaccharidase activity in the small bowel of normal and lactase-deficient subjects. Gastroenterology 19665 k48 1-8.

7 Puls W, Keup U, Krause HP, Thomas G, Hoffmeister F. Glucosidase inhibition: a new approach to the treatment of diabetes, obesity and hyperlipoproteinemia. Natunvissenschaften

8 Puls W, Keup U. Influence of an a-amylase inhibitor (BAY D 7791) on blood glucose, serum insulin and NEFA in starch loading tests in rats, dogs and men. Diabetologia 1973;997-101.

9 Truscheit E, Hillebrand I, Junge B, Miiller L, Puls W, Schmidt D. Microbial a-glucosidase inhibitors: chemistry, biochemistry and therapeutic potential. Prog Clin Biochem Med 1988;717- 99.

10 Schmidt DD, Frommer W, Junge B er al. a-Glucosidase inhibitors. New complex oligosaccharides of microbial origin. Naturwissenschaften 1977;64535-6.

1 I Hillebrand I, Boehme K, Frank G, Fink H, Berchthold P. The effects of the a-glucosidase inhibitor BAY G 5421 (acarbose) on meal-stimulated elevations of circulating glucose, insulin and triglyceride levels in man. Res Exp Med 1979;175:81-6.

12 Puls W, Bischoff H, Schutt H. Pharmacology of amylase- and glucosidase-inhibitors. In: Creutzfeldt W, Folsch UR, eds. Delaying Absorption as a Therapeutic Principle in Metabolic Diseases. Stuttgart: Thieme Verlag, 198370-6.

1977;64:536-7.

13 Bischoff H, Puls W, Krause HP, Schutt H, Thomas G. Pharma- cological properties of the novel glucosidase inhibitors RAY M 1099 (miglitol) and BAY 0 1248. Diabetes Res Clin Pract 1985; Suppl 1:S53.

14 Ikeda H, Odaka H, Matsuo T. Effect of a disaccharidase inhibitor, AO-128, on a high sucrose-diet-induced h y p e r g l y b in female Wistar fatty rats. Jap. Pharmacology Therapeutics

IS Lembcke B, Fdsch U, Creutzfeldt W. Effect of I-desoxynojir- imycin derivatives on small intestinal disaccharidase activities and on active transport in vitro. Digestion 1985;31:120-7.

16 RImsch K, Wetzehberger N, Piitter J et uf. Pharmacokinetics and metabolism of the desoxynojirimycin derivadves BAY M 1099 and BAY 0 1248. Diabetes Res Clin Pract 1985; Suppl. 1 :461(A).

17 Bustamante S, Gasparo M, Kendall K e f al. Increased activity of rat intestinal lactase due to increased intake of a-saccharides (starch, sucrose) in isocaloric diets. J Nutrition 1981;111:943- 53.

18 Yamada K, Bustamante S. Koldovsky 0. Time- and dose- dependency of intestinal lactase activity in adult rats on starch intake. Biochem Biophys Acta 1981;676:108-12.

19 Lec S, Bustamante S, Koldovsky 0. The effect of alpha- glucosidase inhibition on intestinal disaccharidase activity in normal and diabetic mice. Metabolism 1983;32:793-9.

20 Thomas G, Keup U, Krause HP, Puls W. Pharmacological studies on acarbose. 11: Antihyperlipaemic effects. In: Creutz- feldt W ed. Proceedings of the First International Symposium on Acarbose. Amsterdam: Excerpta Medica, 1982151-5.

21 Puls W, Keup U, Krause HP ef a/. Pharmacology of Q-

glucosidase inhibitor. In: Creutzfeldt W ed. Front Hormone Res Vol. 7, Basel: S. Karger. 1980:235-47.

22 Brownlee M, Vlassara H, Cerami A. Nonenzymatic glycosvla-

1991 ; 19:445 1-6.

tion and the pathogenesis of diabetic complica%ons. Lnn Iniern Med 1984;101:527-37.

23 Friedman JE, de Vente JE, Peterson RG et al. Altered expression of muscle glucose transporter GLUT4 in diabetic fatty Zucker rats (ZDF/Drt-fa). Am J Physiol 1991;261:E782-8.

24 Sima AAF, Chakrabarti S. Long-term suppression of postpran- dial hyperglycaemia with acarbose retards the development of neuropathies in the BB/W-rat. Diabetologia 1992;35:325-30.

25 Cohen MP. Klepser H, WU W. Effect ofa-glucosidase inhibition on the nonenzymatic glycation of glomerular basement mem- brane. Gen Pharmac 1991;22:515-9.

26 Cohen MP. Klepser H. a-glucosidase inhibition prevents increased collagen fluorescence in experimental diabetes. Gen Pharmac 1991 ;22:607- 10.

27 Lee SM. The effect of chronic a-glucosidase inhibition on diabetic nephropathy in the db/db mouse. Diabetes

28 Cohen AM, Rosenmann E. Acarbose treatment and diabetic nephropathy in the Cohen diabetic rat. Hormon Metab Res

1982;3 k249-54.

1990;22511-5. 29 Zhang WX, Chakrabarti S , Greene DA el al. Diabetic auto-

nomic neuropathy in BB-rats: the effect of ARI-treatment on heart rate variability and vagus nerve structure. Diabetes

30 McEwen TAJ, Sima AAF. Autonomic neuropathy in the BB- rat. Assessment by an improved method for measuring heart rate variability. Diabetes 1987;36:251-5.

1990;39613-8.