AMYLASE FRAGMENTS FOR BLOOD GLUCOSE CONTROL

TECHNICAL FIELD

The present invention relates to the field of means and methods for controlling blood glucose in a subject, in particular with regard to treatment of hyperglycemic conditions, conditions involving hyperinsulinemia and conditions involving impaired insulin sensitivity.

BACKGROUND TO THE INVENTION

Insulin misregulation in adolescence has a considerable impact on metabolic processes in adults. High carbohydrate consumption and as well as high protein diet results especially when combined with physical inactivity in the overproduction of insulin, leading to the development of insulin resistance, undesirable anabolic activity (including weight gain and obesity), metabolic syndrome and ultimately type 2 diabetes. Existing mainstream treatments for type 2 diabetes include exogenous insulin administration, GLP-1 receptor agonists, metformin, sulfonylureas, meglitinides, DPP-4 inhibitors, thiazolidinediones and SGLT2 inhibitors. Other treatments include exercise and dietary management. None of the existing treatments is without drawbacks and the treatments are not fully efficacious in a substantial number of patients.

The role of the exocrine pancreas in food digestion and nutrient breakdown in the intestine is well-studied and recognized. For a long time, the activity of gut pancreatic enzymes have generally been considered merely a source of the postprandial hyperglycemic condition, which is reflected in studies of different inhibitors on its actions (Bays 2004; McDougall and Stewart 2005). However, alternative aspects of pancreatic enzyme impact on glycemic control and insulin regulation seem to have been mostly overlooked. The control of glucose in the blood is believed to be accomplished by the concerted action of endocrine pancreas, liver, kidneys, skeletal muscle and gastrointestinal hormones acting on insulin secretion.

Glucose intolerance associated with a delayed insulin release has been observed during exocrine pancreatic insufficiency (i.e. in a condition characterized by lack on pancreatic enzyme secretion) in humans and pigs (Knop, et al. 2007; Lozinska, et al. 2013). So far, such physiological changes are recognized as a pancreatogenic type of diabetes (3c type) and suggested to occur together with exocrine pancreatic insufficiency or pancreas injury (Ewald and Hardt 2013). In humans, the influence of pancreatic enzymes replacement therapy on glucose

metabolism remains controversial although impaired fasting glucose was found in patients with exocrine and/or endocrine pancreatic insufficiency. On one hand, improved blood glucose control and reduced glycated hemoglobin were reported in one study (Knop, et al. 2007; Mohan, et al. 1998), where on the other hand other studies found no effect (Ewald, et al. 2007; Glasbrenner, et al. 1990; O'Keefe, et al. 2001).

By using a young pigs with induced exocrine pancreatic insufficiency, the inventors found in a previous study that pancreatic enzymes present in the gut lumen improved direct blood glucose utilization (without reinforcing insulin release) and improved growth performance of the pigs (Lozinska, et al. 2013). Furthermore, utility of amylase in the treatment of diabetes has been mentioned in patent literature in WO2006136161 and GB2468629.

An object of the present invention is to provide improved methods and means for blood glucose control, in particular with regard to insulin sensitivity. Another object of the present invention is to provide improved methods and means for the treatment of hyperglycemic conditions, conditions involving hyperinsulinemia and for conditions involving impaired insulin sensitivity.

DEFINITIONS

The term amylase refers to an enzyme having alpha-amylase activity (EC 3.2.1.1).

The term treatment in the present context refers to treatments resulting in a beneficial effect on a subject afflicted with the condition to be treated, including any degree of alleviation, including minor alleviation, substantial alleviation, major alleviation as well as cure. Preferably, the degree of alleviation is at least a minor alleviation.

The term prevention in the present context refers to preventive measures resulting in any degree of reduction in the likelihood of developing the condition to be prevented, including a minor, substantial or major reduction in likelihood of developing the condition as well as total prevention. Preferably, the degree of likelihood reduction is at least a minor reduction.

The term sequence identity expressed in percentage is defined as the value determined by comparing two optimally aligned sequences over a comparison window, wherein a portion of the sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Unless indicated otherwise, the comparison window is the entire length of the sequence being referred to. I n this context, optimal alignment is the alignment produced by the BLASTP algorithm as implemented online by the US National Center for Biotechnology I nformation (see The NCBI Handbook [I nternet], Chapter 16), with the following input parameters: Word length=3, Matrix=BLOSUM62, Gap cost=ll, Gap extension cost=l.

Any GenBank accession numbers cited herein refer to entries in the most recent version of said database at the date of filing of the present application.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 illustrates effect of amylase and amylase fragments on intravenous glucose tolerance test (IVGTT) glucose clearance. Fragmented amylase is clearly more efficacious than intact amylase.

Figure 2 illustrates effect of amylase and amylase fragments on insulin secretion in IVGTT. Fragmented amylase is clearly more efficacious than intact amylase.

Figure 3 illustrates effect of amylase and amylase fragments on C-peptide in IVGTT.

Fragmented amylase is clearly more efficacious than intact amylase.

I n the figures and corresponding Tables 1-3, mean values with a letter (a, b, c) are statistically significantly different from values in other groups not denoted with the same letter (P <0.05).

SUMMARY OF THE INVENTION

The present invention relates to the following items. The subject matter disclosed in the items below should be regarded disclosed in the same manner as if the subject matter were disclosed in patent claims.

1. A composition comprising one or more effective fragments of an amylase,

wherein the one or more fragments have a greater biological effect in increasing glucose clearance in a mammal after oral administration, compared to intact amylase from which the one or more fragments are derived,

for use as a medicament or a dietary supplement.

The composition according to item 1, for use as a medicament.

The composition according to item 1, for use as a dietary supplement.

The composition according to any of the preceding items, wherein the composition comprises an effective fragmented amylase.

The composition according to any of the preceding items, wherein the composition comprises an effective protease-fragmented amylase.

The composition according to any of the preceding items, wherein the composition comprises an effective trypsin-digested amylase.

The composition according to any of the preceding items, wherein the amylase is selected from a fungal amylase, a mammalian amylase, a vertebrate amylase, an invertebrate amylase, a bacterial amylase or a plant amylase.

The composition according to any of the preceding items, wherein the amylase is selected from a human amylase, a porcine amylase, a fungal amylase, an Aspergillus amylase, preferably an Aspergillus oryzae-a mylase, more preferably a human amylase, most preferably a human salivary amylase.

The composition according to any of the preceding items, wherein the composition comprises an Aspergillus oryzae-a mylase digested with a porcine trypsin.

The composition according to any of the preceding items, wherein the composition comprises an Aspergillus oryzae-a mylase according to SEQ ID NO: 1 digested with a porcine trypsin.

The composition according to any of the preceding items, wherein the composition comprises trypsin-digested human amylase, preferably human salivary amylase. The composition according to any of the preceding items, wherein the composition comprises human amylase, preferably human salivary amylase, digested with porcine trypsin.

The composition according to any of the preceding items, wherein the composition comprises trypsin-digested porcine amylase.

The composition according to any of the preceding items, wherein the composition comprises porcine amylase digested with porcine trypsin. 15. The composition according to any of the preceding items, wherein the composition comprises one or more of the peptides according to any of SEQ ID NOs: 2-29.

16. The composition according to any of the preceding items, wherein the composition further comprises a protease.

17. The composition according to any of the preceding items, wherein the composition further comprises a trypsin, preferably a porcine or human trypsin.

18. The composition according to any of the preceding items, wherein the composition is pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

19. The composition according to any of the preceding items, wherein the composition is included in a medical food further comprising a calorie source.

20. The composition according to any of the preceding items, wherein the composition is formulated for release in the duodenum.

21. The composition according to any of the preceding items, for use in the treatment of a condition selected from the list consisting of

hyperglycemic conditions, conditions involving hyperinsulinemia and

conditions involving impaired insulin sensitivity.

22. The composition according to item 21, wherein the condition is selected from the list consisting of type 1 diabetes, type 2 diabetes, type 3 diabetes, metabolic syndrome, prediabetes, obesity, Alzheimer's disease, Cushing's syndrome.

DETAILED DESCRIPTION

The inventors have studied the role of digestive enzymes, produced by the exocrine pancreas, in blood glucose regulation. The inventors have surprisingly found that one or more fragments of amylase (e.g. amylase digested with a proteolytic enzyme such as trypsin) are significantly more effective in regulating blood glucose than the amylase from which the one or more fragments are derived from in its intact (non-fragmented) state (See Examples 1 and 2).

Effective fragments of amylase

Thus, in a first aspect of the present invention, there is provided a composition comprising one or more effective fragments of an amylase, for use as a medicament or a dietary supplement. The first aspect also includes provision of a method of treatment comprising administering one or more effective fragments of an amylase as a medicament or a dietary supplement to a subject in need thereof. Furthermore, the first aspect includes provision of a use of one or more effective fragments of an amylase in the manufacture of a medicament or a dietary supplement. Preferably, the composition is for use as a medicament.

By effective in this context is meant that the one or more fragments have a greater biological effect in increasing glucose clearance in a mammal after oral administration, compared to intact amylase from which the one or more fragments are derived. Preferably, the term "effective" in this context also entails that the one or more fragments have a greater biological effect in increasing insulin sensitivity in a mammal after oral

administration, compared to intact amylase from which the one or more fragments are derived. The comparison is preferably made at an equimolar basis, e.g. one nmol of the one or more fragments is compared to one nmol of an intact amylase from which the one or more fragments are derived. Needless to say, the composition contains an effective amount of the effective amylase fragments.

Amylase from which the one or more fragments are derived refers to an amylase comprising the corresponding primary structure(s) as the primary structure of the one or more effective fragments. The one or more fragments and the intact amylase may or may not share a common origin or method of manufacture.

By greater biological effect is meant that the difference can be detected with generally accepted statistical significance (e.g. p<0.05). The amplitude of the difference may be that the biological effect of the one or more effective fragments is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 120%, 150%, 200%, or 300% higher than that of the intact amylase.

The one or more fragments are preferably regarded as effective when they have a statistically significantly greater effect on glucose clearance in an intravenous glucose tolerance test compared to intact amylase from which the one or more fragments are derived, wherein the one or more fragments are administered orally lh before the glucose injection. Structural features of effective fragments

The one or more fragments of an amylase may be derived from a non-recombinant amylase (or a part thereof) subjected to a fragmentation treatment, a recombinant amylase (or a part thereof) subjected to a fragmentation treatment or may be prepared recombinantly or via chemical synthesis as ready-made fragments.

The composition may comprise an effective protease-fragmented amylase.

Preferably, the composition comprises an effective trypsin-digested amylase.

Preferably, the composition comprises an effective fragmented amylase, i.e. the complete set of fragmentation products of an amylase subjected to a fragmentation treatment.

The composition may comprise one or more fragments of an Aspergillus oryzae-a mylase digested with porcine trypsin, preferably the entire collection of fragments resulting from porcine trypsin digestion of an Aspergillus oryzoe-amylase.

Preferably, the Aspergillus oryzoe-amylase comprises the sequence according to GenBank accession no. XP_003189619 (SEQ ID NO:l).

The composition may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 of the peptides according to SEQ ID NOs: 2-29 which represent a computer simulation of trypsin digestion of an Aspergillus oryzoe-amylase of XP_003189619. Preferably, the composition comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 of the peptides according to SEQ ID Nos: 2- 29 which are at least 5, 6, 7, 8, 9, 10 or 12 amino-acids long.

Table 7: Peptides resulting from simulation of trypsin digestion of an Aspergillus oryzae- amylase of XP_003189619

SEQ Position Resulting peptide sequence Peptide Peptide of

length mass cleavage

site [aa] [Da]

2 28 MMVAWWSLFLYGLQVAAPALAATPADWR 28 3136.722

3 39 SQSIYFLLTDR 11 1342.514

42 FAR 3 392.458 56 TDGSTTATCN ADR 14 1413.436

57 K 1 146.189 69 YCGGTWQGI IDK 12 1340.516

LDYIQGMGFTAIWITPVTAQLPQTTAYGDAYHGYWQQ 53 6019.605 DIYSLNENYGTADDLK 131 ALSSALHER 983.092 201 GMYLMVDVVA HMGYDGAGSSVDYSVFKPFSSQDYFHP 7906.705

FCLIQNYEDQTQVEDCWLGDNTVSLPDLDT K 205 DVVK 459.543 225 NEWYDWVGSLVSNYSIDGLR 20 2373.563 230 IDTVK 574.675 234 HVQK 510.594 242 DFWPGYNK 1026.116 284 AAGVYCIGEVLDGDPAYTCPYQNVMDGVLNYPIYYPLLNAFK 42 4632.297 301 STSGSMDDLYNMINTVK 17 1876.083 321 SDCPDSTLLG FVENHDNPR 20 2217.351 333 FASYTNDIALAK 1313.473 365 NVAAFIILNDGIPIIYAGQEQHYAGGNDPA R 3412.766 381 EATWLSGYPTDSELYK 16 1860.007 390 LIASANAIR 9 928.099 396 NYAISK 6 694.786 404 DTGFVTYK 8 930.025 410 NWPIYK 6 819.958 418 DDTTIAMR 8 922.021 419 K 146.189 433 GTDGSQIVTILSNK 1432.594

26 482 GASGDSYTLSLSGAGYTAGQQLTEVIGCTTVTVGSDGN 4714.210

VPVPMAGGLPR

27 489 VLYPTEK 848.995

28 494 VLYPTEK 474.558

29 499 ICSSS 495.548

Fragmentation treatments

The fragmentation treatment discussed above may be a chemical treatment or an enzymatic treatment. Preferably the fragmentation treatment is a treatment with a protease enzyme. The protease enzyme may a trypsin or an enzyme with a similar activity, however a large number of different proteases and various chemical treatments also can be used to generate effective fragments. Any of the proteases and chemical treatments listed below in Table 4 may be useful in generating effective fragments. For further information on fragmentation treatments resulting in specific fragmentation, the skilled reader is directed to the ExPASy website "PeptideCutter" (http://web.expasy.org/peptide_cutter/)

The listed enzymes and treatments in Table 4 have the highest likelihood of cleaving a polypeptide at sites where sequences fulfilling the specified rules for cleavage occur. The cleavage site is indicated by listing the surrounding residues as follows:

N-terminal side— P4-P3-P2-P1-P1'-P2'— C-terminal side

Cleavage occurs between PI and PI'. Where the residue is indicated with a hyphen ("-"), the residue may be any residue.

Table 4: Various enzymes and chemical treatments and their cleavage rules

Enzyme/treatment P4 P3 P2 PI PI' P2'

Arg-C proteinase - - - R - -

Asp-N D

endopeptidase BNPS-Skatole - - - W - -

Caspase 1 F, W, Y, or L H, D not P, E,

A D,Q, K or or R T

Caspase 2 D V A D not P, E,

D,Q, K or R

Caspase 3 D M Q D not P, E,

D,Q, K or R

Caspase 4 L E V D not P, E,

D,Q, K or R

Caspase 5 Lor W E H D - -

Caspase 6 V E H D not P, E, orl D,Q, K or R

Caspase 7 D E V D not P, E,

D,Q, K or R

Caspase 8 1 orL E T D not P, E,

D,Q, K or R

Caspase 9 L E H D - -

Caspase 10 1 E A D - -

Chymotrypsin-high - - - ForY not P - specificity (C-term to w not M or [FYW], not before P) P

Chymotrypsin-low - - - F,L or Y not P - specificity (C-term to

W not M or [FYWML], not before

P

P)

M not P or

Y

H not

D,M,P or W

Clostripain R

(Clostridiopeptidase

B)

CNBr - - - M - -

Enterokinase D or E D or E D K

or

E

Factor Xa A,F,G,I,L,T,V D or E G R

or M

Formic acid - - - D - -

Glutamyl E

endopeptidase

GranzymeB 1 E P D - -

Hydroxylamine - - - N G - lodosobenzoic acid - - - W - -

LysC - - - K - -

Neutrophil elastase - - - A or V - - NTCB (2-nitro-5- C thiocyanobenzoic

acid)

Pepsin (pH1.3) not H,K, or R not not R ForL not

P P not H,K, or R not ForL not

P P

Pepsin (pH>2) not H,K or R not not R F,L,Wor not

P Y P not H,K or R not F,L,WorY not

P P

Proline- H,K P not P endopeptidase or

R

Proteinase K A,E,F,I,L,T,V,W

orY

Staphylococcal not E

peptidase 1 E

Thermolysin not D or E A,F,I,L,M

orV

Thrombin - - G R G -

A,F,G,I,L,T,V A,F,G,I,L,T,V,W P R not D or not or M or A E DE

Trypsin (please note - - - KorR not P - the exceptions in

- - W K P - Table 5)

- - M R P - The exception rules:

The above cleavage rules do not apply, i.e. no cleavage occurs, with the following compositions of the cleavage sites:

Table 5: Exception rules for trypsin

Preferably, a protease enzyme in a fragmentation treatment is trypsin or an enzyme with similar activity, most preferably porcine trypsin.

Amylase

The amylase of the first aspect of the invention may be selected from a fungal amylase (such as an Aspergillus amylase), a mammalian amylase, a vertebrate amylase, an invertebrate amylase, a bacterial amylase or a plant amylase.

I n many organisms the amylase enzyme may exist in several isoforms or homologues. For instance, in most mammals the amylase excreted in saliva is not identical (albeit highly similar) as the enzyme excreted by the pancreas. Thus, said mammalian amylase may be a salivary amylase or a pancreatic amylase, preferably a salivary amylase. The fungus

Aspergillus oryzae in turn has three highly similar but not identical amylases.

The amylase may preferably be an Aspergillus oryzoe-a mylase (such as GenBank accession no XP_003189619), more preferably a porcine amylase (such as GenBank accession no NP_999360, SEQ I D NO: 30), and most preferably human salivary a-amylase 1 (such as GenBank accession no NP_001008219, SEQ I D NO: 31), or human pancreatic a-amylase (such as GenBank accession no NP_000690, SEQ ID NO: 34).

The amylase preferably has a degree of sequence identity (see Definitions) with a human amylase (preferably according to GenBank accession no NP_001008219, SEQ I D NO: 31). The sequence identity may be at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100%.

As shown in Table 6, such amylases include porcine amylase (GenBank accession no NP_999360) with 86% identity, a Vulconisoeto distributo DSM14429 amylase (GenBank accession no ADN51908, SEQ ID NO: 32) with 67% identity, a Desulfurococcus fermentans DSM16532 amylase (GenBank accession no AFL66315, SEQ ID NO: 33) with 50% identity and an Aspergillus oryzae amylase (such as GenBank accession no XP_003189619, SEQ ID NO: 1) with 28% identity.

Table 6: Exemplary sequences and their sequence identities with human amylase

Description Max Total Query E Ident Accession no. SEQ ID NO score score cover value alpha-amylase 1063 1063 100% 0 100% BAA14130.1 31

[Homo sapiens] alpha-amylase A 59.7 108 37% 8.00E- 28% XP_003189619.1 1 type-1/2 14

[Aspergillus

oryzae RIB40] pancreatic 939 939 100% 0 86% NP_999360.1 30 alpha-amylase

precursor [Sus

scrofa]

Alpha-amylase 16.2 62.7 21% 2.8 67% ADN51908.1 32

[Vulcanisaeta

distributa DSM

14429]

Alpha-amylase 15 15 1% 7.4 50% AFL66315.1 33

[Desulfurococcus

fermentans DSM

16532] Composition

The composition may further comprise a protease, preferably a trypsin, more preferably porcine or human trypsin.

The composition may have any formulation, and can also be included in foodstuffs. The composition may be a pharmaceutical composition further comprising a pharmaceutically acceptable carrier. The composition may be a dietary supplement or a nutraceutical. The composition may further comprise a calorie source and may be a medical food, a functional food or a part thereof. The calorie source may comprise a protein, a carbohydrate and/or a lipid. The calorie source may contain at least 20 kcal, preferably at least 50 kcal, most preferably at least 100 kcal, per serving.

The composition may be formulated as a powder, a tablet, a capsule, a liquid, as a part of a liquid food item, or as a part of a solid food item.

The composition may be as an enterically coated formulation. Preferably, the composition is formulated for release in the duodenum, to avoid exposing the amylase fragments to conditions in the stomach that might degrade efficacy. Suitable delayed-release

formulations of the kind are well known in the art for instance from US 8562981 B2 or EP 0261213 Bl.

Conditions for treatment and prevention

Since the composition of the present invention increases the capacity for glucose clearance (see Example 1), with apparently lower insulin release needed to achieve the glucose clearance (see Example 2), the composition of the first aspect is useful for treatment for a number of pathological conditions.

Thus, the composition according to the first aspect may be for use in the treatment or prevention of a condition selected from the list consisting of hyperglycemic conditions, conditions involving hyperinsulinemia and conditions involving impaired insulin sensitivity. The subject in the method of treatment of the first aspect may be in need of treatment of a condition selected from the list consisting of hyperglycemic conditions, conditions involving hyperinsulinemia and conditions involving impaired insulin sensitivity. The medicament manufactured according to the first aspect may be for the treatment of a condition selected from the list consisting of hyperglycemic conditions, conditions involving hyperinsulinemia and conditions involving impaired insulin sensitivity.

According to a Mayo Clinic Staff Opinion: "Diabetes and Alzheimer's disease (AD) are connected in ways that aren't yet fully understood. While not all research confirms the connection, many studies suggest people with diabetes, especially type 2 diabetes, are at higher risk of eventually developing Alzheimer's dementia or other dementias"

(http://www,mavocijnic.orR/djseases-conditjons/aizhejmers-dj sease/jn-depth/cliabetes- a nd-a izhei mers/a rt-20046987).

The study of Auriel et al. (Auriel A. Willette, Barbara B. Bendlin, Erika J. Starks, Alex C.

Birdsill, Sterling C. Johnson, Bradley T. Christian, Ozioma C. Okonkwo, Asenath La Rue, Bruce P. Hermann, Rebecca L. Koscik, Erin M. Jonaitis, Mark A. Sager, Sanjay Asthana. Association of Insulin Resistance With Cerebral Glucose Uptake in Late Middle-Aged Adults at Risk for Alzheimer Disease. JAMA Neurology, 2015; DOI : 10.1001/iamaneuroL2015.0613) are the first connecting insulin resistance of people of average age 60, identify a decreased blood sugar use because of insulin resistance related to Alzheimer's and link that to memory decline.

"The link between insulin resistance and Alzheimer's disease is important for prevention and for treatment of AD as well. Problems regulating blood sugar may impact cognitive function at any age. For Alzheimer's, it's not just people with Type 2 diabetes. Even people with mild or moderate insulin resistance who don't have Type 2 diabetes might have an increased risk for Alzheimer's disease because they're showing many of the same sorts of brain and memory relationships" (Auriel et al. supra).

In conclusion, the improved insulin sensitivity achieved by fragmented amylase as shown herein provides a novel therapeutic approach for the treatment of Alzheimer's disease.

Thus, the above-mentioned condition to be prevented or treated may be a condition selected from the list consisting of type 1 diabetes, type 2 diabetes, type 3 diabetes, metabolic syndrome, prediabetes, obesity, Alzheimer's disease, Cushing's syndrome.

The term "comprising" is to be interpreted as including, but not being limited to. All references are hereby incorporated by reference. The arrangement of the present disclosure into sections with headings and subheadings is merely to improve legibility and is not to be interpreted limiting in any way, in particular, the division does not in any way preclude or limit combining features under different headings and subheadings with each other.

EXAMPLES

The following examples are not to be regarded as limiting. For further information on the experimental details, the skilled reader is directed to a separate section titled Materials and Methods.

Example 1: Effect of different supplements on glucose utilization during IVGTT

Intravenous glucose tolerance test (IVGTT) was chosen as convenient and appropriate for testing glucose-stimulated insulin response and capacity for blood glucose clearance. The main advantages of IVGTT are that it can reveal the loss of beta-cell glucose sensitivity and responsiveness at an early stage of metabolic syndrome and type 2 diabetes development by detecting slower blood glucose utilization. Young healthy pigs were used as the experimental model because of their physiological similarity to humans, good reproducibility, and ability to be trained, which decreases stress under experimental conditions.

In general, glucose utilization during IVGTT had a similar pattern during all experimental days and after 30 minutes blood glucose level was reverted to the initial values (Table 1, Fig. 1; mean values with a letter (a, b, c) are statistically significantly different from values in other groups not denoted with the same letter (P <0.05)).

Oral administration of the amylase or amylase fragments (termed amylase-frag herein) lh prior to IVGTT had a significant lowering effect on blood glucose clearance as compared to vehiculum (Control 1 Group) or vehiculum + trypsin (Control 2 Group) loadings (Table 1, Fig. 1).

Oral feeding with amylase or amylase-frag affected blood glucose clearance, amylase caused lower blood glucose level at 5 minutes, compared to control values (p<0.001). However, amylase-frag exhibit significantly more potent lowering effect since postloading values at 5 and 15 for glucose were significantly lower for amylase-frag as compared to amylase alone and to controls (p<0.001). AUCgiucose is informative of the sum of glucose utilization during IVGTT in pigs after the administration of different treatments (Table 1, Fig. 1). There was a significant influence on the total blood glucose utilization of both amylase and amylase-frag, but the effect of amylase-frag was significantly greater than that of amylase.

Example 2: Effect of different supplements on insulin / C-peptide response during IVGTT

The peak of insulin response due to glucose infusion was detected at 5 and 15 minutes, and after 30 minutes returned to the initial level (Table 2, Fig 2). Amylase and amylase-frag oral administration caused lower plasma insulin level at 5 and 15 minutes, following glucose injection, as compared to controls values (p<0.001). Amylase-frag loading had a significantly greater lowering effect as compared to controls and pure amylase loading.

Additional analysis of C-peptide was performed to verify glucose-stimulated insulin response in the pig's plasma after amylase and amylase-frag supplementation. The plasma C-peptide level after oral administration of amylase and amylase-frag was still significantly lower than in the control samples at 5 and 15 minutes (p<0.001), however amylase-frag administration induced the most potent and significantly stronger effect on C-peptide (Table 3, Fig 3).

Total detected insulin and C-peptide response during IVGTT reflected in AUCinsuiin amylase- frag feeding caused significantly lower AUCinsuiin compared to controls values as well as to amylase infusion (p<0.05) (Table 3, Fig 3).

The inventors concluded that oral administration of amylase and in particular its fragments facilitated glucose clearance despite lower insulin release in response to intravenous glucose injection when compared to control feeding.

On the background with immutable glucose utilization dynamics the experiments show that such supplementation facilitates/promotes cell glucose uptake. Insulin sensitivity index S2 was also higher for samples, taken after amylase supplementation, compared to control feeding. S2 was proposed to be relevant for pigs as an accurate representative insulin sensitivity index based on IVGTT. This data indicates that the administration of pancreatic- like amylase but most probably it fragments requires less insulin release and perhaps limit glucose uptake. Table 1. Glucose concentration in blood, mmol/L

Table 2. Insulin concentration in blood, pmol/L insulin,

Insulin concentration in t i!ood, pmo!/L pmol/L*120 min

Area under the

Time, min curve (AUC)

(-60) 0 5 15 30 45 60 75 90 105 120

0.15 0.12 37.56 39.79 2.07 1.26 0.05 0.04 0.04 0.03 0.01

Control 1 ±0.01 ±0.02a ±2.05 a ±3.02 a ±0.11 ±0.05 ±0.01 ±0.01 ±0.01 ±0.03 ±0.01 885.93+34.09 a

0.14 0.15 32.38 34.08 1.98 1.30 0.04 0.02 0.03 0.04 0.04

Control 2 ±0.02 ±0.01a ±3.433 ±5.553 ±0.08 ±0.23 ±0.02 ±0.01 ±0.03 ±0.01 ±0.03 835.56±23.76 a

0.15 0.07 17.67 22.34 2.01 1.25 0.06 0.04 0.05 0.06 0.03

Amylase ±0.01 +0.03b +2.01 b ±2.34 b ±0.14 ±0.16 ±0.01 ±0.02 +0.02 +0.04 +0.02 584.91+11.12 b

Amylase 0.15 0.05 10.09 12.04 2.03 1.20 0.03 0.04 0.02 0.02 0.03

fragments ±0.02 ±0.01b ±1.76 c ±3.13 c ±0.02 ±0.21 ±0.01 +0.01 ±0.01 ±0.01 ±0.01 404.67±25.07 c

Table 3. C-peptide concentration in blood, pmol/L

MATERIALS AND METHODS

Animals

The study was approved by the Malmo/Lund Ethical Committee on Animal Experiments (Sweden). The experiments were performed on crossbred ((Yorkshire ^χ Swedish Landrace) ^χ Hampshire) pigs, obtained from Odarslov research farm, belonging to the Swedish

University of Agricultural Sciences (Alnarp, Sweden). All pigs were housed individually in pens in the same stable at 20 ± 2°C and with light on from 7.00-19.00. The pigs had free access to water via a drinking nipple and were offered a commercial pelleted feed (Vaxtill 320, Lantmannen, Sweden), 4% of their body weight per day (at 11.00-12.00). Pigs were socialized to prevent stress during manipulation and the experiments.

At the age of 7 weeks, 5 weaned pigs (9.6 ± 0.7 kg) were selected for experiment and a catheter was placed in the anterior vena cava, via the external jugular vein, for blood sampling performed during the experiments.

Experiments and design

Amylase enzyme (Sigma-Aldrich: Amylase A9857 from Aspergillus oryzae; was tested in as intact enzyme and after fragmentation (with trypsin) as oral supplements. The dose of the investigated microbial enzyme were correlated to the 4 pancrelipase capsules (Creon ^® 10,000, Abbott Healthcare Products Ltd, Southampton, United Kingdom) (Fedkiv, et al. 2009; Rengman, et al. 2009) and based on the corresponding amylolytic activity. Amylase activity was analysed using ethylidene-pNP-G7 (4,6-ethylidene-p-nitrophenyl-alpha, D- maltoheptaoside) as the substrate, according to the manufacturer's instruction (Infinity Amylase Liquid Stable Reagent; Thermo Electron, Victoria, Australia). The specific activity in 1 mg of individual enzymes were measured at 37^C and compared with the activity of 1 pancrelipase capsule and the appropriate amount (in milligrams) of the investigated enzymes matching corresponding enzymatic activities in 4 capsules of commercial pancrelipase were used as a single dose.

Each portion of the amylase powder were dissolved in 30 ml of water and sweetened with 3 tablets of Hermesetas mini sweeteners (Valora Trade Denmark Food, Herlev, Denmark) as vehiculum. Amylase fragments (also termed Amylase-frag herein) were obtained after adding to amylase solution of 30 ml 2 ml of trypsin (NovoNordisk, Denmark) solution lmg/ml. The solution was placed on water batch 37 C and stirred for 15 minutes. Amylase and amylase-frag solutions was loaded orally through the syringe within 2 minutes and immediately after that pig was watered with another 40ml of tap water to rinse mouth and throat from enzymes. As a control vehiculum (30 ml of water with 3 tablets of sweetener + trypsin 2 mg) was tested by itself without amylase.

Intravenous glucose tolerance test (IVGTT)

The intravenous glucose tolerance test (IVGTT) was performed on pigs that had fasted overnight for about 20 hours, and 1 hour prior to glucose injection, the oral administration of the: 1/ Amylase or 2/ Amylase-Frag, or 3/ vehiculum (Control 1 Group) or 4/ vehiculum + trypsin (Control 2 Group) were performed. Glucose loading was carried out with a sterile 50% D-glucose solution (0.5 g/kg body weight) infused via the venous catheter during 30 seconds. Immediately after infusion of glucose, the catheter was flushed with 5ml of 0,9% sterile saline solution. Blood samples for the glucose tolerance test were drawn at -1 (presented as time point 0'), 5, 15, 30, 45, 60, 120 minutes after infusion. Blood was collected into 5 ml syringes containing EDTA (0.20 mg) and a protease inhibitor, aprotinin (1000 klU, Bayer, Leverkusen, Germany), as described previously (Rantzer, et al. 1995). The blood samples were immediately cooled on ice and then centrifuged at 3.000 ^χ g for 15 min at 4°C. Plasma was separated and stored at -20°C until analyses.

Analysis of blood glucose and plasma insulin and C-peptide

Glucose was measured in the fresh blood samples using a glucose-meter with test strips (Accu-Chek ^s Aviva, Roche Diagnostics, Mannheim, Germany). Plasma insulin and C-peptide levels were measured using porcine insulin or C-peptide ELISA kits, respectively (Mercodia, Uppsala, Sweden), according to the manufacturer's protocols with a lower detection limit of 0.2 pmol/L for insulin and 2 pmol/L for C-peptide.

Statistical analysis and calculations

The incremental area under the curve (AUC) was calculated for the post-load glucose, insulin and C-peptide levels using the trapezoidal rule.

Insulin sensitivity was calculated as a surrogate index S2, using the classic glucose disappearance rate (k) related to released insulin concentration during 0-30minutes, 30*k/o- 3oAUCinsuiin. The glucose disappearance rate is negative slope of the linear regression of the logarithm of glucose in 25-min interval from 5 to 30 min (k = _Ί2 _ _Ί1 ) · The area under the curve (o-3oAUCinsuiin) of insulin concentration from 0 to 30 min was calculated with a trapezoidal rule. The units of the new index are l/min*pM (Christoffersen, et al. 2009).

Statistical data analyses were done using Prism, version 6 (GraphPad Software, I nc, San Diego, CA, USA). The level of statistical significance between time points was determined using repeated measures two-way Anova with Tukey's multiple comparison test. The differences for the AUC and S2 were determined using two-way Anova with Sidak's multiple comparison test. A P-value of < 0.05 was considered to be statistically significant while a P < 0.1 as a tendency. Mean values presented with a letter (a, b, c) are statistically significantly different from values in other groups not denoted with the same letter (P <0.05).

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