WO2014076270A1 - Soluble collagen vi for use in the treatment of hyperglycemia linked diseases and glucose transport disorders. pharmaceutical composition, method and use of a cell extracellular liquid medium for increasing glucose uptake - Google Patents

Abstract

Soluble collagen VI for use in the treatment of hyperglycemia linked diseases, wherein said disease is selected from the group composed by diabetes mellitus, metabolic syndrome, dysfunction of the thyroid gland, dysfunction of the adrenal gland, dysfunction of the pituitary gland, exocrine pancreatic diseases, acute stress, sepsis, infection with hepatitis C. The present invention also relates to soluble collagen VI for use in the treatment of glucose transport disorders. The present invention also relates to a pharmaceutical composition comprising soluble collagen VI, to the use of a cell extracellular liquid medium comprising soluble collagen VI for increasing glucose uptake in mammalian cells and to a kit and a method for increasing glucose uptake in mammalian cells.

Description

SOLUBLE COLLAGEN VI FOR USE IN THE TREATMENT OF HYPERGLYCEMIA LINKED DISEASES AND GLUCOSE TRANSPORT DISORDERS.

PHARMACEUTICAL COMPOSITION, METHOD AND USE OF A CELL EXTRACELLULAR LIQUID MEDIUM FOR INCREASING GLUCOSE UPTAKE

FIELD OF THE INVENTION

The present invention is related to the use of soluble collagen VI for the preparation of a medicament for the treatment of hyperglycemia linked diseases and glucose transport disorders. It is also related to a cell extracellular medium comprising the protein collagen VI as a method for increasing glucose uptake in mammalian cells.

BACKGROUND ART OF THE INVENTION

Collagen VI is a protein found in many extracellular matrices including that of (striated) muscle, tendon, skin, blood vessels and adipose tissue. Collagen VI is encoded by three separate genes, COL6A1 , COL6A2 and COL6A3. Mutations in any of the three collagen VI genes give raise to a spectrum of neuromuscular phenotypes including Ullrich congenital muscular dystrophy (UCMD), Bethlem myopathy (BM), intermediate collagen Vl-related myopathy and myosclerosis myopathy. Ullrich Congenital Muscular Dystrophy (UCMD) is the second most common form of congenital muscular dystrophy. Patients with UCMD present typically at birth or within the first year of life with hypotonia, delayed motor milestones, proximal muscle weakness, distal joint hyperlaxity and proximal joint contractures. The most severe cases never achieve ambulation. Those patients with UCMD who walk independently typically loose ambulation by the age of 12 years. Respiratory insufficiency invariably develops in UCMD and intermediate collagen VI patients who require night-time noninvasive ventilatory support during the teenage years.

Skeletal muscle and adipose tissue are two of the key tissues responsible for the regulation of glucose and lipid metabolism. Collagen VI is the most abundant collagen type in the extracellular matrix of adipose tissue. It has been shown that expression of collagen VI genes increases during adipogenesis and once the protein is secreted surrounds the fat cell. At the transcriptional level, collagen VI genes expression is regulated by glucose levels and diet. Hyperglycemic glucose concentrations and a diet rich in fatty acids increases COL6 mARN levels whilst PPPAR Y agonists have the opposite effect (Berria, R. et al., 2006, Increased collagen content in insulin-resistant skeletal muscle. Am J Physiol Endocrinol Metab, 290(3):E560-5; Khan, T. et al., 2009, Metabolic dysregulation and adipose tissue fibrosis: role of collagen VI. Mol Cell Biol, 29: 1575-91 ; Muona, P. et al., 1993, Hyperglycemic glucose concentrations up-regulate the expression of type VI collagen in vitro; Relevance to alterations of peripheral nerves in diabetes mellitus. Am J Pathol, 142(5): 1586-97). Moreover, COL6A3 mRNA levels positively correlate with body mass index and visceral adipose tissue mass in humans suggesting a role in adipose tissue turnover. UCMD patients are frequently underweight despite gastrostomy and caloric supplementation.

DESCRIPTION OF THE INVENTION

One aspect of the invention is the use of soluble collagen VI, herewith use of soluble collagen VI of the invention, for the preparation of a medicament for the treatment of hyperglycemia linked diseases.

Collagen VI consists of three a chains, namely a1 (VI), a2 (VI) and α3 (VI), encoded by genes COL6A1 , (database accession number OMIM 120220), COL6A2 (database accession number OMIM 120240) and COL6A3 (database accession number OMIM 120250), respectively. Each α (VI) chain is made up of two large globular domains connected by a short triple-helical domain of Gly-Xaa-Yaa amino acid repeat sequences. Heterotrimeric monomers align to form antiparallel dimers, which in turn associate to form tetramers (dimers and tetramers are stabilised by disulfide bonds occurring between cysteine residues in the triple-helical domains). These tetramers are secreted from the cell and associate in an end-to-end fashion to give rise to the final microfilamentous network.

The term "hyperglycemia linked diseases" of the present application refers to a group of diseases and conditions characterised by a common fact, an impairment of glucose uptake. The impairment of glucose uptake causes hyperglycemia, a prolonged excessive concentration of glucose circulating in the blood plasma (above 140 mg/dL according to the American Diabetes Association).

Chronic hyperglycemia, this is a prolonged excessive amount of glucose circulating in the blood plasma, is associated with an increased risk for a number of serious, sometimes life-threatening complications, such as cardiovascular, nervous system, retina and kidney disease, among others. Hyperglycemia is most commonly caused by diabetes mellitus. Type 1 diabetes mellitus is due to a deficient production of insulin subsequent to autoimmune destruction of the pancreatic beta-cells and type 2 diabetes mellitus (T2DM) is triggered by an impaired response to insulin action (or insulin resistance) and beta-cell dysfunction. T2DM is the most common form of diabetes (accounting for 90% of the 346 million people with diabetes across the world according to the world health organization) and its incidence is especially very high in the aged and obese population. Many other diseases can cause hyperglycemia, including exocrine pancreatic diseases, endocrinopathies and some genetic syndromes such as lipodystrophy (The expert committee on the diagnosis and classification of diabetes mellitus, 2002, Report of the Expert Committee on the Diagnosis and Classification of Diabetes Mellitus. Diabetes care 25, suppl 1 ; Fiorenza CG, Chou SH, Mantzoros CS. (201 1 ) Lipodystrophy: pathophysiology and advances in treatment. Nature Reviews Endocrinology 7(3):137-50.).

Several studies have shown that improvement in glycemic control delays or prevents the onset and advance of some adverse complications. Particularly, the benefits in patients with T2DM with regard to microvascular complication, myocardial infarction and death from any cause are proven (Holman, R. R. et al., 2008, 10-year followup of intensive glucose control in type 2 diabetes. New England Journal of Medicine, 359, 1577-1589.).

Current interventions to improve glycemic control in T2DM are life style modifications, such as diet therapy and increased physical activity, and pharmacological therapies, mainly, metformin, sulfonylurea, insulin and incretins. Some of these pharmacological agents may have side effects, and insulin and sulfonylureas predispose to weight gain and can cause hypoglycemia (Blonde, L, 2009, Current antihyperglycemic treatment strategies for patients with type 2 diabetes mellitus. Cleveland Clinic Journal of Medicine 76 suppl 5:s4-1 1 .).

Moreover, there are hereditary diseases that are caused by a defect in the glucose uptake by tissues. This is the case of the deffect of GLUT1 , which is a congenital error of glucose transport to the brain causing cerebral glucose deficiency. The brain, for its high level of cellular activity, is a major consumer of energy that is obtained, mainly, from glucose. The arrival of glucose to the brain should therefore be regularly maintained and controlled. This is especially important in the case of children, in which the brain is immature and needs glucose to develop normally. GLUT-1 is the main glucose transporter in the blood-brain barrier. GLUT-1 transporter deficiency occurs due to mutations (mostly de novo dominants) in the SLC2A1 gene encoding this protein. The diagnosis is carried out analyzing cerebrospinal fluid (CSF) by lumbar puncture. In patients with GLUT-1 deficit, glucose levels in CSF are low while normal in plasma. The classic form presents with early-onset acute epilepsy with poor response to anti-epilepsy drugs and also retardation in cephalic growth with acquired microcephaly, retarded psychomotor development, ataxia and spasticity. At present there are no curative therapies. So far the best results were obtained with a diet based on increased intake of fats: ketogenic diet.

One embodiment is the use of soluble collagen VI of the invention, wherein said disease is selected from the group composed by diabetes mellitus, metabolic syndrome, dysfunction of the thyroid gland, dysfunction of the adrenal gland, dysfunction of the pituitary gland, exocrine pancreatic diseases, acute stress, sepsis, infection with hepatitis C. Particularly, said acute stress is stroke or myocardial infarction. One embodiment is the use of soluble collagen VI of the invention, wherein said disease is diabetes mellitus type 2.

One embodiment is soluble collagen VI for use in the treatment of glucose transport disorders. Particularly, said glucose transport disorder is caused by a defficiency in the brain of the glucose transporter GLUT 1 .

One embodiment is the use of soluble collagen VI of the invention, wherein said hyperglycemia linked disease is hyperglycemia originated by corticosteroid treatment or anti-HIV treatment with protease inhibitors.

Another aspect of the invention is a pharmaceutical composition, herewith pharmaceutical composition of the invention, comprising soluble collagen VI, together with pharmaceutically acceptable excipients. One embodiment is the pharmaceutical composition of the invention, wherein the administration of said composition is selected from the group composed by parenteral, digestive, respiratory and topical administration. Another aspect of the invention is the use of a cell extracellular liquid medium, herewith use of a cell extracelullar liquid medium of the invention, comprising soluble collagen VI for increasing glucose uptake in mammalian cells.

One embodiment is the use of a cell extracelullar liquid medium of the invention, wherein said mammalian cells are human cells.

One embodiment is the use of a cell extracellular liquid medium of the invention, wherein said human cells are selected from the group composed by skeletal muscle cells, adipose cells and liver cells.

One embodiment is the use of a cell extracelular liquid medium of the invention, wherein said collagen VI concentration is in the range of 0.1 to 30 mg/L.

One embodiment is the use of a cell extracelular liquid medium of the invention, wherein said concentration is in the range of 0.5 to 10 mg/L.

Another aspect of the invention is a kit for the preparation of a cell extracellular liquid medium, herewith kit of the invention, comprising collagen VI and other appropriate reagents.

Another aspect of the invention is a method for increasing glucose uptake in mammalian cells, herewith method of the invention, which comprises:

(a) adding collagen VI to a cell extracellular medium in vitro,

(b) culturing mammalian cells in the cell extracelullar liquid medium comprising collagen VI obtained from step (a).

One embodiment is the method of the invention, characterised in that collagen VI concentration is in the range of 0.1 to 30 mg/L. One embodiment is the method of the invention, wherein said concentration is in the range of 0.5 to 10 mg/L.

The therapeutical use of the invention can also be formulated as soluble collagen VI for use in the treatment of hyperglycemia linked diseases.

This aspect can also be formulated as a method of treating hyperglycemia linked diseases, herewith method of treatment of the invention, comprising administering a therapeutically effective amount of soluble collagen VI to a human subject

The preferred embodiment of the invention is a pharmaceutical composition comprising soluble collagen VI, together with pharmaceutically acceptable excipients.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 Stimulation of 2-deoxyglucose uptake in LHCN-M2 skeletal muscle cells treated during different times with soluble collagen VI. LHCN-M2 cells, 5 days post- differentiation, were incubated without collagen VI, control cells (crol), or with 5 mg/l collagen VI for the indicated times (20, 40 minutes, 2 and 16 hours). Value data are mean ± SEM of at least 2 experiments performed in triplicate and are presented as a percentage of the mean value in control cells. The significance of the differences is with respect to control cells # p < 0.001 .

Figure 2. Stimulation of 2-deoxyglucose uptake in LHCN-M2 skeletal muscle cells treated with increasing concentrations of soluble collagen VI. Differentiated LHCN-M2 cells, 5 days post-differentiation, were pre-incubated without collagen VI, control cells (crol), or with the indicated concentrations of collagen VI for 16 h. Then, cells were further incubated without insulin (white bar) or with 0.1 μΜ insulin (INS) for 20 min (black bars). Value data are mean ± SEM of at least 2 experiments performed in triplicate and are presented as a percentage of the mean value in control cells without insulin. The significance of the differences is: with respect to control cells - INS ^*p< 0.001 and ^**p< 0.0001 and with respect to control cells +INS, #p < 0.05, ##p< 0.001 and ###p<0.0001 . Figure 3. Effect of collagen VI as a surface coating on 2-deoxyglucose uptake in LHCN-M2 skeletal muscle cells. Wells of a 24-multiwell tissue culture plate were coated with 400 μΙ of increasing concentrations of collagen VI as stated. LHCN-M2 cells were grown on these plates and confluent cultures were differentiated for 5 days. Then, cells were incubated without insulin (white bar) or with 0.1 μΜ insulin for 20 min (black bars). Value data are mean ± SEM of 2 experiments performed in quadruplicate and are presented as a percentage of the mean value in control cells without insulin. Figures 4a and 4b. Effect of soluble collagen I and V on 2-deoxyglucose uptake in LHCN-M2 skeletal muscle cells. Differentiated LHCN-M2 cells, 5 days post- differentiation, were pre-incubated without collagen, control cells (crol), or with the indicated concentrations of collagen I (Figure 4a) or V (Figure 4b) for 16 h. Then, cells were further incubated without insulin (white bar) or with 0.1 μΜ insulin (INS) for 20 min (black bars). Value data are mean ± SEM of 2 experiments performed in quadruplicate and are presented as a percentage of the mean value in control cells without insulin. The significance of the differences is: with respect to control cells with insulin #p < 0.05. Figure 5. Effect of soluble collagen I, V and VI on 2-deoxyglucose uptake in C2C12 mouse skeletal muscle cell line. Differentiated C2C12 cells, 5 days post- differentiation, were pre-incubated without collagen, control cells (crol), or with 10 mg/l of the indicated collagens I, V o VI for 16 h. Then cells, were further incubated without insulin (white bar) or with 0.1 μΜ insulin (INS) for 20 min (black bars). Value data are mean ± SEM of 2 experiments performed in quadruplicate and are presented as a percentage of the mean value in control cells without insulin. The significance of the differences is: with respect to control cells without insulin ^*p < 0.001 ; with respect to control cells with insulin #p < 0.05. Figure 6. Effect of soluble collagen I, V and VI on 2-deoxyglucose uptake in L6 rat skeletal muscle cell line. Differentiated L6 cells, 4 days post-differentiation, were pre- incubated without collagen, control cells (crol), or with 10 mg/l of the indicated collagens I, V or VI for 16 h. Cells were further incubated without insulin (white bar) or with 0.1 μΜ insulin (INS) for 20 min (black bars). Value data are mean ± SEM of 2 experiments performed in quadruplicate and are presented as a percentage of the mean value in control cells without insulin. The significance of the differences is: with respect to control cells without insulin ^*p < 0.001 ; with respect to control cells with insulin #p < 0.05. Figure 7. Effect of soluble collagen VI on 2-deoxyglucose uptake in mouse 3T3-L1 adipocytes. Differentiated 3T3-L1 adipocytes, 4 days post-differentiation, were pre- incubated without collagen, control cells (crol), or with 5 mg/ml soluble collagen VI for 16 h. Then, cells were further incubated without insulin (white bar) or with 0.1 μΜ insulin (INS) for 20 min (black bars). Value data are mean ± SEM of 2 experiments performed in quadruplicate and are presented as a percentage of the mean value in control cells without insulin. The significance of the differences are: with respect to control cells -INS ^*p< 0.001 and with respect to control cells +INS #p < 0.001 .

Figure 8. Effect of soluble collagen VI in the phosphorylation of PKB/Akt. Myotubes LHCN-M2, 5 days post-differentiation, were pre-incubated without collagen and without insulin (COL6-/INS-), or with insulin (COL6-/INS+) 0.1 μΜ for 20 min, or with collagen VI (COL6+/INS-) 5 mg/ml for 16 hours, or with collagen VI and insulin (COL6+/INS+). The amount of phosphorylated PKB/Akt in each of the conditions was analyzed by immunoblot using monoclonal antibodies that detects the phosphorylated form of PKB/Akt in Ser743. The band intensity was adjusted in relation to the intensity of the correspondent bands of a control protein, which concentration is not altered (alpha-tubulin). The band intensity was measured by densitometry with the software ImageJ and it is expressed as intensity units. EXAMPLES OF THE INVENTION

Methodological Example 1 . Measurement of 2-deoxy-d-[³H]glucose uptake.

Purified collagens (extracted from human placenta) were purchased from BD Biosciences: Collagen I (ref 354265), Collagen V (ref 354246) and Collagen VI (ref 354261 ). Collagen VI used consists of a chains crt (VI), α2 (VI) and α3 (VI), encoded by genes COL6A1 , COL6A2 and COL6A3, respectively.

Cells (LHCN-M2, C2C12, L6 or 3T3-L1 ) were grown in 24-well tissue culture plates (16 mm each) under the conditions mentioned above. Collagen VI was diluted in 1 M NaCI, 1 .25 mM Tris (pH 8.0) solution and eventually the same volume of this solution was added to control cells. Collagen I and V were diluted in distilled water. Collagens were added to cell culture medium at a dilution ratio 1 :50. Cell monolayers were incubated in PBS, 0.1 mmol/l CaCI(2), and 0.1 % BSA. Insulin was included when stated, and the mixture was incubated for 20 min at 37 °C. 2-Deoxy-d-fH] glucose uptake was then measured by the addition of 0.5 Ci/well and unlabeled 2- deoxyglucose at the concentration of 0.1 mmol/l, followed by three washes in ice-cold PBS after 6 min of exposure. Cells were subsequently lysed with 1 % SDS, and aliquots were measured for radioactivity in 5 ml of the cocktail (Optiphase, LKB).

Example 2. Glucose intolerance and insulin resistance in Ullrich Congenital Muscular Dystrophy (UCMD) patients.

We have conducted a pilot study measuring the insulin response and glucose levels in 5 UCMD patients (using the 2 h glucose tolerance test). In said test, a drink containing from 75 to 100 g glucose is administered and blood is extracted at regular intervals of time up to two hours.

Reference basal and postprandial glucose levels as determined by the American Diabetes Association were used. These are as follows:

- Basal: <100 mg/dL normal, 100-126 mg/dL fasting glucose intolerance, >126 mg/dL diabetes

- Postprandial (2 hours after glucose intake): <140 normal, 140-200 mg/dL intolerance, >200 mg/dL diabetes

We found that glucose levels remained elevated (>140mg/dL) after two hours despite appropriate insulin secretion in three out of the five patients (Table 1 ). This finding indicates that genetically determined collagen deficiency is associated with intolerance to glucose.

Table 1 . Summary of Oral Glucose Tolerance Test in Ullrich Congenital Muscular Dystrophy patients

Age 0 min 30 min 60 min 90 min 120 min

P1 Glucose

15 yrs 91 .8 129.8 138.6 1 17 138.6

(mg/mL) Insulin

6.8 69.6 81 .9 51 .1 121 (mU/L)

Glucose

90 165.6 154.8 145.8 145.8

P2 (mg/mL

8 yrs

Insulin

1 .3 37.8 49.2 64.6 78.2 (mU/L)

Glucose

84.6 106.2 167.4 190.8 190.8

P3 (mg/mL

19 yrs

Insulin

<0.5 1 .3 9.7 17.3 38.8 (mU/L)

Glucose

P4 8 yrs 97.2 160.2 109.8 1 1 1 .6 127.8

(mg/mL

Insulin

8 124 34.2 28.4 90.1 (mU/L)

Glucose

100.8 1 1 1 .6

P4^* (mg/mL

7.5 yrs

Insulin

<0.5 29.1 (mU/L)

^* O^'Sullivan^'s test which is a simplified version of Oral Glucose Tolerance Test, where fasting of the patient is not needed. In this case 50 g glucose was administered and blood levels were measured after two hours. Conclusion: These results provide in vivo evidence that inherited collagen VI deficiency (as in the UCMD patients studied here) is associated with hyperglycemia and glucose intolerance.

Example 3. Soluble collagen VI increases glucose uptake in cultured human skeletal muscle cells.

We treated LHCN-M2 myotubes (human immortalized skeletal muscle cell line, kind gift of Woodring Wright, UT Southwestern, US) with 5 mg/ml of soluble collagen VI (purified from placenta, BD) for increasing periods of time. After 16 hours treatment, glucose uptake increased very significantly (p < 0.001 ) (Fig. 1 ). Treating LHCN-M2 myotubes with increasing concentrations of soluble collagen VI (0.5 mg/L, 1 mg/L, 5 mg/L and 10mg/L) for 16 h resulted in a dose dependent increase in glucose uptake by LHCN-M2 cells (Fig. 2) with and without insulin pre- treatment.

Conclusion: 16 h treatment with soluble collagen VI increases significantly glucose uptake by LHCN-M2 skeletal muscle cells and this effect is dose dependent.

Example 4 (comparative example). Collagen I and collagen V are not able to stimulate glucose uptake in skeletal muscle cells.

We applied either soluble purified human collagen type I or collagen V to LHCN-M2 cells at different doses (0.5mg/L, 1 mg/L, 2.5mg/L and 5mg/L). In either case, we did not observe any dose-response effect on basal or insulin stimulated glucose uptake (Figs. 4a and 4b).

Conclusion: The effect described above is specific for collagen VI and cannot be reproduced using other proteins also abundant in the extracellular matrix (at least from the sources used).

Example 5. Immobilised collagen VI does not affect glucose utilisation in skeletal muscle cells.

When the same source of collagen VI was used as a surface coating and LHCN-M2 cells plated on top of it, grown and differentiated (instead of being added to the medium in a soluble form) we did not observe any effect on basal (data not shown) or in insulin-stimulated glucose uptake (Figure 3) relative to cells grown on plastic.

Conclusion: the positive effect of collagen VI on glucose uptake may be mediated by a soluble fragment derived from the total protein.

Example 6. Collagen VI also induces glucose uptake in murine skeletal muscle cells lines. Similar experiments in C2C12 (mouse skeletal muscle cell line, Fig. 5) and in L6 cells (rat skeletal muscle cell line, Fig. 6) show that human collagen VI does significantly increase glucose transport (with and without insulin pre-treatment) whereas collagen I and V do not (Fig. 6 and 7).

Conclusion: Collagen VI can induce glucose uptake in several skeletal muscle cell lines which lends evidence for its role on regulation of glucose utilisation.

Example 7. Collagen VI induces glucose uptake in adipocytes

Similar experiments were conducted in 3T3-L1 differentiated adipocytes (Fig 7).

Conclusion: Collagen VI does significantly increase glucose uptake in adipocytes. Example 8. The collagen VI treatment reduces the phosphorylation of PKB/Akt molecule in human muscle cells.

The treatment of human myotubes LHCN-M2 with soluble collagen for 16 hours results in a quantitative reduction of the phosphorylated form of kinase protein B, PKB/Akt, in cells LHCN-M2 treated with insulin (Fig. 8).

Conclusion: The 16-hour treatment with soluble collagen VI inhibits the activation of the insulin/Akt route.

Claims

1 . Soluble collagen VI for use in the treatment of hyperglycemia linked diseases.

2. Soluble collagen VI for use according to claim 1 , characterised in that said disease is selected from the group composed by diabetes mellitus, metabolic syndrome, dysfunction of the thyroid gland, dysfunction of the adrenal gland, dysfunction of the pituitary gland, exocrine pancreatic diseases, acute stress, sepsis and infection with hepatitis C.

3. Soluble collagen VI for use according to claim 1 , characterised in that said hyperglycemia linked disease is hyperglycemia originated by corticosteroid treatment or anti-HIV treatment with protease inhibitors.

4. Soluble collagen VI for use according to claim 2, characterised in that said acute stress is stroke or myocardial infarction.

5. Soluble collagen VI for use in the treatment of glucose transport disorders.

6. Soluble collagen VI for use according to claim 5, characterised in that said glucose transport disorder is caused by a defficiency in the brain of the glucose transporter GLUT 1 .

7. A pharmaceutical composition comprising soluble collagen VI, together with pharmaceutically acceptable excipients.

8. The pharmaceutical composition according to claim 7, characterised in that the administration of said composition is selected from the group composed by parenteral, digestive, respiratory and topical administration.

9. Use of a cell extracellular liquid medium comprising soluble collagen VI for increasing glucose uptake in mammalian cells.

10. Use according to claim 9, characterised in that said mammalian cells are human cells.

1 1 . Use according to claim 10, characterised in that said human cells are selected from the group composed by skeletal muscle cells, adipose cells and liver cells.

12. Use according to any of claims 9 to 1 1 , characterised in that collagen VI concentration is in the range of 0.1 to 30 mg/L.

13. Use according to claim 12, characterised in that said concentration is in the range of 0.5 to 10 mg/L.

14. A kit for the preparation of a cell extracellular liquid medium, comprising collagen VI and other appropriate reagents.

15. A method for increasing glucose uptake in mammalian cells, which comprises:

(a) adding collagen VI to a cell extracellular medium in vitro, (b) culturing mammalian cells in the cell extracelullar liquid medium comprising collagen VI obtained from step (a).

16. Method according to claim 15, characterised in that collagen VI concentration is in the range of 0.1 to 30 mg/L.

17. Method according to claim 16, characterised in that said concentration is in the range of 0.5 to 10 mg/L.