C.79-Hepatitis C virus and type 2 diabetes: Molecular homology between HCV E2 envelope and glucagon

C.79-Hepatitis C virus and type 2 diabetes: Molecular homology between HCV E2 envelope and glucagon

Poster39 presented at the Viral Hepatitis Conference, March 25-26 2013, New York Academy of Science, New York, USA

TRAN GMK

31 avenue du bois, 92290Chatenay Malabry, France

Association « Positifs » BP 230,75865 Paris Cedex 18

Background

200 millions people were infected with HCV in the world. Prevalence of type 2 diabetes in chronic HCV ranges 24-50%, a frequency 5 times greater than the rest of hepatic cirrhosis. Prevalence of hepatitis C among diabetes is 5 to 12%, versus 0.1 to 2% in the general population (Mehta SH, 2001). A direct involvement of HCV core has been hypothetized inducing a high level of Tumor Necrosis Factor-alpha and suppression of tyrosine phosphorylation of insulin receptor substrate-1 (Shintani Y, 2004).

We analyse the mechanism of action of HCV in diabetes/insulino-resistence production.

Methods

Basic Local Alignment Search Tool Protein (BLASTP) comparison between human glucagon and HCV proteins [followed by visual examination of 2073 HCV (genotypes 1, 2, 3, 4, 5 and 6) E2 envelopes from the European database of Pr Gilbert Deleage (Lyon) (euhcvdb.ibcp.fr)], and analysis of glucagon structure-function relationship and three–dimensional (3D) structure (Koth CM, 2012).

Results

We found a homology between human glucagon, especially miniglucagon (19-29),

and HCV E2 protein chimera; the glucagon motif H……………RR..DFVQ W LM

was conserved in HCV E2 motif H… … …….RR..DF .QGW.PI,

including the complete receptor binding motif located at the COOH-terminus and containing the residues D15, A19, F22, W25, L26, M27, T29 (M27 replaced by I; and linearly D15 is missing, A19 replaced by V):

Receptor binding residues * * * * * * *

Numbering 15 19 22 25262729

Glucagon 15-DSRRAQDFVQ–W–LMNT-29

HCV E2 chimera 459-CRRVE DFVQGWGL I N-473

HCV E2 variant T-474

17-RR-18 is a protease cleavage site.

The 2 gaps for glycines G (=Gly) are also encountered in a phylogenetically related glucagon in insects, the Adipokinetic Hormone pQVNFTPGWGTG (Gade G, 1997; Tran GMK, 2011), an 11 residues long peptide, which possesses the GWG motif of HCV E2.

Adipokinetic Hormone pQVNFTPGWGT (pQ = pyroGlu)

HCV 2b E2 E- DFRI GWGT

Mini-glucagon (19-29) AQDFVQWLMNT is 1000 fold more powerful than glucagon itself (Dalle S, 2002). It is a very potent (ID50 close to 0.1 pmol/l) inhibitor of insulin release from beta-cell.

The glucagon (1-21) and des-(22-26) glucagon have potencies of <0.0001% and 0.0006% respectively (Frandsen EK, 1985, 1981) pointing to the crucial role of the short 22-FVQWLMNT-29 COOH-terminus.

In fact, the 3D structure of mini-glucagon is an alpha-helix, so in the space glucagon A19 is near F22, and if we consider this spatial glucagon helical A19-F22 proximity, HCV E2 contains an Alanine A464 near F465, matching with the glucagon A19 and F22; HCV contains also an aspartic D461 matching with the glucagon D15:

Spatial 3D structure superimposition

Glucagon numbering 15 19 22

Glucagon DSRRAQDF

HCV E2 D I K A F

HCV E2 numbering 461 464465

HCV genotype classification is based on 5′ untranslated region, core, E1, non structural 5B (ns5B). This classification is not based on E2, where is located the glucagon-like sequence.

Among the 2073 HCV sequences studied, the most homologous HCV E2 to glucagon are some HCV E2 genotype 1b; this is accordance with the results of Knobler H (2000) and Rudoni S (1999) who found an increase of HCV genotype 1b in diabetics. However as glucagon-like E2 is not strictly linked to genotypes, the result of Mason AL (1999), who found an increase of HCV genotype 2a, may be also valid. For example, some genotypes 2a has the sequence EDFRIGWGTLQ which is glucagon-like. Moucari R (2008) in France found an association between insulino-resistance and genotype 4: This agrees rather well with the sequence of genotype 4 EDFPQGWGPLT which contains a proline P 471, instead of Leucine L 471, corresponding to L 26 in glucagon; genotype is also glucagon-like, but to a lesser degree than genotype 1b, thus there is not diabetes, but only insulin-resistance.

Genotype 3 has not been associated with diabetes, but with steatosis (Neuschwander–Tetri BA, 2008). Genotype 3 has a serine S 471 instead of Leucine L 471 and also an aromatic (F, Y) or big (H) residue before F 465, that could induce steric hindrance effect. So genotype 3 does not look like a functional glucagon.

Conclusions

The link between HCV and diabetes is explained by a molecular homology between HCV E2 envelope (mainly of genotype 1b, to a lesser extent genotype 2a and 4), and human glucagon, especially miniglucagon (19-29), which impairs insulin secretion and induces diabetes. In the case of anti-protease side effect (severe anemia) or resistance to interferon-α ribavirine, treatment of HCV by

high doses of intravenous silymarin from Milk Thistle (silybum marianum), (Ferenci P, 2008; Biermer M, 1999), or

oral silybin-phosphatidylcholine [(Siliphos)(Indena Laboratory)],

intravenous alpha-lipoic acid (or ALA, or thioctic acid)(Berkson B, 1999, 1979) [Reed L and Gunsalus I isolated only a few milligrams of ALA from many tons of liver tissue in 1953],

Desmodium Adscendens (Dr Tubery Pierre)(Netlab Pharma) from Cameroon,

can counteract HCV-induced diabetes.

ALA is also efficient in diabetes neuropathy (Golbidi S, 2011; Poh ZX, 2009).

Bibliography

Alpha-lipoic acid (or ALA, or thioctic acid) recommanded by Dr Berkson B were intravenous Thioctacid 600T (AstaMedica) and ALA products by Metabolic Maintenance (www.metabolicmaintenance.com) and Bio Tech Pharmacal (www.bio-tech-pharm.com)

Berkson BM. A conservative triple antioxidant approach to the treatment of hepatitis C. Combination of alpha lipoic acid (thioctic acid), silymarin, and selenium: three case histories. Med Klin (Munich) 1999, 94 suppl 3: 84-9.

Berkson BM. Thioctic acid in treatment of hepatotoxic mushroom (Phalloides) poisoning. N Engl J Med 1979, 300: 371.

Biermer M, Berg T. Rapid suppression of hepatitis C viremia induced by intravenous silibinin plus ribavirin. Gastroenterology 2009, 137: 390-1.

Dalle S, Fontés G, Lajoix AD et al. Miniglucagon (glucagon 19-29): a novel regulator of the pancreatic islet physiology. Diabetes 2002, 51: 406-12.

Desmodium Adscendens (Dr Tubery Pierre)(Netlab Pharma).

Ferenci P, Scherzer TM, Kerschner H et al. Silibinin is a potent antiviral agent in patients with chronic hepatitis C not responding to pegylated interferon/ribavirin therapy. Gastroenterology 2008, 135: 1561-7.

Frandsen EK, Thim L, Moody AJ, Markussen J. Structure-function relationships in glucagon. Re-evaluation of glucagon-(1-21). J Biol Chem 1985, 260: 7581-4.

Frandsen EK, Grønvald FC, Heding LG et al. Glucagon: structure-function relationships investigated by sequence deletions. Hoppe Seylers Z Physiol Chem 1981, 362: 665-77.

Gade G, Hoffmann KH, Spring JH. Hormonal regulation in insects: facts, gaps, and future directions. Physiol Rev 1997, 77: 963-1032.

Golbidi S, Badran M, Laher I. Diabetes and alpha lipoic Acid. Front Pharmacol 2011, 2:69. doi: 10.3389/fphar.2011.00069. Epub 2011 Nov 17.

Knobler H, Schihmanter R, Zifroni A et al. Increased risk of type 2 diabetes in noncirrhotic patients with chronic hepatitis C virus infection. Mayo Clin Proc 2000, 75: 355-9.

Koth CM, Murray JM, Mukund S et al. Molecular basis for negative regulation of the glucagon receptor. Proc Natl Acad Sci U S A 2012, 109: 14393-8.

Mason AL, Lau JY, Hoang N et al. Association of diabetes mellitus and chronic hepatitis C virus infection. Hepatology 1999, 29: 328-33.

Mehta SH, Strathdee SA, Thomas DL. Association between hepatitis C virus infection and diabetes mellitus. Epidemiol Rev 2001, 23: 302-12.

Moucari R, Asselah T, Cazals-Hatem D et al. Insulin resistance in chronic hepatitis C: association with genotypes 1 and 4, serum HCV RNA level, and liver fibrosis. Gastroenterology 2008, 134: 416-23.

Neuschwander–Tetri BA. Hepatitis C Virus-Induced Insulin Resistance: Not All Genotypes Are the Same. Gastroenterology 2008, 134: 619-22.

Poh ZX and Goh KP. A current update on the use of alpha lipoic acid in the management of type 2 diabetes mellitus. Endocr. Metab. Immune Disord. Drug Targets 2009, 9: 392–8.

Reed LJ, Gunsalus IC, Schnakenberg GHF et al. Isolation, Characterization and Structure of α-Lipoic Acid. J Am Chem Soc 1953, 75: 1267–70.

Rudoni S, Petit JM, Bour JB et al. HCV infection and diabetes mellitus: influence of the use of finger stick devices on nosocomial transmission. Diabetes Metab 1999, 25: 502-5.

Siliphos (Indena) www.indena.com

Shintani Y, Fujie H, Miyoshi H et al. Hepatitis C virus infection and diabetes: direct involvement of the virus in the development of insulin resistance. Gastroenterology 2004, 126: 840-8.

Tran GMK, Caprani A, Gerbaud L. Aids lipodystrophy and anti-proteases: Molecular homology of HIV-1 Gag proteolytic site with ADipokinetic Hormone (ADH). Oral presentation at the 13th European Aids Clinical Society (EACS) Conference, Belgrade, Serbia, 13.10.2011. Free on Positifs.org C70

Update (24.3.13)

Whereas there is a consensus on the prevalence of type 2 diabetes among chronic HCV which frequency (24-50%) is admitted,

on the contrary the HBV diabetes/insulino-resistance link is controversial: It is rare [prevalence of insulino-resistance among HBV is 5% in France (Moucari R, 2008)] or less well documented: We found 3 publications from East Europe (HBV genotype D): Poland [in diabetic children (Korczowski R, 1972)], Czechoslovakia [a study on 788 diabetics (Meluzin F, 1974)], North Moravia (Plesník V, 1976). In South Africa (HBV genotype A), Kew MC (1976) found no association between well controlled Diabetes and HBV.

However, a study in Beirut, Lebanon (a Mediterranean country, genotype D), found that 51% moderately controlled diabetics (versus 25% controls) were HBV+ (p less than 0.001); the controls were themselves hospital personnel, with relatively already high risk HBV exposure (Khuri KG, 1985): Thus, among different HBV geographical genotypes, Mediterranean and East European HBV genotype D seemed more diabetogenic, whereas South African genotype A is not.

Classen JB (1996) found a 60% rise in type 1 diabetes after HBV vaccination. Such a causal link was not accepted in France by Thivolet C (1999) on 28 cases. The general opinion is to vaccinate diabetics, in order to protect them against HBV.

We analyse the mechanism of action of HBV Mediterranean and East European genotype D in diabetes/insulino-resistance production. We propose that diabetes different prevalence may be explained by geographical differences between HBV genotypes.

Methods: Basic Local Alignment Search Tool Protein (BLASTP) comparison between human glucagon and HBV surface antigen HBs. Nucleotidic comparison was assessed by BLASTN (N= Nucleotide).

Results:

Glucagon 16-SRRAQDFVQWLM-NT-29

HBV HBs genotype D ayw 179-FVQWLMGLS-187 (chimera of embl:X77308 and gb AAV90939)

Nucleotidic glucagon tttgtgcagtggttgatg

HBV HBs tttgttcagtggttcatg (embl:X77308)

There is 16/18 (88,89%) nucleotide identity.

The glucagon (1-21) and des-(22-26) glucagon have potencies of <0.0001% and 0.0006% respectively (Frandsen EK, 1985, 1981), pointing to the crucial role of 22-FVQWLMNT-29 COOH-terminus.

The most homologous to glucagon are HBV HBs genotype D subtype ayw4 (gb AAV90939, Siberia/Estonia), ayw (embl X77308, Cagliari, Italy, chronicity), ayw3 (gb AY859852, Madrid, Spain), adw2 (hepatoma) and HBV Chinese subtype adrq+ (gb FJ787441, Bejing, China, chronicity). The diabetogenic HBV is also associated with more severe pathologies: Hepatic transplantation (gb AF289998, Australia), HIV-1 infection (gb KC113409, Brazil), adefovir chemoresistance (gb EU 027323, France).

It is noteworthy that China has 200 millions of diabetics and is also a high HBV prevalence country, and that Chinese HBV genotype (gb FJ787441, subtype adrq+) is found here to be glucagon-like.

As negative controls, other HBV genotypes or subtypes were not homologous to glucagon. For example, genotype A [South Africa, negative study of Kew C (1976) concerning the diabetes-HBV link] is not glucagon-like: This is coherent with the postulate that only glucagon-like HBV are diabetogenic genotype/subtypes.

Conclusion (preliminary): As for HCV, some HBV HBs, but not all of them, seem also implicated in insulino-resistance/diabetes by a molecular homology with mini-glucagon, which is 1000 fold more powerful than glucagon itself. These diabetogenic HBV seem to be genotypes D and C, and are restricted to some geographical areas in the world: Mediterranean area (genotype D) and China (genotype C). Genotype D is also present in other areas (India, East and West Europe, although less frequently in USA). Further studies are needed to precisely clarify this point: An analysis of the glucagon-like sequence, in addition to genotyping, in each diabetes-associated HBV would be necessary.

HBV major group a is based on the HBs sequence 124-147, and HBV subtyping is based on HBs residues 122 (y/d), 126 and 160 (w/r) (Servant-Delmas A, 2007), so they are not strictly linked to the HBs glucagon-like region 179-187, which is located downstream.

Implications (preliminary): From our molecular homology, we would perhaps predict the absence [or a low level] of association between Diabetes and HBV in some geographical regions, where only non-diabetogenic (= non glucagon-like) are prevalent, [or associated with diabetogenic glucagon-like HBV]:

1°) no association:

South Africa genotype A (non diabetogenic)

Central Africa genotype E (non diabetogenic, non glucagon-like)

2°) low or variable level of association:

South America, Alaska genotype F non diabetogenic, with rarely genotype D diabetogenic

USA association of 4 genotypes: 2 diabetogenic D and C,

2 non diabetogenic A and B

West Europe association of 2 genotypes: diabetogenic D

and non diabetogenic A

This would be an analysis case by case, for the genotype/subtypes.

Most probably, those with a more severe clinical status/prognosis would be infected by diabetogenic glucagon-like HBV genotype/subtypes: Chronicity, hepatoma, transplantation, adefovir resistance, HIV-1 infection.

The second implication concerns possible side effects of HBV vaccine: It may be possible that in some susceptible individuals, the homology with mini-glucagon would promote the occurence of a diabetes in vaccinated people. Our preliminary data (updated 24.3.13) cannot draw any conclusion for the moment and need further investigation (ongoing) on Recombinax vaccine (subtype adw), Genhevac (subtype ayw) and GenerixB vaccines amino acid sequences.

Bibliography

Classen JB. New Z J Med 1996

Frandsen EK, Thim L, Moody AJ, Markussen J. Structure-function relationships in glucagon. Re-evaluation of glucagon-(1-21). J Biol Chem 1985, 260: 7581-4.

Frandsen EK, Grønvald FC, Heding LG et al. Glucagon: structure-function relationships investigated by sequence deletions. Hoppe Seylers Z Physiol Chem 1981, 362: 665-77.

Kew MC, MacKay ME, Mindel A et al. Prevalence of hepatitis B surface antigen and antibody in white and black patients with diabetes mellitus. J Clin Microbiol 1976, 4: 467–9.

Korczowski R. [Australia antigen in diabetic children]. Polish. Wiad Lek 1972, 25: 1229-31.

Khuri KG, Shamma’a MH, Abourizk N. Hepatitis B virus markers in diabetes mellitus. Diabetes Care. 1985,8: 250-3.

Meluzín F, Kubecová F, Spicka J, Slavík V. [Australia antigen (HBAg) in medical personel and diabetic patients (author’s transl)]. [Article in Czech] Cas Lek Cesk 1974, 113: 1387-9. Cited by Kew MC (1976).

Plesník V, Machálek J, Hejná J. [Finding of hepatitis B antigen (HBsAg) in diabetics in the North Moravian region].[Article in Czech]. Vnitr Lek 1976, 22: 786-91.

Servant-Delmas A, Mercier M, Girault A, Laperche S. Impact clinique, thérapeutique et diagnostique de la diversité génétique du virus de l’hépatite B. Virologie 2007, 11 : 297-307.

Thivolet C, Vialettes B, Boitard C, Bringer J. Absence de preuves pour un lien possible entre vaccination contre l’hépatite virale et survenue d’un diabète de type 1. Diabetes et Metabolisme 1999, 25: 441.

C.79-Hepatitis C virus and type 2 diabetes: Molecular homology between HCV E2 envelope and glucagon

C.79-Hepatitis C virus and type 2 diabetes: Molecular homology between HCV E2 envelope and glucagon

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