Patent Application
20190282630
The content of the electronically submitted sequence listing in ASCII text file (Name: 4139_0020002_Seqlisting_ST25.txt; Size: 6,231 bytes; and Date of Creation: Feb. 25, 2019) is incorporated herein by reference in its entirety.
The present invention relates to the treatment of metabolic disorders, such as, for example, metabolic disorders related to overweight and obesity, such as, for example, Diabetes Mellitus or high cholesterol. The present invention more specifically relates to a composition comprising Akkermansia spp or fragments thereof for treating a metabolic disorder.
Obesity is a worldwide problem, with an estimated number of obese adults of about 250 million. This epidemic of obesity is correlated with a great increase in the prevalence of obesity-related disorders, such as, for example, Diabetes, hypertension, cardiac pathologies and liver diseases. Due to these highly disabling pathologies, obesity is currently considered in western countries as one of the most important public health problem. There is thus a real need of compositions and methods for treating or preventing obesity and/or obesity-related disorders.
Obesity and obesity-related diseases are associated with (i) metabolic dysfunctions (with an impact on glucose homeostasis and lipid metabolism for example); (ii) low grade inflammatory state associated to higher blood lipopolysaccharides (LPS) levels (also referred as metabolic endotoxemia); and (iii) impaired gut barrier function (i.e. increased gut permeability). In order to treat obesity, impact on at least one, preferably 2 and more preferably 3 of these 3 factors is thus needed.
The human gut is colonized by a diverse, complex and dynamic community of microbes representing over 1000 different species, which continuously interact with the host (Zoetendal, Rajilic-Stojanovic and de Vos, Gut 2008, 57: 1605-1615). The homeostasis of the gut microbiota is dependent on host characteristics (age, gender, genetic background . . . ) and environmental conditions (stress, drugs, gastrointestinal surgery, infectious and toxic agents . . . ), but also on the day-to-day dietary changes. Growing evidences support the role of gut microbiota in the development of obesity and related disorders (Delzenne & Cani, Annu. Rev. Nutr. 2011, 31: 15-31).
Therefore, treatment with products that target the gut microbiota appeared as promising therapeutic tools for treating obesity and related disorders. These products may consist of living microbes, such as in the case of most probiotics, or contain dead microbes or fragments thereof. In addition, these products may comprise substrates that are used by the gut microbiota, such as in the case of prebiotics, or contain compounds that change the balance of the intestinal microbiota, such as specific antimicrobial compounds.
For example, WO 2008/076696 describes the gut microbiota as a therapeutic target for treating obesity and related disorders. WO 2008/076696 specifically describes methods for altering the abundance of Bacteroides and/or Firmicutes in the gut of a subject, by administering antibiotics and/or probiotics to the subject.
Moreover, EP 2 030 623 relates to the prevention and/or treatment of metabolic disorders, such as, for example, obesity related disorders, by regulating the amount of Enterobacteria in the gut. EP 2 030 623 discloses reducing the amount of Enterobacteria in the gut by administering probiotic bacteria, such as, for example, Bifidobacterium, Lactococcus, Streptococcus, Enterococcus or Lactobacillus.
Furthermore, the Applicant described that the gut microbiota is modified in prebiotic-treated obese mice (Everard et al., Diabetes, 2011 November; 60(11):2775-86). Moreover, prebiotics (1) improve glucose and lipid metabolisms in obese mice, (2) reduce plasma LPS and improve gut barrier function (e.g. reduction of inflammation) in obese mice, (3) induce an increased enteroendocrine L-cell number in obese mice, and (4) improve leptin sensitivity and glucose homeostasy in diet-induced obese and diabetic mice.
Among the modification induced by prebiotic treatment of obese mice is a considerable alteration of the gut microbiota composition, characterized by (i) a decreased abundance of Bacteroidetes, Lactobacillus spp and bacteria of the Bacteroides-Prevotella group; and (ii) an increased abundance of Bifidobacterium spp., of bacteria of the Erectale/C. coccoides group and of Akkermansia muciniphila, belonging to the Verrucomicrobia. A. muciniphila, a bacteria firstly identified in 2004 by the Applicant, represents approximately 1 to 3% of the total microbiota of healthy adults (Derrien et al, International Journal of Systematic and Evolutionary Microbiology, 2004, 54:1469-1476; Derrien et al Applied Environmental Microbiology 2008, 74, 1646-1648.).
Indirect evidences suggested a relationship between the abundance of A. muciniphila and intestinal dysfunctions or obesity-related disorders. For example, WO 2011/107481 describes that the absence of Akkermansia muciniphila in the gut of a subject, combined with the presence of Bacteroides capillosus and Clostridium leptum indicates that this subject is suffering from ulcerative colitis. Moreover, Hansen and colleagues showed that the administration of an antibiotic, Vancomycin, to neonatal NOD mice (the NOD mouse model is a model for Diabetes) suppresses clinical onset of Diabetes and propagates Akkermansia muciniphila (Hansen et al., Diabetologia, 2012 August; 55(8):2285-94). However, this may be an indirect effect due to the insensitivity of intestinal Akkermansia spp. to the used antibiotic.
These results thus showed that the complete composition of the gut microbiota is modified following the administration of prebiotic in mice. More specifically, no evidence suggested a specific role of one bacterial species, such as, for example, Akkermansia muciniphila, in the beneficial response to prebiotics administration. Moreover, to the Applicant knowledge, no beneficial effect of the direct administration of Akkermansia muciniphila has never been described, nor suggested.
Here, the Applicant surprisingly showed that repeated administration of Akkermansia muciniphila alone impacts the three underlying dysfunctions associated with obesity and related disorders, i.e. metabolic dysfunctions, low grade inflammatory state associated to higher blood lipopolysaccharides (LPS) levels and impaired gut barrier function. The present invention thus relates to the use of Akkermansia muciniphila or fragments thereof for treating obesity and related disorders.
The present invention thus relates to Akkermansia muciniphila or fragments thereof for treating, or for use in treating, a metabolic disorder in a subject in need thereof. In one embodiment of the invention, said metabolic disorder is obesity. In another embodiment of the invention, said metabolic disorder is selected from the group comprising metabolic syndrome, insulin-deficiency or insulin-resistance related disorders, Diabetes Mellitus (such as, for example, Type 2 Diabetes), glucose intolerance, abnormal lipid metabolism, atherosclerosis, hypertension, cardiac pathology, stroke, non-alcoholic fatty liver disease, hyperglycemia, hepatic steatosis, dyslipidemia, dysfunction of the immune system associated with overweight and obesity, cardiovascular diseases, high cholesterol, elevated triglycerides, asthma, sleep apnoea, osteoarthritis, neuro-degeneration, gallbladder disease, syndrome X, inflammatory and immune disorders, atherogenic dyslipidemia and cancer.
The present invention also relates to Akkermansia muciniphila or fragments thereof for increasing energy expenditure of a subject, preferably without impacting the food intake of said subject. The present invention also relates to Akkermansia muciniphila or fragments thereof for increasing satiety in a subject.
In one embodiment of the invention, viable cells of Akkermansia muciniphila are administered to the subject in need thereof.
In one embodiment of the invention, Akkermansia muciniphila is orally administered.
In one embodiment of the invention, an amount of Akkermansia muciniphila ranging from about 1·102 to about 1·1015 cfu, preferably from about 1·104 to about 1·1012 cfu, more preferably from about 1·105 to about 1·1010 cfu, and even more preferably from about 1·106 to about 1·109 cfu is administered to the subject. In another embodiment of the invention, an amount of Akkermansia muciniphila ranging from about 1·104 to about 1·1012 cfu, more preferably from about 1·105 to about 1·1011 cfu, and even more preferably from about 1·106 to about 1·1010 cfu is administered to the subject.
In one embodiment of the invention, Akkermansia muciniphila is administered at least once a day. In one embodiment of the invention, Akkermansia muciniphila is administered at least three times a week. In another embodiment of the invention, Akkermansia muciniphila is administered at least once a week.
In one embodiment of the invention, Akkermansia muciniphila is co-administered with another probiotic strain and/or with one or more prebiotics.
Another object of the invention is a composition for treating, or for use in treating, a metabolic disorder, or for increasing energy expenditure or for increasing satiety in a subject comprising Akkermansia muciniphila or fragments thereof as described hereinabove in association with an excipient. In one embodiment, said composition is a nutritional composition. In one embodiment of the invention, said composition is orally administered.
The present invention also relates to a pharmaceutical composition for treating, or for use in treating, a metabolic disorder or for increasing energy expenditure or for increasing satiety in a subject comprising Akkermansia muciniphila or fragments thereof as hereinabove described in association with a pharmaceutically acceptable vehicle.
Another object of the present invention is a medicament for treating, or for use in treating, a metabolic disorder or for increasing energy expenditure or for increasing satiety in a subject comprising Akkermansia muciniphila or fragments thereof as hereinabove described.
Another object of the present invention is the use of Akkermansia muciniphila or fragments thereof for promoting weight loss in a subject in need thereof.
The present invention also relates to a cosmetic composition comprising Akkermansia muciniphila or fragments thereof for promoting weight loss in a subject in need thereof.
In the present invention, the following terms have the following meanings:
This invention relates to Akkermansia muciniphila or a fragment thereof for treating, or for use in treating, metabolic disorders in a subject in need thereof.
As used herein, a metabolic disorder is a disorder related to an altered metabolic homeostasis, such as, for example, an altered glucidic or lipidic homeostasis.
In one embodiment of the invention, said metabolic disorder is obesity.
Examples of other metabolic disorders include, but are not limited to, metabolic syndrome, insulin-deficiency or insulin-resistance related disorders, Diabetes Mellitus (such as, for example, Type 2 Diabetes), glucose intolerance, abnormal lipid metabolism, atherosclerosis, hypertension, cardiac pathology, stroke, non-alcoholic fatty liver disease, hyperglycemia, hepatic steatosis, dyslipidemia, dysfunction of the immune system associated with overweight and obesity, cardiovascular diseases, high cholesterol, elevated triglycerides, asthma, sleep apnoea, osteoarthritis, neuro-degeneration, gallbladder disease, syndrome X, inflammatory and immune disorders, atherogenic dyslipidemia and cancer.
In another embodiment, said metabolic disorder is an overweight and/or obesity related metabolic disorder, i.e. a metabolic disorder that may be associated to or caused by overweight and/or obesity. Examples of overweight and/or obesity related metabolic disorder include, but are not limited to metabolic syndrome, insulin-deficiency or insulin-resistance related disorders, Diabetes Mellitus (such as, for example, Type 2 Diabetes), glucose intolerance, abnormal lipid metabolism, atherosclerosis, hypertension, cardiac pathology, stroke, non-alcoholic fatty liver disease, hyperglycemia, hepatic steatosis, dyslipidemia, dysfunction of the immune system associated with overweight and obesity, cardiovascular diseases, high cholesterol, elevated triglycerides, asthma, sleep apnoea, osteoarthritis, neuro-degeneration, gallbladder disease, syndrome X, inflammatory and immune disorders, atherogenic dyslipidemia and cancer.
In one embodiment, said metabolic disorder is Diabetes Mellitus, preferably Type 2 Diabetes. In another embodiment, said metabolic disorder is hypercholesterolemia (also known as high cholesterol). In one embodiment, hypercholesterolemia corresponds to a plasma cholesterol concentration superior or equal to 2 g/L or 5 mmol/L. In another embodiment, hypercholesterolemia corresponds to a ratio plasma concentration of total cholesterol:plasma concentration of HDL (high density lipoprotein cholesterol) superior or equal to 4.5:1, preferably 5:1.
In one embodiment of the invention, living strains of Akkermansia muciniphila are used in the present invention, preferably the living strains are derived from cells in stationary phase of growth.
In one embodiment of the invention, Akkermansia muciniphila may be in the form of viable cells. In another embodiment of the invention, Akkermansia muciniphila may be in the form of non-viable cells.
In one embodiment, metabolically active Akkermansia muciniphila cells are used in the present invention. In one embodiment, strains of Akkermansia muciniphila are not metabolically inactivated, wherein metabolic inactivation may result, for example, from autoclave treatment.
In one embodiment, Akkermansia muciniphila or fragment thereof is substantially purified. As used herein, the term “substantially purified” means that Akkermansia muciniphila or fragment thereof is comprised in a sample wherein it represents at least about 50%, preferably at least about 60, 70, 80, 85, 90, 95, 99% or more of the bacterial strains or fragment thereof of said sample.
The present invention also relate to a composition comprising an effective amount of Akkermansia muciniphila or a fragment thereof for treating, or for use in treating, a metabolic disorder.
In one embodiment of the invention, the effective amount of Akkermansia muciniphila corresponds to the amount of the bacteria sufficient for restoring a normal amount and/or proportion of Akkermansia muciniphila within the gut of the subject. Indeed, the Applicant showed that the gut of obese or overweight subject is depleted in Akkermansia muciniphila (see Examples). In one embodiment of the invention, the normal amount and/or proportion of Akkermansia muciniphila corresponds to the amount, and/or to the proportion of Akkermansia muciniphila present in the gut of a healthy subject.
As used herein, the term “healthy subject” is used to define a subject which is not affected by the disease to be treated. For example, if Akkermansia muciniphila or a fragment thereof is used for treating obesity, the healthy subject is not affected by obesity. Preferably, the healthy subject shares common characteristics with the subject to be treated, such as, for example, same gender, age, sex, diet, drugs intake or geolocation.
In one embodiment of the invention, the normal proportion of Akkermansia muciniphila in the gut ranges from about 0.1% to about 10% (in number of Akkermansia muciniphila cells to the total number of bacteria cells of the gut), preferably from about 0.3% to about 5%, more preferably from about 1% to about 3%.
In one embodiment of the invention, the effective amount of Akkermansia muciniphila ranges from about 1·102 to about 1·1015 cfu, preferably from about 1·104 to about 1·1012 cfu, more preferably from about 1·105 to about 1·1010 cfu, and even more preferably from about 1·106 to about 1·109 cfu, wherein cfu stands for “colony forming unit”.
In another embodiment of the invention, the effective amount of Akkermansia muciniphila ranges from about 1·106 to about 1·1010 cfu, preferably from about 1·108 to about 1·1010 cfu, more preferably from about 1·109 to about 1·1010 cfu.
In another embodiment of the invention, the effective amount of Akkermansia muciniphila ranges from about 1·106 to about 1·1010 cfu, preferably from about 1·106 to about 1·109 cfu, more preferably from about 1·108 to about 1·109 cfu.
In one embodiment of the invention, the effective amount of a fragment of Akkermansia muciniphila ranges from fragments derived from about 1·102 to about 1·1015 cfu, preferably from about 1·104 to about 1·1012 cfu, more preferably from about 1·105 to about 1·1010 cfu, and even more preferably from about 1·106 to about 1·109 cfu, wherein cfu stands for “colony forming unit”. In another embodiment of the invention, the effective amount of a fragment of Akkermansia muciniphila ranges from fragments derived from about 1·106 to about 1·1010 cfu, preferably from about 1·108 to about 1·1010 cfu, more preferably from about 1·109 to about 1·1010 cfu. In another embodiment of the invention, the effective amount of a fragment of Akkermansia muciniphila ranges from fragments derived from about 1·106 to about 1·1010 cfu, preferably from about 1·106 to about 1·109 cfu, more preferably from about 1·108 to about 1·109 cfu.
In one embodiment of the invention, the composition of the invention comprises an amount of Akkermansia muciniphila ranging from about 1·102 to about 1·1015 cfu/g of the composition, preferably from about 1·104 to about 1·1012 cfu/g of the composition, more preferably from about 1·105 to about 1·1010 cfu/g of the composition and even more preferably from about 1·106 to about 1·109 cfu/g of the composition. In one embodiment of the invention, the composition of the invention comprises an amount of Akkermansia muciniphila ranging from about 1·102 to about 1·1015 cfu/mL of the composition, preferably from about 1·104 to about 1·1012 cfu/mL of the composition, more preferably from about 1·105 to about 1·1010 cfu/mL of the composition and even more preferably from about 1·106 to about 1·109 cfu/mL of the composition. In another embodiment of the invention, the composition of the invention comprises an amount of Akkermansia muciniphila ranging from about 1·106 to about 1·1010 cfu/g or cfu/mL of the composition, preferably from about 1·108 to about 1·1010 cfu/g or cfu/mL, more preferably from about 1·109 to about 1·1010 cfu/g or cfu/mL. In another embodiment of the invention, the composition of the invention comprises an amount of Akkermansia muciniphila ranging from about 1·106 to about 1·1010 cfu/g or cfu/mL of the composition, preferably from about 1·106 to about 1·109 cfu/g or cfu/mL, more preferably from about 1·108 to about 1·109 cfu/g or cfu/mL.
In one embodiment of the invention, the composition of the invention comprises an amount of fragments of Akkermansia muciniphila ranging from fragments derived from about 1·102 to about 1·1015 cfu/g or mL of the composition, preferably from about 1·104 to about 1·1012 cfu/g or mL of the composition, more preferably from about 1·105 to about 1·1010 cfu/g or mL of the composition and even more preferably from about 1·106 to about 1·109 cfu/g or mL of the composition. In another embodiment of the invention, the composition of the invention comprises an amount of fragments of Akkermansia muciniphila ranging from fragments derived from about 1·106 to about 1·1010 cfu/g or cfu/mL of the composition, preferably from about 1·108 to about 1·1010 cfu/g or cfu/mL, more preferably from about 1·109 to about 1·1010 cfu/g or cfu/mL. In another embodiment of the invention, the composition of the invention comprises an amount of fragments of Akkermansia muciniphila ranging from fragments derived from about 1·106 to about 1·1010 cfu/g or cfu/mL of the composition, preferably from about 1·106 to about 1·109 cfu/g or cfu/mL, more preferably from about 1·108 to about 1·109 cfu/g or cfu/mL.
The present invention also relates to a pharmaceutical composition comprising an effective amount of Akkermansia muciniphila or a fragment thereof and at least one pharmaceutically acceptable excipient. In one embodiment of the invention, the pharmaceutical composition of the invention is for treating a metabolic disorder. In another embodiment of the invention, the pharmaceutical composition is for restoring a normal proportion of Akkermansia muciniphila in the gut of a subject in need thereof.
As used herein the term “pharmaceutically acceptable excipient” refers to an excipient that does not produce an adverse, allergic or other untoward reaction when administered to an animal, preferably a human. It may include any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents and the like. For human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologics standards.
The present invention also relates to a medicament comprising an effective amount of Akkermansia muciniphila or a fragment thereof. In one embodiment of the invention, the medicament of the invention is for treating a metabolic disorder. In another embodiment of the invention, the medicament is for restoring a normal proportion of Akkermansia muciniphila in the gut of a subject in need thereof.
The present invention also relates to a method for treating a metabolic disorder in a subject in need thereof, wherein said method comprises administering an effective amount of Akkermansia muciniphila or a fragment thereof to the subject.
Another object of the invention is a method for restoring a normal proportion of Akkermansia muciniphila in the gut of a subject in need thereof, wherein said method comprises administering an effective amount of Akkermansia muciniphila or a fragment thereof to the subject.
In one embodiment, the method of the invention comprises administering an effective amount of the composition, of the pharmaceutical composition or of the medicament of the invention to the subject.
In one embodiment of the invention, Akkermansia muciniphila or a fragment thereof, or the composition, pharmaceutical composition or medicament is administered at least once a week, preferably at least twice a week, more preferably at least three times a week, and even more preferably three times a week. In another embodiment, Akkermansia muciniphila or a fragment thereof, or the composition, pharmaceutical composition or medicament is administered at least once a day, and preferably at least twice a day.
In one embodiment, Akkermansia muciniphila or a fragment thereof, or the composition, pharmaceutical composition or medicament of the invention is administered during 1 week, preferably during 2, 3, 4, 5, 6, 7 or 8 weeks or more.
In one embodiment, Akkermansia muciniphila or a fragment thereof, or the composition, pharmaceutical composition or medicament of the invention is administered for a period that lasts until the desired outcome is achieved (e.g. weight loss, metabolic disorder treatment, decrease of cholesterol plasma level . . . ).
In one embodiment, the administration of Akkermansia muciniphila or a fragment thereof, or the composition, pharmaceutical composition or medicament of the invention is permanent, i.e. is not limited in time.
In one embodiment of the invention, the daily amount of Akkermansia muciniphila administered per day ranges from 1·102 to about 1·1015 cfu/day, preferably from about 1·104 to about 1·1012 cfu/day, more preferably from about 1·105 to about 1·1010 cfu/day and even more preferably from about 1·106 to about 1·109 cfu/day.
In another embodiment of the invention, the daily amount of Akkermansia muciniphila administered per day ranges from 1·106 to about 1·1010 cfu/day, preferably from about 1·108 to about 1·1010 cfu/day, more preferably from about 1·109 to about 1·1010 cfu/day.
In another embodiment of the invention, the daily amount of Akkermansia muciniphila administered per day ranges from 1·106 to about 1·1010 cfu/day, preferably from about 1·106 to about 1·109 cfu/day, more preferably from about 1·108 to about 1·109 cfu/day.
In one embodiment of the invention, the daily amount of fragments of Akkermansia muciniphila administered per day ranges from fragments derived from 1·102 to about 1·1015 cfu/day, preferably from about 1·104 to about 1·1012 cfu/day, more preferably from about 1·105 to about 1·1010 cfu/day and even more preferably from about 1·106 to about 1·109 cfu/day. In another embodiment of the invention, the daily amount of fragments of Akkermansia muciniphila administered per day ranges from fragments derived from 1·106 to about 1·1010 cfu/day, preferably from about 1·108 to about 1·1010 cfu/day, more preferably from about 1·109 to about 1·1010 cfu/day. In another embodiment of the invention, the daily amount of fragments of Akkermansia muciniphila administered per day ranges from fragments derived from 1·106 to about 1·1010 cfu/day, preferably from about 1·106 to about 1·109 cfu/day, more preferably from about 1·108 to about 1·109 cfu/day.
In one embodiment of the invention, the subject is overweight. In another embodiment, the subject is obese.
In one embodiment of the invention, the subject is diagnosed with a metabolic disorder, such as, for example, with an overweight and/or obesity related metabolic disorder.
In another embodiment, the subject is at risk of developing a metabolic disorder, such as, for example, an overweight and/or obesity related metabolic disorder. In one embodiment, said risk is related to the fact that the subject is overweight or obese. In another embodiment, said risk corresponds to a predisposition, such as, for example, a familial predisposition to a metabolic disorder, such as, for example, to an overweight and/or obesity related metabolic disorder.
In one embodiment of the invention, the subject presents a deregulation of the gut microbiota composition. Preferably, the gut microbiota of said subject is depleted in Akkermansia muciniphila strains. In one embodiment, the proportion of Akkermansia muciniphila in the gut of the subject is inferior to 1%, preferably inferior to 0.5%, more preferably inferior to 0.1%, in number of Akkermansia muciniphila cells to the total number of bacterial cells in the gut.
The present invention also relates to the cosmetic use of Akkermansia muciniphila or a fragment thereof for promoting weight loss in a subject.
Another object of the invention is thus a cosmetic composition comprising a cosmetically effective amount of Akkermansia muciniphila or a fragment thereof, and the use thereof for promoting weight loss in a subject. As used herein, a “cosmetically effective amount” refers to the amount of a cosmetic composition necessary and sufficient for promoting a cosmetic effect, such as, for example, for inducing weight loss in a subject.
The present invention also relates to a method for promoting weight loss in a subject in need thereof, wherein said method comprises administering a cosmetically effective amount of Akkermansia muciniphila or a fragment thereof to said subject.
In one embodiment, the method of the invention comprises administering a cosmetically effective amount of the composition or of the cosmetic composition of the invention to the subject.
In one embodiment of the invention, the cosmetically effective amount of Akkermansia muciniphila ranges from about 1·102 to about 1·1015 cfu, preferably from about 1·104 to about 1·1012 cfu, more preferably from about 1·105 to about 1·1010 cfu and even more preferably from about 1·106 to about 1·109 cfu. In another embodiment of the invention, the cosmetically effective amount of Akkermansia muciniphila ranges from about 1·106 to about 1·1010 cfu, preferably from about 1·108 to about 1·1010 cfu, more preferably from about 1·109 to about 1·1010 cfu. In another embodiment of the invention, the cosmetically effective amount of Akkermansia muciniphila ranges from about 1·106 to about 1·1010 cfu, preferably from about 1·106 to about 1·109 cfu, more preferably from about 1·108 to about 1·109 cfu.
In one embodiment of the invention, the cosmetically effective amount of fragments of Akkermansia muciniphila ranges from fragments derived from about 1·102 to about 1·1015 cfu, preferably from about 1·104 to about 1·1012 cfu, more preferably from about 1·105 to about 1·1010 cfu and even more preferably from about 1·106 to about 1·109 cfu. In another embodiment of the invention, the cosmetically effective amount of fragments of Akkermansia muciniphila ranges from fragments derived from about 1·106 to about 1·1010 cfu, preferably from about 1·108 to about 1·1010 cfu, more preferably from about 1·109 to about 1·1010 cfu. In another embodiment of the invention, the cosmetically effective amount of fragments of Akkermansia muciniphila ranges from fragments derived from about 1·106 to about 1·1010 cfu, preferably from about 1·106 to about 1·109 cfu, more preferably from about 1·108 to about 1·109 cfu.
In one embodiment of the invention, Akkermansia muciniphila or a fragment thereof, or the composition or cosmetic composition is administered at least once a week, preferably at least twice a week, more preferably at least three times a week, and even more preferably three times a week. In another embodiment, Akkermansia muciniphila or a fragment thereof, or the composition or cosmetic composition is administered at least once a day, and preferably at least twice a day.
In one embodiment, Akkermansia muciniphila or a fragment thereof, or the composition or cosmetic composition of the invention is administered during 1 week, preferably 2, 3, 4, 5, 6, 7 or 8 weeks or more.
In one embodiment, Akkermansia muciniphila or a fragment thereof, or the composition or cosmetic composition of the invention is administered for a period that lasts until the desired outcome is achieved (e.g. weight loss . . . ).
In one embodiment, the administration of Akkermansia muciniphila or a fragment thereof, or the composition or cosmetic composition of the invention is permanent, i.e. is not limited in time.
In one embodiment of the invention, the daily amount of Akkermansia muciniphila administered per day ranges from 1·102 to about 1·1015 cfu/day, preferably from about 1·105 to about 1·1012 cfu/day, more preferably from about 1·108 to about 1·1010 cfu/day, and even more preferably from about 1·109 to about 1·1010 cfu/day. In another embodiment of the invention, the daily amount of Akkermansia muciniphila administered per day ranges from 1·106 to about 1·1010 cfu/day, preferably from about 1·108 to about 1·1010 cfu/day, more preferably from about 1·109 to about 1·1010 cfu/day. In another embodiment of the invention, the daily amount of Akkermansia muciniphila administered per day ranges from 1·106 to about 1·1010 cfu/day, preferably from about 1·106 to about 1·109 cfu/day, more preferably from about 1·108 to about 1·109 cfu/day.
In one embodiment of the invention, the daily amount of fragments of Akkermansia muciniphila administered per day ranges from fragments derived from about 1·102 to about 1·1015 cfu/day, preferably from about 1·105 to about 1·1012 cfu/day, more preferably from about 1·108 to about 1·1010 cfu/day, and even more preferably from about 1·109 to about 1·1010 cfu/day. In another embodiment of the invention, the daily amount of fragments of Akkermansia muciniphila administered per day ranges from fragments derived from about 1·106 to about 1·1010 cfu/day, preferably from about 1·108 to about 1·1010 cfu/day, more preferably from about 1·109 to about 1·1010 cfu/day. In another embodiment of the invention, the daily amount of fragments of Akkermansia muciniphila administered per day ranges from fragments derived from about 1·106 to about 1·1010 cfu/day, preferably from about 1·106 to about 1·109 cfu/day, more preferably from about 1·108 to about 1·109 cfu/day.
In one embodiment, said subject is not an obese subject. In another embodiment, said subject is overweight.
In one embodiment of the invention, the composition, the pharmaceutical composition, the cosmetic composition or the medicament further comprises additional probiotic strains or species, such as, for example, bacterial probiotic strains or species;
prokaryotes probiotics other than bacteria; or fungal strains or species, preferably yeast strains or species. In one embodiment, said additional probiotic strains or species are selected from those naturally present in the gut of the subject, preferably in the human gut, more preferably in the gut of healthy human subjects.
Examples of bacterial probiotic strains or species that may be used in the present invention include, but are not limited to Lactobacillus, Lactococcus, Bifidobacterium, Veillonella, Desemzia, Coprococcus, Collinsella, Citrobacter, Turicibacter, Sutterella, Subdoligranulum, Streptococcus, Sporobacter, Sporacetigenium, Ruminococcus, Roseburia, Proteus, Propionobacterium, Leuconostoc, Weissella, Pediococcus, Streptococcus, Prevotella, Parabacteroides, Papillibacter, Oscillospira, Melissococcus, Dorea, Dialister, Clostridium, Cedecea, Catenibacterium, Butyrivibrio, Buttiauxella, Bulleidia, Bilophila, Bacteroides, Anaerovorax, Anaerostopes, Anaerofilum, Enterobacteriaceae, Fermicutes, Atopobium, Alistipes, Acinetobacter, Slackie, Shigella, Shewanella, Serratia, Mahella, Lachnospira, Klebsiella, Idiomarina, Fusobacterium, Faecalibacterium, Eubacterium, Enterococcus, Enterobacter, Eggerthella.
Examples of prokaryote strains or species that may be used in the present invention include, but are not limited to Archaea, Firmicutes, Bacteroidetes (such as, for example, Allistipes, Bacteroides ovatus, Bacteroides splachnicus, Bacteroides stercoris, Parabacteroides, Prevotella ruminicola, Porphyromondaceae, and related genus), Proteobacteria, Betaproteobacteria (such as, for example, Aquabacterium and Burkholderia), Gammaproteobacteria (such as, for example, Xanthomonadaceae), Actinobacteria (such as, for example, Actinomycetaceae and Atopobium), Fusobacteria, Methanobacteria, Spirochaetes, Fibrobacters, Deferribacteres, Deinococcus, Thermus, Cyanobacteria, Methanobrevibacteria, Peptostreptococcus, Ruminococcus, Coprococcus, Subdolingranulum, Dorea, Bulleidia, Anaerofustis, Gemella, Roseburia, Dialister, Anaerotruncus, Staphylococcus, Micrococcus, Propionobacteria, Enterobacteriaceae, Faecalibacteria, Bacteroides, Parabacteroides, Prevotella, Eubacterium, Bacilli (such as, for example, Lactobacillus salivarius and related species, Aerococcus, Granulicatella, Streptococcus bovis and related genus and Streptococcus intermedius and related genus), Clostridium (such as, for example, Eubacterium hallii, Eubacterium limosum and related genus) and Butyrivibrio.
Examples of fungal probiotic strains or species, preferably yeast probiotic strains or species that may be used in the present invention include, but are not limited Ascomycetes, Zygomycetes and Deuteromycetes, preferably from the groups Aspergillus, Torulopsis, Zygosaccharomyces, Hansenula, Candida, Saccharomyces, Clavispora, Bretanomyces, Pichia, Amylomyces, Zygosaccharomyces, Endomycess, Hyphopichia, Zygosaccharomyces, Kluyveromyces, Mucor, Rhizopus, Yarrowia, Endomyces, Debaryomyces, and/or Penicillium.
The Applicant herein shows that the beneficial effects observed after Akkermansia muciniphila administration are specific of this bacterial strain. Indeed, it is shown in the Examples that the administration of Lactobacillus plantarum WCSF-1 does not have the same beneficial effects.
In one embodiment of the invention, the composition, the pharmaceutical composition, the cosmetic composition or the medicament does not comprise the bacterial strains Lactobacillus-Enterococcus, Bacteroides and/or Atopobium.
In one embodiment of the invention, the only one microbial strain or species, preferably bacterial strain or species, comprised in the composition, pharmaceutical composition, cosmetic composition or medicament is Akkermansia muciniphila.
In one embodiment of the invention, the composition, pharmaceutical composition, cosmetic composition or medicament consists of Akkermansia muciniphila.
In another embodiment of the invention, the composition, pharmaceutical composition, cosmetic composition or medicament consists essentially of Akkermansia muciniphila, wherein “consisting essentially of” herein means that Akkermansia muciniphila is the only microbial strain or species, preferably the only bacterial strain or species comprised in the composition, pharmaceutical composition, cosmetic composition or medicament.
In one embodiment of the invention, Akkermansia muciniphila or a fragment thereof activates or inhibits the growth and/or biological activity of other bacterial strain(s) or species of the gut microbiota.
In one embodiment of the invention, the composition, the pharmaceutical composition, the cosmetic composition or the medicament further comprises a prebiotic.
Examples of prebiotics that may be used in the present invention include, but are not limited to, inulin and inulin-type fructans, oligofructose, xylose, arabinose, arabinoxylan, ribose, galactose, rhamnose, cellobiose, fructose, lactose, salicin, sucrose, glucose, esculin, tween 80, trehalose, maltose, mannose, mellibiose, mucus or mucins, raffinose, fructooligosaccharides, galacto-oligosaccharides, amino acids, alcohols, and any combinations thereof.
Other non-limiting examples of prebiotics include water-soluble cellulose derivatives, water-insoluble cellulose derivatives, unprocessed oatmeal, metamucil, all-bran, and any combinations thereof.
Examples of water-soluble cellulose derivatives include, but are not limited to, methylcellulose, methyl ethyl cellulose, hydroxyethyl cellulose, ethyl hydroxyethyl cellulose, cationic hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxyethyl methylcellulose, hydroxypropyl methylcellulose, and carboxymethyl cellulose.
Akkermansia muciniphila or a fragment thereof or the composition, pharmaceutical composition, cosmetic composition or medicament of the invention may be administered by several routes of administration. Examples of adapted routes of administration include, but are not limited to, oral administration, rectal administration, administration via esophagogastroduodenoscopy, administration via colonoscopy, administration using a nasogastric or orogastric tube and the like.
According to an embodiment, Akkermansia muciniphila or a fragment thereof or the composition, pharmaceutical composition, cosmetic composition or medicament of the invention is in a form adapted to oral administration. According to a first embodiment, the form adapted to oral administration is a solid form selected from the group comprising tablets, pills, capsules, soft gelatin capsules, sugarcoated pills, orodispersing/orodispersing tablets, effervescent tablets or other solids. According to a second embodiment, the form adapted to oral administration is a liquid form, such as, for example, a drinkable solution, liposomal forms and the like.
In one embodiment, the composition, pharmaceutical composition, cosmetic composition or medicament of the invention further comprises excipients, diluent and/or carriers selected with regard to the intended route of administration. Examples of excipients, diluent and/or carriers include, but are not limited to, water, phosphate buffer saline, anaerobic phosphate buffer saline, sodium bicarbonate, juice, milk, yogurt, infant formula, dairy product, coloring agents, such as, for example, titane dioxide (E171), iron dioxide (E172) and brilliant black BN (E151); flavoring agents; thickeners, such as, for example, glycerol monostearate; sweeteners; coating agents, such as, for example, refined colza oil, soya oil, peanut oil, soya lecithin or fish gelatin; diluting agents, such as, for example, lactose, monohydrated lactose or starch; binding agents, such as, for example, povidone, pregelatinized starch, gums, saccharose, polyethylene glycol (PEG) 4000 or PEG 6000; disintegrating agents, such as, for example, microcrystalline cellulose or sodium carboxymethyl starch, such as, for example, sodium carboxymethyl starch type A; lubricant agents, such as, for example, magnesium stearate; flow agent, such as, for example, colloidal anhydrous silica, etc. . . . .
In one embodiment of the invention, the composition, pharmaceutical composition, cosmetic composition or medicament is in the form of a nutritional composition, i.e. comprises liquid or solid food, feed or drinking water. In one embodiment of the invention, the composition, pharmaceutical composition, cosmetic composition or medicament is a food product, such as, for example, dairy products, dairy drinks, yogurt, fruit or vegetable juice or concentrate thereof, powders, malt or soy or cereal based beverages, breakfast cereal such as muesli flakes, fruit and vegetable juice powders, cereal and/or chocolate bars, confectionary, spreads, flours, milk, smoothies, confectionary, milk product, milk powder, reconstituted milk, cultured milk, yoghurt, drinking yoghurt, set yoghurt, drink, dairy drink, milk drink, chocolate, gels, ice creams, cereals, reconstituted fruit products, snack bars, food bars, muesli bars, spreads, sauces, dips, dairy products including yoghurts and cheeses, drinks including dairy and non-dairy based drinks, sports supplements including dairy and non-dairy based sports supplements.
In one embodiment of the invention, the composition, pharmaceutical composition, cosmetic composition or medicament is in the form of a food additive, drink additive, dietary supplement, nutritional product, medical food or nutraceutical composition.
Akkermansia muciniphila is a strictly anaerobic bacterium. Therefore, in the embodiment where viable or living strains are used, prolonged contact with oxygen should be avoided. Examples of means for avoiding prolonged contact with oxygen include, but are not limited to, freeze of the bacterial cells or packaging in a sealed container and the like.
The Applicant herein showed that obesity and related disorders are associated with an increased gut permeability and with impaired mucus production, epithelium barrier, immune system and/or antibacterial compounds production by the subject; and that the administration of A. muciniphila may restore these parameters.
Therefore, the present invention also relates to Akkermansia muciniphila or a fragment thereof for decreasing gut permeability and/or for restoring impaired mucus production and/or for restoring epithelium barrier and/or for restoring immune system and/or for decreasing the production of antibacterial compounds. Another object of the invention is a method for decreasing gut permeability and/or for restoring impaired mucus production and/or for restoring epithelium barrier and/or for restoring immune system and/or for decreasing the production of antibacterial compounds in a subject in need thereof, comprising administering an effective or cosmetically effective amount of Akkermansia muciniphila or a fragment thereof to a subject in need thereof.
The Applicant surprisingly showed that the administration of A. muciniphila controls gut barrier by regulating mucus layer thickness and the production of colon antimicrobial peptides (such as, for example, RegIIIgamma). In addition, they also showed that A. muciniphila regulates the production of acylglycerols that belongs to the endocannabinoids family involved in the control of inflammation, gut barrier and gut peptides secretion (GLP-1 and GLP-2). GLP-1 and GLP-2 are involved in a great variety of functions, including improving insulin signalling, decreasing inflammation, and promoting satiety.
Therefore, the present invention also relates to Akkermansia muciniphila or a fragment thereof for controlling gut barrier function, and to a method for controlling gut barrier function comprising administering an effective or cosmetically effective amount of Akkermansia muciniphila or a fragment thereof to a subject in need thereof. In one embodiment, Akkermansia muciniphila or a fragment thereof regulates mucus layer thickness (which may be decreased in obesity or other metabolic disorders). In another embodiment, the administration of Akkermansia muciniphila or a fragment thereof induces the production of colon antimicrobial peptides, such as, for example, RegIIIgamma. In another embodiment, the administration of Akkermansia muciniphila or a fragment thereof induces the production of compounds of the endocannabinoids family, such as, for example, acylglycerols selected from the group comprising 2-oleoylglycerol, 2-palmitoylglycerol and 2-arachidonoylglycerol. In another embodiment, the administration of Akkermansia muciniphila or a fragment thereof regulates mucus turnover.
Another object of the invention concerns Akkermansia muciniphila or a fragment thereof for use in treating metabolic dysfunction associated with or caused by a metabolic disorder. Still another object of the invention is thus a method for treating metabolic dysfunction associated with or caused by a metabolic disorder in a subject in need thereof, comprising administering an effective amount or a cosmetically effective amount of Akkermansia muciniphila or a fragment thereof to a subject in need thereof.
The Applicant also showed that the administration of Akkermansia muciniphila controls fat storage and adipose tissue metabolism. Therefore, another object of the invention concerns Akkermansia muciniphila or a fragment thereof for use in controlling fat storage and adipose tissue metabolism. Another object of the invention is also a method for controlling fat storage and adipose tissue metabolism comprising administering an effective amount or a cosmetically effective amount of Akkermansia muciniphila or a fragment thereof to a subject in need thereof. In one embodiment, said control does not involve any change in food intakes. In one embodiment of the invention, administration of Akkermansia muciniphila or a fragment thereof abolishes metabolic endotoxemia. In another embodiment, administration of Akkermansia muciniphila or a fragment thereof lowers fat mass. In another embodiment, administration of Akkermansia muciniphila or a fragment thereof increases mRNA expression of adipocyte differentiation and lipid oxidation, preferably without affecting lipogenesis.
The Applicant also showed that the administration of Akkermansia muciniphila regulates adipose tissue metabolism and glucose homeostasis. The present invention thus relates to Akkermansia muciniphila or a fragment thereof for use in the regulation of adipose tissue metabolism and glucose homeostasis; and to a method for regulating adipose tissue metabolism and glucose homeostasis comprising administering an effective amount or a cosmetically effective amount of Akkermansia muciniphila or a fragment thereof to a subject in need thereof. In one embodiment of the invention, the administration of Akkermansia muciniphila or a fragment thereof reverses diet-induced fasting hyperglycemia. In another embodiment, the administration of Akkermansia muciniphila or a fragment thereof induces a reduction of at least 10%, preferably of at least 30%, more preferably of at least 40% of hepatic glucose-6-phosphatase expression. In another embodiment, the administration of Akkermansia muciniphila or a fragment thereof induces a reduction of the insulin-resistance index. In one embodiment, said reduction of the insulin-resistance index is of at least 5%, preferably of at least 10%, more preferably of at least 15%.
Moreover, the Applicant showed that the administration of Akkermansia muciniphila leads to the normalization of adipose tissue CD11c subpopulation of macrophages. The amount of cells of this population of macrophages is increased in metabolic disorders such as, for example, obesity and Type 2 Diabetes, and is a hallmark of inflammation related to these metabolic disorders. Therefore, the present invention also relates to Akkermansia muciniphila or a fragment thereof for treating inflammation, preferably low grade inflammation, associated with or caused by metabolic disorders; and to a method for treating inflammation related to metabolic disorders comprising administering an effective amount or a cosmetically effective amount of Akkermansia muciniphila or a fragment thereof to a subject in need thereof. In one embodiment of the invention, the administration of Akkermansia muciniphila or a fragment thereof decreases the amount of CD11c macrophages in the adipose tissue.
Finally, the Applicant showed that the administration of Akkermansia muciniphila decreases plasma cholesterol in high-fat diet fed mice. Therefore, the present invention also relates to Akkermansia muciniphila or a fragment thereof for decreasing plasma cholesterol; and to a method for decreasing plasma cholesterol comprising administering an effective amount or a cosmetically effective amount of Akkermansia muciniphila or a fragment thereof to a subject in need thereof.
In one embodiment of the invention, the administration of Akkermansia muciniphila or a fragment thereof to a subject has no impact on food intake of said subject.
In one embodiment of the invention, the administration of Akkermansia muciniphila or a fragment thereof to a subject increases energy expenditure of said subject, preferably without impacting the food intake of said subject.
The present invention thus also relates to a method of increasing energy expenditure of a subject, comprising administering Akkermansia muciniphila or a fragment thereof, or a composition, pharmaceutical composition, cosmetic composition or medicament of the invention to the subject, preferably in a therapeutically or cosmetically effective amount. Preferably, the method of the invention does not comprise or further comprise modulating the food intake of said subject. In one embodiment of the invention, the method of the invention increases energy expenditure, thereby inducing durable weight loss in the subject, and thereby treating metabolic disorders in said subject, such as, for example, obesity related metabolic disorders.
In one embodiment, the administration of Akkermansia muciniphila or a fragment thereof to a subject increases satiety in said subject. Consequently, according to this embodiment, the method of the invention increases satiety in a subject, thereby inducing durable weight loss in the subject, and thereby treating metabolic disorders in said subject, such as, for example, obesity related metabolic disorders.
The present invention is further illustrated by the following examples.
Materials and Methods
Mice
Ob/Ob Experiment:
ob/ob versus lean study: Six-week-old ob/ob (n=5/group) mice (C57BL/6 background, Jackson-Laboratory, Bar Harbor, Me., USA) were housed in a controlled environment (12-h daylight cycle, lights-off at 6-pm) in groups of two or three mice/cage, with free access to food and water. The mice were fed a control diet (A04, Villemoisson-sur-Orge, France) for 16 weeks. Cecal content was harvested immersed in liquid nitrogen, and stored at −80° C., for further Akkermansia muciniphila analysis.
Ob/Ob Prebiotic Study:
Six-week-old ob/ob (n=10/group) mice (C57BL/6 background, Jackson-Laboratory, Bar Harbor, Me., USA) were housed in a controlled environment (12-h daylight cycle, lights-off at 6-pm) in groups of two mice/cage, with free access to food and water. The mice were fed a control diet (Ob-CT) (A04, Villemoisson-sur-Orge, France) or a control diet supplemented with prebiotics, such as oligofructose (Ob-Pre) (Orafti, Tienen, Belgium) for 5-weeks as previously described (Everard et al. Diabetes 60, 2775-2786 (2011)). This set of mice has been previously characterized in Everard et al (Everard et al. Diabetes 60, 2775-2786 (2011)).
High-Fat Prebiotic Experiment:
A set of 10-week-old C57BL/6J mice (40 mice, n=10/group) (Charles River, Brussels, Belgium) were housed in groups of five mice/cage, with free access to food and water. The mice were fed a control diet (CT) (A04, Villemoisson-sur-Orge, France) or a control diet and treated with prebiotics, such as oligofructose (Orafti, Tienen, Belgium) (0.3 g/mouse/day) added in tap water (CT-Pre), or fed a high-fat diet (HF) (60% fat and 20% carbohydrates (kcal/100 g), D12492, Research diet, New Brunswick, N.J., USA) or a HF diet and treated with oligofructose (0.3 g/mouse/day) added in tap water (HF-Pre). Treatment continued for 8 weeks.
HFD Akkermansia muciniphila Treatment:
A set of 10-week-old C57BL/6J mice (40 mice, n=10/group) (Charles River, Brussels, Belgium) were housed in groups of 2 mice/cage, with free access to food and water. The mice were fed a control diet (CT) (AIN93Mi; Research diet, New Brunswick, N.J., USA) or a high-fat diet (HF) (60% fat and 20% carbohydrates (kcal/100 g), D12492, Research diet, New Brunswick, N.J., USA). Mice were treated with an oral administration of Akkermansia muciniphila by oral gavage at the dose 2·108 cfu/0.2 ml suspended in sterile anaerobic phosphate buffer saline (CT-Akk and HF-Akk) and control groups were treated with an oral gavage of an equivalent volume of sterile anaerobic phosphate buffer saline (CT and HF). Treatment continued for 4 weeks.
A. muciniphila MucT (ATTC BAA-835) was grown anaerobically in a mucin-based basal medium as previously described (Derrien et al Int J Syst Evol Microbiol 54, 1469-1476 (2004)) and then washed and suspended in anaerobic phosphate buffer saline, including 25% (v/v) glycerol, to an end concentration of 1·1010 cfu/ml.
HFD Akkermansia muciniphila Alive Treatment Vs Heat-Killed and Lactobacillus platarum WCFS1:
A set of 10-week-old C57BL/6J mice (40 mice, n=8/group) (Charles River, Brussels, Belgium) were housed in groups of 2 mice/cage, with free access to food and water. The mice were fed a control diet (CT) (AIN93Mi; Research diet, New Brunswick, N.J., USA) or a high-fat diet (HF) (60% fat and 20% carbohydrates (kcal/100 g), D12492, Research diet, New Brunswick, N.J., USA). Mice were treated daily with an oral administration of Akkermansia muciniphila by oral gavage at the dose 2·108 cfu/0.2 ml suspended in sterile anaerobic phosphate buffer saline A. muciniphila was heat-killed/inactivated by autoclaving (15 min, 121° C., 225 kPa). A viability check by culturing on mucin-containing medium confirmed the absence of any viable cells. Lactobacillus plantarum WCFS1 was grown anaerobically in MRS medium (Difco Lactobacilli MRS broth; BD), washed, concentrated, and manipulated an identical ways as the A. muciniphila preparation. The two controls group (CT and HF) were treated daily with an oral gavage of an equivalent volume of sterile anaerobic PBS containing a similar end concentration of glycerol (2.5%) (reduced with one drop of 100 mM titanium citrate) as the treatment groups for 4 weeks.
Food and water intake were recorded once a week. Body composition was assessed by using 7.5 MHz time domain-nuclear magnetic resonance (TD-NMR) (LF50 minispec, Bruker, Rheinstetten, Germany).
All mouse experiments were approved by and performed in accordance with the guidelines of the local ethics committee. Housing conditions were specified by the Belgian Law of Apr. 6, 2010, regarding the protection of laboratory animals (agreement number LA1230314).
Tissue Sampling
The animals have been anesthetized with isoflurane (Forene®, Abbott, Queenborough, Kent, England) before exsanguination and tissue sampling, then mice were killed by cervical dislocation. Adipose depots (epididymal, subcutaneous and mesenteric), liver were precisely dissected and weighed; the addition of the three adipose tissues corresponds to the adiposity index. The intestinal segments (ileum, cecum and colon), the cecal content and the adipose tissues were immersed in liquid nitrogen, and stored at −80° C., for further analysis.
Mucus Layer Thickness
Proximal colon segments were immediately removed and fixed in Carnoy's solution (ethanol-acetic acid-chloroform, 6/3/1 v/v/v) for two hours at 4° C. Then the samples were immersed in ethanol 100% for 24 hours prior to processing for paraffin embedding. Paraffin sections of 5 μm were stained with alcian blue. A minimum of 20 different measurements were made perpendicular to the inner mucus layer per field by an investigator blinded to the experimental groups. 5 to 19 randomly selected fields were analyzed for each colon for a total of 2146 measurements by using an image analyzer (Motic-image Plus 2.0 ML, Motic, China).
RNA Preparation and Real-Time qPCR Analysis
Total RNA was prepared from tissues using TriPure reagent (Roche). Quantification and integrity analysis of total RNA was performed by running 1 μl of each sample on an Agilent 2100 Bioanalyzer (Agilent RNA 6000 Nano Kit, Agilent).
cDNA was prepared by reverse transcription of 1 μg total RNA using a Reverse Transcription System kit (Promega, Leiden, The Netherlands). Real-time PCRs were performed with the StepOnePlus™ real-time PCR system and software (Applied Biosystems, Den Ijssel, The Netherlands) using Mesa Fast gPCR™ (Eurogentec, Seraing, Belgium) for detection according to the manufacturer's instructions. RPL19 was chosen as the housekeeping gene. All samples were run in duplicate in a single 96-well reaction plate, and data were analyzed according to the 2−ΔCT method. The identity and purity of the amplified product was checked through analysis of the melting curve carried out at the end of amplification. Primer sequences for the targeted mouse genes are presented in the Table 1 below.
Insulin Resistance Index
Insulin resistance index was determined by multiplying the area under the curve (0 min and 15 min) of both blood glucose and plasma insulin obtained following an oral glucose load (2 g of glucose per kg of body weight) performed after 4 weeks (A. muciniphila study) of treatment. Food was removed two-hours after the onset of the daylight cycle and mice were treated after 6-h-fasting period as previously described (Everard et al. Diabetes 60, 2775-2786 (2011)).
Biochemical Analyses
Portal vein blood LPS concentration was measured by using Endosafe-MCS (Charles River Laboratories, Lyon, France) based on the Limulus amaebocyte Lysate (LAL) kinetic chromogenic methodology that measures color intensity directly related to the endotoxin concentration in a sample. Serum were diluted 1/10 with endotoxin free buffer to minimize interferences in the reaction (inhibition or enhancement) and heated 15 min at 70° C. Each sample was diluted 1/70 or 1/100 with endotoxin-free LAL reagent water (Charles River Laboratories) and treated in duplicate and two spikes for each sample were included in the determination. All samples have been validated for the recovery and the coefficient variation. The lower limit of detection was 0.005 EU/ml. Plasma insulin concentration was determined in 25 μl of plasma using an ELISA kit (Mercodia, Upssala, Sweden) according to the manufacturer instructions.
Fecal IgA levels were determined using an ELISA kit (E99-103, Bethyl Laboratories, Montgomery, Tex.). Freshly collected feces were frozen at −80° C. then diluted in 50 mM Tris, pH 7.4, 0.14M NaCl, 1% bovine serum albumin, 0.05% Tween 20. A 1/250 dilution was used to measure IgA by ELISA following the manufacturer instructions.
DNA Isolation from Mouse Cecal Samples
The cecal content of mice collected post mortem was stored at −80° C. Metagenomic DNA was extracted from the cecal content using a QIAamp-DNA stool mini-kit (Qiagen, Hilden, Germany) according to manufacturer's instructions.
Measurement of Endocannabinoids Intestinal Levels
Ileum tissues were homogenised in CHCl3 (10 ml), and deuterated standards were added. The extraction and the calibration curves were generated as previously described (Muccioli et al, Mol Syst Biol, 2010, 6, 392), and the data were normalised by tissue sample weight.
qPCR: Primers and Conditions
The primers and probes used to detect Akkermansia muciniphila were based on 16S rRNA gene sequences: F-Akkermansia muciniphila CCTTGCGGTTGGCTTCAGAT (SEQ ID NO: 29), R-Akkermansia muciniphila CAGCACGTGAAGGTGGGGAC (SEQ ID NO: 30). Detection was achieved with StepOnePlus™ real-time PCR system and software (Applied Biosystems, Den Ijssel, The Netherlands) using Mesa Fast gPCR™ (Eurogentec, Seraing, Belgium) according to the manufacturer's instructions Each assay was performed in duplicate in the same run. The cycle threshold of each sample was then compared to a standard curve (performed in triplicate) made by diluting genomic DNA (five-fold serial dilution) (DSMZ, Braunshweig, Germany). The data are expressed as Log of bacteria/g of cecal content.
MITChip: PCR Primers and Conditions
DNA extracted from cecal contents was analyzed using the Mouse Intestinal Tract Chip (MITChip), a phylogenetic microarray consisting of 3,580 different oligonucleotides probes targeting two hypervariable regions of the 16S rRNA gene (V1 and V6 regions). Analysis of the MITChip were performed as previously described (Everard et al. Diabetes 60, 2775-2786 (2011); Geurts et al. Front Microbiol. 2, 149 (2011)). Briefly, Microbiota analysis was carried out on level 2 corresponding to genus-like level. Multivariate analysis was performed by representational difference analysis (RDA) as implemented in the CANOCO 4.5 software package (Biometris, Wageningen, The Netherlands) on average signal intensities for 99 bacterial groups (levels 2). All environmental variables were transformed as log (1+X). A Monte-Carlo permutation test based on 999 random permutations was used to test the significance. P values<0.05 were considered significant.
Measurement of Plasma Cholesterol
Plasma samples were assayed for cholesterol by measuring cholesterol present after enzymatic hydrolysis of ester cholesterol, using a commercial kit (DiaSys, Condom, France).
Statistical Analysis
Data are expressed as means±s.e.m. Differences between two groups were assessed using the unpaired two-tailed Student's t-test. Data sets involving more than two groups were assessed by ANOVA followed by Newman-Keuls post hoc tests. Correlations were analyzed using Pearson's correlation. Data with different superscript letters are significantly different P<0.05, according to the post-hoc ANOVA statistical analysis. Data were analyzed using GraphPad Prism version 5.00 for windows (GraphPad Software, San Diego, Calif., USA). Results were considered statistically significant when P<0.05.
Results
We found that abundance of A. muciniphila was 3300-fold lower in leptin-deficient obese mice versus their lean littermates (
To address this question, A. muciniphila was orally administered to control or HF-fed mice during four weeks, thereby increasing A. muciniphila (
We found that A. muciniphila treatment normalized diet-induced metabolic endotoxemia, adiposity and adipose tissue CD11c marker (
We next discovered that colonization with A. muciniphila completely reversed diet-induced fasting hyperglycemia, by a mechanism associated with a 40% reduction of hepatic glucose-6-phosphatase expression (
To further elucidate how A. muciniphila colonization affects gut barrier function; we measured the expression of Paneth and epithelial cells antibacterial markers in the ileum. We found that A. muciniphila increased Reg3g (regenerating islet-derived 3-gamma, RegIIIγ) expression under control diet, whereas this effect was not observed in HF-fed mice (
Recent evidences support that interactions between gut microbiota and mucus layer are dynamic systems affecting the biology of mucus barrier (Belzer et al, ISME J 6, 1449-1458 (2012); Johansson et al, Proc Natl Acad Sci USA 108 Suppl 1, 4659-4665 (2011)). Thus we investigated the impact of A. muciniphila colonization on the inner mucus layer thickness. Remarkably, we found a 46% thinner mucus layer in HF-fed mice (
To further demonstrate whether A. muciniphila has to be alive to exert its metabolic effects, we have compared the impact of viable A. muciniphila administration (2·108 bacterial cells suspended in 200 μl of sterile anaerobic phosphate-buffered saline (PBS)) to heat-killed/inactivated A. muciniphila (autoclaving, 15 minutes, 121° C., 225 kPa). We found that viable and metabolically active A. muciniphila counteracted diet-induced metabolic endotoxemia, fat mass development and altered adipose tissue metabolism (
These results thus confirm that that HF diet-induced obesity is associated with changes in gut microbiota composition, however, antimicrobial peptides in the ileum were not affected by the treatments. In contrast, Reg3g expression in colon epithelial cells was significantly reduced by approximately 50% in HF and heat-inactivated A. muciniphila treated mice, whereas metabolically active A. muciniphila treatment completely blunted this effect and increased Reg3g expression upon HF diet (
We then wanted to test if A. muciniphila administration was still efficient during a prolonged high-fat diet treatment (8 weeks) and if the administration of A. muciniphila 3 times a week (instead of daily) was sufficient to protect against diet-induced obesity. The preparations as well as the doses of A. muciniphila were similar to those presented in the protocol using daily oral gavage or metabolically inactivated A. muciniphila.
We found that A. muciniphila treatment reduces body weight gain (
In order to confirm that these results were specific of A. muciniphila, we then treated HF-fed mice with a probiotic (i.e. Lactobacillus plantarum WCFS1). We found that L. plantarum administration did not change fat mass development, adipose tissue metabolism, mucus layer thickness, colon Reg3g mRNA, and metabolic endotoxemia (
Hypercholesterolemia is known as a key factor involved in cardiovascular diseases. We therefore tested the effect of A. muciniphila administration on plasma cholesterol of mice fed a high-fat diet. As shown in
In summary, our findings not only provide substantial insights into the intricate mechanisms by which A. muciniphila regulates the crosstalk between the host and the gut microbiota, but also provide a rationale for considering the development of a treatment using this human mucus-colonizer for the prevention or the treatment of obesity and associated metabolic disorders such as, for example, hypercholesterolemia.
| Number | Date | Country | Kind |
|---|---|---|---|
| PCT/EP2012/073011 | Nov 2012 | EP | regional |
This application is a divisional application of U.S. application Ser. No. 14/443,829, filed May 19, 2015, currently pending; which is a 371 of International Application PCT/EP2013/073972, filed Nov. 15, 2013; which claims priority to PCT/EP2012/073011, filed Nov. 19, 2012. The contents of all above-named applications are incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 14443829 | May 2015 | US |
| Child | 16285088 | US |
