NZ720422B - Process and compositions for achieving mammalian energy balance - Google Patents
Process and compositions for achieving mammalian energy balanceInfo
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- NZ720422B NZ720422B NZ720422A NZ72042215A NZ720422B NZ 720422 B NZ720422 B NZ 720422B NZ 720422 A NZ720422 A NZ 720422A NZ 72042215 A NZ72042215 A NZ 72042215A NZ 720422 B NZ720422 B NZ 720422B
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
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- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/35—Fat tissue; Adipocytes; Stromal cells; Connective tissues
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/04—Anorexiants; Antiobesity agents
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/06—Antihyperlipidemics
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
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- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
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- C12N5/0653—Adipocytes; Adipose tissue
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- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/475—Assays involving growth factors
- G01N2333/51—Bone morphogenetic factor; Osteogenins; Osteogenic factor; Bone-inducing factor
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- G01N2333/515—Angiogenesic factors; Angiogenin
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
Abstract
Disclosed is a method of achieving optimal mammalian energy balance using forskolin on a particular physiological and developmental stage of the mammalian cellular system.
Description
PROCESS AND COMPOSITIONS FOR ACHIEVING MAMMALIAN ENERGY BALANCE
CROSS-REFERENCE TO RELATED PATENT APPLICATIONS
This application is the PCT filing drawing priority from U.S. non-provisional patent application
14936830 filed on November 10, 2015.
BACKGROUND OF THE INVENTION
[Para 001] Field of the invention: The disclosure in general relates to dietary supplements. More
specifically, the present disclosure relates to a method of achieving optimal mammalian energy
balance using forskolin on a particular physiological and developmental stage of the mammalian
cellular system.
[Para 002] Description of Prior Art: Disruption of mammalian energy balance has been implicated as
the cause for worldwide epidemics of metabolic diseases that calls for modifications in life style and
food habits and also therapeutic intervention. Current diet regimens, exercise, health care awareness
or drug strategies however are often unable to tackle homeostasis of energy in the mammalian body
where optimally, a perfect balance between energy accumulation and energy expenditure is sought
(Elattar.S and Satyanarayana, “Can Brown Fat Win the Battle against White Fat?”, J Cell Physiol.
2015 Mar 11, Zafrir B, “Brown adipose tissue: research milestones of a potential player in human
energy balance and obesity”, Horm Metab Res. 2013 Oct;45(11):774-85). An impetus to the
understanding of critical biological processes controlling brown adipocyte activity and differentiation
has been in vogue in view of developing brown adipose tissue (BAT) focussed therapies for energy
homeostasis (Giralt M, “White, brown, beige/brite: different adipose cells for different functions?
Endocrinology. 2013 Sep; 154(9):2992-3000) where undue energy abundance is effectively countered
by optimal energy expenditure. The present disclosure discusses the potential of forskolin to mediate
mammalian energy balance. Accordingly, it is the principle objective of the present invention to
disclose,
A. The ability of forskolin to prevent the formation of lipids within adult adipocytes during the
differentiation of pre-adipocytes to adipocytes wherein the adipogenesis (fat deposition)
inhibition is remarkably enhanced when forskolin is administered (brought into contact) to
pre-adipocytes rather than to mature adipocytes;
B. The ability of forskolin to enhance the expression of secreted factors that selectively recruit
brown adipose tissue (BAT) like bone morphogenetic protein-7 (BMP-7), bone
morphogenetic protein-4 (BMP-4), vascular endothelial growth factor (VEGF-A) and
mitochondrial uncoupling protein (UCP1) wherein said enhanced expression of secreted
factors that selectively recruit brown adipose tissue (BAT) is remarkably more enhanced
when forskolin is administered (brought into contact) to pre-adipocytes than to mature
adipocytes. In other words, forskolin treated pre-adipocytes are selectively able to
differentiate into BAT.
[Para 003] The present invention fulfils the aforesaid objectives and provides further related
advantages.
SUMMARY OF THE INVENTION
[Para 004] In a first embodiment the present invention provides a method comparatively evaluating
adipogenesis inhibition potential, comprising, adding forskolin to pre-adipocytes before
differentiation by the steps of:
a) Seeding mammalian adipocyte precursor cells (pre-adipocytes) in wells of microplates
wherein approximately 60×10 cells are seeded for 48-72 hours to get 70-80% confluence;
b) Adding forskolin at concentrations of 50µg/ml and 100µg/ml respectively in different wells
of the pre-seeded microplates of step a) consisting of undifferentiated pre-adipocytes and
thereafter incubating the pre-adipocytes for 72 hours in a medium added to the wells of the
microplates to form wells treated with forskolin;
c) Incubating the cells treated with forskolin in step b) for 48 hours in a humidified atmosphere
at 37 C with 5% CO and 95% air;
d) Fixing the cells of step c) by adding 100 μl of 10% formalin and staining using the Oil Red O
technique;
e) Reading the optical density of cells of step d) at 492 nm in a microplate reader and expressing
the results as inhibitory concentration (IC50); and
f) Calculating the percentage inhibition of adipogenesis in the cells of steps d) and e) using the
formula, C-T/T×100, wherein C is the absorbance of Oil Red O in
differentiating/undifferentiated cells and T is the absorbance of Oil Red O in sample treated
differentiating/undifferentiated cells;
adding forskolin to mature adipocytes by the steps of:
g) Seeding mammalian adipocyte precursor cells (pre-adipocytes) in wells of microplates
wherein approximately 60×10 cells are seeded for 48-72 hours to get 70-80% confluence;
h) After 72 hours of incubation of the pre-adipocytes in a medium added to the wells of the
microplates, adding graded concentrations of forskolin of 50µg/ml and 100µg/ml to the wells
of step g);
i) Incubating the cells treated with forskolin in step h) for 48 hours in a humidified atmosphere
C with 5% CO and 95% air;
at 37 2
j) Fixing the cells of step i) by adding 100 μl of 10% formalin and staining using the Oil Red O
technique;
k) Reading the optical density of cells of step j) at 492 nm in a microplate reader and expressing
the results as inhibitory concentration (IC50) values; and
l) Calculating the percentage inhibition of adipogenesis in the cells of steps j) and k) using the
formula, C-T/T×100, wherein C is the absorbance of Oil Red O in
differentiating/undifferentiated cells and T is the absorbance of Oil Red O in sample treated
differentiating/undifferentiated cells; and
m) Comparing the percentage inhibition of adipogenesis in the cells of steps f) and l).
[Para 004a] In a second embodiment the present invention provides a method of promoting the
expression of secreted factors that selectively recruit brown adipose tissue (BAT) said method
comprising:
a) Seeding mammalian adipocyte precursor cells (pre-adipocytes) in wells of
microplates wherein approximately 60×10 cells are seeded for 48-72 hours to get 70-80%
confluence;
b) Adding forskolin at concentrations of 50µg/ml and 100µg/ml respectively in different
wells of the pre-seeded microplates of step a) consisting of undifferentiated pre-adipocytes
and therefore incubating the pre-adipocytes for 72 hours in a medium added to the wells of
the microplates to form cells treated with forskolin;
c) Incubating the cells treated with forskolin in step b) for 48 hours in a humidified
atmosphere at 37 C with 5% CO and 95% air; and
d) Quantitatively determining by immunoassay techniques the expressions of bone
morphogenic protein-7 (BMP-7), bone morphogenic protein-4 (BMP-4), and vascular
epithelial growth factor-A (VEGF-A) in the cell supernatant; wherein
the secreted factors that selectively recruit brown adipose tissue (BAT) comprise one
or more members selected from the group consisting of BMP-7, BMP-4 and VEGF-A, and
the expression of the secreted factors that selectively recruit brown adipose tissue
(BAT) is enhanced as measured when forskolin is administered to pre-adipocytes compared to mature
adipocytes.
[Para 004b] Also described is,
(A) The ability of forskolin to prevent the formation of lipids within adult adipocytes during
the the differentiation of pre-adipocytes to adipocytes wherein the adipogenesis (fat
deposition) inhibition is remarkably more enhanced when forskolin is administered
(brought into contact) to pre-adipocytes than to mature adipocytes;
(B) The ability of forskolin to enhance the expression of secreted factors that selectively
recruit brown adipose tissue (BAT) like bone morphogenetic protein-7 (BMP-7), bone
morphogenetic protein-4 (BMP-4), vascular endothelial growth factor-A (VEGF-A) and
mitochondrial uncoupling protein (UCP1) wherein said enhanced expression of secreted
factors that selectively recruit brown adipose tissue (BAT) is remarkably enhanced when
forskolin is administered (brought into contact) to pre-adipocytes than to mature
adipocytes. In other words, forskolin treated pre-adipocytes are selectively able to
differentiate into BAT.
[Para 005] The advantages of the present invention includes the demonstration of a method to achieve
mammalian energy balance using forskolin on a particular physiological and developmental stage of
the mammalian cellular system wherein forskolin evinces increased potential to (i) inhibit
adipogenesis; and (ii) enhance the expression of secreted factors that selectively recruit brown adipose
tissue (BAT) like bone morphogenetic protein-7 (BMP-7), bone morphogenetic protein-4 (BMP-4),
vascular endothelial growth factor-A (VEGF-A) and mitochondrial uncoupling protein (UCP1) when
brought into contact or administered to pre-adipocytes rather than to mature adipocytes.
[Para 006] Other features and advantages of the present invention will become apparent from the
following more detailed description, taken in conjunction with the accompanying images, which
illustrate, by way of example, the principle of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[Para 007] Fig.1 shows the graphical representation of BMP-7 in cell culture supernatant of cultured
3T3-L1 adipocytes when forskolin (50µg/ml and 100µg/ml) are respectively added before the
differentiation of pre-adipocytes to adipocytes and after the differentiation of pre-adipocytes to
adipocytes.
[Para 008] Fig.2 shows the graphical representation of BMP-4 in cell culture supernatant of cultured
3T3-L1 adipocytes when forskolin (50µg/ml and 100µg/ml) are respectively added before the
differentiation of pre-adipocytes to adipocytes and after the differentiation of pre-adipocytes to
adipocytes.
[Para 009] Fig.3 shows the graphical representation of VEGF-A in cell culture supernatant of cultured
3T3-L1 adipocytes when forskolin (50µg/ml and 100µg/ml) are respectively added before the
differentiation of pre-adipocytes to adipocytes and after the differentiation of pre-adipocytes to
adipocytes.
[Para 0010] Fig.4 shows the graphical representation of UCP1 in cell culture supernatant of cultured
3T3-L1 adipocytes when forskolin (50µg/ml and 100µg/ml) are respectively added before the
differentiation of pre-adipocytes to adipocytes and after the differentiation of pre-adipocytes to
adipocytes.
DETAILED DESCRIPTION OF THE MOST PREFERRED EMBODIMENTS
(Figs. 1, 2, 3 and 4)
[Para 0011] In the most preferred embodiment, the present disclosure relates to a method of achieving
mammalian energy balance using forskolin in a process of adipogenesis inhibition wherein forskolin
is added seperately to pre-adipocytes before differentiation and also to mature adipocytes to
comparatively evaluate adipogenesis inhibition potential of, said process comprising steps of:
a) Seeding mammalian adipocyte precursor cells (pre-adipocytes) in wells of microplates
wherein approximately 60×10 cells are seeded for 48-72 hours to get 70-80% confluence;
b) Adding forskolin at concentrations of 50µg/ml and 100µg/ml in the pre-seeded microplates of
step a consisting of undifferentiated pre-adipocytes;
c) Adding 200 μl of freshly prepared Adipogenesis induction medium to the wells;
d) Adding 200 μl of freshly prepared Adipogenesis progression medium after 72 hours of
incubation with the Adipogenesis induction medium in step c;
e) Incubating the cells treated with forskolin (step b), adipogenesis induction medium (step c)
and adipogenesis progression medium (step d) for 48 hours in a humidified atmosphere (37
deg. C.) of 5% CO2 and 95% air;
f) Fixing the cells of step e by adding 100 μl of 10% formalin and staining using the Oil Red O
technique;
g) Reading the optical density of cells of step f at 492 nm in a microplate reader and expressing
the results as inhibitory concentration (IC50) values using the graph pad prism software;
h) Calculating the percentage inhibition of adipogenesis in the cells of steps f and g using the
formula, C-T/T×100, wherein C is the absorbance of Oil Red O in
differentiating/undifferentiated cells and T is the absorbance of Oil Red O in sample treated
differentiating/undifferentiated cells.
i) Adding 200 μl of freshly prepared Adipogenesis induction medium to the wells of step a.
j) Adding 200 μl of freshly prepared Adipogenesis progression medium comprising graded
concentrations of forskolin(50µg/ml and 100µg/ml respectively) to the wells of step i after 72
hours of incubation with the Adipogenesis induction medium;
k) Incubating the cells treated with forskolin (step j), adipogenesis induction medium (step i) and
adipogenesis progression medium (step j) for 48 hours in a humidified atmosphere (37 deg.
C.) of 5% CO2 and 95% air;
l) Fixing the cells of step l by adding 100 μl of 10% formalin and staining using the Oil Red O
technique;
m) Reading the optical density of cells of step m at 492 nm in a microplate reader and expressing
the results as inhibitory concentration (IC50) values using the graph pad prism software;
n) Calculating the percentage inhibition of adipogenesis in the cells of steps m and n using the
formula, C-T/T×100, wherein C is the absorbance of Oil Red O in
differentiating/undifferentiated cells and T is the absorbance of Oil Red O in sample treated
differentiating/undifferentiated cells; and
o) Comparing percentage inhibition of adipogenesis in the cells of steps h and o.
[Para 0012] In another most preferred embodiment, the present disclosure also relates to a method of
mammalian energy balance using forskolin in a process of promoting the expression of secreted
factors that selectively recruit brown adipose tissue (BAT) like bone morphogenetic protein-7 (BMP-
7), bone morphogenetic protein-4 (BMP-4), vascular endothelial growth factor-A (VEGF-A) and
mitochondrial uncoupling protein (UCP1) wherein said expression of secreted factors that selectively
recruit brown adipose tissue (BAT) is remarkably enhanced as measured when forskolin is
administered (brought into contact) to pre-adipocytes than to mature adipocytes, said method
incorporating the steps of ,
a) Seeding mammalian adipocyte precursor cells (pre-adipocytes) in wells of microplates
wherein approximately 60×10 cells are seeded for 48-72 hours to get 70-80% confluence;
b) Adding forskolin at concentrations of 50µg/ml and 100µg/ml in the pre-seeded microplates of
step a consisting of undifferentiated pre-adipocytes;
c) Adding 200 μl of freshly prepared Adipogenesis induction medium to the wells;
d) Adding 200 μl of freshly prepared Adipogenesis progression medium after 72 hours of
incubation with the Adipogenesis induction medium in step c;
e) Incubating the cells treated with forskolin (step b), adipogenesis induction medium (step c)
and adipogenesis progression medium (step d) for 48 hours in a humidified atmosphere (37
deg. C.) of 5% CO2 and 95% air;
f) Quantitatively determining by appropriate immunoassay techniques the expressions of BMP-
7, BMP-4, VEGF-A and UCP-1 in the cell supernatant;
g) Adding 200 μl of freshly prepared Adipogenesis induction medium to the wells of step a;
h) Adding 200 μl of freshly prepared Adipogenesis progression medium comprising graded
concentrations of forskolin(50µg/ml and 100µg/ml respectively) to the wells of step g after 72
hours of incubation with the Adipogenesis induction medium;
i) Incubating the cells treated with forskolin (step h), adipogenesis induction medium (step g)
and adipogenesis progression medium (step h) for 48 hours in a humidified atmosphere (37
deg. C.) of 5% CO2 and 95% air; and
j) Quantitatively determining by appropriate immunoassay techniques the expressions of BMP-
7, BMP-4, VEGF-A and UCP-1 in the cell supernatant.
[Para 0013] In yet another most preferred embodiment, the present disclosure relates to a method of
achieving energy balance in mammalian adipose cellular systems, said method comprising step of
administering forskolin in effective amounts targeted towards mammalian pre-adipocytes to achieve
effects of (a) increased inhibition of adipogenesis and (b) increased expression of secretory factors
that function individually or in combination to specifically recruit brown adipocytes or brown like
(beige or brite) adipocytes. In specific embodiments, the secretory factors are selected from the group
consisting of bone morphogenetic protein-7 (BMP-7), bone morphogenetic protein-4 (BMP-4),
vascular endothelial growth factor-A (VEGF-A) and mitochondrial uncoupling protein (UCP1).
[Para 0014] In yet another most preferred embodiment, the disclosure pertains to forskolin for use in
therapy for obesity wherein said therapy involves achieving energy balance in mammalian adipocytes
by administering forskolin in effective amounts targeted towards mammalian pre-adipocytes to bring
about the effects of (a) increased inhibition of adipogenesis and (b) increased expression of secretory
factors that function individually or in combination to specifically recruit brown adipocytes or brown
like (beige or brite) adipocytes. In specific embodiments, the secretory factors are selected from the
group consisting of bone morphogenetic protein-7 (BMP-7), bone morphogenetic protein-4 (BMP-4),
vascular endothelial growth factor-A (VEGF-A) and mitochondrial uncoupling protein (UCP1)
[Para 0015] Also described is a method to induce the brown like phenotype (beige or brite adipocytes)
in white adipocyte depots in mammals said method comprising step of administering effective amount
of forskolin to obese mammals with depots of fully differentiated white adipocytes to achieve effect
of increase in secretory factors that bring about the development of brown like phenotype (beige or
brown adipocytes) within white adipocyte depots. In specific embodiment, the secretory factors are
vascular endothelial growth factor-A (VEGF-A) and mitochondrial uncoupling protein (UCP1). Also
described is Forskolin for use in the therapy of obesity characterised in that forskolin is administered
in effective amounts targeting mammalian white adipocyte depots to achieve effect of increased
expression of secretory factors vascular endothelial growth factor-A (VEGF-A) and mitochondrial
uncoupling protein (UCP1) that cause the development of brown like phenotype (beige or brite
adipocytes) in white adipocyte depots in mammals.
[Para 0016] Also described is a method of achieving energy balance in mammalian adipose cellular
systems, said method comprising step of administering forskolin in effective amounts targeted
towards mammalian pre-adipocytes to bring about of the effect of enhanced expression of
mitochondria uncoupling protein 1 (UCP-1) to result in increased mitochondrial thermiogenesis in
differentiated brown adipocytes and brown like (beige or brite) adipocytes.
[Para 0017] Also described is forskolin for use in therapy for obesity wherein said therapy involves
achieving energy balance in mammalian adipocytes by administering forskolin in effective amounts
targeted towards mammalian pre-adipocytes to bring about the effects of enhanced expression of
secretory factor mitochondria uncoupling protein 1 (UCP-1) to result in increased mitochondrial
thermiogenesis in differentiated brown adipocytes and brown like (beige or brite) adipocytes.
ILLUSTRATIVE EXAMPLES
[Para 0018] As illustrative examples of the most preferred embodiments outlined herein above in
paragraphs [0011]-[0017], the following results are presented to show that forskolin when
administered in increasing concentration is more effective in (a) preventing adipogenesis and (b) also
in promoting the expression of secreted factors like BMP-7, BMP-4, VEGF-A and UCP-1 that recruit
the brown adipocytes thereby creating energy balance in mammalian cell systems, when administered
at the pre-adipocyte stage than once the transformation of pre-adipocytes to adipocytes has occurred.
RESULT 1-Prevention of adipogenesis
Table A
CONCENTRATION (µg/ml) % inhibition of adipogenesis % inhibition of adipogenesis
when forskolin is added at the when forskolin is added after
pre-adipocyte stage (before differentiation of pre-adipocytes
differentiation into the to the adipocyte stage
adipocyte stage)
6.25 10.2 1.2
12.50 12.8 6.8
19.7 10.6
50 35.5 12.9
100 41.8 18.5
[Para 0019] Table A shows that at each tested concentration of forskolin, the administration of
forskolin at the mammalian pre-adipocyte stage has a profound effect on preventing adipogenesis that
when administered after the differentiation of pre-adipocytes to adipocytes. Double or more than
double the % inhibition of adipogenesis was observed when forskolin was administered at the pre-
adipocyte stage as compared to administration at the adipocyte stage.
RESULT 2-Expression of secretory proteins that recruit brown adipocytes
A. BMP-7
[Para 0020] The biological role of BMP-7 as a recruiter of the brown adipocyte lineage has been
discussed in the following scientific literature.
1. Mathew Harms and Patrick Seale, “Brown and beige fat: development, function and
therapeutic potential”, Nature Medicine, Volume 19, Number 10, October 2013, pages
1252-1263;
2. BMP7 Activates Brown Adipose Tissue and Reduces Diet-Induced Obesity at Sub
thermoneutrality .Mariëtte R. Boon Published: September 16, 2013; PLOS One.
3. New role of bone morphogenetic protein 7 in brown adipogenesis and energy
expenditure. Tseng et al. Nature. 2008 Aug 21; 454(7207):1000-4. doi:
.1038/nature07221.
4. Transcriptional Control of Brown Fat Development; Kajimure et al. Cell Metabolism;
Volume 11, Issue 4, 7 April 2010, Pages 257–262.
[Para 0021] Immunoassays (Enzyme linked immunosorbent assay) for the quantification of BMP-7 in
the cell culture supernatant when forskolin (50µg/ml and 100 µg/ml) was administered at the pre-
adipocyte stage and once the differentiation to adipocytes occurred indicated that forskolin profoundly
increased BMP-7 expression in at the pre-adipocyte stage than at the adipocyte stage.
[Para 0022] Thus, in correlation with the literature cited above, it may be deduced that forskolin
evinces greater potential for brown fat conversion of pre-adipocytes (Fig.1) rather than fully
differentiated white adipocytes.The example exemplified by Fig.1 provides substantiation to the
disclosed most preferred embodiment that forskolin directs the selective differentiation of mammalian
pre-adipocytes to brown adipocytes by allowing the expression of secretory factor BMP-7.
B. BMP-4
[Para 0023] Acting along with BMP-7, BMP-4 is a new adipokine and acts on adipogenesis and white
to brown transition (Qian S W et al Proc Natl Acad Sci USA 110: E798-807, 2013). Immunoassays
(Enzyme linked immunosorbent assay) for the quantification of BMP-4 in the cell culture supernatant
when forskolin (50µg/ml and 100µg/ml) was administered at the pre-adipocyte stage and once the
differentiation to adipocytes occurred indicated that forskolin profoundly increased BMP-4 expression
in at the pre-adipocyte stage than at the adipocyte stage.
[Para 0024] Thus, in correlation with the literature cited above, it may be deduced that forskolin
evinces greater potential for conversion of white pre-adipocytes to the brite/beige adipocyte (brown
adipocyte like) (Fig.2) by the combined increased expressions and biological actions of secretory
factors BMP-4 and BMP-7. The example exemplified by Fig.2 provides substantiation to the most
preferred embodiment that forskolin brings about the transformation of white pre-adipocytes to brite
or beige adipocytes.
C. VEGF-A
[Para 0025] VEGF-A over expression leads to an increase in brown adipose tissue (BAT)
thermogenesis and also promotes a “BAT-like” phenotype in white adipose tissue depots. In diet-
induced obese mice, introducing VEGF-A locally in BAT rescues capillary rarefaction, ameliorates
brown adipocyte dysfunction, and improves deleterious effects on glucose and lipid metabolism
caused by a high-fat diet challenge. These results demonstrate a direct positive role of VEGF-A in the
activation and expansion of BAT. VEGF-A over expression also exerts its action on macrophages by
increasing the recruitment of M2 anti-inflammatory macrophages to fat depots. The decreased obesity
and the anti-inflammatory milieu induced by VEGF-A in adipose tissue is responsible for the
reduction of insulin resistance in transgenic mice (Bagchi et al, “Vascular endothelial growth factor is
important for brown adipose tissue development and maintenance”, FASEB J. 27, 3257-3271 (2013).
Immunoassays (Enzyme linked immunosorbent assay) for the quantification of VEGF-A in the cell
culture supernatant when forskolin (50µg/ml and 100µg/ml) was administered at the pre-adipocyte
stage and once the differentiation to adipocytes occurred indicated that forskolin profoundly increased
VEGF-A expression in the pre-adipocyte stage than at the adipocyte stage. Thus, in correlation with
the literature cited above, it may be deduced that forskolin evinces greater potential for conversion of
white pre-adipocytes to the brown adipocyte like (brite or beige) cells (Fig. 3) among white adipocyte
depots in the mammalian body.
D. Uncoupling Protein-1(UCP-1)
[Para 0026] A system of thermogenesis that evolved to protect the body from hypothermia is based
upon the uncoupling of oxidative phosphorylation in brown adipocytes by the mitochondrial
uncoupling protein (UCP-1). It has been shown that up-regulation of UCP1 by genetic manipulations
or pharmacological agents can reduce obesity and improve insulin sensitivity (International Journal of
Obesity (2008) 32, S32–S38; doi:10.1038/ijo.2008.236 UCP1: its involvement and utility in obesity.
L P Kozak and R Anunciado-Koza). Immunoassays (Enzyme linked immunosorbent assay) for the
quantification of UCP-1 in the cell culture supernatant when forskolin (50µg/ml and 100µg/ml) was
administered at the pre-adipocyte stage and once the differentiation to adipocytes occurred indicated
that forskolin profoundly increased UCP-1 expression in the pre-adipocyte stage than at the adipocyte
stage. Thus, in correlation with the literature cited above, it may be deduced that forskolin evinces
greater potential for conversion of pre-adipocytes to the BAT-like or brown adipocytes and enhanced
UCP-1 expression in these cells can be expected to enhance bringing about energy balance through
appropriate energy expenditure (Fig. 4).
[Para 0027] It is already reported that administration of forskolin in humans apparently does not cause
clinically significant side effects (Shonteh Henderson et al, Effect of Coleus forskolii supplementation
on body composition and haematological profiles in mildly overweight women, J Int Soc Sports Nutr.
2005; 2(2): 54–62). The study elucidates that supplementation with forskolin dietary supplement
Forslean® [250 mg of 10% Coleus forskolii extract, 25 mg of forskolin] two times a day for 12 weeks
apparently had no clinical side effects. It may thus be inferred that the illustrative in-vitro examples
included herein above to achieve energy balance in mammalian adipocyte systems is also applicable
in vivo studies in animals (mammals) including human beings.
[Para 0028] Although the foregoing invention has been described in some detail by way of illustration
and example for purposes of clarity of understanding, it will be obvious that certain changes and
modifications may be practiced within the scope of the appended claims.
Claims (2)
1. A method comparatively evaluating adipogenesis inhibition potential, comprising, adding forskolin to pre-adipocytes before differentiation by the steps of: a) Seeding mammalian adipocyte precursor cells (pre-adipocytes) in wells of microplates wherein approximately 60×10 cells are seeded for 48-72 hours to get 70-80% confluence; b) Adding forskolin at concentrations of 50µg/ml and 100µg/ml respectively in different wells of the pre-seeded microplates of step a) consisting of undifferentiated pre-adipocytes and thereafter incubating the pre-adipocytes for 72 hours in a medium added to the wells of the microplates to form wells treated with forskolin; c) Incubating the cells treated with forskolin in step b) for 48 hours in a humidified atmosphere C with 5% CO and 95% air; at 37 2 d) Fixing the cells of step c) by adding 100 μl of 10% formalin and staining using the Oil Red O technique; e) Reading the optical density of cells of step d) at 492 nm in a microplate reader and expressing the results as inhibitory concentration (IC50); and f) Calculating the percentage inhibition of adipogenesis in the cells of steps d) and e) using the formula, C-T/T×100, wherein C is the absorbance of Oil Red O in differentiating/undifferentiated cells and T is the absorbance of Oil Red O in sample treated differentiating/undifferentiated cells; adding forskolin to mature adipocytes by the steps of: g) Seeding mammalian adipocyte precursor cells (pre-adipocytes) in wells of microplates wherein approximately 60×10 cells are seeded for 48-72 hours to get 70-80% confluence; h) After 72 hours of incubation of the pre-adipocytes in a medium added to the wells of the microplates, adding graded concentrations of forskolin of 50µg/ml and 100µg/ml to the wells of step g); i) Incubating the cells treated with forskolin in step h) for 48 hours in a humidified atmosphere at 37 C with 5% CO and 95% air; j) Fixing the cells of step i) by adding 100 μl of 10% formalin and staining using the Oil Red O technique; k) Reading the optical density of cells of step j) at 492 nm in a microplate reader and expressing the results as inhibitory concentration (IC50) values; and l) Calculating the percentage inhibition of adipogenesis in the cells of steps j) and k) using the formula, C-T/T×100, wherein C is the absorbance of Oil Red O in differentiating/undifferentiated cells and T is the absorbance of Oil Red O in sample treated differentiating/undifferentiated cells; and m) Comparing the percentage inhibition of adipogenesis in the cells of steps f) and l).
2. A method of promoting the expression of secreted factors that selectively recruit brown adipose tissue (BAT) said method comprising: a) Seeding mammalian adipocyte precursor cells (pre-adipocytes) in wells of microplates wherein approximately 60×10 cells are seeded for 48-72 hours to get 70-80% confluence; b) Adding forskolin at concentrations of 50µg/ml and 100µg/ml respectively in different wells of the pre-seeded microplates of step a) consisting of undifferentiated pre-adipocytes and therefore incubating the pre-adipocytes for 72 hours in a medium added to the wells of the microplates to form cells treated with forskolin; c) Incubating the cells treated with forskolin in step b) for 48 hours in a humidified C with 5% CO and 95% air; and atmosphere at 37 2 d) Quantitatively determining by immunoassay techniques the expressions of bone morphogenic protein-7 (BMP-7), bone morphogenic protein-4 (BMP-4), and vascular epithelial growth factor-A (VEGF-A) in the cell supernatant; wherein the secreted factors that selectively recruit brown adipose tissue (BAT) comprise one or more members selected from the group consisting of BMP-7, BMP-4 and VEGF-A, and the expression of the secreted factors that selectively recruit brown adipose tissue (BAT) is enhanced as measured when forskolin is administered to pre-adipocytes compared to mature adipocytes. Fig.1 ■ ForskolinSOpg/ml Forskolin 100pg/ml ^ 200 & 160 | 120 80 i £ 40 I Addition of forskolin before the Addition of forskolin after the differentiation of pre-adipocytes differentiation of pre-adipocytes to to adipocytes adipocytes Page 1 of 4
Applications Claiming Priority (3)
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US14/936,830 US10085963B2 (en) | 2015-11-10 | 2015-11-10 | Process and compositions for achieving mammalian energy balance |
US14/936830 | 2015-11-10 | ||
PCT/US2015/060176 WO2017082896A1 (en) | 2015-11-10 | 2015-11-11 | Process and compositions for achieving mammalian energy balance |
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NZ720422B true NZ720422B (en) | 2019-12-03 |
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