1. Metabolism and Cancer (continue)

2. glioblastoma

4. Darwin: genetics,Nuclear somatic mutation
Cancer progression is more consistent with Lamarckian than Darwinian evolution
Darwin: genetics,Nuclear somatic mutation
Lamarck:epigenetics, inheritance of acquired adaptability.
Mitochondria,cytoplasmic inheritance
这些后天获得的性状能够遗传给后代（即获得性遗传），这样经过一代代的积累，就会形成生物的新类型。

5. Ketoacidosis>15 mmol
The ketogenic diet is a high-fat, adequate-protein, low-carbohydrate diet that in medicine is used primarily to treat difficult-to-control (refractory) . Most dietary fat is made of molecules called long-chain triglycerides (LCTs). However, medium-chain triglycerides (MCTs)—made from fatty acids with shortercarbon chains than LCTs—are more ketogenic. A variant of the classic diet known as the MCT ketogenic diet uses a form of coconut oil, which is rich in MCTs, to provide around half the calories. As less overall fat is needed in this variant of the diet, a greater proportion of carbohydrate and protein can be consumed, allowing a greater variety of food choices.[3][4]

7. Metabolism and epigenetics

8. HAT: Histone acetyltransferase
HDAC: Histone deacetylase

11. Metabolism and Stem Cell

13. Energy metabolism and immunology

16. Exercise and metabolism

17. Skeletal Muscle PGC-1α1 Modulates Kynurenine Metabolism and
Mediates Resilience to Stress-Induced Depression.
Cell, 25 September 2014; DOI: /j.cell

19. L-KYNA (L-kynurenic acid)
L-KYN ( L-kynurenine )

22. PGC-1 and NT-PGC1

23. What’s PGC1 PGC-1 --PPAR  (gamma) coactivator-1
PPAR---Peroxisome proliferator-activated receptor 
PPAR  agonist: Thiazolidinediones(TZDs): Rosiglitazone (Avandia),Pioglitazone (Actos)
From above introdution,we have to know what is PGC1 and what is the PGC1 function ?
PGC1 is PPARg coactivator ,PGC1 means the abbreviation of first capital alphabet of PPARg coactivator.
What is the PPARg,it is one of nuclear receptors.called peroxisome proliferator activated receptor gamma.it is too longer name,
I am sure all of you here know the PPARg agonist,TZD compounds.Thiazolidinediones.Rosiglitazone and pioglitazone
PPARg is like steroid hormone receptor, glucocorticoid ,estrogen,Androgen receptor
They are called insulin sensitizer which can promote blood glucose to enter into cells ,like muscle cells ,liver cells and adipocytes to lower the blood glucose concentration.Muscle ,liver and adipose ,the three organs ,are major organs to dispose glucose (disposition of glucose)
Glitazone: Rosiglitazone,pioglitazone,troglitazone.ciglitazone,englitazone

24. Brown Adipose Tissue (BAT) and White Adipose Tissue (WAT)
Fat is storaged in fat tissue and fat cells which is called adipose tissue and adipocyte.
There are two types of adipose tissue,one is called brown adipose tissue (BAT),another is called white adipose tissue (WAT).
They have different characteristics,or called different functions in the body.
The left carton is BAT ,the cells in BAT is smaller and full of mitochondria.
The right is WAT,the cell size is larger than BAT,filled with fat .
WAT
BAT

25. Functions of White and Brown Adipocytes
The white adipocyte (left) functions as a fuel tank,storage of fat.
And the brown adipocyte (right) is like a furnace,to burn the fat or free fatty acid from white adipose tissue to release energy heat .
So we can summarize that two kink of fat cells have totally different function.

26. PGC1a as a protein docking platform
TRAP/DRIP
TFIID
TFIIE
TFIIA
TFIIB
TFIIF
SRC-1
RNAPII
PGC-1
PPAR
Ligand
RXRa
9-Cis-RA
CBP/p300
TBP
UCP1
PPRE
TATA
PGC1 and PPARg work together cooperatively synergistically to increase the expression of their target genes.
For example,here is UCP1 structural gene,upstream -25 ,promoter,TATA box ,this sequence is for general transcriptional machinery.It will bind TFs (transcriptional factors,TATA binding protein,IIA,B,D,E,F et al).This is just for basal transcription.
There is another upstream sequence ,called enhancer,Here is the PPAR nuclear receptor binding site,named PPRE.
PPAR nuclear binds with RXRa to form a heterodimer ,and sit in PPRE site.they also bind their own ligands ,TZD,or 9-Cis-RA.
Then ,they recruit other coactivators (CBP,SRC,), bridges the transcription factor to the basal transcriptional initiation complex
to enhance the basal transcriptional machinery ,then make higher expression of UCP1.
So this model show that PPARg and PGC1 work together to increase target gene expression.
When PGC1 bound to a transcription factor,recruits histone modifying enzymes,bridges the transcription factor to the basal transcriptional initiation complex and takes part in processing of the target gene mRNA .

27. PGC1a as an inducible protein
Handschin C,Spiegelman B,Endocrine Reviews,2006 December 7

28. and oxidative metabolism
Master regulator of mitochondrial function
and oxidative metabolism
PGPC-1
NRF1 ?
Mitochondrial
Biogenesis,other?
PPAR TR
PPAR
Thermogenesis
Mitochondrial
Biogenesis,
PPAR
NRF
Mitochodrial
biogenesis
Gluconeogenesis
HNF4
FOXO1 GR
NRF,PPAR
MEF2,ERR
Mitonchodrial
Biogenesis,
Slow-twitch fiber
Master Gene:Little Molecules with Big Goals Bert W.O’Malley,Science 2006 Nov.
PGC1a is capable of coactivating nearly all known nuclear receptors,
as well as many other transcription factors.
Bruce M.Spiegelman,et al,PNAS,April 27,2004,vol.101,p6570
PGC-1a was first reported by Dr.Spiegelman group in 1998.now ,It is a well-known coactivator.
In Brown adipose tissue ,it is induced by cold exposure and b-adrenoceptor stimulation.PGC-1a interact with PPARg PPARa and TR to increase adaptive thermogenesis and mitochondrial biogenesis.
In liver,PGC-1a was induced by fasting,and it induces gluconeogenesis.
In Muscle , heart and brain, PGC-1 promotes mitochondrial biogenesis

29. PGC1a and Diseases Obesity: --PGC1a is reduced in WAT
Diabete:-- PGC1a is reduced muscle,but PGC1a is increased in liver and pancreas (rodent models)
Parkinson’s Disease,Alzheimer’s disease,Huntington’s disease

30. PGC1a and Drug Development
Selective PPAR  modulator (SPPAR Ms)
PGC1a plays a crucial role in “selecting” the sebsut of genes that make up a given biological program,e.g.UCP1 and aP2.
TZD: Rosiglitazone (Avandia®) and Pioglitazone (Actos®)
A novel partial agonist of PPAR  recruits PGC1a ,prevents triglyceride accumulation,and potentiates insulin signaling in vitro
---Burgermeister E.et al,Molecular Endocrinology,April 2006,20(4):
Glucocorticoid:Cortisone,Dexamethasone
A novel antiinflammatory maintains glucocorticoid efficacy with
Reduced side effects.
---Coghlan,M.,et al,Molecular Endocrinology,May 2003,17(5):860
aP2(FABP4) is strongly induced by PPARg in WAT,UCP1 are the main PPARg targets in BAT.PGC1a confers transcriptional specificity to PPARg by coactivating this nuclear receptor on the UCP1 but not on the aP2 promoter.
Insulin sensitization and body weight gain (TG storage )
PA-802 ,isoquinoline,preferentially recruits PGC1a/ERRa/OXPHOS-pathway,enhance insulin sensitivity and or energy expenditure.And antagonized rosiglitazone-driven TG accumulation .
AL-438,PGC1a/Glucocordicoid stimulate glucose production in liver ,diabetes and metabolic syndrome X.
GRIP (GR-interacting protein I coactivator play in GR-mediated transcritional repression of the inflammatory mediator collagenase.
Thus,Coregulators may play distinct roles in mediating the positive and negative effects of GCs.
A ligand that altered the interaction between the receptor and specific coactivators may exhibit tissue-and /or gene-specific activity.

31. NT-PGC-1

33. TTG,TTT,TTA,TAA,ATG,TGC,CAT,ATC,TTC,CAG,T
DNA Sequence of NT-PGC1
TTG,TTT,TTA,TAA,ATG,TGC,CAT,ATC,TTC,CAG,T
Leu-Phe-Leu ***
To investigate the effect of leptin on PGC1 expression,I designed experiment to clone the PGC1 .
I found a new transcripts ,not on purpose.At that time I was excited and surprised,I told my mentor the discovery .
Then I focused on this project for three years.
We identified the NTPGC1 from mouse Brown adipose tissue cDNA library one year ago,the primers of RT-PCR was designed based on the known PGC1 5’ and 3’ end sequence.We got a new transcripts containing an extra insertion 31bp between 256 and 233 of PGC1 cDNA.
We analyzed the new transcripts sequence,we found this stop codon “TAA” is in-frame of PGC1 start codon.there is new “open reading frame”,the start codon is same as the PGC1 and stop codon locates in the insertion region.

34. Alternative Splicing of PGC1
DNA 5’ 1 2 3 4 5 6 7 8 9 12 13 10 11 3’
Transcription
hnRNA 5’ 3’
Alternative Splicing
insertion from intron 6
mRNA
31bp 5’ 1 2 3 4 5 6 7 8 9 10 11 12 13 3’ 5’ 1 2 3 4 5 6 7 8 9 10 11 12 13 3’
Translation
Translation
This is the genomic sequence of mouse PGC1,Mouse PGC1 is is composed of 13 exons,encode a 91-kD protein.
This new transcript is resulted from the PGC1 gene alternative splice expression.
The new transcript mRNA contains an 31 bases insertion and has a in-frame stop codon.so this transcript is translated into short form of protein.
It is composed of 270 amino acid residues.
on chromosome 4p15.1 ,approximately 67kb,
According to the ORF ,the MW of NTPGC is supposed to 30Kd. PGC1—100kd.
Pere puigserver and Bruce M Spiegelman identified two isoform of transcription :5Kb and 8Kb in mouse
Esterbauer et al.(1999) cloned the human homolog of PGC1 from kindey tissue.The gene spans a genomic region of approximately 67kb on chromosome 4p15.1,is composed of 13 exons,and encodes a 91kD protein that exhibits 94% amino acid identify with the mouse ortholog.the OPR predicted a protein of 798 amino acids.Two mRNA species of 6.4 and 5.3 kb,which arise through utilization of 2 polyadenylation signals,are transcribed from the TATA-less promoter.
We don’t know the whole length of each transcripts.Pere puigserver reported there are two isoform of transcripts:5Kb and 8kb ,which arise through 2 polyadenylation signals and transcribed from the same TATA-less promoter.
Protein N C N C
PGC1 (797aa)
NTPGC1 (270aa)
Alternative Splicing of PGC1

35. Primers for PGC1 and NT-PGC1
Based on the sequence of these two transcripts,I designed the sepecific primers to detect the expression and localization of NT-PGC1.
The top this pair of primer is for full-length PGC1
And the lower pair of primer is for NT-PGC1.
They have the same 5’forward primer,different 3’reverse primer.so we can predict that the bottom primer PCR product will be a little longer than top primer products.

36. PCR for PGC1 and NT-PGC1 NTPGC1 PGC1a 5’ 3’ RT-PCR 269bp 5’ 3’ 300bp4 5 6 7 8 9 10 11 12 13 5’ 3’
RT-PCR
269bp
PGC1a 5’
insertion from intron 6
31bp 1 2 3 4 5 6 7 8 9 10 11 12 13 3’
300bp
RT-PCR
NTPGC1
300bp
400bp
500bp
200bp
100bp 1 2 3
Lane 1 DNA Marker
Lane 2 PGC1 product
Lane 3 NTPGC1 product
BAT
WAT
105 Kd
35Kd
Protein expresion
Why:run PCR reaction:
Here is the PCR product picture.lane 1 is DNA size marker,lane 2 for PGC1 and Lane 3 is for NT-PGC1.
we can see lane 3 has a little larger size for NT-PGC1 than lane 2 for full-length PGC1.
This picture shows the western blot result,we can see the MW of PGC1 is about 105 Kd and NT-PGC is about 35 Kd.
Conclusion:according to these results,we can confirm that the exist of NT-PGC1
Specific primers for PGC1 and NTPGC1 were designed based on the cDNA sequence and splicing sites.
We selected same 5’-forward primer cross exon 4 and exon 5 for PGC1 and NTPGC1.
The 3’-reverse primer for PGC cross exon7 and 6.The 3’reverse primer for NTPGC cross exon7 and insertion.
This picture shows the PCR products,the size of band is same as the expected MW.NTPGC-1 is little more than PGC1 .
PCR for PGC1 and NT-PGC1

37. Tissue Distribution
To determine the expression pattern of the newly cloned NT-PGC1 expression. We did Tissue distribution.
C57/BL6 white mice ,6-8 weeks age, were sacrificed by CO2 inhalation.Three mice were pooled for each sample.
Total RNA were isolated from different tissues,and Real-time PCR were performed using our designed primers.
We can see that NT-PGC1 has a higher expression in Brain,heart and BAT,lower expression in liver,wat and spleen,et al.
And this picture shows the PGC1 expression pattern as control.it is different from NT-PGC1 expression pattern.
Real-time PCR were performed on RNA isolation from various mouse tissues.PCR primers are same as mentioned above,spaning the exon interface.
NTPGC and PGC share similar mRNA expression pattern.
NTPGC1 is expressed in a highly tissue-selective manner as shown by-Real time PCR.NTPGC1 mRNA is most abundantly present in brown adipose tissue ,muscle,heart and brain,both notable for containing very high concentrations of mitochondria.These results strongly suggest that like PGC1,NTPGC may play an important role in mitochondria function,respiration ,and/or thermogenesis.

38. Western Blotting for NT-PGC1
To confirm the NT-PGC tissue mRNA expression pattern,we did the protein expression pattern by western blotting.
We took the tissues ,and homogenized and run PAGE electrophoresis,and used PGC1 antibody as primary antibody
We can see that NT-PGC1 protein has high content in Brain,BAT and kidney tissues.lower expression in liver.
Conclusion: This picture indicates that protein expression has a similar pattern with mRNA expression .

39. We know that PGC1 is induced by cold exposure,we have a question:Doee NT-PGC1 is induced by cold exposure ,same as pgc1?
We did the cold exposure experiment,place the C57/Bl6 mouse at 4 degree centegrade cold room for 6 hours.
Then sacrificed mouse by CO2 inhalation,took bat tissue,then check the mRNA expression by real time PCR.
We can see that 6 hours cold exposure induced NT-PGC and PGC expression in mouse BAT tissue.This is our expected and predicted result.

40. Protein expression after cold exposure
We also need to check protein expression by cold exposure.
We did the similar experiment,just took tissues and homogenized and test protein by western blotting.
We can see that 4 degree centigrade induced NT and PGC1 protein levels,compared with room temperature.

41. Cellular Localization
To determine the cellular localization of NT-PGC1,
we conducted immunofluorescent experiment.(this experiment was done by similar method with western blotting).
Fixed cells in coverslip with formaldehyde,and make cell membrane permeable with triton X-100,and primary antibody ,incubation overnight at 4 ,and washed cells and add fluorescent (Cy3 or 5 or fitz?)labelled secondary antibody,then check images with fluorescent microscope.
We labelled second antibody with green fluorescent .so we can see that green image is in cytosol,that meams NT-PGC1 mainly locates in cytosol.
B picture indicates that treatment of cells with cAMP analogs affect NT-PGC1 localization.we can see green fluorescent equivalently distribution in nucleus and cytosol.
We treated cells with CAMP analog and PKA inhibitor H89,the green color locates in cytosol, same as control ,this phenomenon demonstrated that NT-PGC1 distribution was controlled by PKA pathway.
D picture shows that cells were treated by cAMP and p38 MAPK inhibitor SB ,the green color is equivalent distribution in cytosol and nucleus. This phenomenon indicates NT-PGC1 cellular localization is independent on p38 MAPK pathway.
Dr.Spiegelman group has early verified that PGC1 locates mainly in nucleus.from above experiment ,we can see the difference between NTPGC1 and PGC1 localization. This has an important physiological function.We conclude that the function of NT-PGC1 is dependent on PKA signalling .
Many physiological response activates intracellular PKA activation.For example,sympathetic norepinepherine? Adrenaline

42. Immortalization of Fat cell
5 day old mouse
Primary cell culture
To explore the function of NT-PGC1 in vitro,
The primary BAT cells were isolated from interscapular BAT ,Immortalized fat cell line was established by retroviral infection of primary fat cells.
Immortalized fat cells can be differentiated into fat cells with more fat droplets.
To isolate the BAT primary cells,the interscapular BAT of 5 day old mouse were minced, and incubated in a digestion solution composed of 0.1% Type IV Collagenase (Boehringer Mannheim), 0.02% DNase I, 0.01% Type VI Protease (Sigma), and 0.1% Trypsin (GIBCO-BRL) at 37° C for 30 min. The tissue/enzyme solution was then centrifuged at 500 × g for 3 min. The supernatant was discarded, The cell pellet was rinsed three times with HBSS, resuspended in complete culture medium, and expanded in 6-well-cell-culture plates.
Infection the primary cells with retrovirus containing SV40 early antigen and select transformed cells by puromycin ,and the immortalization of fat cells was accomplished .
The imrortalized fat cells were induced to differerentiation by the induction medium containing Glucocorticord 1uM Dexamethasone,0.5mM Isobutyl-methyl-Xanthine,125uM Indomethacin,17nM Insulin,and 1nM T3 for 48 hours.
Results:the immortalized fat cell lines were established.
To verify the NTPGC1 expression in vitro during the fat cell differentiation.
SV40-Immortalization
Differentiation

43. Preadipocyte Day4 differentiation Day2 differentiation
These pictures show the morphological change of fat cell during the differentiation.
This is the preadipocyte cell.Day two after differentiation,Day 4,Day 6 and Day 8
Results:We can see fat droplets were appeared more and more during the differentiation processing.

44. Ectopic Expression of NTPGC in FVB cell
Character of FVB transfected stable cell
NTPGC-HA
Empty
PGC-V5
IB: V5
IB:HA
115 Kd
38 Kd
Ectopic Expression of NTPGC in FVB cell
FVB cell
Lentivirus
Transfection
To explore the function of NT-PGC-1,we generated three stably transfected fat cells ,Using Lentivirus system,
One is FVB stably transfected Empty vector genes
One is FVB cell transfected NTPGC-HA gene ,called FVB_NTPGC1 cell line
Another is FVB cell stably transfected PGC-1-V5 gene.
We use WB checking the exogene expression.These result show we have got the expected cell lines.
we made the constructs:NTPGC-HA,PGC-V5,and empty construct as control,then produced lentivirus containing target constructs
Transfected FVB cell line,this cell line is derived from FVB mouse brown fat cell and immortalized with SV40 T-antigen .After lentivirus transfection,cells were selected by puromycin for 10 days.we got the stably transfected FVB cell lines.
Then we did western bloting for confirming the transfected cell line,there is a V5 expression in FVB-PGC
HA in FVB –NTPGC,nothing in FVB-Empty
FVB-Empty
FVB-NTPGC1-HA
FVB-PGC1-V5

45. Western Blotting
To confirm the stably transfected cell line ,we run western blot to check the exogene expression.
These first three cell line are empty vector (control group without exogene)
The middle three cell lines stands for NT-PGC1 ,and last cell lines means PGC1 stably transfected cell lines.compared with control group we can see that pgc1 expression.

46. Induction of UCP1 expression by coactivation of NTPGC-1 and PGC1
UCP1 mRNA fmol /ug RNA
We have known that UCP1 was the first identified PGC-1-coactivating target gene in Brown adipose tissue by Dr.Spiegelman.
To determine whether NTPGC-1 coactivates the transcriptional activity of PPARs on the UCP1 promoter.
We diffentiated these three FVB stable cell lines.On the day 5 of differentiation,the differentiated FVB stable cells were treated with PPARa,RXRa or PPAR g and RXRa agonist cocktail for overnight (16hrs).
Then the cells were harvested , the total cell lysate was subjected to western blotting and real-time PCR.
We can see that there is higher expression of UCP1 in tNTPGC-1 transduced FVB stable cell line at either protein level or mRNA levels than PGC-1 and empty transduced FVB stable cell lines.
This slide indicates that the NTPGC-1 has more effect on the UCP1 expression than PGC-1.
Induction of UCP1 expression by coactivation of NTPGC-1 and PGC1

47. Mitochondrial Biogenesis
To examine mitochondrial copy number and mass among the 3 cell lines,
We measured the ratio of mtDNA to nuclear DNA in each cell line.
Differentiated adipocytes were also imaged with Mitofluor Red 589 ,and fluorescent intensity was measured using fluorescense microscopy with TRITC filters .
In NT-PGC1 versus empty vector transformed adipocytes,the mtDNA/nuDNA ratio was 2.1 fold higher and the Mitofluor red intensity was 1.9 fold higher.
The PGC1 transformed line showed a similar increase in mitochondrial number and mass compared to the empty vector line with both methods.
This data illustrate that NT-PGC1 is also capable of supporting mitochondrial biogenesis in brown adipocytes.

48. To test the function of NT-PGC1 in vivo,We generated adipose tissue-specific and inducible expression of NT-PGC-1 in Transgenic mice.
Tetracycline-sensitive rtTA transcriptional activator only in adipose tissue (aP2-rtTA)
pTRE-NT-PGC-1,the pTRE-NT-PGC-1 promoter is silent in the absence of both rtTA and doxycycline.
Maximal induction of the transgene occurs 4 d after adding doxycylin (0.5 mg/ml) to drinking water.
Tara did the animal experiments,she observed the NT-PGC-1 trangenic mice increased oxygen comsuption.

49. Body Weight and Fat Weight

50. Expression of CPT 1β in Tg mice
After 7 weeks doxycycline treatment,mice were sacrificed ,we used real-time PCR analyzed the fat pad CPT1b expression.
we found CPT1b expression was significantly induced by Doxycycline in the BAT of transgenic mice.
.there are an increased expression of CPT1b in Epidydimal,inguanal,retroperitoneal WAT,but not significantly.

51. NT-PGC-1α （muscle）
Ruas JL,et al., Cell, 2012,151:1319–1331

53. Mitochondria-targeted antioxidants

54. 16,569 bp 13 proteins 2 rRNAs 22 tRNAs LHON Mutation 11,778
Leber hereditary optic neuropathy, LHON
FIGURE 19-38a Mitochondrial genes and mutations. (a) Map of human mitochondrial DNA, showing the genes that encode proteins of Complex I, the NADH dehydrogenase (ND1 to ND6); the cytochrome b of Complex III (Cyt b); the subunits of cytochrome oxidase (Complex IV) (COI to COIII); and two subunits of ATP synthase (ATPase6 and ATPase8). The colors of the genes correspond to those of the complexes shown in Figure Also included here are the genes for ribosomal RNAs (rRNA) and for some mitochondrion-specific transfer RNAs; tRNA specificity is indicated by the one-letter codes for amino acids. Arrows indicate the positions of mutations that cause Leber's hereditary optic neuropathy (LHON) and myoclonic epilepsy and ragged-red fiber disease (MERRF). Numbers in parentheses indicate the position of the altered nucleotides (nucleotide 1 is at the top of the circle and numbering proceeds counterclockwise).
LHON
Mutation
11,778 54

55. Mitochondrial ROS and pathology

56. TPP is a mitochondria-targeted agent
-30~ - 60 mV
△ψm
-150~ -180 mV
Small molecules conjugated to lipophilic cations such as tetraphenylphosphonium (TPP) result in their accumulation in the mitochondrial matrix. Lipophilic conjugated small molecules will first traverse across the plasma membrane and accumulate in the cytosol. This process is driven by the plasma membrane potential (Δψp). Subsequently, the mitochondrial membrane potential (Δψm) will drive the accumulation of these molecules into the mitochondria by several hundredfold.
S.E.Weinberg and N.S.Chandel, Nat Chem Biol. 2015,11(1):9

57. TPP-conjugated mitochondria-targeted compounds
Resveratrol-TPP
TPP-Nanoparticle
S.Marrache and S.Dhar,Chemical Science,2015,6:1832

58. SS peptide ,Szeto and Schiller
SS H-Dmt-D-Arg-Phe-Lys-NH2

59. Berberine Mitochondria-targeted agent
BBR
Berberine (BBR) is an isoquinoline alkaloid isolated from
several Chinese herbal medicines, such as Coptis chinensis,
Berberis aristata, and Coptis japonica. It has been used in traditional
oriental medicine for the treatment of gastroenteritis
and secretory diarrhea [8]. Multiple pharmacological effects
have been attributed to BBR and its related derivatives, such as
antidiarrheic, antimicrobial, anticancer, antiinflammatory, and
antiarrhythmic actions [9–13]. Recently, BBR was identified as
a promising lipid-lowering drug, able to effectively up-regulate
hepatic LDLR expression in liver cells and vascular cells, and
it has been shown to decrease both serum triglycerides and
cholesterol [14–16].
Antonina V.et al.Mitochondrion 2013;13:520

60. Metabolites of BBR through CYP in vivo
Cytochrome P450 CYP
DMB
Y.Li ,J.D.Jiang, J of Translational Medicine,2011,9:62

61. Antioxidant capacity of DMB in vitro

62. Antioxidant capacity of DMB in HepG2
HepG2 cell

63. Mitochondrial distribution of DMB

64. DMB protects liver from chronic ethanol exposure
Pengcheng Zhang,et.al,J Pharmacol Exp Ther,2015,352:139-47

65. DMB supresses ethanol-induced mitochondrial oxidative stress

66. DMB Recovers MMP Flow cytometric histograms by
Rhodamine123 fluorescence

67. DMB ameliorates mitochondrial morphology
Mitochondrial swelling test
Electron microscopy

68. DMB decreases hepatosteatosis

69. DMB increases lipid catabolism

70. DMB inhibits CPY2E1 in mitochondria

71. Summary DMB is a natural mitochondria-targeted antioxidant
DMB is an AMPK agonist
DMB is a potential agent for treating chronic liver diseases (ALD,NAFLD,liver fibrosis)

75. FIGURE 19-1 Biochemical anatomy of a mitochondrion
FIGURE 19-1 Biochemical anatomy of a mitochondrion. The convolutions (cristae) of the inner membrane provide a very large surface area. The inner membrane of a single liver mitochondrion may have more than 10,000 sets of electron-transfer systems (respiratory chains) and ATP synthase molecules, distributed over the membrane surface. The mitochondria of heart muscle, which have more profuse cristae and thus a much larger area of inner membrane, contain more than three times as many sets of electron-transfer systems as liver mitochondria. The mitochondrial pool of coenzymes and intermediates is functionally separate from the cytosolic pool. The mitochondria of invertebrates, plants, and microbial eukaryotes are similar to those shown here, but with much variation in size, shape, and degree of convolution of the inner membrane.

76. Cristae

77. FIGURE 19-18 ROS formation in mitochondria and mitochondrial defenses
FIGURE ROS formation in mitochondria and mitochondrial defenses. When the rate of electron entry into the respiratory chain and the rate of electron transfer through the chain are mismatched, superoxide radical (･O2–) production increases at Complexes I and III as the partially reduced ubiquinone radical (･O–) donates an electron to O2. Superoxide acts on aconitase, a 4Fe-4S protein, to release Fe2+. In the presence of Fe2+, the Fenton reaction leads to formation of the highly reactive hydroxyl free radical (･OH). The reactions shown in blue defend the cell against the damaging effects of superoxide. Reduced glutathione (GSH; see Figure 22-27) donates electrons for the reduction of H2O2 and of the oxidized Cys residues (—S—S—) of enzymes and other proteins, and GSH is regenerated from the oxidized form (GSSG) by reduction with NADPH. 77

78. FIGURE 19-41a Heteroplasmy in mitochondrial genomes
FIGURE 19-41a Heteroplasmy in mitochondrial genomes. (a) When a mature egg cell is fertilized, all of the mitochondria in the resulting diploid cell (zygote) are maternal; none come from the sperm. If some fraction of the maternal mitochondria have a mutant gene, the random distribution of mitochondria during subsequent cell divisions yields some daughter cells with mostly mutant mitochondria, some with mostly wild-type mitochondria, and some in between; thus the daughter cells show varying degrees of heteroplasmy. 78

79. Pathological conditions that involve
oxidative stress
Inflammation
Atherosclerosis
Ischemia/reperfusion injury
Cancer
Aging