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The Chronicles of Berberine
PCSK9, AMPK, and the LDL receptor
Berberine in Lipid Management
JAMES C. ROBERTS MD, FACC, FAARFM
Principal Investigator: EECP Registry Study
MME in diabetic neuropathy and low back pain
Trial to Assess Chelation Therapy
Dal-Outcomes (Dalcetrapib post-ACS)
Relox (Relox in post-CVA strength recovery)
ENGAGE-AF (Edoxaban vs. Warfarin in Atrial Fib)
CIRT (Chronic Inflammation Reduction Trial)
THE CHRONICLES of BERBERINE C. S. Lewis and R. H. Poling
PCSK9, AMPK, and the LDL receptor
Berberine in lipid management
Berberine and the White Witches (Cytokines, FFAs, & IKK) of IR
Resistance Berberine in diabetes management and weight loss
Dr. Poling’s Nephew
Berberine in Inflammation, Auto-Immunity, Malignancy, and Toxicity
Voyage of the Plaque Buster
Berberine in the Treatment of Cardiovascular Disease
DECISION to GENERATE CHOLESTEROL
Decision to Respond to Infection (Real or Perceived):
ROS Inflammatory Cytokines
Cellular Proliferation Immune Upregulation
QUEEN IKB KINASE
Coronary Angiogram
Percutaneous Intervention
Bypass Surgery
Drug Therapy
ALLOPATHIC CARDIOLOGY
Anatomy - Supply and Demand
CORONARY RISK FACTORS - 2013
Non-modifiable risks Modifiable metabolic risks Metabolic toxins
Male sex High LDL(ox) Homocysteine
Advancing age Low HDL Smoking
High Trigs. Trans-Fats
Free radical burden
Inflammatory States Elevated Lipoprotein(a) Iron/ Metal Overload
Th1/Th17 Dysregulation Organic Pollutants
Sleep Apnea Endothelial Dysfunction
Chronic Infection Vitamin deficiency states
Autonomic Dysfunction Anti-oxidant vitamins
Spiritual risk factors Anti-oxidant minerals
Chronic Stress Elevated Viscosity Essential fatty acids 3 & 6
Lack of Social Connectedness Bioflavonoids
Lack of Belief Hormone Deficiency Vitamin K2
Vitamin D
Bioenergetic deficiency Hypertension B Vitamins
Co-Enzyme Q10 Overweight Folic Acid
Carnitine Diabetes Genomic Defects
Magnesium Insulin Insensitivity Methyl Cycle
Vascular Biochemistry and Cell Biology
CORONARY RISK FACTORS - 2013
Non-modifiable risks Modifiable metabolic risks Metabolic toxins
Male sex High LDL(ox) Homocysteine
Advancing age Low HDL Smoking
High Trigs. Trans-Fats
Free radical burden
Inflammatory States Elevated Lipoprotein(a) Iron/ Metal Overload
Th1/Th17 Dysregulation Organic Pollutants
Sleep Apnea Endothelial Dysfunction
Chronic Infection Vitamin deficiency states
Autonomic Dysfunction Anti-oxidant vitamins
Spiritual risk factors Anti-oxidant minerals
Chronic Stress Elevated Viscosity Essential fatty acids 3 & 6
Lack of Social Connectedness Bioflavonoids
Lack of Belief Hormone Deficiency Vitamin K2
Vitamin D
Bioenergetic deficiency Hypertension B Vitamins
Co-Enzyme Q10 Overweight Folic Acid
Carnitine Diabetes Genomic Defects
Magnesium Insulin Insensitivity Methyl Cycle
Vascular Biochemistry and Cell Biology
BERBERINE
Quaternary protoberberine-type alkaloid with a dibenzoa,gquinolizidine ring
MW 353.36 Bright yellow
Found in rhizomes, stem, and bark of medicinal plants
Berberidaceae and Ranunculaceae families
Human dose is 5-20 mg/kg/day (350-1500 mg/day)
Peak level 2.4 hours after 1.2 gm po, and Cmax is 395 ng/ml
LD50 is 400 mg/kg in mice and above 1000 mg/kg in rats
C20H19NO5
Berberine Coptis chinensis French
In TCM, atherosclerosis with phlegm, blood stasis, and toxin accumulation.
Chinese herbs that clear heat and detox may inhibit atherosclerosis
via anti-inflammatory and immunodepressive actions.
C. chinensis: Clear heat & dry dampness; purging fire to eliminate toxin.
USE in TCM and HERBAL MEDICINE
Huanglian (Rhizoma Coptidis or Chinese goldthread)
Huangbai (Cortex Phellodendri )
Umbellatine (Berberine)
Oregon Grape Tree Turmeric Barberry
(Berberis aquifolium) (Indian Barberry)
USE in TCM and MODERN MEDICINE
Huanglian (Rhizoma Coptidis or Chinese goldthread)
“Note of Elite Physicians” by Hongjing Tao 500 AD – Hypoglycemic effect
Shennong’s Herbal : Coptidis to treat dysentery and infectious diarrhea
Use in DM: 1988 report of BBR to treat diarrhea in diabetic pts in China
Kong 2004: LDL receptor upregulation
2013 – Universal value in prevention and treatment of CV disease
BERBERINE and POLING’S POSTULATES
1. The microorganism must be found in abundance in all organisms suffering from the
disease, but should not be found in healthy organisms.
2. The microorganism must be isolated from a diseased organism and grown in pure
culture.
3. The cultured microorganism should cause disease when introduced into a healthy
organism.
4. The microorganism must be reisolated from the inoculated, diseased experimental
host and identified as being identical to the original specific causative agent.
POLING’S POSTULATES for CV NUTRITIONAL MEDICINE
Atherosclerotic Risk Factors and MetS/DM II:
Insulin insensitivity Hyperlipidemia
Overweight Hypertension
Endothelial function and oxidative stress
Inflammation (Th1/Th17 immune dysregulation)
Without damping appropriate immune response
Preferably with an anti-microbial effect
Blunting of auto-immunity and collateral tissue damage
Defined mechanisms at levels of transcription and translation
Limited toxicity and reasonable cost
Synergy with pharmaceutical and mechanical interventions
Published studies to document mechanisms and efficacy
BERBERINE – PHYSIOLOGIC EFFECTS
Complex I inhibition Glycolysis AMP/ATP PKC AMPK
Lipid Metabolism
pHMG Co-A Reductase Decrease cholesterol biosynthesis
LDLR expression ed and PCSK9 ed Decrease in serum LDL
pACC Carboxylase Decrease in FA synthesis and increase in FA oxidation
Decrease in triglycerides
Glucose Metabolism
Insulin receptor expression ed
Insulin resistance due to SFAs, LPS, and Th1 cytokines blunted
Improved insulin sensitivity
and decrease in serum glucose
BERBERINE and AMP–SENSITIVE PROTEIN KINASE
Need for Energy
Oxidative Stress
Don’t Build – Burn
BERBERINE and AMP–SENSITIVE PROTEIN KINASE
Caloric Restriction, Exercise,
Leptin, Adiponectin, Metformin, and
Berberine
Energy (Caloric) Excess
Inflammatory Cytokines
BERBERINE – PHYSIOLOGIC EFFECTS
Complex I inhibition Glycolysis AMP/ATP PKC AMPK
Lipid Metabolism
pHMG Co-A Reductase Decrease cholesterol biosynthesis
LDLR expression ed and PCSK9 ed Decrease in serum LDL
pACC Carboxylase Decrease in FA synthesis and increase in FA oxidation
Decrease in triglycerides
Glucose Metabolism
Insulin receptor expression ed
Insulin resistance due to SFAs, LPS, and Th1 cytokines blunted
Improved insulin sensitivity
and decrease in serum glucose
BERBERINE – PHYSIOLOGIC EFFECTS
Complex I inhibition Glycolysis AMP/ATP PKC AMPK
Weight Physiology
Adipocyte differentiation delayed Smaller cells with healthier Adipokine profile
GI tract flora altered Calories absorbed from CHO blunted
LPS translocation blunted
pACC Carboxylase Increased FA oxidation
Weight reduction
Oxidative Stress
NADPH oxidase is restrained Superoxide production deceased
Uncoupling protein is up regulated ROS production with in SOD expression
Protective vs. ROS/ischemic tissue damage in animal models (protects mitochondria)
Apoptosis of malignant cells enhanced
BERBERINE – PHYSIOLOGIC EFFECTS
Complex I inhibition Glycolysis AMP/ATP PKC AMPK
Endothelial Function
AMPK eNOS NO cGMP vasodilation, platelet, and VSMC effects
NO pIKKB NF-B restrained Th1 cytokine production decreased
ICAM, VCAM, and MCP-1 decreased Mononuclear infiltration blunted
Endothelial progenitor cell count rises and endothelial microparticle count falls
VSMC proliferation and MMP activity blunted
CV Physiology
Endothelial independent vasodilation
ACE Inhibition
Beneficial electrophysiological effects (Ito and Ica currents preserved)
VSMC proliferation is inhibited
Vascular elasticity improves
Autoimmune and Inflammatory Conditions
Antioxidant (indirect) and anti-inflammatory effects demonstrated
Beneficial effects in animal models of auto-immunity
BERBERINE and AMP–SENSITIVE PROTEIN KINASE
ENDOTHELIAL FUNCTION and OUTCOME
Poling’s Dictum Poling’s Corollary
As Goes the Endothelium Control of the Endothelium
So Goes the Patient Controls the CV Density of the Patient
BERBERINE – PHYSIOLOGIC EFFECTS
Complex I inhibition Glycolysis AMP/ATP PKC AMPK
Endothelial Function
AMPK eNOS NO cGMP vasodilation, platelet, and VSMC effects
NO pIKKB NF-B restrained Th1 cytokine production decreased
ICAM, VCAM, and MCP-1 decreased Mononuclear infiltration blunted
Endothelial progenitor cell count rises and endothelial microparticle count falls
VSMC proliferation and MMP activity blunted
CV Physiology
Endothelial independent vasodilation
ACE Inhibition
Beneficial electrophysiological effects (Ito and Ica currents preserved)
VSMC proliferation is inhibited
Vascular elasticity improves
Autoimmune and Inflammatory Conditions
Antioxidant (indirect) and anti-inflammatory effects demonstrated
Beneficial effects in animal models of auto-immunity
BERBERINE – CLINICAL EFFECTS
Complex I inhibition Glycolysis AMP/ATP PKC AMPK
LDL and triglycerides decrease with minimal change in HDL
Glucose falls and insulin sensitivity improves
Weight loss may occur
Elevated BP may fall
Hepatic steatosis improves
Diabetic nephropathy may improve
Endothelial function improves
Post PCI for ACS Lower cytokine elaboration and improved outcome
Heart Failure Rise in EF, functional status, and reduced arrhythmia
Berberine synergizes with and adds to standard pharmaceutical measures
BERBERINE in LOW CV RISK INDIVIDUALS
♥ 137 overtly healthy Italian men and women
Cholesterol 200-240 mg/dl BMI 25-29
Normotensive Non-smokers
1mmol/l (38 mg/dl) drop in LDL decreases 1 and 2 CV event risk by 20%
Drug therapy may not be cost-effective here and has side-effect risk
Will nutritional therapy get the job done at lower cost and lower risk?
BERBERINE in LOW CV RISK INDIVIDUALS
Run-in over six months:
Regular physical activity
50% carb, 30% fat, 20% protein ; chol. < 300 mg & fiber > 35 g/day
Randomize to:
Run-in protocol + placebo
Run-in protocol + Berberine 500 mg twice a day (lunch and dinner)
BERBERINE in LOW CV RISK INDIVIDUALS
166.5
159
154
156
158
160
162
164
166
168
Baseline 6 Month Run-in
Weight (lbs.)
26.8
26.4
26.2
26.3
26.4
26.5
26.6
26.7
26.8
26.9
Baseline 6 Month Run-in
BMI (kg/m2)
92
164
98 90
159
85
0
20
40
60
80
100
120
140
160
180
FBS LDL Triglycerides
Fasting Glucose & Lipid Values
Baseline 6 Month Run-in
BERBERINE in LOW CV RISK INDIVIDUALS
166.5
159
155.3 156 156
148
150
152
154
156
158
160
162
164
166
168
Baseline 6 MonthRun-in
3 MonthsBerberine
Washout 2nd 3 MonthsBerberine
Weight (lbs.)
26.8
26.4
25 25.1 25.1
24
24.5
25
25.5
26
26.5
27
Baseline 6 MonthRun-in
3 MonthsBerberine
Washout 2nd 3 MonthsBerberine
BMI (kg/m2)
BERBERINE in LOW CV RISK INDIVIDUALS
92
90 90
92
89
87.5
88
88.5
89
89.5
90
90.5
91
91.5
92
92.5
Baseline 6 MonthRun-in
3 MonthsBerberine
Washout 2nd 3 MonthsBerberine
Fasting Blood Glucose (mg/dl)
164 157
133
174
134
0
50
100
150
200
Baseline 6 MonthRun-in
3 MonthsBerberine
Washout 2nd 3 MonthsBerberine
LDL Cholesterol (mg/dl)
98
85
67
96
72
0
20
40
60
80
100
120
Baseline 6 MonthRun-in
3 MonthsBerberine
Washout 2nd 3 MonthsBerberine
Triglycerides (mg/dl)
CHOLESTEROL LEVEL and FUTURE MORTALITY
Risk of cerebral hemorrhage is inversely related to serum cholesterol
Japanese researchers reported in 1971
Confirmed in the US
In CHF, outcome inversely related to cholesterol level
45
28 25
23
0
5
10
15
20
25
30
35
40
45
50
< 172 172-202 202-234 > 235
Quartiles of Serum Cholesterol
Deaths per 100 person-years
CHOLESTEROL LEVEL and FUTURE MORTALITY
Risk of cerebral hemorrhage is inversely related to serum cholesterol
Japanese researchers reported in 1971
Confirmed in the US
National Heart, Lung, Blood Institute study in 1992
Analyzed nineteen prospective cohort studies (9-30 year follow-up)
Cardiovascular death rate
Overall death rate
Death related to non-cardiovascular disease conditions
Optimal range for cholesterol was 160-199 mg/dl
Calculate long-term risk relative to reference range for cholesterols:
♦ < 160 ♦ 200 - 239 ♦ > 240
CHOLESTEROL LEVEL and MORTALITY
SYSTEMIC CHOLESTEROL METABOLISM
SYSTEMIC CHOLESTEROL METABOLISM
HEPATIC CHOLESTEROL METABOLISM
LDL Receptor
Synthesis and Expression of the LDLR Hepatic Need for Cholesterol
HEPATIC CHOLESTEROL METABOLISM
SCAP – SREBP – SRE Interaction
SCAP (SREBP Cleavage Activating Protein) conditional chaperone of SREBP
SREBP (Sterol Regulatory Element Binding Protein) binds to SRE
SRE (Sterol Regulatory Element ) promoter site for transcription of LDLR
LDLR Synthesis Hepatic Need for Cholesterol
HEPATIC CHOLESTEROL METABOLISM
LDLR Synthesis Hepatic Need for Free Cholesterol
HEPATIC CHOLESTEROL METABOLISM
SREBP – SRE Interaction
LDL Receptor
HMG Co-A Reductase
PCSK9 (Proprotein Convertase Subtilisin/Kexin type 9)
HEPATIC CHOLESTEROL METABOLISM
PCSK9
HEPATIC CHOLESTEROL METABOLISM
PCSK9
System designed for primitive man
Primary cause of hepatic hypolipidemia was dietary insufficiency
PCSK9 defeats drug approaches to lipid reduction in modern man
HEPATIC CHOLESTEROL METABOLISM
PCSK9 Up Regulation
Gain of function SNIP – 3rd gene of Familial Hyperlipidemia
Any cause of hepatic hypolipidemia:
Decreased intake or absorption
HMG Co-A Reductase inhibition (Statins, RYRE, Policosanol)
PCSK9 Down Regulation
Loss of function SNIP – Low LDL and reduced CV risk
Monoclonal Ab vs. PCSK9
Berberine
FOAM CELLS
LDL is FOOD
MONOCYTE CHOLESTEROL METABOLISM
SREBP – SCAP – SRE Interactions
High intracellular free cholesterol:
LDLR synthesis and expression blunted
Cholesterol synthesis turned off
MONOCYTE CHOLESTEROL METABOLISM
SREBP – SCAP – SRE – LDLR interaction deals with native LDL
Altered (oxidized, glycated, acetylated) LDL internalized via:
Scavenger receptor
CD 36 receptor
LOX receptor
MONOCYTE CHOLESTEROL METABOLISM
SREBP – SCAP – SRE – LDLR negative feedback system
Bypassed (down regulated) by inflammation
Cross talk between:
IKK-NF-B
SCAP-SREBP
Low Chol LPS LDL LPS + LDL
CHOLESTEROL SYNTHETIC DECISION
PERCEIVED INFECTION HYPERLIPIDEMIA
1.7
2.8
4.1
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
Minimal Dz Moderate Dz Severe Dz
C-Reactive Protein in Periodontal Disease
115 117
121
126 128
105
110
115
120
125
130
Zero One Two Three Four
CPITN Score
LDL Cholesterol (mg/dl)
Th1 Cytokine Elaboration:
Il-6 Il-1 TNF-
Hyperlipidemia and Hyperglycemia
AMP SENSITIVE PROTEIN KINASE
BERBERINE and AMP SENSITIVE PROTEIN KINASE
♥ L6 myotubes
Incubates with:
Vehicle Ionomycin (positive control) Berberine
Measure :
AMPK and pAMPK ACC and pACC
BERBERINE and AMP SENSITIVE PROTEIN KINASE
♥ Rat muscle mitochondria
Measure oxygen consumption with Complex I substrate pyruvate
No effect on Complex-II related respiration with substrate succinate
BERBERINE and AMP SENSITIVE PROTEIN KINASE
♥ Male Syrian golden hamsters
All receive high fat diet (0.12% cholesterol and 10% coconut oil) for two weeks
Then treat for two weeks with:
Vehicle
Vehicle + Berberine 100 mg/kg
Then obtain labs and liver tissue
BERBERINE and AMP SENSITIVE PROTEIN KINASE
150
22
90
22 0
20
40
60
80
100
120
140
160
LDL HDL
Plasma Lipids (mg/dl)
Control Berberine
39.4
25.7 28.8
19.8
0
10
20
30
40
50
Cholesterol Triglycerides
Hepatic Lipids (nmol/mg)
Control Berberine
710
596
520
540
560
580
600
620
640
660
680
700
720
Control Berberine
Hepatic Fatty Acids (nmol/mg)
BERBERINE and AMP SENSITIVE PROTEIN KINASE
♥ HepG2 cells maintained in tissue culture
Incubate with 14C over six hours with:
Berberine at varying concentrations
Vehicle
Measure :
Incorporation of 14C into cholesterol and triglycerides
Secretion of 14C cholesterol and triglycerides in to the media
Will Berberine blunt lipid synthesis?
BERBERINE and AMP SENSITIVE PROTEIN KINASE
100% 100% 90% 92%
70% 70%
55% 57%
0%
20%
40%
60%
80%
100%
120%
Cholesterol Triglycerides
Berberine (ug/ml)
Intracellular 14C Lipids (% Control)
0 4 7 15
100% 100%
75% 75% 70% 60% 60%
40%
55%
35%
0%
20%
40%
60%
80%
100%
120%
Cholesterol Triglycerides
Berberine (ug/ml)
Secreted 14C Lipids (% Control)
0 2 4 7 15
BERBERINE and AMP SENSITIVE PROTEIN KINASE
♥ HepG2 cells maintained in tissue culture
Incubate with 14C acetate over six hours with:
Berberine at 10 ug/ml
With or without PD98059 (ERK ½ inhibitor)
BBR ERK ½ pAMPK pHMG Co-A Reductase Cholesterol Synthesis
BBR ERK ½ LDLR mRNA stabilization LDLR expression
Cholesterol synthesis and LDLR expression Serum Cholesterol
Berberine ERK ½ pAMPK ACC Carboxylase inhibition FA/Trigs.
PD98059 blocks ERK ½ (and should blunt BBR lipid effect)
BERBERINE and AMP SENSITIVE PROTEIN KINASE
100% 100%
65%
85%
0%
20%
40%
60%
80%
100%
120%
Vehicle PD98059 (ERK Inhibitor)
Intracellular 14C Triglycerides (% Control)
Control Berberine 10 ug/ml
100% 100%
60%
97%
0%
20%
40%
60%
80%
100%
120%
Vehicle PD98059 (ERK Inhibitor)
Intracellular 14C Cholesterol (% Control)
Control Berberine 10 ug/ml
BERBERINE and AMP SENSITIVE PROTEIN KINASE
♥ HepG2 cells maintained in tissue culture
Incubate with 14C acetate or 14C glycerol over six hours with:
Berberine at 15 ug/ml
AICAR at 2 Mm (AMPK mimic)
HMG Co-A Reductase stimulates cholesterol synthesis
ACC Carboxylase stimulates FA synthesis (and inhibits FA oxidation)
AMPK Inhibition of HMG Co-A Reductase and ACC Carboxylase
AMPK is activated by low AMP/ATP (and oxidative stress)
Berberine AMPK Inhibition of HMG Co-A Reductase & ACC Carboxylase
AICAR is an AMPK mimic:
HMG Co-A Reductase and ACC Carboxylase inhibition
AMP/ATP independent
BERBERINE and AMP SENSITIVE PROTEIN KINASE
100% 100%
47% 50% 42%
30%
0%
20%
40%
60%
80%
100%
120%
Cholesterol Triglycerides
14C Acetate Lipid Synthesis (% Control)
Control Berberine AICAR
100%
32%
50%
0%
20%
40%
60%
80%
100%
120%
Control Berberine AICAR
14C Glycerol Triglyceride Synthesis (% Control)
Control Berberine AICAR
BERBERINE and AMP SENSITIVE PROTEIN KINASE
Vehicle
ERK ½ AMPK Inhibitor
BERBERINE and AMP SENSITIVE PROTEIN KINASE
Berberine ERK ½ (upregulation) pAMPK (activation)
pHMG Co-A Reductase (down regulation) decreased LDL synthesis
pACC Carboxylase (down regulation) decreased FA synthesis
Berberine ERK ½ (upregulation) LDLR mRNA stabilization
BERBERINE and LDLR mRNA DYNAMICS
Hamsters
Feed regular or HFHC chow for two weeks
Baseline labs
Treat HFHC hamsters with:
Control
Berberine 50 mg/kg
Berberine 100 mg/kg
Evaluate lipids and liver histology at day 10
BERBERINE and LDLR mRNA DYNAMICS
97
181
139
116
0
20
40
60
80
100
120
140
160
180
200
Control Chow HFHC Chow HFHC + Berberine50 mg/kg
HFHC + Berberine100 mg/kg
Cholesterol (mg/dl)
1
2.5
3.5
0
0.5
1
1.5
2
2.5
3
3.5
4
HFHC Chow HFHC + Berberine50 mg/kg
HFHC + Berberine100 mg/kg
LDLR mRNA (fold of HFHC group)
BERBERINE and LDLR mRNA DYNAMICS
Extracts of 700 herbs used in Chinese Medicine herbs
Incubate with HepG2 cells:
Cholesterol depleted
Cholesterol replete medium
Evaluate LDLR mRNA expression
Berberine most effective in a time
and dose-dependent fashion
1
1.4 1.7
2.4 2.4 2.6
0.7
1.3 1.6 1.5
1.7
1.3
0
0.5
1
1.5
2
2.5
3
0 2 4 6 8 24
Hours
LDLR mRNA (fold of control)
Cholesterol Deficient Cholesterol RepleteLDLR protein surface expression
BERBERINE and LDLR mRNA DYNAMICS
1 1.2
1.4 1.6
2.2
3
4.6
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
0 0.5 ug/ml 2.5 ug/ml 5 ug/ml 7.5 ug/ml 10 ug/ml 15 ug/ml
Berberine Concentration
LDLR mRNA (fold of control)
LDL uptake
BERBERINE and LDLR mRNA DYNAMICS
How does Berberine Upregulate LDLR Expression?
Low cholesterol is sensed by SCAP
SCAP-SREBP translocates from ER to Golgi apparatus
SREBP released to translocate into the nucleus
SREBP binds to SRE-1 site on LDL gene promoter region
LDLR mRNA transcription
Treat HepG2 cells with berberine with/without Lovastatin
1
2.2
3.3 3.3
4.8 4.9
0
1
2
3
4
5
6
Control Lovastatin0.5 uM
Lovastatin1 uM
Berberine10 ug/ml
Berberine +Lov. 0.5
Berberine +Lov. 1.0
LDLR mRNA (fold of control)
BERBERINE and LDLR mRNA DYNAMICS
Could berberine lead to translocation of SREBP into the nucleus?
GW707 splits SREBP from its chaperone SCAP
SREBP translocation into the nucleus
LDLR mRNA transcription
C
N
BERBERINE and LDLR mRNA DYNAMICS
Could berberine bind to the LDLR promoter SRE-1?
Treat HepG2 cells with berberine or the SREBP agonist GW707
GW707 splits SREBP from SCAP
SREBP translocates to the nucleus
SREBP binds to SRE-1 site on LDL gene promoter region
LDLR mRNA transcription
0.25 0.29
0.8
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
Control Berberine 10 ug/ml GW707
LDLR promoter activity (units)
BERBERINE and LDLR mRNA DYNAMICS
Berberine is not:
Blocking cholesterol synthesis (via HMG Co-A Reductase inhibition)
Translocating SREBP into the nucleus
Directly stimulating SRE-1 within the LDLR gene promoter region
So berberine is not (directly) increasing transcription of LDLR mRNA
But berberine does increase:
LDLR mRNA expression
LDLR protein expression
LDLR mediated LDL uptake
How is this possible?
BERBERINE and LDLR TRANSCRIPTION
HepG2 cells treated with Actinomycin D +/- berberine
LDLR mRNA contains non-translated region
that mediate rapid turnover of LDLR mRNA
Berberine interacts with non-translated region
Stabilizes mRNA and prolongs its half-life
More LDLR protein will be translated
Increased LDLR protein expression
Serum LDL will be lower
BERBERINE and LDLR mRNA DYNAMICS
Berberine activates the MEK1-ERK ½ pathway LDLR mRNA stabilization
U0126 inhibits MEK-1 (which up regulates ERK via phosphorylation)
1 0.8
2.8
0.4 0
0.5
1
1.5
2
2.5
3
Control Control + U0126 Berberine Berberine + U0126
LDL mRNA (fold of control)
BERBERINE for LIPID CONTROL in the ELDERLY
♥ 80 elderly non-diabetic hyperlipidemic subjects with statin intolerance/refusal:
Age > 75 years Cholesterol > 200 & LDL > 160 mg/dl
60% hypertensive 25% smokers
84% secondary prevention 45% coronary disease
33% vascular disease
Baseline measurements
Randomize to receive:
Red Yeast Rice 200 mg, Policosanol 10 mg, and Berberine 500 mg
Co-Enzyme Q10 2 mg, Folate 0.2 mg, and Astaxanthin 0.5 mg
Placebo
Repeat baseline measurements over twelve months
BERBERINE for LIPID CONTROL in the ELDERLY
173 172 175
119
0
20
40
60
80
100
120
140
160
180
200
Placebo BBR/RYR/P
LDL Cholesterol (mg/dl)
Baseline One Year
253 252 255
201
0
50
100
150
200
250
300
Placebo BBR/RYR/P
Cholesterol (mg/dl)
Baseline One Year
BERBERINE for LIPID CONTROL in the ELDERLY
179 179 177
162
150
155
160
165
170
175
180
185
Placebo BBR/RYR/P
Triglycerides (mg/dl)
Baseline One Year
44 44
45
49
41
42
43
44
45
46
47
48
49
50
Placebo BBR/RYR/P
HDL Cholesterol (mg/dl)
Baseline One Year
BERBERINE for LIPID CONTROL in the ELDERLY
91
94
90 89
86
88
90
92
94
96
Placebo BBR/RYR/P
Fasting Glucose (mg/dl)
Baseline One Year
5.6%
5.7%
5.6%
5.3%
5.1%
5.2%
5.3%
5.4%
5.5%
5.6%
5.7%
5.8%
Placebo BBR/RYR/P
HbA1c (%)
Baseline One Year
1.48
1.68
1.48 1.51
1.35
1.4
1.45
1.5
1.55
1.6
1.65
1.7
Placebo BBR/RYR/P
HOMA-IR (units)
Baseline One Year
BERBERINE for LIPID CONTROL in the ELDERLY
20%
31%
10% 10% 10%
0%
5%
10%
15%
20%
25%
30%
35%
Cholesterol LDL HDL Triglycerides HOMA
Clinical Improvement (%)
Safety
No change in liver chemistry or CPK values
Mild side-effects in 10% treatment group vs. 18% placebo group
No events or mortality in either group
BERBERINE – STATIN SYNERGY
♥ 63 hyperlipidemic subjects (previously untreated)
Baseline measurements
Randomize to receive over two months:
Berberine 500 mg twice a day
Simvastatin 20 mg/day
Combination therapy
Repeat lab assessment
BERBERINE – STATIN SYNERGY
253 238
260
231
186 184
100
120
140
160
180
200
220
240
260
280
Simvastatin Berberine Combination
Cholesterol (mg/dl)
Baseline Eight Weeks
165
147
168
142
112 115
60
80
100
120
140
160
180
Simvastatin Berberine Combination
LDL Cholesterol (mg/dl)
Baseline Eight Weeks
BERBERINE – STATIN SYNERGY
202
172
241
179
134 147
60
80
100
120
140
160
180
200
220
240
260
Simvastatin Berberine Combination
Triglycerides (mg/dl)
Baseline Eight Weeks
44 47
56
46 44
52
0
10
20
30
40
50
60
Simvastatin Berberine Combination
HDL Cholesterol (mg/dl)
Baseline Eight Weeks
BERBERINE – STATIN SYNERGY
♥ HepG2 cells maintained in tissue culture
Incubate with:
Berberine
Simvastatin
BERBERINE – STATIN SYNERGY
LDL Receptor Expression on Cell Membrane
BERBERINE – STATIN SYNERGY
♥ Male Wistar strain rats (180 gm.)
Feed over eight weeks:
Control diet
HFHC (2% chol, 10% egg yolk, 15% lard, and 0.2% sodium cholate)
Randomize the HFHC rats into five treatment groups:
Saline (control group)
Simvastatin 6 mg/kg/day
Berberine 90 mg/kg/day
Simvastatin 6 and Berberine 90 mg/kg/day
Simvastatin at 12 mg/kg/day
At 30 days evaluate:
Lipid levels
Hepatic LDLR mRNA expression
Hepatic histology
BERBERINE – STATIN SYNERGY
Normal diet
HFHC diet
Simvastatin 6 mg/kg/day
BBR 90 mg/kg/day
BBR 90 + Simvastatin 6 mg/kg/day
Simvastatin 12 mg/kg/day
BERBERINE – STATIN SYNERGY
1
2.2 2.1
3.7 3.7
0
0.5
1
1.5
2
2.5
3
3.5
4
HFHC Diet SIMVA6 mg/kg
BBR90 mg/kg
BBR 90 mg/kg +SIMVA 6 mg/dk
SIMVA12 mg/kg
LDLR mRNA (vs. HFHC)
1
1.9
1
1.95
2.95
0
0.5
1
1.5
2
2.5
3
3.5
HFHC Diet SIMVA6 mg/kg
BBR90 mg/kg
BBR 90 mg/kg +SIMVA 6 mg/dk
SIMVA12 mg/kg
HMG-CoA Reductase mRNA (vs. HFHC)
Statin therapy
is
self-defeating
BERBERINE – STATIN SYNERGY
3.7
14.3
9.7 9.7
6.9 7.2
0
2
4
6
8
10
12
14
16
Control Diet HFHC Diet SIMVA6 mg/kg
BBR90 mg/kg
BBR 90 +SIMVA 6
SIMVA12 mg/kg
Hepatic Cholesterol (umol/g)
2.2
12.2
9.2 8.5
6.5 6.8
0
2
4
6
8
10
12
14
Control Diet HFHC Diet SIMVA6 mg/kg
BBR90 mg/kg
BBR 90 +SIMVA 6
SIMVA12 mg/kg
Hepatic Triglycerides (umol/g)
BERBERINE/RYRE/P add on therapy in HeFH
♥ 30 subjects with HeFH:
12 with receptor defective LDLR gene mutation
33% known CADz
25% prior revascularization
18 with receptor negative LDLR gene mutation
61% known CADz
33% prior revascularization
All on stable doses of maximally tolerated statin +/- ezetimibe therapy
Baseline measurements
Add on Berberine 500 mg, Red Yeast Rice Extract 200 mg, & Policosanol 10 mg
Repeat baseline measurements at three months
BERBERINE/RYRE/P add on therapy in HeFH
305
173
141
0
50
100
150
200
250
300
350
Baseline Statinor Statin + Ezitimibe
Plus BBR/RR/P
LDL Cholesterol (mg/dl)
384
245 214
0
50
100
150
200
250
300
350
400
450
Baseline Statinor Statin + Ezitimibe
Plus BBR/RR/P
Cholesterol (mg/dl)
BERBERINE/RYRE/P add on therapy in HeFH
131
113
98
0
20
40
60
80
100
120
140
Baseline Statinor Statin + Ezitimibe
Plus BBR/RR/P
Triglycerides (mg/dl)
50 50 51
0
10
20
30
40
50
60
Baseline Statinor Statin + Ezitimibe
Plus BBR/RR/P
HDL Cholesterol (mg/dl)
36%
43%
18%
44%
53%
23%
0%
10%
20%
30%
40%
50%
60%
Cholesterol LDL Triglycerides
Percent Reduction
Statin or Statin + Ezitimibe Plus BBR/RR/P
BERBERINE/RYRE/P add on therapy in HeFH
36% 43%
18%
44%
53%
23%
0%
10%
20%
30%
40%
50%
60%
Cholesterol LDL Triglycerides
Percent Reduction
Statin or Statin + Ezitimibe Plus BBR/RR/P
“It’s all about PCSK9”
HEPATIC CHOLESTEROL METABOLISM
PCSK9
SREBP mediates PCSK9 as well as LDLR transcription
PCSK9 Up Regulation Statin Dosing
Berberine selectively inhibits PCSK9 transcription
BERBERINE and PCSK9 EXPRESSION
HepG2 cells – Measure LDL receptor mRNA
1
1.5
2
2.8 3.2
3.4
0
0.5
1
1.5
2
2.5
3
3.5
4
Vehicle BBR 2.5ug/ml
BBR 5ug/ml
BBR 10ug/ml
BBR 15ug/ml
BBR 25ug/ml
LDLR mRNA
1 1.5 2
2.6 3
1 1.4
3.3
4.8
6
0
1
2
3
4
5
6
7
Baseline 4 Hours 8 Hours 12 Hours 24 Hours
LDLR mRNA
Vehicle Berberine 15 ug/ml
BERBERINE and PCSK9 EXPRESSION
1
0.6 0.54
0.3 0.22 0.15
0
0.2
0.4
0.6
0.8
1
1.2
Vehicle BBR 2.5ug/ml
BBR 5ug/ml
BBR 10ug/ml
BBR 15ug/ml
BBR 25ug/ml
PCSK9 mRNA
1 1.8 2.1
3.8
9.5
1 1.7 1.6 1.7
3.8
0
2
4
6
8
10
Baseline 4 Hours 8 Hours 12 Hours 24 Hours
PCSK9 mRNA
Vehicle Berberine 15 ug/ml
1
0.12 0
0.2
0.4
0.6
0.8
1
1.2
Vehicle Berberine 15 ug/ml
PCSK9 in media
BERBERINE and PCSK9 EXPRESSION
1
0.31
0
0.2
0.4
0.6
0.8
1
1.2
Vehicle Berberine 15 ug/ml
PCSK9 Transcription Promoter Activity
Berberine selectively inhibits PCSK9 transcription
BERBERINE and PCSK9 EXPRESSION
1
1.9 1.8
2.6
0
0.5
1
1.5
2
2.5
3
Vehicle Mevastatin Berberine Mevastatin+ Berberine
LDLR Protein
1
2
0.7
1
0
0.5
1
1.5
2
2.5
Vehicle Mevastatin Berberine Mevastatin+ Berberine
PCSK9 mRNA
1 2
4
6.8
0
1
2
3
4
5
6
7
8
Vehicle Mevastatin Berberine Mevastatin+ Berberine
LDLR mRNA
BERBERINE, RYRE, & POLICOSANOL vs. EZITIMIBE
♥ 228 hyperlipidemic subjects with:
Intolerance to statin therapy, or Statin therapy declined
None with DM II
None with coronary ischemia or carotid plaque > 40%
Low cholesterol, low saturated fat diet for three months
Baseline measurements
Randomize to receive:
Berberine 500 mg, Red Yeast Rice Extract 200 mg, & Policosanol 10 mg
Ezetimibe 10 mg/day
Repeat baseline measurements at six months
14/80 Ezitimibe Group stopped: 8 for poor compliance & 6 due to GI side-effects
0/148 Nutraceutical Group stopped or experienced side-effects
BERBERINE, RYRE, & POLICOSANOL vs. EZITIMIBE
298 295
241 228
0
50
100
150
200
250
300
350
Ezitimibe BBR/RR/P
Cholesterol (mg/dl)
Baseline Six Months
207 207
154 141
0
50
100
150
200
250
Ezitimibe BBR/RR/P
LDL Cholesterol (mg/dl)
Baseline Six Months
BERBERINE, RYRE, & POLICOSANOL vs. EZITIMIBE
148
129 127
99
0
20
40
60
80
100
120
140
160
Ezitimibe BBR/RR/P
Triglycerides (mg/dl)
Baseline Six Months
61 60 61 59
0
10
20
30
40
50
60
70
Ezitimibe BBR/RR/P
HDL Cholesterol (mg/dl)
Baseline Six Months
19%
25%
15%
24%
32%
20%
0%
5%
10%
15%
20%
25%
30%
35%
Cholesterol LDL Triglycerides
Percent Reduction
Ezitimibe BBR/RR/P
BERBERINE, RYRE, & POLICOSANOL plus EZITIMIBE
♥ 26 subjects with limited response to monotherapy
LDL reduction below the study median (-29%)
New baseline measurements
Assign to combination therapy
Repeat lab studies monthly over three months
BERBERINE, RYRE, & POLICOSANOL plus EZITIMIBE
299
245
216
0
50
100
150
200
250
300
350
Baseline Monotherapy Combination Therapy
Cholesterol (mg/dl)
211
162
133
0
50
100
150
200
250
Baseline Monotherapy Combination Therapy
LDL Cholesterol (mg/dl)
BERBERINE, RYRE, & POLICOSANOL plus EZITIMIBE
61 61 62
0
10
20
30
40
50
60
70
Baseline Monotherapy Combination Therapy
HDL Cholesterol (mg/dl)
131
99 97
0
20
40
60
80
100
120
140
Baseline Monotherapy Combination Therapy
Triglycerides (mg/dl)
18%
24%
19%
28%
37%
23%
0%
5%
10%
15%
20%
25%
30%
35%
40%
Cholesterol LDL Triglycerides
Percent Reduction
Monotherapy Combination Therapy
MONOCYTE CHOLESTEROL METABOLISM
SREBP – SCAP – SRE – PCSK9 – LDLR interaction deals with native LDL
Altered (oxidized, glycated, acetylated) LDL internalized via:
Scavenger receptor
CD 36 receptor
LOX receptor
BERBERINE and NADPH OXIDASE O2- GENERATION
♥ Human macrophages in tissue culture
Pre-incubate for 20 minutes with:
Vehicle
Berberine (25 umol/l)
Stimulate the macrophages with LPS (10 ug/l) over increasing time periods
100%
180% 170% 170%
110%
150%
60%
0%
20%
40%
60%
80%
100%
120%
140%
160%
180%
200%
Control LPS LPS + BBR LPS LPS + BBR LPS LPS + BBR
One Hour Six Hours Twelve Hours
Superoxide Anion (% Control)
BERBERINE and NADPH OXIDASE O2- GENERATION
♥ Human macrophages in tissue culture
Pre-incubate for 20 minutes with:
Vehicle
Berberine at increasing concentrations (10-50 mol/l)
Stimulate the macrophages with LPS (10 ug/l) over fixed (six hours) time period
100% 95%
180%
165%
115%
80%
0%
20%
40%
60%
80%
100%
120%
140%
160%
180%
200%
Control BBR 25 LPS LPS +BBR 10
LPS +BBR 25
LPS +BBR 50
Superoxide Anion at Six Hours (% of control)
BERBERINE and NADPH OXIDASE O2- GENERATION
♥ Human macrophages in tissue culture
Pre-incubates with:
Vehicle
Berberine (25 umol/l)
Apocynin - NADPH oxidase inhibitor (300 umol/l)
Stimulate the macrophages with LPS (10 ug/l)
100% 95%
180%
110%
70%
0%
50%
100%
150%
200%
Control BBR 25 LPS LPS + BBR LPS +Apocynin
Superoxide Anion (% of control)
100%
220%
60% 40%
0%
50%
100%
150%
200%
250%
Control LPS LPS + BBR LPS + Apocynin
NADPH Oxidase activity (% control)
BERBERINE and NADPH OXIDASE O2- GENERATION
100%
270%
180%
0%
50%
100%
150%
200%
250%
300%
Control LPS LPS + BBR
gp91phos mRNA expression (% control)
100%
240% 230%
0%
50%
100%
150%
200%
250%
300%
Control LPS LPS + BBR
p22phox mRNA expression (% control)
Control LPS LPS + BBR
Berberine had no effect on
p22phox, p67phox, p47phox , or RAC
siRNA for gp91phox blunted LPS
mediated superoxide production
RHO KINASE INHIBITION
Rho Kinase upregulates NADPH Oxidase Superoxide
LDL OXIDATION and ENDOTHELIAL TOXICITY
Berberine and Trolox protect against LDL oxidation
Incubate LDL with CuSO4
LDL OXIDATION and ENDOTHELIAL TOXICITY
Berberine and Trolox protect against Malondialdehyde formation
LDL OXIDATION and ENDOTHELIAL TOXICITY
Berberine and Trolox protect against ApoB fragmentation
LDL OXIDATION and ENDOTHELIAL TOXICITY
Trolox is a strong (LDL-free) antioxidant
Berberine is a weak (LDL-free) antioxidant
LDL OXIDATION and ENDOTHELIAL TOXICITY
Berberine and Trolox protect against oxLDL-induced cytotoxicity
Incubate HUVECs with oxLDL
LDL OXIDATION and ENDOTHELIAL TOXICITY
Berberine and Trolox blunt oxLDL-induced ROS formation nuclear damage
Incubate HUVECs with oxLDL
LDL OXIDATION and ENDOTHELIAL TOXICITY
Berberine maintains the
mitochondrial transmembrane
potential in the presence of oxLDL
Incubate HUVECs with oxLDL
High Low
Membrane Potential
LDL OXIDATION and ENDOTHELIAL TOXICITY
oxLDL damage to mitochondria releases
Cytochrome C to the cytoplasm,
activating (cleaving) caspase 3,
which activates (cleaves) PARP,
with increased Bax and
decreased Bcl-2 expression
Berberine
provides dose-related
protection against
(ROS/mitochondrial dysfunction)
mediated activation of these
apoptotic pathways
Incubate HUVECs with oxLDL
MONOCYTE CHOLESTEROL METABOLISM
SREBP – SCAP – SRE – LDLR negative feedback system
Bypassed (down regulated) by inflammation
Cross talk between:
IKK-NF-B
SCAP-SREBP
Low Chol LPS LDL LPS + LDL
BERBERINE in LPS-INDUCED DYSLIPIDEMIA
♥ 40 female C57BL/6J mice
Randomize to receive over four weeks:
Chow diet control group
Chow diet interventional group
Chow + Berberine 10 mg/kg
Chow + Berberine 30 mg/kg
5th week three interventional
group mice receive high
cholesterol diet over seven days
Then treat interventional group
mice with LPS 5 mg/kg ip
110
330
240
130
0
50
100
150
200
250
300
350
Control LPS LPS + BBR 10 LPS + BBR 30
Plasma 8-Isoprostane (pg/ml)
BERBERINE in LPS-INDUCED DYSLIPIDEMIA
1.5
1
1.35
1.5
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
Control LPS LPS + BBR 10 LPS + BBR 30
LDLR mRNA (fold vs. 36B4)
1.4
0.8
1.1
1.3
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
Control LPS LPS + BBR 10 LPS + BBR 30
LDLR Protein Expression (fold vs. -actin)
Hepatic
BERBERINE in LPS-INDUCED DYSLIPIDEMIA
0.4
1.1
0.7
0.45
0
0.2
0.4
0.6
0.8
1
1.2
Control LPS LPS + BBR 10 LPS + BBR 30
PCSK9 mRNA (fold vs. 38B4)
97 48 68
160
257
132
33
321
147
69 46
202
112
51 56
168
20
70
120
170
220
270
320
370
Cholesterol LDL HDL Triglycerides
Serum Lipids (mg/dl)
Control LPS LPS + BBR 10 LPS + BBR 30
DECISION to GENERATE CHOLESTEROL
Decision to Respond to Infection (Real or Perceived):
ROS Inflammatory Cytokines
Cellular Proliferation Immune Upregulation
BERBERINE in LPS-INDUCED DYSLIPIDEMIA
0.4
1.1
0.7
0.45
0
0.2
0.4
0.6
0.8
1
1.2
Control LPS LPS + BBR 10 LPS + BBR 30
PCSK9 mRNA (fold vs. 38B4)
11.1
24.2
14.5 12.6
0
5
10
15
20
25
30
Control LPS LPS + BBR 10 LPS + BBR 30
Interferon- (ng/ml)
32.9
86.6
49.9
36.7
0
10
20
30
40
50
60
70
80
90
100
Control LPS LPS + BBR 10 LPS + BBR 30
Interleukin-1 (ng/ml)
0.86
1.59
1.2
0.96
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
Control LPS LPS + BBR 10 LPS + BBR 30
TNF (ng/ml)
INFLAMATION and LDL RECEPTOR DYSFUNCTION
♥ Human coronary VSMCs
Incubate in tissue culture with/without:
LDL 200 ug/ml (saturate cells to sequester SCAP-SREBP)
EDTA and BHT (block LDL oxidation)
Il-1 5 ng/ml (Th1 cytokine)
Heparin (prevents LDL attachment to cell membrane)
MB47 (blocks the LDL receptor)
Measure VSMC cholesterol content:
Stain with Oil Red 0 to measure lipid accumulation
Direct measure of free and esterified cholesterol
INFLAMATION and LDL RECEPTOR DYSFUNCTION
100% 105% 110% 90% 80%
100%
145%
200% 210% 195%
0%
50%
100%
150%
200%
250%
Control LDL 200 ug/ml LDL + Il-1b5 ng/ml
LDL + Il-1b10 ng/ml
LDL + Il-1b20 ng/ml
VSMC Cholesterol Accumulation
Free Cholesterol Esterified Cholesterol
100% 105% 90%
110% 110% 100%
145%
210%
50% 30% 0%
50%
100%
150%
200%
250%
Control LDL 200 ug/ml LDL + Il-1b10 ng/ml
LDL + Il-1b10 ng/ml+ Heparin
LDL + Il-1b10 ng/ml+ MB47
VSMC Cholesterol Accumulation
Free Cholesterol Esterified Cholesterol
100% 125% 120%
250% 240%
0%
50%
100%
150%
200%
250%
300%
Control LDL 200ug/ml
LDL + Il-1b5 ng/ml
LDL + Il-1b10 ng/ml
LDL + Il-1b20 ng/ml
ACAT1 Activity
INFLAMATION and LDL RECEPTOR DYSFUNCTION
100%
45%
275%
100%
0%
50%
100%
150%
200%
250%
300%
Control LDL 200 ug/ml Il-1b 5 ng/ml LDL + Il-1b 5 ng/ml
LDL Receptor mRNA
200%
150% 160%
190%
0%
50%
100%
150%
200%
250%
LDLR SCAP SREBP ACAT1
Il-1 Effect on mRNA Expression (low LDL)
INFLAMATION and LDL RECEPTOR DYSFUNCTION
200%
150% 160%
190%
0%
50%
100%
150%
200%
250%
LDLR SCAP SREBP ACAT1
Il-1 Effect on mRNA Expression (low LDL)
100% 35% 50% 55% 75%
100%
60% 75%
65%
90%
100%
60% 60%
75% 80%
0
0
0
1
1
1
1
Control LDL 200 ug/ml LDL + Il-1b0.5 ng/ml
LDL + Il-1b5 ng/ml
LDL + Il-1b10 ng/ml
Il-1 Effect on mRNA Expression (LDL Loaded)
LDLR SCAP SREBP
INFLAMATION and LDL RECEPTOR DYSFUNCTION
LDL 200 ug/ml LDL 200 ug/ml
+ Il-1 5 ng/ml
LDL + Il-1 + Scavenger LDL 200 ug/ml
Receptor Blockade + Heparin
INFLAMATION and LDL RECEPTOR DYSFUNCTION
SCAP Golgi Overlay
Lipid
Depleted
Media
LDL
200 ug/ml
LDL
+ Il-1
TLR4/MyD88 CROSS-TALK with SCAP-SREBP
♥ Human Monocyte derived Macrophages
Incubate in tissue culture with/without:
LDL 200 ug/ml (saturate cells to sequester SCAP-SREBP)
LPS 1 ug/ml (TLR4/MyD88 IKK NF-B activation)
LDL + LPS
siRNA MyD88 (blocks TLR4/MyD88 cascade)
Measure Macrophage cholesterol content:
Stain with Oil Red 0 to measure lipid accumulation
Direct measure of esterified cholesterol
X
TLR4/MyD88 CROSS-TALK with SCAP-SREBP
MyD88 siRNA blunts trafficking through TLR4/MyD88 pathway
TNF-
AngII
AT1 TLR4/MyD88
LPS, FFA
TLR4/MyD88 CROSS-TALK with SCAP-SREBP
TLR4/MyD88 CROSS-TALK with SCAP-SREBP
150
220 200
280
140
170 180 190
0
50
100
150
200
250
300
Low Chol Media LPS LDL LPS + LDL
Cholesterol Ester (ug/mg)
No siRNA MyD88 siRNA
No
siRNA
MyD88
siRNA
Low Chol LPS LDL LPS + LDL
TLR4/MyD88 CROSS-TALK with SCAP-SREBP
1
2
0.5
1.1
0.7 0.9
0.4 0.5
0
0.5
1
1.5
2
2.5
Low Chol Media LPS LDL LPS + LDL
LDLR mRNA (fold of control)
No siRNA MyD88 siRNA
1
1.9
0.6
1.1
0.7
1
0.5 0.6
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
Low Chol Media LPS LDL LPS + LDL
HMGCoA Reductase mRNA (fold of control)
No siRNA MyD88 siRNA
Opposite of
Hepatic
Response
TLR4/MyD88 CROSS-TALK with SCAP-SREBP
LPS and Il-1B Increase TNF Increases
LDLR & HMG-CoA Reductase Expression LDLR & HMG-CoA Reductase
Effect blunted by MyD88 Blockade Not blunted by MyD88 Blockade
TLR4/MyD88 CROSS-TALK with SCAP-SREBP
TNF-alpha binds TNFR – not TLR4/MyD88
TLR4/MyD88 CROSS-TALK with SCAP-SREBP
1
1.8
0.8
1.3
0.9 1
0.7 0.7
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
Low Chol Media LPS LDL LPS + LDL
SCAP mRNA (fold of control)
No siRNA MyD88 siRNA
1
1.7
0.7
1.2
0.8
1.1
0.6 0.7
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
Low Chol Media LPS LDL LPS + LDL
SREBP mRNA (fold of control)
No siRNA MyD88 siRNA
TLR4/MyD88 CROSS-TALK with SCAP-SREBP
Low Chol
LPS
LPS +
MyD88
siRNA
LDL
LDL
+ LPS
LDL+ LPS
+ MyD88
siRNA
SCAP Golgi Nucleus SCAP-Golgi
1
1.3
0.5 0.4
0.9
0.5
0
0.2
0.4
0.6
0.8
1
1.2
1.4
Low CholMedia
LPS LPS +siMyD88
LDL LDL + LPS LDL + LPS+ siMyD88
SCAP-Golgi Co-Localization
TLR4/MyD88 CROSS-TALK with SCAP-SREBP
BMS inhibits IKK and has no effect on MyD88
TLR4/MyD88 CROSS-TALK with SCAP-SREBP
1
0.05
2.5
0.1
1
0.1
2
0.15 0
0.5
1
1.5
2
2.5
3
Low Chol Media BMS LPS LPS + BMS
Il-6 and TNF-alpha mRNA (fold of control)
IL-6 TNF-alpha
1 0.9
1.8
1.2
1
0.8
1.7
1.1
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
Low Chol Media BMS LPS LPS + BMS
LDLR and HMGCo-A Reductase Expression
LDLR HMGCo-A Reductase
TLR4/MyD88 CROSS-TALK with SCAP-SREBP
1
0.3
1.8
0.4
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
Control siRNA SCAP LPS LPS + siSCAP
SCAP Expression
1
0.4
1.8
1
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
Low Chol Media Low Chol + siSCAP LPS LPS + siSCAP
LDLR Expression
1
0.5
1.8
0.9
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
Low Chol Media Low Chol + siSCAP LPS LPS + siSCAP
HMGCo-A Reductase Expression
TLR4/MyD88 CROSS-TALK with SCAP-SREBP
Threat NF-B Cytokines ACAT Free Chol SCAP
SCAP nSREBP HMGCo-A Reductase & LDLR
Foam Cells Atherosclerosis
BERBERINE FULLFILLS POLING’S POSTULATES
Atherosclerotic Risk Factors and MetS/DM II:
Insulin insensitivity Hyperlipidemia
Overweight Hypertension
Endothelial function and oxidative stress
Inflammation (Th1/Th17 immune dysregulation)
Without damping appropriate immune response
Preferably with an anti-microbial effect
Blunting of auto-immunity and collateral tissue damage
Defined mechanisms at levels of transcription and translation
Limited toxicity and reasonable cost
Synergy with pharmaceutical and mechanical interventions
Published studies to document mechanisms and efficacy
BERBERINE – PHYSIOLOGIC EFFECTS
Complex I inhibition Glycolysis AMP/ATP PKC AMPK
Lipid Metabolism
pHMG Co-A Reductase Decrease cholesterol biosynthesis
LDLR expression ed and PCSK9 ed Decrease in serum LDL
pACC Carboxylase Decrease in FA synthesis and increase in FA oxidation
Decrease in triglycerides
Glucose Metabolism
Insulin receptor expression ed
Insulin resistance due to SFAs, LPS, and Th1 cytokines blunted
Improved insulin sensitivity
and decrease in serum glucose
BERBERINE – PHYSIOLOGIC EFFECTS
Complex I inhibition Glycolysis AMP/ATP PKC AMPK
Weight Physiology
Adipocyte differentiation delayed Smaller cells with healthier Adipokine profile
GI tract flora altered Calories absorbed from CHO blunted
LPS translocation blunted
pACC Carboxylase Increased FA oxidation
Weight reduction
Oxidative Stress
NADPH oxidase is restrained Superoxide production deceased
Uncoupling protein is up regulated ROS production with in SOD expression
Protective vs. ROS/ischemic tissue damage in animal models (protects mitochondria)
Apoptosis of malignant cells enhanced
BERBERINE – PHYSIOLOGIC EFFECTS
Complex I inhibition Glycolysis AMP/ATP PKC AMPK
Endothelial Function
AMPK eNOS NO cGMP vasodilation, platelet, and VSMC effects
NO pIKKB NF-B restrained Th1 cytokine production decreased
ICAM, VCAM, and MCP-1 decreased Mononuclear infiltration blunted
Endothelial progenitor cell count rises and endothelial microparticle count falls
VSMC proliferation and MMP activity blunted
CV Physiology
Endothelial independent vasodilation
ACE Inhibition
Beneficial electrophysiological effects (Ito and Ica currents preserved)
VSMC proliferation is inhibited
Vascular elasticity improves
Autoimmune and Inflammatory Conditions
Antioxidant (indirect) and anti-inflammatory effects demonstrated
Beneficial effects in animal models of auto-immunity
BERBERINE – CLINICAL EFFECTS
Complex I inhibition Glycolysis AMP/ATP PKC AMPK
LDL and triglycerides decrease with minimal change in HDL
Glucose falls and insulin sensitivity improves
Weight loss occurs
Elevated BP may fall
Hepatic steatosis improves
Diabetic nephropathy may improve
Endothelial function improves
Post PCI for ACS Lower cytokine elaboration and improved outcome
Heart Failure Rise in EF, functional status, and reduced arrhythmia
Berberine synergizes with and adds to standard pharmacologic measures
BLUNTING the DECISION to GENERATE CHOLESTEROL
Blunting the Decision to Respond to Perceived Infection:
ROS Inflammatory Cytokines
Cellular Proliferation Immune Upregulation
The Chronicles Will Continue
THE CHRONICLES of BERBERINE C. S. Lewis and R. H. Poling
PCSK9, AMPK, and the LDL receptor
Berberine in lipid management
Berberine and the White Witches (Cytokines, FFAs, & IKK) of IR
Berberine in diabetes management and weight loss
Dr. Poling’s Nephew
Berberine in Inflammation, Auto-Immunity, Malignancy, and Toxicity
Voyage of the Plaque Buster
Berberine in the Treatment of Cardiovascular Disease