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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


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

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


Caloric Restriction, Exercise,

Leptin, Adiponectin, Metformin, and

Berberine

Energy (Caloric) Excess

Inflammatory Cytokines



Lipid Metabolism





Glucose Metabolism







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



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

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









CV Physiology


ACE Inhibition







BERBERINE – CLINICAL EFFECTS


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?


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)


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


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



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


3 MonthsBerberine


BMI (kg/m2)


92

90 90

92

89

87.5

88

88.5

89

89.5

90

90.5

91

91.5

92

92.5


3 MonthsBerberine


Fasting Blood Glucose (mg/dl)

164 157

133

174

134

0

50

100

150

200


3 MonthsBerberine


LDL Cholesterol (mg/dl)

98

85

67

96

72

0

20

40

60

80

100

120


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

HEPATIC CHOLESTEROL METABOLISM

LDL Receptor

Synthesis and Expression of the LDLR Hepatic Need for Cholesterol


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


LDLR Synthesis Hepatic Need for Free Cholesterol


SREBP – SRE Interaction

LDL Receptor

HMG Co-A Reductase

PCSK9 (Proprotein Convertase Subtilisin/Kexin type 9)


PCSK9


PCSK9

System designed for primitive man

Primary cause of hepatic hypolipidemia was dietary insufficiency

PCSK9 defeats drug approaches to lipid reduction in modern man


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


SREBP – SCAP – SRE – LDLR interaction deals with native LDL

Altered (oxidized, glycated, acetylated) LDL internalized via:

Scavenger receptor

CD 36 receptor

LOX receptor


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


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


♥ Rat muscle mitochondria

Measure oxygen consumption with Complex I substrate pyruvate

No effect on Complex-II related respiration with substrate succinate


♥ 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


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)


♥ 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?


100% 100% 90% 92%

70% 70%

55% 57%

0%

20%

40%

60%

80%

100%

120%


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%


Berberine (ug/ml)

Secreted 14C Lipids (% Control)

0 2 4 7 15



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)


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



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


100% 100%

47% 50% 42%

30%

0%

20%

40%

60%

80%

100%

120%


14C Acetate Lipid Synthesis (% Control)

Control Berberine AICAR

100%

32%

50%

0%

20%

40%

60%

80%

100%

120%


14C Glycerol Triglyceride Synthesis (% Control)



Vehicle

ERK ½ AMPK Inhibitor


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


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)


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


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


LDL uptake


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



Could berberine lead to translocation of SREBP into the nucleus?

GW707 splits SREBP from its chaperone SCAP

SREBP translocation into the nucleus


C

N


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


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 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 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


173 172 175

119

0

20

40

60

80

100

120

140

160

180

200

Placebo BBR/RYR/P


Baseline One Year

253 252 255

201

0

50

100

150

200

250

300

Placebo BBR/RYR/P

Cholesterol (mg/dl)

Baseline One Year


179 179 177

162

150

155

160

165

170

175

180

185

Placebo BBR/RYR/P


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


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


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)


Randomize to receive over two months:

Berberine 500 mg twice a day

Simvastatin 20 mg/day

Combination therapy

Repeat lab assessment


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





202

172

241

179

134 147

60

80

100

120

140

160

180

200

220

240

260




44 47

56

46 44

52

0

10

20

30

40

50

60






Incubate with:

Berberine

Simvastatin


LDL Receptor Expression on Cell Membrane


♥ 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


Normal diet

HFHC diet


BBR 90 mg/kg/day

BBR 90 + Simvastatin 6 mg/kg/day



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


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


Add on Berberine 500 mg, Red Yeast Rice Extract 200 mg, & Policosanol 10 mg

Repeat baseline measurements at three months


305

173

141

0

50

100

150

200

250

300

350

Baseline Statinor Statin + Ezitimibe

Plus BBR/RR/P


384

245 214

0

50

100

150

200

250

300

350

400

450


Plus BBR/RR/P

Cholesterol (mg/dl)


131

113

98

0

20

40

60

80

100

120

140


Plus BBR/RR/P


50 50 51

0

10

20

30

40

50

60


Plus BBR/RR/P


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


36% 43%

18%

44%

53%

23%

0%

10%

20%

30%

40%

50%

60%


Percent Reduction

Statin or Statin + Ezitimibe Plus BBR/RR/P

“It’s all about PCSK9”


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


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


1

0.12 0

0.2

0.4

0.6

0.8

1

1.2


PCSK9 in media


1

0.31

0

0.2

0.4

0.6

0.8

1

1.2


PCSK9 Transcription Promoter Activity

Berberine selectively inhibits PCSK9 transcription


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


PCSK9 mRNA

1 2

4

6.8

0

1

2

3

4

5

6

7

8


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


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


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


Baseline Six Months


148

129 127

99

0

20

40

60

80

100

120

140

160

Ezitimibe BBR/RR/P


Baseline Six Months

61 60 61 59

0

10

20

30

40

50

60

70

Ezitimibe BBR/RR/P


Baseline Six Months

19%

25%

15%

24%

32%

20%

0%

5%

10%

15%

20%

25%

30%

35%


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


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




61 61 62

0

10

20

30

40

50

60

70



131

99 97

0

20

40

60

80

100

120

140



18%

24%

19%

28%

37%

23%

0%

5%

10%

15%

20%

25%

30%

35%

40%


Percent Reduction

Monotherapy Combination Therapy


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)



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)



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)


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%


p22phox mRNA expression (% control)


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


Berberine and Trolox protect against Malondialdehyde formation


Berberine and Trolox protect against ApoB fragmentation


Trolox is a strong (LDL-free) antioxidant

Berberine is a weak (LDL-free) antioxidant


Berberine and Trolox protect against oxLDL-induced cytotoxicity

Incubate HUVECs with oxLDL


Berberine and Trolox blunt oxLDL-induced ROS formation nuclear damage



Berberine maintains the

mitochondrial transmembrane

potential in the presence of oxLDL


High Low

Membrane Potential


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



SREBP – SCAP – SRE – LDLR negative feedback system

Bypassed (down regulated) by inflammation

Cross talk between:

IKK-NF-B

SCAP-SREBP


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)


1.5

1

1.35

1.5

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6


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


LDLR Protein Expression (fold vs. -actin)

Hepatic


0.4

1.1

0.7

0.45

0

0.2

0.4

0.6

0.8

1

1.2


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)


DECISION to GENERATE CHOLESTEROL

Decision to Respond to Infection (Real or Perceived):




0.4

1.1

0.7

0.45

0

0.2

0.4

0.6

0.8

1

1.2


PCSK9 mRNA (fold vs. 38B4)

11.1

24.2

14.5 12.6

0

5

10

15

20

25

30


Interferon- (ng/ml)

32.9

86.6

49.9

36.7

0

10

20

30

40

50

60

70

80

90

100


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


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


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


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)


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


LDL 200 ug/ml LDL 200 ug/ml

+ Il-1 5 ng/ml

LDL + Il-1 + Scavenger LDL 200 ug/ml

Receptor Blockade + Heparin


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


MyD88 siRNA blunts trafficking through TLR4/MyD88 pathway

TNF-

AngII

AT1 TLR4/MyD88

LPS, FFA

http://upload.wikimedia.org/wikipedia/commons/5/5e/NFKB_mechanism_of_action.png


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



1

2

0.5

1.1

0.7 0.9

0.4 0.5

0

0.5

1

1.5

2

2.5




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


HMGCoA Reductase mRNA (fold of control)


Opposite of

Hepatic

Response


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


TNF-alpha binds TNFR – not TLR4/MyD88


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


SCAP mRNA (fold of control)


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


SREBP mRNA (fold of control)



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


BMS inhibits IKK and has no effect on MyD88


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


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


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



Lipid Metabolism





Glucose Metabolism







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









CV Physiology


ACE Inhibition







BERBERINE – CLINICAL EFFECTS


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:



The Chronicles Will Continue

THE CHRONICLES of BERBERINE C. S. Lewis and R. H. Poling


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

the chronicles of berberineassets.a4m.com/assets/webcasts/webcast_pdfs/2013-11-18...2013/11/18 ·...

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