1. Karen Jonscher, PhD
Department of Anesthesiology
University of Colorado Anschutz Medical Campus
PQQ prevents developmental programming of microbial dysbiosis and macrophage polarization to attenuate liver fibrosis in obese mice    
CCTSI Summit, August 2017

2. The obesity epidemic
~2/3 of women in the US of childbearing age are overweight or obese
Maternal BMI ≥ 30 increased risk for early obesity in offspring (by age 4) and obesity in adulthood.
By 2020, 60 million children will classify as overweight/obese
Obesity in early life accelerates onset of obesity-related diseases: NAFLD
Whitaker RC Pediatrics 2004;114:29
(2009) In Weight Gain During Pregnancy: Reexamining the Guidelines
Baird J, BMJ 2005;331:929
de Onis, M., Am Soc Nutrition 2010

3. NAFLD - #1 liver disease worldwide
~ 100 million US adults (90% of obese population)
Half of the children with NAFLD have NASH at the time of diagnosis.
75%
25%
NAFLD
NASH
steatosis
steatosis + inflammation + scarring
reversible
irreversible
Currently, NAFLD affects about 100 million people in the US, with a prevalency of 80-90% in obese people. About ¾ of those with NAFLD have steatosis, or fat in their liver, with or without inflammation. This is generally reversible. However ¼ of NAFLD patients have steatohepatitis, the more severe form of the disease, that generally includes fibrosis and can progress to liver cancer. NAFLD affects almost 10% of all children in the US, mostly in late adolescence, and therefore have a high risk for progressing to severe liver disease in adulthood. Over the past decade there has been an enormous increase in the number of liver transplants due to NASH and in many places it is now the #1 cause for liver transplants. There is no therapy that currently exists to reverse NASH and clinical and preclinical studies focus on inhibiting the 3 main lesions of NAFLD – hepatic fat accretion, inflammation and fibrosis.
NASH is the #1 cause for liver transplants in many areas of the US
No effective pharmaceutical therapy currently exists
Goyal NP, Schwimmer JB. Clin Liver Dis;2016.
Cholankeril G, et al. Hepatology;2016.
Brumbaugh DE, Friedman JE. Pediatr Res;2014.
Anderson EL, et al. PLoS One;2015.

4. The macrophage may promote or resolve liver injury
Resident Macrophages
Maintain homeostasis
Monocyte
Resident macrophage
Recruited macrophage
Recruited Macrophages
Respond to liver injury
Are responsible for wound healing but may promote fibrosis if metabolically/ pathologically activated or polarized
Activated by microbial products - LPS v
Stem cells

5. Hypothesis and study design
Western-style diet-induced gastrointestinal dysbiosis leads to metabolic activation of liver macrophages and NASH in offspring.
Moms
Offspring
3 wks of age
20 wks of age WD CH
Mat. diet
Chow
Western-style Diet
+/- PQQ
- PQQ
NASH (fat accumulation, inflammation, fibrosis)
Bone marrow-derived macrophage polarization
Dysbiotic microbiota

6. WD-fed mice have detrimental age-related changes in microbiota composition
macrophage
Analyzed cecal contents from 3 wk old and 20 wk old offspring.
WD-fed mice
Increase in OTU associated with NASH
Decrease in OTUs considered beneficial

7. Macrophages are polarized in WD-fed offspring
Liver
WD: increased inflammation, oxidative stress, macrophage infiltration
Attenuated by PQQ
Bone marrow-derived macrophages
WD: increased pro-inflammatory response at baseline and when stimulated with LPS
Baseline inflammatory response attenuated by PQQ
Co-stimulation (LPS + PQQ) blocked inflammatory response

8. 20 wk old WD-fed offspring have NASHCH WD
H & E
WDPQQ
WDPQQ/WD
NASH Score
Conclusions: PQQ protects WD-fed offspring from development of NASH, even when withdrawn at weaning. Mechanism may be via modulation of the microbiota to affect macrophage inflammatory polarization.
H & E staining
Coherent anti-Stokes Raman Spectroscopy
Second Harmonic Generation Microscopy
Stimulated and Spontaneous Raman Spectroscopy
Picrosirius red staining
ALT/AST in serum
Hepatic and serum triglycerides
qPCR for mRNA expression of pro-inflammatory genes

9. Thank you! PQQ Metabolic Core Paul MacLean UC Denver Friedman Group
Matt Jackman
UC Denver
Friedman Group
Jed Friedman
Rachel Janssen
Becky de la Houssaye
Microbiome Core
Dan Frank
Diana Ir
Advanced Light Microscopy Core
Radu Moldovan
Greg Glazner
Metabolomics Core
Angelo D’Alessandro
Julie Haines
El Kasmi Group
Karim El Kasmi
Aimee Anderson
UC Irvine
Potma Group
Eric Potma
Alba Alfonso-Garcia
Jeff Suhalim
Alexander Fast
I’d like to thank everyone who contributed to this work, especially my K award mentor, Jed Friedman, and present and past members of his group. Michael and Margaret did the fetal mice studies. Moshe is my co-mentor and his lab has been amazing for training and resources. I did some CARs here at the ALMC with help from Radu and Greg and have much more microscopy planned! The metabolic chamber experiments and qMRI were done in the metabolic core under the direction of Paul MacLean. I had some amazing undergrads who volunteered in the lab and really helped out. The imaging was mostly done at UC Irvine in collaboration with Eric Potma and his talented graduate students, and metabolomics at UC Davis. I am deeply grateful for my funding, including my K award, some funds from Mitsubishi, the department, and UCH.
Students
Ellen Wiitala
Garrett Florey
Raleigh Jonscher
Angelina Miley
PQQ

11. Pyrroloquinoline quinone (PQQ) in food
“RDA” ~ 300 μg
Sources of PQQ
μg/100 g fresh weight or 100 mL volume
Approximate intake (μg)/g dry food consumed based on 2000 Cal/day diet
Legumes
2-72 (25-50 g)
Potatoes
1-1.7
1-14 (10 g)
Vegetables (parsley, celery, spinach)
5-324 (60 g)
Fruits (banana, kiwi, papaya)
2-108 (30-50 g)
Soy
6.2
62 (10 g)
Milk
128 (bovine)
80 (human)
PQQ is in interstellar dust and in the soil and is made by bacteria, although not the ones in our gut, and is found in foods. It is particularly enriched in beans, especially soy, and potatoes, veggies such as parsley, celery and spinach, fruits and, interesting, milk. Based on rodent studies, an RDA is estimated at about 300 ug which you can get from eating a normal balanced diet. Unfortunately, meat, wine and beer are all very low in PQQ.
Revised from

12. Macrophage recruitment & polarization are increased by WD & attenuated by PQQ
LIVER
mRNA expression of genes associated with
Recruitment of macrophages from bone marrow (Ccr2, Cd11b, Ly6c)
Inflammation (Il1b, Tnf, Il6)
Oxidative stress (Nos2, Hmox1)
WD PQQ
Bone marrow-derived macrophages (BMDM)
mRNA expression of
Pro-fibrotic genes (Arg1)
Pro-inflammatory genes (Il6)
in unstimulated cells
in LPS-stimulated cells WD
PQQ
suppressed inflammatory response when given ex vivo