Josh Rabinowitz: Metabolomics, NAD+, and Cancer Metabolism

Joshua Rabinowitz’s Career Path

• Started in industry straight out of medical school 
  • Co-​​founded Lexopharmaceuticals, focused on fast drug delivery through inhalation
  • One FDA approved drug for acute agitation
• Moved to academia, joined Princeton faculty without doing a postdoc 
  • Noticed many important medications work via metabolism
  • Started lab with a focus on measuring classic metabolites and their activities

Metabolism Research in the Late 90s

• According to Navdeep Chandel, metabolism research was considered uninteresting 
  • Ranked bottom 10 percentile in terms of scientific interest
  • Genomics, immunology, and other fields were considered more “sexy”
• Despite this, Chandel and Rabinowitz found metabolism research interesting and pursued it
  Metabolism and Metabolomics
• Metabolism: process that converts food into usable energy and building blocks for growth/​​regeneration
• Metabolomics: study of metabolites (e.g. glucose, amino acids, fats) involved in metabolism
• About 100 core metabolites involved in making usable energy
• Metabolites exist at all levels of life, from bacteria to humans
• Around 1,000 metabolites with clear biological function

Regulation of Metabolites

• Many important metabolites have a preferred range of 2–3 fold in the bloodstream
• The body uses mass action (linear consumption) as a primary regulatory principle 
  • When a metabolite is present, use it up
• Insulin is a key regulatory hormone in mammals 
  • Controls blood sugar by promoting glucose uptake and preventing glucose production
  • Also signals when it’s safe to not release fat from adipose tissue (when enough carbohydrates are available)

Metabolomics in Medicine

• Metabolomics can help understand how the body works and how to choose the right diet for health
• Studying metabolomics can potentially help address metabolic syndrome and other health problems
• More research needed to understand the full scope and potential of metabolomics in medicine
  Metabolomics and Fluxomics
• Metabolomics: study of metabolites, intermediates in the process of converting food into usable energy and protein 
• Fluxomics: study of the movement and derivatives of metabolites with respect to time
• Metabolites are low in abundance and flow very fast, meant to be made and used within a second to an hour

Glucose and Diabetes

• Glucose is essential for life and offers a portrait in velocity
• Healthy fasting glucose levels are around 80–100 milligrams per deciliter (mg/​​dL)
• Type 2 diabetes occurs when fasting glucose levels reach around 180 mg/​​dL
• The difference between healthy and diabetic glucose levels is relatively small, but it has significant health consequences

Randall Hypothesis

• Over 50 years old
• Suggests that there is competition between carbohydrates (glucose) and fat for burning in tissues
• Fat is a preferred fuel for tissues, and when it is available, glucose tends not to be burned effectively, potentially leading to diabetes

Recent Evidence Supporting Randall Hypothesis

• Experiments show that fat suppresses glucose use in tissues
• Turning off lipolysis rapidly induces glucose consumption
• Alternative fuels, such as lactate, also compete with glucose and suppress its use
• Competitive nutrient environment plays a central role in determining glucose clearance and levels

Lactate

• Important circulating fuel that competes with glucose
• Generated when demand for ATP is high and quick, taking a quicker but less efficient path that doesn’t require the same cellular oxygen as the Krebs cycle
  Lactate as a Fuel
• Lactate is a major circulating nutrient with a fast turnover 
  • Constantly being made, released into the bloodstream, and consumed
  • Serves as a universal nutrient, with transporters carrying it into virtually any cell in the body
• Glucose penetration into tissues is heavily regulated 
  • Lactate is the universally available form of carbohydrate
• Healthy heart in the fasted state will not touch glucose, but will use lactate, free fatty acids, and ketone bodies
• Lactate usage in the brain is still unclear 
  • Uncertain which cell types in the brain are lactate consumers versus lactate producers
  • Lactate allows for flexibility in the brain’s activities

Evolutionary Reason for Lactate as a Fuel

• Eukaryotic life has been wired to use lactate as a fuel 
  • Default is to spit out redox-​​balanced waste (lactate in humans, ethanol in yeast)
  • Allows for flexibility in running glycolysis and using glucose
• Lactate helps solve the redox problem by distributing it across the body 
  • Constant trading of lactate in and out of cells
  • Allows cells that need carbohydrate energy to use lactate
• System would be less flexible if only the liver could clean up lactate 
  • Advantageous for the heart to use lactate during burst exercise
  • Decision to use lactate is regulated at each cell and based on physical chemistry
    Lactate and Metabolic Health
• Fasting lactate levels in the population can indicate metabolic health 
  • Higher fasting lactate levels in insulin-​​resistant individuals (around 2 millimole)
  • Healthy individuals have lower fasting lactate levels (below 0.5 millimole)
• Lactate levels can indicate fuel partitioning and metabolomics issues 
  • Correlation between fasting glucose and fasting lactate
  • Lactate clearance system not working well due to competition between lactate and fat to be burned
  • Lactate dysregulation could be an early indicator of metabolic syndrome

Electron Transport Chain and Redox

• Electron transport chain is a series of proteins in the inner mitochondrial membrane 
  • Protons are pumped out of the mitochondria, creating electrical energy
  • As protons flow back in, they turn a turnstile, converting electrical energy back to chemical energy (ATP)
• Krebs cycle (also known as the citric acid cycle or TCA cycle) is crucial for metabolism 
  • Takes in two-​​carbon units from carbohydrates, fats, and proteins
  • Spits out carbon dioxide and passes electrons to NAD, creating NADH
  • NADH feeds into the electron transport chain

Metabolic Syndrome and Lactate

• Metabolic syndrome is diagnosed too late, with hypertension, obesity, dyslipidemia, and hyperglycemia
• Lactate dysregulation could be an earlier indicator of metabolic issues
• Preventing metabolic syndrome is more important than diagnosing it
  Carbon to Oxygen Bonds and Energy
• Carbon to oxygen double bonds are high energy bonds 
  • Physics and chemistry flow towards creating these high energy bonds
  • Carbon, carbon, and carbon, hydrogen bonds have potential energy
  • This energy is liberated through electron transferring processes

Oxidation and Reduction

• Oxidation and reduction are always coupled 
  • They refer to the movement of electrons
  • When electrons go from substance A to substance B, the one that gives up the electrons is oxidized
  • The one that receives the electrons is reduced

Redox Pairing: NAD and NADH

• NAD is the oxidized form, NADH is the electron holding or reduced form 
  • Normally exists in a biased ratio towards a lot of NAD and a small amount of NADH
  • NAD is a decent electron acceptor, prepared to pick up electrons from intermediates of the TCA cycle
  • NADH feeds into the electron transport chain

Metformin and Complex One Inhibition

• Metformin is believed to inhibit complex one of the electron transport chain 
  • This slows the conversion of NADH back to NAD
  • Inhibition of complex one can lead to an increase in NADH levels, which can cause various issues 
    • Too many electrons in the electron transport chain can lead to free radical production
    • Gums up metabolism, leading to problems with ATP production
    • Can cause signaling issues

NADP and NADPH

• NADP and NADPH are important cofactors with different chemical handles than NAD and NADH 
  • NADP and NADPH have a more even pairing, allowing for more driving force to dump electrons off
  • NADPH is a master energetic building material, second only to ATP 
    • Used to assemble fat, fight reactive oxygen species, and intentionally create oxidative stress when needed

NAD Supplementation and Aging

• Cellular NAD levels decline as we age 
  • This has led to interest in supplementing with NAD to counteract this decline
    NAD Importance and Measurement
• NAD plays a central role in energy generation 
• NAD is depleted with aging, but depletion is subtle (10–20% reduction)
• NAD measurement is not as difficult as ATP measurement 
  • NADH measurement is more difficult
  • NAD tends to sit around for hourish timescale
• NAD is a tissue metabolite, not a circulating metabolite 
  • Need biopsy specimens to measure it
  • NAD levels in human tissues not as well-​​studied due to difficulty in obtaining biopsies

NAD Restoration Hypotheses

1. Restoring NAD levels in old organisms to young organism levels will make old organisms feel and perform like young organisms
2. Inducing supranormal levels of NAD in any organism will make them feel supernormal

Intravenous NAD

• NAD and its precursors are broken down in the gastrointestinal tract
• Intravenous NAD bypasses the liver’s first pass effect
• NAD breaks down into NR and NMN in the vascular system 
  • These can be taken up by some cells
• NR or NMN can be reconstituted into NAD within cells 
  • Energetically favored reaction
  • Not an expensive process

Oral NAD Precursors: NR and NMN

• Most efforts to increase intracellular NAD are done through oral precursors
• NR and NMN are considered equivalent approaches
• Oral NR and NMN can be broken down and reconstituted into NAD within cells
  Gastrointestinal Tract and NAD Precursors
• NR and NMN are broken down to nicotinic acid or niacin 
  • Main way they enter the body
  • Possible local effects or impact on the microbiome
• Niacin pro drugs 
  • Delayed absorption forms of niacin
  • Better tolerated

NR and NMN in Tissues

• Converted to niacin 
  • Raise niacin in the liver and portal circulation
• Effect on boosting circulating levels is subtle or vanishing 
  • Remain in the bloodstream less than nicotinamide
  • No clear route for oral NR or NMN to produce circulating levels high enough to compete with nicotinamide

Potential Misleading Factors

• Local effects of NR or NMN on the intestine
• Impact on the microbiome
• Small amounts of NR reaching specific cells that prefer it over nicotinamide

Restoring NAD Levels

• IV administration is a promising way to restore NAD levels
• Chronic administration of NR or NMN could have different effects
• Restoring intracellular NAD levels might not necessarily improve performance or health

Hormone Replacement Therapy Misinterpretation

• Women’s Health Initiative study misinterpreted 
  • Estrogen only group showed non-​​significant reduction in breast cancer risk
  • Estrogen plus MPA group showed barely significant increase in breast cancer risk
  • Absolute risk change was small (0.1%)
  • MPA could be the issue, not estrogen
• Preferred route of administration for hormone replacement therapy is now a patch 
  • Reduces cardiovascular mortality
    Intravenous NAD and Cellular Health
• Intravenous NAD may indirectly get into some cells 
• No convincing clinical study in humans using NR or NMN
• Need to map basic pharmacology of NAD in animals and humans
• Develop technologies to look at cellular resolution of NAD levels
• Successful clinical experiments needed
• NIH and Biotech interested in funding NAD research

Cancer Metabolism

• Cancer cells tend to be glucose users
• Programmed internally to feel like they’re always seeing insulin
• Leads to positive FDG Pet scans
• Uncontrolled growth requires uncontrolled nucleic acid synthesis
• Targeted by medications like Pematrexid for lung cancer
• Inducing mutations through metabolic stress on nucleotide system can make immunotherapy work better
  Cancer Metabolism and Fuel Usage
• Cancer cells can use various types of fuels depending on availability 
• Starving cancer is difficult due to:
• Glucose always being present in circulation
• Cancer cells having access to internally stored fuel (glycogen, amino acids, fat, lactate, ketones)
• Strategies to control cancer:
• Interrupt cancer cells’ ability to synthesize DNA, leading to more mutations and increased immune response
• Apply strong stress to cancer while putting pressure on fuel supply (e.g., chemotherapy paired with ketogenic diet)

Pancreatic Adenocarcinoma

• Fourth leading cause of cancer death in men and women
• Highly lethal, with only a 5% survival rate
• Difficult to treat due to:
• Early metastases
• Anatomical location, allowing for easy local invasion and access to the portal system
• Metabolic challenges, driven by Ras oncogene mutations, leading to scavenging nutrients from the environment and nonstandard nutrient intake
• Pancreatic cancer cells don’t need to be very metabolically active to be lethal

Potential Strategies for Pancreatic Cancer

• Make cancer a chronic disease with maintenance therapy
• Target cancer metabolism to create nucleotide imbalances, leading to mutations and immune response
• Combine chemotherapy with a ketogenic diet to lower glucose in tumors and improve outcomes
• Focus on early diagnosis and more effective treatments to increase the duration of response
  Metabolic Approaches to Cancer Therapy
• Metabolic approaches to cancer therapy show promise 
  • Combination of directed metabolic immune supplements and diet
  • Work with therapy to treat cancer
  • Potential to make immunotherapy work for a majority of patients
• Potential dietary strategies 
  • Amino acid restriction or nutrient restriction
  • Type of fat (saturated vs. unsaturated) can play different roles in cancer 
    • Higher saturated fat ketogenic diet could be more tumor suppressive in some contexts
  • Timing of macronutrients
  • Connection to the microbiome (fiber)
• Challenges in studying dietary strategies 
  • Converting animal diet and human diet
  • Aligning diets to isolate variables
  • Clinical trials and adherence to dietary strategies
• Future outlook
  • Building momentum in animal models and clinical work
  • Potential impact on patients’ lives within 5–10 years
  • Simplifying dietary strategies for clinical actionability
  • Nutritional supplements and acute dietary changes
  • Exploring ketosis in cancer patients with low carbohydrate diets and SGLT2 inhibitors

Princeton University and Medical Schools

• Princeton does not have a medical school 
  • Focus on undergraduate education
  • Pure intellectual environment
  • No business school or law school either
  • Distinct and special place for education
    Discussion with Josh
• Josh is a fan of Oppenheimer 
  • Read a biography about him
  • Matt Damon filming a movie about Oppenheimer on campus
• Josh has three of Feynman’s books 
  • Table of integrals from high school
  • Advanced calculus books from Princeton and Cornell
  • Books have Feynman’s notes and signatures
• Josh’s kids went to nursery school in the building where von Neumann built the first computer
• Looking forward to seeing each other in person again
• Appreciative of the work Josh has done over the past 20+ years