SLU-PP-332

Research Overview

SLU-PP-332 serves as a valuable research tool for investigating mitochondrial uncoupling mechanisms and their effects on cellular metabolism in laboratory settings. Mitochondrial uncouplers dissociate oxidative phosphorylation from ATP synthesis by increasing proton leak across the inner mitochondrial membrane, resulting in increased oxygen consumption, substrate oxidation, and heat production without proportional ATP generation.

Unlike classical mitochondrial uncouplers such as 2,4-dinitrophenol (DNP) or FCCP that show steep dose-response curves and narrow therapeutic windows, SLU-PP-332 demonstrates more favorable characteristics for research applications. The compound exhibits milder uncoupling activity, allowing investigation of subtle metabolic effects without the severe mitochondrial dysfunction associated with higher-potency uncouplers.

Research applications span mitochondrial bioenergetics, metabolic regulation, thermogenesis, obesity research, diabetes research, and investigations of the role of mitochondrial function in various disease models. Laboratory studies examine SLU-PP-332’s effects on mitochondrial respiration, membrane potential, ATP production, substrate utilization, gene expression, and whole-organism metabolic parameters in cell culture and preclinical models.

Molecular Characteristics

Complete Specifications:

• Molecular Weight: 456.5 Da
• Molecular Formula: C₂₄H₂₈N₄O₅
• Chemical Class: Synthetic mitochondrial uncoupler
• Appearance: Powder (appearance varies by specific formulation)
• Solubility: DMSO, ethanol (organic solvents), limited aqueous solubility
• Mechanism: Increases proton leak across inner mitochondrial membrane
• Selectivity: Shows milder uncoupling compared to classical uncouplers

The molecular structure of SLU-PP-332 contains functional groups enabling proton shuttling across biological membranes. Lipophilic characteristics allow membrane partitioning while ionizable groups facilitate proton binding and release, the fundamental mechanism of chemical uncoupling. The specific structural features of SLU-PP-332 result in its characteristic mild uncoupling profile.

Mechanisms of Mitochondrial Uncoupling

Classical Uncoupling Mechanism:
Mitochondrial uncouplers function as lipophilic weak acids that shuttle protons across the inner mitochondrial membrane, bypassing ATP synthase:

1. Protonated Form: The neutral, protonated form of the uncoupler diffuses across the lipid bilayer from the intermembrane space (where proton concentration is high due to electron transport chain activity) into the mitochondrial matrix.

2. Deprotonation: In the more alkaline matrix environment, the uncoupler releases its proton, becoming negatively charged.

3. Return Cycle: The charged, deprotonated form crosses back across the membrane (either through the lipid bilayer or via membrane transporters), where it can pick up another proton to repeat the cycle.

This futile cycle dissipates the proton-motive force that normally drives ATP synthesis, converting energy into heat instead of ATP.

SLU-PP-332 Characteristics:
SLU-PP-332 demonstrates milder uncoupling activity compared to classical uncouplers like DNP or FCCP. Research suggests:

• Dose-dependent increase in oxygen consumption
• Modest effects on mitochondrial membrane potential at lower concentrations
• Less dramatic ATP depletion compared to higher-potency uncouplers
• Better tolerability in cell culture and potentially animal models
• Suitable for investigating subtle metabolic shifts

These properties make SLU-PP-332 valuable for research examining mild metabolic stimulation without severe mitochondrial stress.

Research Applications

Mitochondrial Bioenergetics Research

SLU-PP-332 enables investigation of fundamental mitochondrial function and bioenergetic principles:

Mitochondrial Respiration Studies:

• Oxygen consumption rate (OCR) measurement using Seahorse XF analyzer or Clark-type electrode
• Assessment of basal respiration, ATP-linked respiration, and maximal respiratory capacity
• Dose-response characterization of uncoupling effects
• Comparison with classical uncouplers (DNP, FCCP, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone)
• Substrate-specific respiration (complex I, complex II, fatty acid oxidation)

Membrane Potential Measurements:

• TMRM (tetramethylrhodamine methyl ester) or JC-1 fluorescent probes
• Flow cytometry analysis of mitochondrial polarization in cell populations
• Confocal microscopy of membrane potential in live cells
• Relationship between uncoupler concentration and membrane potential changes
• Assessment of mitochondrial depolarization thresholds

ATP Production Analysis:

• Cellular ATP content measurement using luciferase-based assays
• ATP/ADP ratio determination
• Real-time ATP production rate measurement
• Relationship between oxygen consumption and ATP synthesis (P/O ratio)
• Effects on cellular energy charge

Mitochondrial Morphology and Dynamics:

• Live-cell imaging of mitochondrial network structure
• Assessment of fusion and fission dynamics
• Effects of mild uncoupling on mitochondrial morphology
• Relationship between bioenergetic state and mitochondrial dynamics
• Mitochondrial protein import and quality control

Research protocols typically employ isolated mitochondria, permeabilized cells, intact cells, or precision-cut tissue slices to characterize SLU-PP-332’s effects on mitochondrial function across different biological complexity levels.

Metabolic Research Applications

SLU-PP-332 enables investigation of metabolic regulation and energy balance:

Substrate Utilization Studies:

• Glucose oxidation and glycolytic flux measurements
• Fatty acid oxidation assessment using radiolabeled substrates
• Amino acid catabolism studies
• Metabolic flexibility testing (substrate switching capacity)
• Nutrient sensor activation (AMPK, mTOR, sirtuins)

Thermogenesis Research:

• Heat production measurement using calorimetry
• Brown adipose tissue (BAT) activation studies
• UCP1-independent thermogenesis investigation
• Cold-induced thermogenesis models
• Diet-induced thermogenesis studies

Energy Expenditure Analysis:

• Whole-body oxygen consumption and CO₂ production (metabolic cages)
• Respiratory exchange ratio (RER) determination
• Activity-corrected energy expenditure
• Fuel preference analysis
• 24-hour metabolic profiling

Metabolic Disease Models:

• Diet-induced obesity models
• Insulin resistance and type 2 diabetes models
• Non-alcoholic fatty liver disease (NAFLD) studies
• Metabolic syndrome investigations
• Aging and metabolic decline studies

Experimental approaches include cell culture models (adipocytes, hepatocytes, myocytes), tissue explants, and rodent models with comprehensive metabolic phenotyping.

Obesity and Diabetes Research

Mild mitochondrial uncoupling represents a potential research approach for metabolic disease:

Adipose Tissue Research:

• White adipocyte lipolysis and fat oxidation
• Brown adipocyte activation and thermogenic capacity
• Beige/brite adipocyte induction in white adipose tissue
• Adipose tissue gene expression (UCP1, PGC-1α, PPARs)
• Adipokine secretion profiles

Hepatic Metabolism:

• Hepatic glucose production and gluconeogenesis
• Fatty acid synthesis and oxidation balance
• Triglyceride accumulation and lipid droplet dynamics
• Hepatic insulin sensitivity
• Mitochondrial function in NAFLD models

Skeletal Muscle Metabolism:

• Muscle glucose uptake and insulin sensitivity
• Mitochondrial biogenesis and oxidative capacity
• Exercise-mimetic effects
• Muscle fiber type characteristics
• Myokine secretion

Whole-Body Glucose Homeostasis:

• Glucose tolerance tests (GTT, IPGTT)
• Insulin tolerance tests (ITT)
• Hyperinsulinemic-euglycemic clamp studies
• Fasting and postprandial glucose profiles
• HbA1c and fructosamine measurements

Research investigates whether mild metabolic stimulation via uncoupling improves metabolic parameters without causing adverse effects associated with higher-potency uncouplers.

Cellular Stress and Adaptive Response Research

Mild mitochondrial uncoupling activates cellular stress response pathways:

AMPK Activation:

• AMPK phosphorylation status (Thr172)
• Downstream target phosphorylation (ACC, TBC1D1)
• AMPK-dependent gene expression changes
• Metabolic consequences of AMPK activation
• Comparison with pharmacological AMPK activators

Mitochondrial Biogenesis:

• PGC-1α expression and activity
• Mitochondrial DNA copy number
• Mitochondrial protein expression (OXPHOS complexes)
• Citrate synthase and COX enzyme activities
• Mitochondrial mass measurements (MitoTracker, citrate synthase)

Mitochondrial Quality Control:

• Mitophagy induction and flux
• PINK1-Parkin pathway activation
• Mitochondrial protein import efficiency
• Mitochondrial unfolded protein response (UPRmt)
• Proteolytic pathways (Lon protease, ClpP)

Oxidative Stress and Antioxidant Responses:

• Reactive oxygen species (ROS) production measurement
• Antioxidant enzyme expression (SOD, catalase, GPx)
• Glutathione and NADPH levels
• Lipid peroxidation markers
• Nrf2 pathway activation

Research examines how mild metabolic stress from uncoupling activates adaptive pathways potentially conferring long-term benefits.

Aging and Longevity Research

Mitochondrial function plays central roles in aging processes:

Mitochondrial Aging:

• Age-related changes in mitochondrial function
• Mitochondrial DNA mutations and damage
• Decline in oxidative capacity with aging
• Mitochondrial-derived reactive oxygen species (mtROS)
• Effects of mild uncoupling on aging parameters

Healthspan and Lifespan Studies:

• Longevity assessments in model organisms (C. elegans, Drosophila, mice)
• Age-related disease onset and progression
• Physical performance and frailty measurements
• Cognitive function assessments
• Inflammatory markers and immunosenescence

Caloric Restriction Mimetics:

• Comparison of mild uncoupling effects with caloric restriction
• Overlapping molecular pathways (sirtuins, AMPK, mTOR)
• Metabolic reprogramming
• Stress resistance and resilience
• Potential caloric restriction-mimetic properties

Research investigates whether mild metabolic stress from uncoupling activates pathways similar to those induced by caloric restriction, potentially extending healthspan.

Neuroscience Research Applications

Mitochondrial function significantly influences neuronal health and function:

Neuronal Bioenergetics:

• Neuronal ATP production and energy status
• Synaptic transmission energy requirements
• Axonal transport energy dependence
• Effects of mild uncoupling on neuronal function
• Neuroprotection vs. neurotoxicity thresholds

Neurodegenerative Disease Models:

• Parkinson’s disease models (mitochondrial complex I deficiency)
• Alzheimer’s disease models (mitochondrial dysfunction, amyloid beta effects)
• Huntington’s disease models (mutant huntingtin mitochondrial effects)
• ALS models (mitochondrial impairment in motor neurons)
• Potential neuroprotective effects of mild uncoupling

Neuroinflammation:

• Microglial metabolic phenotypes
• Inflammatory mediator production
• Effects of metabolic modulation on neuroinflammation
• Blood-brain barrier integrity
• Neuron-glia metabolic coupling

Research examines whether mild metabolic stress improves neuronal resilience or potentially provides therapeutic benefits in neurodegenerative models.

Laboratory Handling and Storage Protocols

Powder Storage:

• Store at -20°C in sealed container with desiccant
• Protect from light exposure (use amber container if available)
• Maintain desiccated environment
• Stability data available for storage conditions
• Minimize opening frequency to prevent moisture exposure
• Record receipt and opening dates

Stock Solution Preparation:

• Dissolve in DMSO (primary solvent) to create concentrated stock solutions
• Typical stock concentrations: 10-100 mM depending on solubility
• Vortex thoroughly ensuring complete dissolution
• Verify complete dissolution visually before use
• May require gentle warming (37°C water bath) or brief sonication
• Never use open flame heating
• Document stock concentration, preparation date, solvent, lot number

Stock Solution Storage:

• Aliquot immediately after preparation (50-200 μL volumes)
• Store at -20°C in polypropylene tubes
• Protect from light (amber tubes or aluminum foil wrap)
• Avoid repeated freeze-thaw cycles (maximum 2-3 cycles)
• Single-use aliquots recommended
• Label with compound name, concentration, solvent, date, lot

Working Solution Preparation:

• Dilute stock solutions into appropriate cell culture medium, buffer, or vehicle for animal studies
• Add DMSO stock slowly with mixing
• Final DMSO concentration in cell culture: ≤0.1-0.5%
• For animal studies, appropriate vehicles required (PEG, Tween, cyclodextrin)
• Prepare fresh working solutions for each experiment when possible
• Filter sterilize for cell culture applications (0.22 μm)
• Include vehicle controls at equivalent concentration

Handling Precautions:

• Appropriate personal protective equipment (lab coat, gloves, safety glasses)
• Handle in fume hood or well-ventilated area
• Avoid skin contact and inhalation
• Follow institutional chemical safety protocols
• Dispose as hazardous chemical waste
• Clean spills immediately
• Wash hands thoroughly after handling

Special Considerations:

• Mitochondrial uncouplers can affect cellular viability at higher concentrations
• Establish concentration-response relationships in each experimental system
• Start with lower concentrations and titrate upward
• Monitor cell viability alongside metabolic measurements
• Consider temperature effects (uncoupling increases with temperature)

Quality Assurance and Analytical Testing

Each SLU-PP-332 batch undergoes comprehensive analytical characterization:

Purity Analysis:

• High-Performance Liquid Chromatography (HPLC): ≥98% purity
• Reversed-phase HPLC with appropriate column and mobile phase
• UV detection at compound-specific wavelength
• Multiple peak integration for accurate purity determination
• Impurity identification and quantification

Structural Verification:

• Electrospray Ionization Mass Spectrometry (ESI-MS): Confirms molecular weight (456.5 Da)
• High-resolution mass spectrometry for exact mass determination
• Nuclear Magnetic Resonance (NMR): ¹H-NMR and ¹³C-NMR structural verification
• Infrared spectroscopy for functional group confirmation
• Comparison with reference standards

Contaminant Testing:

• Bacterial endotoxin: <5 EU/mg (LAL method) for cell culture applications
• Heavy metals: Below detection limits per USP standards
• Residual solvents: GC-MS quantification within acceptable limits
• Water content: Karl Fischer titration
• Related substances and degradation products by HPLC

Documentation:

• Certificate of Analysis (COA) provided with each batch
• Analytical chromatograms and spectra available upon request
• Third-party analytical verification available
• Batch-specific QC results traceable by lot number
• Stability data for recommended storage conditions

Research Considerations

Experimental Design Factors:

1. Concentration Selection: Determine appropriate concentration ranges through preliminary dose-response studies. SLU-PP-332 typically shows effects at μM concentrations in cell culture. Start with broad concentration ranges (e.g., 0.1-100 μM) and narrow based on initial results.

2. Time Course: Effects may be immediate (bioenergetic changes) or delayed (gene expression, adaptive responses). Design time courses appropriate for endpoints measured. Acute measurements (minutes to hours) assess direct uncoupling effects. Chronic treatments (days to weeks) examine adaptive responses.

3. Temperature Control: Uncoupling activity temperature-dependent. Maintain consistent temperature during measurements. Consider temperature effects when comparing in vitro and in vivo results.

4. Cell Type Considerations: Different cell types show varying sensitivity to uncouplers based on metabolic characteristics. Highly metabolic cells (neurons, cardiomyocytes, hepatocytes) may show greater sensitivity than less metabolic cells (fibroblasts).

5. Vehicle Controls: Always include vehicle controls matching DMSO or other solvent concentration. Vehicle effects should be characterized in initial experiments.

6. Positive Controls: Include well-characterized uncouplers (FCCP, DNP at appropriate concentrations) as positive controls to validate experimental systems.

Mechanism Validation:

Confirm mitochondrial uncoupling mechanism through multiple complementary approaches:

Direct Mitochondrial Effects:

• Oxygen consumption increase
• Membrane potential decrease
• ATP production decrease
• Proton leak rate increase (measured in isolated mitochondria or permeabilized cells)
• Dose-response relationships for above parameters

Cellular Metabolic Consequences:

• Increased substrate oxidation
• AMPK activation
• Compensatory glycolysis upregulation
• Heat production increase
• Nutrient sensor pathway activation

Specificity Controls:

• Effects prevented by electron transport chain inhibitors (confirms dependence on respiration)
• No direct effects on ATP synthase (confirmed by maintaining effects in presence of oligomycin)
• Effects on mitochondria-rich vs. mitochondria-poor cells
• Comparison with structural analogs and inactive compounds

Compliance and Safety Information

Regulatory Status:
SLU-PP-332 is provided as a research chemical for in-vitro laboratory studies and preclinical research only. This compound has not been approved by FDA for human therapeutic use, dietary supplementation, or medical applications.

Intended Use:

• In-vitro cell culture studies of mitochondrial function
• In-vivo preclinical research in approved animal models
• Laboratory investigation of metabolic regulation
• Academic and institutional research applications
• Pharmaceutical research studying mitochondrial uncoupling

NOT Intended For:

• Human consumption or administration
• Therapeutic treatment or diagnosis
• Dietary supplementation or weight loss products
• Performance enhancement
• Veterinary therapeutic applications without appropriate oversight

Safety Protocols:
Researchers should follow standard laboratory safety practices:

• Use appropriate personal protective equipment
• Handle in well-ventilated areas or fume hood
• Follow institutional biosafety and chemical safety guidelines
• Avoid skin contact and inhalation
• Dispose of waste according to hazardous chemical regulations
• Consult safety data sheet (SDS) for specific safety information

Research Safety Considerations:

• Mitochondrial uncouplers can show toxicity at higher concentrations
• Establish safe concentration ranges in each experimental system
• Monitor cellular and animal health parameters carefully
• Classical uncouplers (DNP) have caused human fatalities – exercise appropriate caution
• While SLU-PP-332 shows milder activity, treat with appropriate respect as a bioactive compound

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