Lecture 12: Interventions Comabting Obesity

How do gut hormones regulate hunger/satiety?

• Ghrelin secretions rise between meals → hunger signal (homeostatic drive).

  • Ghrelin antagonists: not effective for long-term weight loss → poor target.

• Enteroendocrine cells release hormones in response to nutrients in the gut:

  • CCK → contributes to satiation.

    • CCK analogues: limited efficacy for weightless; likely useful only as co-therapy.

  • PYY → contributes to satiety.

• Incretins (GLP-1, GIP) are released with meals:

  • GLP-1/GIP analogues: effective treatments for obesity and weight loss.

What Are Incretins and How Do They Work

• GLP-1 and GIP are gut hormones released after a meal from L and K cells, respectively and enhance insulin secretion from the pancreas

• This promotes the uptake of glucose and fatty acids in the liver, adipose tissue and skeletal muscle, regulating blood glucose after eating

What is the Incretin Effect?

• After a meal, a rise in blood glucose stimulates insulin release from pancreatic beta cells

• GLP-1 and GIP are released from the gut in response to nutrients and travel through the portal vein to the pancreas to enhance glucose-dependent insulin secretion.

• This regulates blood glucose levels after a meal

• GLP-1 is rapidly degraded by the enzyme dipeptidyl peptidase IV → short half-life (unlikely to act in an endocrine fashion at other organs)

  • Native GLP-1 not effective as a drug

• Thought to be a good treatment target for diabetes

How Has the Incretin Effect Been Exploited Theraputically

• Sitagliptin (Januvia by Merck, Sharpe and Dohme) → Blocks dipeptidyl peptidase → increases GLP-1 half life → prolonged effect

  • Acts as a dipeptidyl peptidase antagonist

• Modifications of the GLP-1 amino acid sequence or the addition of a fatty acid side chain that binds albumin → peptide more resistant to breakdown, increasing its half-life e.g.

  • Extendin-4 (Exanitide): first peptide mimetic approved for diabetes → modified GLP-1 protein found in Glia monster lizards

What is Liraglutide (Victoza)?

• A more stable and potent analogue of GLP-1 → fatty acid chain added to bind with albumin and extend drug half-life

• Used in the treatment of diabetes, as daily injections (well tolerated)

• Unexpected weight reduction seen → normally drugs increasing insulin secretion result in weight gain marketed due to increased glucose and fatty acid uptake into tissue

• Problematic side effect: Nausea → problems with compliance and drug discontinuation

• Marketed by Novo Nordisk

How is Liraglutide Used as a Treatment for Obesity

• Marketed as Saxenda (approval in 2014).

• Available as injectable or oral daily formulations.

• NICE criteria: BMI >30, or >27 with comorbidities (e.g., diabetes, hypertension).

• Acts via appetite reduction, not increased energy expenditure

• Problematic side effect: Nausea → problems with compliance and drug discontinuation

What is Semaglutide?

• A higher dose GLP-1 analogue released in 2021 by Novo Nordisk

• Approved for overweight or obese adults with at least one weight-related co-morbidity

• Marketed as Ozempic (diabetes) and Wegovy (obesity).

• Modified GLP-1 peptide with extended half-life, allowing weekly injections.

• Causes ~15% weight loss.

• Generally well tolerated, but nausea and sickness can occur → may reduce compliance or lead to discontinuation.

What is Tirzepatide (Monjaro)

• A dual GLP-1 and GIP receptor agonist developed by Eli Lilly

  • single molecule with dual affinity for GLP-1 and GIP receptors

• Results in 20% weight loss in obese patients (clinical trials)→ More effective than semaglutide and similar to that of bariatric surgery

• Lower rates of nausea → better patient adherence compared to other incretin-based drugs

• Mechanisms affecting appetite and weight loss are not fully understood

Do systemically administered GLP-1 receptor agonists (e.g., semaglutide, tirzepatide) readily penetrate the brain?

• No — fluorescently tagged tirzepatide shows minimal brain penetration.

• Entry occurs only near circumventricular organs (CVOs), which lack a blood–brain barrier.

• These CVOs include:

  • OVLT (organum vasculosum of the lamina terminalis)

  • Subfornical organ

  • Median eminence (next to ARC)

  • ‘Area postrema (brainstem CVO)’

• Drugs have limited access via these regions

Where in the Brain Do GLP-1 Receptor Agonists Exert Their Main Effect?

• Strong binding occurs in the area postrema (brainstem CVO) → likely the main entry site.

• Effective doses activate brain regions beyond CVOs, even though drugs cannot directly reach them.

• This suggests that the drugs act on a distributed, downstream neural network, not just through local receptor activation.

• Many activated regions have receptors for brain-derived GLP-1

• The main site of action is within the brainstem, and other areas are downstream, which are activated transynaptically

What does cFos activation mapping show after semaglutide or tirzepatide administration?

• cFos expression appears in many brain areas that the drug cannot physically access → areas of activation are different to the areas of administration

• Indicates indirect activation via downstream circuits rather than direct receptor binding.

• Reinforces the idea that area postrema and other CVOs are entry points, with widespread activation occurring trans-synaptically.

Why is Brainstem GLP-1 Receptor Essential for Weight Loss Effects of GLP-1 Receptor Agonist GLP-140?

• Floxed GLP-1R mice + Cre recombinase (in AAV) injected into NTS/brainstem → selectively deletes GLP-1R in brainstem neurons → no weight loss

• WT mice injected with the virus retain GLP-1R → dose response effect seen → reduction in body weight

• This suggests that brainstem GLP-1 receptors are the primary site of action for GLP-1R agonist-induced weight loss.

• GLP-1R is expressed in multiple neuron types in the brainstem.

What Are Pre-Proglucagon (PPG) Neurons and How Do They Relate To GLP-1

• PPG: precursor for peptide hormone GLP-1, is expressed in multiple tissues.

  • In the pancreas, converted into glucagon.

  • In the gut, Gut, converted into GLP-1, which mediates the incretin effect.

• PPG neurons are located in the nucleus of the tractus solitarius of the brainstem, distinct from nearby PrRP neurons.

How can PrRP_NTS neurons be selectively activated using chemogenetics?

• Transgenic mouse lines expressing Cre recombinase in PrRP or PPG neurons (PPG-Cre or PrRP-Cre mice)

• AAV virus carrying an inverted DREADD flanked by lox sites is bilateral injected into the NTS

• In cells expressing Cre recombinase, Cre flips the DREADD transgene into the correct orientation, allowing it to exclusively express a designer receptor that can be activated by a designer drug or DREADD. As a result, PrRP neurons express hM3Dq.

• hM3Dq receptor activated exclusively by designer drug; linked to mCherry fluorescent marker for visualisation.

How is chemogenetic activation of PrRP_NTS Neurons Achieved In Vivo?

• Systemic injection of CNO (Clozapine N-oxide) activates the designer receptor (hM3Dq) expressed in PrRP neurons

• Activation can be visualised by FOS induction

• Allows for the selective activation of a single population of neurons in the brainstem

What happens when PrRP or PPG neurons in the NTS are chemogenetically activated at night in hM3Dq Transgenic mice?

• Activation with CNO at “lights out” reduces night-time food intake.

• Control mice injected with saline show normal cumulative food intake.

• Both PrRP and PPG neurons block feeding when activated → demonstrating that PrRP neurons are key in regulating food intake

  • These neurons project to similar brain areas.

How Do PrRP and PPG Neurons in the NTS Respond to Feeding Related Stimuli

• PrRP neurons: Activated by normal-sized meals, gentle stomach stretch, lipid infusion directly into the stomach, CCK, or oleoylethanolamide injection.

  • Mediate physiological satiety signals.

  • Possess GLP-1 receptors

• PPG neurons: Not activated by normal meals.

  • Respond to large/unexpected meals, intense stomach stretch, or nausea-inducing chemicals (e.g., lithium chloride).

  • Mediate protective/aversive satiety (stop eating to avoid illness).

What happens when PrRP and peptides are injected into the mouse brain?

• PrRP injection → reduces feeding.

• GLP-1 injection → paucicity in feeding → reduces feeding and produces conditioned aversion.

• Neither PrRP nor PPG neurons express GLP-1 receptors — they are not primary sensing neurons.

• Both neuron types ultimately stop feeding, but for different reasons: physiological vs protective.

Where are GLP-1 and GIP Receptors Located in Mice?

• Expressed within the brainstem, particulalry the area postream

What are GFRAL Neruons

• A distinct subset of neurons located in the area postream and dorsal NTS

  • distinct from PrRP and PPG neurons

• They express GLP1-R and a receptor for the cytokine GDF15, which is released at low levels by damaged or diseased cells, e.g. in cancer → can travel to the AP

  • When injected in mice, GDF15 induces a sickness-like behaviour

• GFRAL (GDNF-family receptor α-like) is only expressed in AP and dorsal NTS, nowhere else in the brain or body

• Also, co-expresses CCK, which acts as an NT in the brain

Which neurons are activated by the GLP-1 analogue Exendin-4, and how does this occur?

• GLP1R/CCK/GFRαl neurons in the area postrema (AP) and NTS are activated

• The short-acting GLP-1 analogue depolarises these neurons, increasing firing rate, even in the presence of TTX (synaptic blockade) → GLP-1 direct activates the neurons

• Significance:

  • GLP1R activation → reduced food intake.

  • GFRAL responds to GDF15, released from peripheral tissue during sickness and disease (peripheral sickness signal). → causes anorexia and nausea

  • This explains the anorexia/nausea side effects of GLP-1 receptor agonists.

• CCK is a widely distributed neurotransmitter in the brain, and its neuron population is heterogeneous in the brainstem; some co-express GLP1R and GFRAL, respectively

Which neurons are involved in the anorectic effect of GDF15 and Exendin-4?

• Brainstem CCK neurons are essential.

• Evidence:

  • CCK-Cre mice injected with control virus

  • Single dose of GDF15 or Ex4 reduces food intake for 24 hours.

  • Silencing CCK neurons with tetanus toxin (TetTox virus) prevents anorexia without killing neurons (transmitter release blocked) → neurons disabled

• CCK neurons mediate the anorexic effects of systemic GLP-1 receptor agonists and GDF15.

How Do Systemic GLP-1 Receptor Agonists (Ex-4) and GDF15 Act to Cause Anorexia?

• Drugs injected systemically and circulate for extended periods → reach and act on brainstem neurons that normally respond to GLP-1.

• They activate PPG neurons, generating anorexic effects and nausea.

• CCK neurons act as the critical downstream mediator of reduced food intake.

  • Silencing these neurons blocks drug-induced anorexia.

What Happens When GFRAL Neurons are Chemogenetically Activated in Mice

• GFRAL-Cre mice injected with a virus expressing a designer receptor,

• Designer receptor activated by CNO.

• Activation produces:

  • Strong anorexia → feeding is blocked.

  • Conditioned taste avoidance → mice avoid sucrose on conditioning day.

• Shows that GFRAL neurons can mediate effects similar to GLP-1 receptor agonists.

How does Tirzepatide act, and why does it reduce nausea compared to GLP-1 analogues alone?

• It is a dual GLP-1 and GIP receptor agonist, activating both receptors to produce synergistic weight loss.

• It shows reduced nausea as:

  • GIP activates inhibitory GABA neurons in the area postrema.

  • These neurons synapse onto and inhibit CCK/GFRAL neurons, lowering nausea.

How does GIP affect Exendin-4–induced brainstem activation and anorexia?

• Exendin-4 (GLP-1RA) alone activates CCK neurons with GLP-1 receptors → increases cFos, reduces food intake.

• GIP alone: little effect on food intake or CCK neurons.

• Co-administration (GIP + Exendin-4):

  • Reduces activation of CCK/GLP-1 neurons in the area postrema.

  • Explains why co-agonist therapy reduces nausea/aversion seen with GLP-1 receptor agonists.

• Synergistic effect on weight loss when both incretins are combined.

How Does Extendin 4 Induce Conditioned Taste Avoidance in Mice?

• Two-flavour conditioned taste test used → mice were acclimated to banana milkshake over a week

  • Steady increase in intake

• Conditioning day:

  • chocolate milkshake + EX4 → intake drastically reduced.

  • chocolate milkshake + saline → minimal effect on intake.

• Test day: chocolate milkshake given without the drug → mice given EX4 drink less, showing aversion/nausea.

• Conclusion: EX4 causes negative valence, leading to conditioned taste avoidance.

What role do CCK neurons play in EX4-induced anorexia and taste aversion?

• In CCK-Cre mice, brainstem CCK neurons were disabled via tetanus toxin.

• EX4 reduces intake on conditioning day in both CCK-Cre mice and controls

• On test day, mice with disabled CCK neurons do not avoid chocolate milkshake, unlike controls→ not formed conditioned avoidance (continue to drink milkshake)

• CCK neurons are necessary for both anorexia and aversive conditioning induced by GLP-1 receptor agonists.

How does co-administration of GIP affect GLP-1 receptor agonist (EX4)-induced aversion in the two-flavour conditioned taste avoidance test?

• GIP alone: minimal effect on chocolate milkshake intake (no aversion).

• EX4 alone: causes conditioned taste avoidance (aversion to chocolate milkshake).

• EX4 + GIP: aversion is reduced/attenuated.

• This explains why dual incretin agonists (e.g., tirzepatide/Mounjaro) cause less nausea while maintaining weight loss → synergistic effect.

Why is understanding GIP and GLP-1 co-agonism important for obesity drug development?

• Co-agonism provides a synergistic effect on weight loss and reduced nausea → dual agonists are now widely marketed for the treatment of obesity

• This mechanism is not fully understood, but it may include direct effects of GIP on adipose tissue which lead to reduced weight overtime

• Insight into their mechanism of action helps guide the development of safer and more effective dual incretin drugs

How does the Triple Agonist LY343943 (retatrutide) compare to tirzepatide in pre-clinical and clinical studies?

• Targets GLP-1, GIP, and glucagon receptors → multi-pathway approach.

• Glucagon receptor important for reducing peripheral fat deposition.

• Compound further developed into retatrutide and has been shown to be more effective than tirzepatide in clinical trials (Eli Lilly).

What is Cagrilintide?

• A long-acting amylin (and calcitonin) receptor agonist produced by Novo Nordisk → amylin co-released with insulin from pancreatic beta cells post prandially to reduce circulating glucose levels (affects appetite and digestion)

  • Alone: reduce weight

  • Cagrilintide + Semaglutide → synergy, with greater weight loss seen (phase 3 trials)

    • Reason for Synergy unknown

• Side effects: nausea → activation of the calcitonin receptor

  • Difficult to target amylin receptor alone as they are from the same family

• Effects not as strong as tirzeptide → new drugs need improved efficacy, tolerance, or fewer side effects.

What is the purpose and effect of LSL-PrRP x Cre transgenic mice in studying PrRP function?

• In LSL-PrRP, the mouse loxSTOPlox codon is in front of PrRP gene and blocks its transcription (PrRP null phenotype).

  • Does not respond to the anorexic effects of CCK.

  • Exhibits late-onset obesity.

• Re-expression of PrRP using Cre-Lox:

  • In Nestin-Cre mice crossed with LSL-PrRP mice, PrRP expression is restored in all three normal brain regions.

  • In TH-Cre mice crossed with LSL-PrRP mice PrRP expression was only restored in brainstem neurons (co-localised with tyrosine hydroxylase).

• It allows the study of region-specific roles of PrRP neurons in appetite and body weight regulation.

How does re-expression of PrRP in LSL-PrRP mice affect the anorectic response to CCK and leptin?

• WT Offspring: CCK reduces food intake (anorectic effect)

• In LSL-PrRP null mice: No PrRP → CCK cannot reduce food intake.

• Nestin-Cre x LSL-PrRP: PrRP expression restored in whole brain → CCK reduces food intake.

• TH-Cre x LSL-PrRP: PrRP expression restored only in brainstem → CCK effect restored, showing brainstem PrRP is key for CCK-induced anorexic effect.

• Hypothalamic PrRP is important for leptin-mediated regulation of food intake

What is the Effect of GPR10 Knock Out In Mice?

• GPR10 is the receptor for PrPR

• Knock-out mice become obese with age

• Little difference in food intake (slightly reduced)

• The cause of obesity was due to low energy expenditure, not overeating

  • This was confirmed using indirect calorimetry shows reduced oxygen consumption/CO₂ → confirms low energy expenditure

• Energy expenditure was low before maturity → main cause of early onset obesity

What is the significance of mutations in the human GPR10 gene?

• Exome sequencing in obese patients revealed 15 SNPs in human GPR10 gene

• Mutations lead to varying degrees of obesity, often early-onset morbid obesity

• Patients also show low blood pressure → suggests an underperforming sympathetic nervous system (SNS)

• Example: Proline → Serine substitution at residue 193 is one of the most common variants

What are the effects of the GPR10 P193S humanised mutation in mice?

• Both heterozygous and homozygous mice are overweight compared to wild-type mice

  • Not as extreme as GPR10 null

• Daily food intake is normal, but they have low energy expenditure → similar to GPR10 null mice

• Phenotype mirrors the most common human GPR10 gene variant

• Implication: GPR10 could be a target for pharmacological intervention in obesity, as it affects both energy intake and expenditure if treatments are safer and effective

How Does the PrRP Analogue NN501 Access the Brain After Systemic Injection

• Only enters the brain at the circumventricular organs (CVOs):

  • Forebrain: organum vasculosum of the lamina terminalis (OVLT), subfornical organ (SFO), median eminence (near arcuate nucleus)

  • Brainstem: area postrema and underlying nucleus of the tractus solitarius (NTS)

• It is s een throughout blood vessels

How does the PrRP analogue NN501 affect body weight and food intake compared with semaglutide?

• NN501 and semaglutide produce similar body-weight loss.

• NN501 only moderately reduces food intake, while semaglutide strongly suppresses feeding.

• After stopping semaglutide treatment, there is a large rebound effect → Hyperphagia and rapid weight regain occur.

• After stopping NN501: no rebound feeding and slower weight regain

What Metabolic Changes Indicate NN501 Increases Fatty Acid Oxidation?

• Pair-fed control mice lost less weight, suggesting NN501 has effects beyond reduced intake.

• Indirect calorimetry revealed no major change in overall energy expenditure.

• But NN501 causes a large, sustained drop in respiratory exchange ratio (RER) - CO2 produced: O2 consumed → indicates the substrates used for metabolism.

  • Low RER value = shift from carbohydrate to fat utilisation → NN501 increases fat utilisation and oxidation in mice.

What Evidence Shows NN501-Induced Weight Loss Depends on Fatty Acid Oxidation?

• NN501-induced weight loss is attenuated by etomoxir, which blocks fatty-acid oxidation.

  • This suggests fat-oxidation pathways are required for NN501’s effect.

• NN501 acts via different mechanisms than semaglutide, raising the possibility of synergy when combined

What Happens When Sub-Optimal Doses of NN501 and Semaglutide are Co-administered?

• They produce an additive loss of body weight.

• Indicates complementary mechanisms and suggests the two drugs may be effective as a combination therapy for obesity.

What Are The Main Stratergies For Combating Obesity?

• Lifestyle changes: Genes haven’t changed; environment has changed → behaviour has a substantial effect

  • Diet or exercise alone is often ineffective due to homeostatic compensation (protection of body adiposity)

• Government & public health interventions:

  • Prevent inital weight gain (e.g. sugar tax, reduced salt intake)

• Bariatric surgery: Most effective for long-term weight loss

  • Expensive, risky, and reserved for severe obesity

• Pharmacological interventions:

  • Target neurotransmitters or gut hormones (often in combination)

  • Can cause side effects (e.g. nausea, SNS activation due to interference of whole transmitter systems)

  • Mimicking natural hormones may be safer

• Education & prevention:

  • Teaching children and families lifestyle changes

  • Considered the most effective long-term solution against the obesity epidemic