npb 130 midterm 2

Growth Hormone

• GH is secreted from anterior pituitary

• controlled by hypothalamus

• acts on multiple tissues (liver, muscle, adipose, bone)

• works via direct effects + IGF-mediated effects

what triggers GH release

stimulators:

• GHRH (from the hypothalamus)

• sleep

• exercise

• hypoglycemia (lower glucose level)

• fasting

inhibitors:

• somatostatin (from the hypothalamus)

• hyperglycemia

• elevated IGF-1

• obesity

what peptides regulate GH

growth hormone-releasing hormone (GHRH)

• produced by GHRH neurons in the hypothalamus

somatostatin

• produced by somatostatin neurons in the hypothalamus (periventricular nucleus, PeN)

both released into hypophyseal portal system

where is GH is from?

anterior pituitary

• secreted by somatotrophs (GH also known as somatotrophin)

• ~50% of anterior pituitary cells

• stored in secretory vesicles/granules

• released via exocytosis

what is released? (GH)

• Hormone: Growth hormone (GH)

• Type: protein hormone (~191 amino acids)

• water-soluble

• circulates partially bound to GH-binding protein (GHBP) —> BP wants to grab GH to increase half-life

• half-life: 20-30 mins

GH Secretion

• pulsatile (long term)

• highest release during sleep

• higher in children and adolescents

• declines with aging

Target tissue of GH

GH acts on:

• liver

• muscle

• adipose tissue

• bone (needs to become longer as growth)

Growth hormone receptor (GHR)

• single-pass transmembrane protein

• cytokine receptor family (conserved signaling method)

• signals via JAK-STAT pathway

GH Mechanism: JAK-STAT signaling

1. GH binds GHR

2. receptor dimerization

3. JAK2 activation

4. STAT phosphorylation

5. STAT dimer enters nucleus

6. transcription of target genes (including IGF-1)

JAK1 and JAK2 phosphorylate each other —> each have intrinsic kinase activity

JAK phosphorylates STAT5 and receptor (via box1) too —> receptor needs to be phosphorylated to change the binding site for STAT5

Direct effects of GH on liver

Increase gluconeogenesis (maintains blood glucose availability, helping preserve circulating glucose for glucose-dependent tissues, like brain)

Direct effects of GH on muscle

• increase protein synthesis (supports tissue growth)

• decrease glucose uptake (preserves circulating glucose for tissues that depend on it, such as brain)

Direct effects of GH on adipose

• increase lipolysis (provides fatty acids that can be burned for energy)

• decrease glucose uptake (preserves circulating glucose for tissues that depend on it, such as brain)

Indirect effects of GH via IGF-1

IGF-1:

• produced mainly by liver, induced by GH

• acts via IGF1R (a receptor tyrosine kinase)

• activates PI3K-Akt and MAPK pathways

Promotes:

• chondrocyte proliferation in growth plate

• bone elongation

• tissue growth

Dwarfism vs Laron Syndrome (Primary Defect)

Dwarfism (GH deficiency): Decreased GH secretion from anterior pituitary (don’t produce enough GH)

Laron Syndrome (GH resistence): mutation in GH receptor (GHR) (even if you have enough the tissues don’t respond to it)

Dwarfism vs Laron Syndrome (GH level)

Dwarfism: Low

Laron Syndrome: High (loss of negative feedback)

Dwarfism vs Laron Syndrome (IGF-1 level)

Dwarfism: low

Laron Syndrome: low

Dwarfism vs Laron Syndrome (growth plate status)

Dwarfism: Open (childhood)

Laron Syndrome: Open (childhood)

Dwarfism vs Laron Syndrome (height)

Dwarfism: short, proportionate

Laron Syndrome: short, proportionate

Dwarfism vs Laron Syndrome (mechanism)

Dwarfism: not enough GH —> insufficient IGF-1 production

Laron syndrome: GH present but receptor nonfunctional —> no IGF-1 production

Dwarfism vs Laron Syndrome (treatments)

Dwarfism: recombinant GH

Laron Syndrome: recombinant IGF-1 (mecasermin)

gigantism vs acromegaly

GH excess before vs after epiphyseal closure

gigantism vs acromegaly (primary defect)

gigantism: GH-secreting pituitary adenoma (childhood)

acromegaly: GH secreting pituitary adenoma (adulthood)

gigantism vs acromegaly (GH level)

gigantism: high

acromegaly: high

gigantism vs acromegaly (IGF-1 level)

gigantism: high

acromegaly: high

gigantism vs acromegaly (growth plate status)

gigantism: open

acromegaly: closed

gigantism vs acromegaly (height)

gigantism: very tall (excess linear growth)

acromegaly: normal height (no further lengthening)

gigantism vs acromegaly (main clinical features)

gigantism: proportionate tall stature

acromegaly: enlarged hands, feet, jaw; soft tissue overgrowth (cartilage)

gigantism vs acromegaly (treatments)

• adenoma removal surgery

• somatostatin analogs

• GH receptor antagonist (pegvisomant)

GH axis (i.e., somatotropic axis) overview

Hypothalamus

—> GHRH (+)

—> somatostatin (-)

Anterior pituitary

—> GH

Liver

—> IGF-1

Negative feedback

• IGF-1 inhibits GH

• GH inhibits GHRH, stimulates somatostatin

endocrine function of the pancreas

pancreatic islet (~2% of the pancreas) —> major endocrine part of pancreas

alpha cell

• releases glucagon

beta cell

• releases insulin

gamma cell

• pancreatic polypeptide

delta cell

• somatostaitn

collagen

duct

ductal cell

centroacinar cell

erythrocyte

blood vessel

acinar cell

• trypsin

• amylase

where does insulin come from?

pancreas and goes to other metabolic organs

Control of pancreatic insulin secretion

secretion is simulated by:

• increase plasma glucose

• parasympathetic NS

secretion is inhibited by:

• decrease in plasma glucose

• sympathetic NS

• stressed —> sympathetic so NO INSULIN

How does plasma glucose cause beta cells to secrete insulin?

BETA CELL SECRETES INSULIN

1. glucose enters pancreatic beta-cells through glucose transporters (primarily GLUT1 in human B-cells; GLUT2 is especially important in rodents)

2. Glucose metabolism increases ATP production, raising the intracellular ATP/ADP ratio

3. ATP-sensitive K+ channels close, reducing K+ efflux

4. The B-cell membrane depolarizes because less positive charge leaves the cell

5. voltage-gated Ca2+ channels open, allowing Ca2+ to enter the cell

6. increased intracellular Ca2+ triggers exocytosis of insulin-containing secretory granules

increased glucose —> increased ATP

How does plasma glucose cause beta cells to secrete insulin?

BETA CELL AT REST

1. low glucose levels in blood

2. metabolism slow

3. ATP decreases

4. K_ATP channels open

5. cell at resting membrane potential —> no insulin is released

   • voltage gated Ca2+ channel is closed

   • no insulin secretion

effect on insulin on target (simplified version)

insulin is the key that unlocks the glucose channel —> glucose channel opens and glucose to enter the cell

what does insulin do?

acts on adipose tissue and skeletal muscles to increase glucose uptake

• in the absence of insulin, glucose cannot enter the cell

• insulin signals the cell to insert GLUT 4 transporters into the membrane, allowing glucose to enter cell

what pathway is insulin-stimulated GLUT4 trafficking from?

Insulin-stimulated GLUT4 trafficking via the PI3K-AKT pathway

IRS = insulin receptor substrate —> phosphorylated by receptor

phorphorylated IRS activares PI3K by conformational change

convert PIP2 —> PIP3

PIP3 recruits PDK1 and phosphorylates the AKT

phosphorylated AKT activated and acts on AS160

AS160 inhibits Rab

Rab carries GLUT4 and this inhibition allows it to move to the cell surface

what does insulin act on to activate hexokinase?

insulin acts on hepatocytes (liver) to activate hexokinase

• before insulin binding

  • glucose concentration high both inside and outside cell

• after insulin binding, liver cell takes up glucose

  • glucose concentration high outside cell but low inside

  • hexokinase-mediated conversion of glucose to G-6-P keeps intracellular [glucose] low

GLUT2 —> liver

GLUT 2 already on cell surface

glucose homeostasis overview

why it matters:

• glucose is the main fuel for cellular function and ultimately organism survival

• glucose —> organs

glucose homeostasis: balance between input (dietary glucose, hepatic output) and utilization (uptake, storage, oxidation)

glucose input - glucose utilization = change in circulating glucose

key target organs of glucose regulating hormones

adipose tissue, skeletal muscle, liver

effects of insulin on target organs

main goal: store ingested nutrients

• increase glucose uptake (muscle, adipose; liver to a lesser degree)

• increase glycogenesis, decrease glycogenolysis (liver, muscle)

• increase lipogenesis (adipose, liver)

• increase protein synthesis (muscle; liver to a lesser degree)

effect of glucagon on target organs

secretion triggered by: decrease glucose

receptor signaling via: GPCR, cAMP-PKA pathway

effects of glucagon on its primary target organ:

main goal: increase hepatic glucose output

• glycogenolysis (liver)

• gluconeogenesis (liver)

the glucagon: insulin ratio is critical

fed state:

insulin outweighs glucagon

• increase glucose oxidation

• increase glycogen synthesis

• increase fat synthesis

• increase protein synthesis

fasted state:

glucagon outweighs insulin

• increase glycogenolysis

• increase gluconeogenesis

• increase ketogenesis

examples of hormones that influence circulating glucose levels through primary mechanisms

growth hormone

• source: anterior pituitary

• target organs: liver, muscle, adipose

• key function related to glucose metabolism: increase gluconeogenesis (liver), decrease glucose uptake (muscle, adipose)

• blood glucose: increase

T3 (triiodothyronine)

• source: thyroid gland (formed from T4 conversion in tissues)

• target organs: liver, muscle, adipose

• key function related to glucose metabolism: increase gluconeogenesis and glycogenolysis (liver), increase glucose uptake

• blood glucose: increase

glucocorticoids

• source: adrenal cortex

• target organs: liver, muscle, adipose

• key function related to glucose metabolism: increase gluconeogenesis (liver), decrease glucose uptake (muscle, adipose, tissue)

• blood glucose: increase

examples of hormones that influence circulating glucose levels through secondary mechanisms

GLP-1 (glucogan-like peptide-1)

• source: intestinal L-cells

• target organs: pancreas

• key function related to glucose metabolism: increase insulin secretion, decrease glucagon secretion

• blood glucose: decrease

estrogen

• source: reproductive and metabolic tissues

• target organs: liver, muscle, adipose

• key function related to glucose metabolism: increase insulin sensitivity

• blood glucose: decrease

progesterone

• source: reproductive and metabolic tissues

• target organs: liver, muscle, adipose

• key function related to glucose metabolism: decrease insulin sensitivity

• blood glucose: high

how does insulin regulate blood glucose?

by inducing glucose uptake and metabolism in metabolic organs

diabetes epidemiology - humans

• 11.6% of americans have diabetes (data from 2023)

• 1.2 million americans are diagnosed each year

• every 26 seconds, someone in the US is diagnosed with diabetes

• diabetes isn’t just a disease - it’s an epidemic, affecting over 38 million americans

• 528 million (11%) worldwide

diabetes epidemiology - animals

companion animals

• dogs: ~0.3-0.6%

• cats: ~0.2-1%

livestock

• cattle and small ruminants: <0.01%

• horses: <0.003%

Type 1 diabetes

• insulin insufficiency

• also called “insulin-dependent”

• ~5% of the diagnosed diabetes

• low/absent blood insulin levels

• typically begins early in life

• causes: genetic, auto-immune

• destruction of beta-cells —> inability to produce insulin

• treatment: insulin replacement

type 2 diabetes

• insulin resistance

• also called “insulin-independent”

• ~85% of the diagnosed diabetes

• low, normal, or high insulin levels

• typically appears later in adulthood

• cause: genetic, obesity, lifestyle

• loss of sensitivity of peripheral tissues to insulin (receptor, IRS, AKT)

• treatment: multifactorial

gestational diabetes

• cause: hormonal changes during pregnancy cause insulin resistance

• may resolve after birth

secondary diabetes

due to:

• pancreatic disease (pancreatitis, tumor)

• endocrine disorders (Cushing’s)

• drugs (glucocorticoids)

symptoms of diabetes

polyuria

• meaning: excessive urination

• mechanism: when blood glucose exceeds the renal threshold (~180 mg/dL in humans), glucose spills into urine. Glucose is osmotically active —> pulls water into the urine —> large urine volume

polydipsia

• meaning: excessive thirst

• mechanism: water loss from polyuria —> dehydration and increased plasma osmolarity —> hypothalamic thirst center activated —> increased drinking behavior

polyphagia

• meaning:excessive hunger

• mechanism: despite high glucose in blood, cells can’t use glucose effectively (lack of insulin or insulin resistance) —> cells sense “energy starvation” —> hypothalamus triggers increased appetite

symptoms and complications of diabetes

• increased thirst

• slow-healing cuts and sores

• fatigue

• blurred vision

• frequent urination

• unexplained weight loss

symptoms and complications of diabetes (animal specific)

human: polyuria, polydipsia, polyphagia, weight loss, blurry vision, numb limbs, dry skin

cattle: weight loss, poor hair coat, glucosuria, ketonuria, and hyperglycemia

sheep: excessive fluid consumption, excessive thirst

horse: weight loss, drinking excessively, increased urination, laminitis

dog: excessive water drinking and increased urination, weight loss, cloudy eyes, recurring infections

diagnosis of diabetes

fasting plasma glucose (FPG): checks fasting blood glucose levels after not having anything to eat or drink (except water) for at least 8 hours before the test

• normal: <100 mg/dL

• pre-diabetes: 100-126 mg/dL

• diabetes: >126mg/dL

oral glucose tolerance test (OGTT): checks blood glucose levels before and two hours after drinking a glucose-rich liquid

• normal: <140 mg/dL

• pre-diabetes: 140-200 mg/dL

• diabetes: >200 mg/dL

A1C test: detects glycated hemoglobin, reflects ~3 months avg glucose

• normal: <5.7%

• pre-diabetes: 5.7-6.5%

• diabetes: >6.5%

treatments for diabetes

T1D

• problem: little or no insulin production

• solution: insulin injections or pumps

T2D:

• problem: insulin resistence

• solution: metformin, semaglutide, etc.

what is adipose tissue (body fat) composed of?

1. adipocytes (fat cells)

   • white adipocytes: contain lipid droplet

   • brown/beige adipocytes: contains lipid droplet + abundant mitochondria

2. stromal vascular fraction (SVF)

   • preadipocytes

   • endothelial cells

   • fibroblasts

   • immune cells

3. vascular and nerve networks

what fat is less abundant than white fat?

brown fat (used for generating heat —> babies have more brown fat)

white fat —> facial, periarticular, pericardial, intramuscular, bone marrow, and others, subcutaneous, visceral, retroperitoneal, inguinal, gonadal

brown fat —> cervical, supraclavicular, axillary, periaortic, paravertebral, perirenal, surrounding organs, inguinal

types of adipocytes

brown adipocytes

• mitochondrial density: high

• primary function: thermogenesis, endocrine

• mitochondria gives the adipose tissue the brown color

white adipocytes

• mitochondrial density: low

• primary function: energy storage, endocrine

brite/beige adipocytes

• mitochondrial density: medium

• primary function: thermogenesis? endocrine?

Leptin

“I have enough energy stored”

• from: white adipose tissue

• gene: Lep

• receptor: LepRb

• target:

  • hypothalamus: food consumption

• circulating level: proportional to adipose amount

Leptin mechanism

signaling pathway: JAK2-STAT3

1. leptin binds LepRb on the POMC (proopiomelanocortin) neuron’s membrane in the arcuate nucleus (ARC) —> activates the associated JAK2

2. JAK2 phosphorylates the receptor, creating a docking site for STAT3 which is then phosphorylated

3. phosphorylated STAT3 forms a dimer and enters the nucleus, where it promotes POMC gene transcription

4. the neuron makes more POMC precursor protein, which is processed into peptides, including α-MSH

5. released α-MSH activates MC4 receptors on downstream neurons in the paraventricular nucleus (PVN) —> reduces

what happens in obesity?

obesity —> increase leptin —> leptin resistence —> increase food uptake and decrease energy expenditure

knockout models for obesity

ob/ob mouse: mutation in leptin

db/db mouse: mutation in leptin receptor

same phenotype:

obesity, hyperphagia, insulin resistance

multidisciplinary methods to discover new secreted proteins and their functions

• sample collection

• biochemical purification

• mass spectrometry

• bioinformatic screen

• bioactivity assay

identification of isthmin from mouse fat cells

white adipocytes —> conditioned media —> TMT-labeling —> mass spectrometry —> isthmin secretion

beige adipocytes —> conditioned media —> TMT-labeling —> mass spectrometry —> isthmin secretion

isthmin

an understudied secreted protein

what are isthmin-ko mice intolerant to?

glucose

what does isthmin not act on?

insulin receptors

what reveals distinct signaling actions by isthmin and insulin?

phosphoproteomic mapping

what does isthmin trigger?

phosphorylation of multiple proteins involved in protein translation and muscle function

what does isthmin induce?

protein synthesis in mouse myotubes (muscle fibers)

how do isthmin-KO mice compare to WT

isthmin-KO have smaller muscle fiber areas compared to WT

they also have lower muscle protein synthesis

how does isthmin modulate metabolic health?

by improving muscle proteostatis and systemic glucose metabolism

buffet eating strategy?

don’t eat fatty food first!

• cholecystokinin (CKK) response

• slows gastric emptying

• can produce early fullness

big picture of the gut

• gut is the body’s largest endocrine organ

• releases many peptide hormones

• communicates with other organs

• integrates feeding and metabolism

structure of the gut

• layers

  • mucosa

  • submucosa

  • muscularis

  • serosa

• endocrine cells are mainly scattered throughout the epithelium within the mucosa

histology of the gut

• mucosa

• epithelium

• lamina propia

• muscularis mucosa

• submucosa

• muscularis externa

enteroendocrine (EE) cells

• specialized hormone-secreting epithelial cells

• apical side senses nutrients in gut lumen

• basolateral side secretes hormones into blood

general signaling mechanisms

• mostly peptide and protein hormones

• mostly bind membrane GPCRs

• endocrine (left villus)

• paracrine and neural actions (right villus)

gut-brain axis

• gut hormones communicate with hypothalamus

• signals hunger and satiety

• neural and endocrine communication

ghrelin

• secreted mainly from stomach

• major circulating hunger hormone

• increases before meals

• stimulates appetite

ghrelin signaling

• acts on hypothalamus

• activates NPY/AgRP neurons

• increases food intake

• receptor: GHS-R1 (a GPCR): growth hormone secretagogue Receptor 1a

peptide YY (PYY)

• secreted from L cells

• released after meals

• promotes satiety

• receptor:

  • Y2 receptor (Y2R)

  • a GPCR

DECREASES APPETITE

incretins

• gut hormone enhancing insulin secretion

• released after oral nutrients

• oral vs IV glucose effect

incretin not released when glucose given through infusion

GLP-1 is a type of incretin

• glucagon-like peptide-1 (GLP-1) —> glucagon + GLP-1 derived from the same precursor

  • glucagon —> increases glucose

  • GLP-1 —> DECREASES glucose

• secreted from L cells

• distal intestine and colon

GLP-1 signaling and functions

• receptor: GLP-1R

• PCR signaling, increased cAMP —> GPCR family

• functions:

  • pancreas:

    • enhances insulin release

    • suppresses glucagon release

  • main brain regions affected: hypothalamus

    • activates satiety neurons (POMC)

    • inhibits hunger neurons (NPY/AgRP)

  • stomach:

    • slows gastric emptying

GLP-1 functions

food —> intestine —> GLP-1 released —> …

brain

• reduces appetite

• induces fullness feeling

stomach

• slows gastric movement

• slows gastric emptying

pancreas

• stimulates insulin

• suppresses glucagon

GLP-1 as a drug target

• GLP-1 receptor agonists:

  • liraglutide (first-generation GLP-1 drug)

  • semaglutide (stronger long-acting GLP-1 drug)

  • tirzepatide [dual incretin (GLP-1 + GIP) drug]

• used for:

  • type 2 diabetes

  • obesity

summary of gut endocrine physiology

• gut hormones regulate:

  • digestion

  • insulin secretion

  • appetite

  • energy balance

• gut acts as sensory endocrine organ

liver big picture

the liver as a communication hub

• senses nutrient status

• releases endocrine factors

• coordinates whole-body metabolism

communicates with

• brain

• muscle

• adipose tissue

• pancreas

• bone (IGF-1 —> GH)

what makes an endocrine organ?

criteria for an endocrine organ

• produces signaling molecules

• releases them into into circulation

• signals act on distant target tissues

• produces physiological effects

the liver satisfies all criteria

major liver cell types

hepatocytes

• main metabolic and secretory cells

kupffer cells (immune cells in the liver)

• resident macrophages

hepatic stellate cells

• vitamin A storage

• fibrosis in disease

liver sinusoidal endothelial cells

• fenestrated capillaries

  • have lots of pores —> so nutrients can get into the liver

hepatocytes as secretory cells

hepatocyte structure

• high protein synthesis capacity

• large rough ER and Golgi

• close contact with blood

sinusoids

• fenestrated capillaries

• rapid secretion into circulation

hepatokines

• IGF-1

• Hepcidin

• FGF21

• Thrombopoietin

• angiotensinogen

• ANGPTLs

not all heptokines are hormones

IGF-1

• peptide hormone

• mainly produced by the liver

• secreted primarily by hepatocytes

• targets: bone, muscle, adipose tissue, and many other organs

• production stimulated by growth hormone (GH)

IGF-1 signaling and function

• receptor: IGF-1 receptor (receptor tyrosine kinase)

• signaling: PI3K-AKT and MAPK pathways

• promotes body growth and protein synthesis

• supports bone elongation and muscle growth

• helps regulate metabolism and cell survival

• low IGF-1: growth failure, reduced muscle mass

angiotensinogen

• peptide precursor protein

• produced mainly by the liver

• secreted primarily by hepatocytes

• released into circulation

• targets blood vessels, adrenal gland, kidney, and brain after conversion to angiotensin II (active peptide hormone)

renin-angiotension-aldosterone system

main goal: regulate blood pressure and fluid balance by stimulating vasoconstriction and sodium/water reabsoption

• angiotensinogen —> produced by liver

• the kidneys release renin, which cleaves angiotensinogen into angiotensin

• ACE (angiotensin-converting enzyme) found in the lungs converts angiotensin I to angiotensin II

• two major actions of angiotensin II are vasoconstriction (acts on vascular smooth muscle to constrict blood vessels) and aldosterone release (acts on the adrenal gland to stimulate the release of aldosterone

• main action of aldosterone on the kidneys: acts on the kidneys to stimulate the reabsorption of sodium and water, increasing blood volume and pressure

step-by-step pathway of the RAAS pathway

1. liver —> angiotensinogen

2. kidneys release renin —> converts angiotensinogen to angiotensin I

3. lungs release ACE —> converts angiotensin I to angiotensin II

4. angiotensin II causes vasoconstriction AND stimulates adrenal gland to release aldosterone

5. aldosterone causes sodium/water reabsorptioann in the kidneys