Week 4B: Histopathology Pancreas, Regulation Gene Expression, Nuclear Hormone Receptors & Iron Metabolism Flashcards
HC27-29
Keratin in skin
-Subaceous glands (talgklieren)
-Epithelium cells (plaveisel)
-Hair follicles
-Sweat glands
From which germ later are the endocrine cells of the pancreas (islets) derived?
Endoderm
Secretion in pancreas
-Exocrine: to lumen duodenum through duct
-Endocrine: give hormones to blood
Formation pancreas out of endoderm
Pancreas is a bulge (uitstulping) of primary endoderm tube, but contact with for islets outside world was lost during embryogenesis.
> can differentiate back
> Islets are epithelial cells
HE staining acini of exocrine pancreas: blue on basal cytoplasm (nucleic acids) and red on apical side (protein)
Mitochondrial DNA but a lot of ribosomes with rRNA at basal cytoplasm for production of enzymes
> made as zymogens
Enzyme production in pancreas types
-Zymogens like trypsinogen
-Active enzymes like lipase (needs cofactor, not immediately active), alpha-amylase
Pancreatic ductal epithelium make a lot of water and bicarbonate, why?
To rise the pH from incoming acidic food in duodenum (neutralize)
> apical granules for secretion
How well are islets vascularized compared to exocrine pancreas?
not very distinct
> portal vein structure of blood flow in pancreas
> oxygen use of one part influences potential to use oxygen for downstream part
The best vascularized endocrine pancreatic cells are the
beta cells
Why gap junctions in islets of Langerhans?
Connects cytoplasms
> calcium waves released upon increased ATP/ADP ratio > processing glucose through GLUT2 increases insulin secretion through gap junction. > quick transduction signals
Protects cells against symptoms diabetes mellitus
- sensitive cells
Functions tight junctions
Sorting proteins to PM, and regulate permeability of epithelium (diffusion barrier)
Streptozotocine can target endocrine pancreatic cells, how?
Partly glucose analog > through GLUT2
Has nitrosurea group (urea connected to nitroso group (R-NO) > DNA alkylating agent > toxic for DNA
> also: depletion NAD+ and oxidative stress
> damaged beta cells > autoantibodies
> DM in mice
> ratio alpha/beta cells to 50/50 from 5/95 in healthy
> beta cells affected: take up glucose
Are there active beta cells in DM?
Yes, but DM has threshold vale
Measuring insulin production
C-peptide levels
> insulin made as large pro-hormone by pancreas
> Oxidation of the large C-peptide > 2 intra chain disulfide bonds formed and one exra intra chain linkage disulfide (disulfide bonds in the mature insulin (A and B chains) > hydrogen peroxide made > oxidative stress when too much
Insulomas make much
insulin
Do insulomas of alpha cells exist
yes
Interactions that regulate secretion of insulin an and glucagon
-Plasma glucose
-Neurons > synaptophysin (neuroendocrine cells)
-Autonomous regulation of islets in neuronal
Beta cells take up glucose through GLUT2 and make insulin to inhibit the cAMP and PKA signaling in alpha cells to secrete glucagon release. Other regulations
-Parasympatic neurons can activate it (activated through glucose)
-Adrenaline can bind beta-adrenergic regulation: stimulate glucagon secretion
-Pancreatic islet delta cells take up glucose through GLUT1,3 and insulin binding by SGLT2 > Somatostatin binds receptor on alpha cells: inhibit cAMP and glucagon secretion.
-Glucose uptake through GLUT1/3 by alpha cells
Epsilon cells of pancreas make the hunger hormone:
Ghrelin
Stages of T1DM
1: beta cell autoimmunity but still normoglycemia and presymptomatic
2: dysglucermia and presymptomatic
3: symptomatic
Pancreatitis
exocrine pancreas damaged: autoinflammation
> islets not targeted
HC28: Hierarchy of metabolic regulation
Top down >
- Transcription-translation
- Expression isozymes or isotypes
- Protein synthesis/breakdown
- Proteolytic activation
- PTMs like phosphorylation
- Allosteric control by metabolites
< Bottom up
Regulating steps gene expression
-Transcriptional control
-RNA processing control
-RNA transport control
-Translation control protein activity control
Gene expression profiles reflect:
Function of organs
like exretion digestive enzymes by pancreas and lipid transport by liver
Nucleosome
Complex formed by histone octamer and 145 bp DNA fragment
Histone octamer
(H3)2(H4)2 tetramer and pair of H2A-H2B dimers
Epigenetic regulation of chromatin accessibility
-DNA methylation
-Histone modification (acetylation, methylation)
Compact chromatin effect
Block gene expression
Promotor
DNA stretch located in front of transcription initiation, where RNA polymerase II will bind.
DNA methylation effect on transcription
Makes heterochromatin > correlated with methylation of cytosines in DNA
Which enzyme methylates cytosines in DNA?
DNA methyl transferases
Which parts of histones are modified?
The tails
General principle methylation and methylation of histone tails
-Acetylation of lysines (K): activation
Trimethylation of histone tails occurs at ?
Promotors for repression
Histone acetylation means … and is done by…
Activation
> Histoen acetyltransferases (HATs)
> Catalyze the transfer of acetyl-groups of acetyl-CoA to specific Lys residues in amino terminal tails of histones
> costs energy: acetyl-CoA with energy rich thioester bond
Which enzymes deacetylate histone tails?
Histone deacetulases (HDACs)
Why does acetylation of histones promote gene expression
-DNA is negatively charged (phosphate group) and histones positively charged: tight packaging
-Acetylation reduced number of positively charged groups on histones and weaken strength of interaction with negatively charged DNA
> promoter / enhancer becomes more accessible
Are HATs and HDACs specific for histone tails?
No, they can acetylate lysines of proteins, also of transcription factors which leads to either activation or inactivation
HATs aid TFs like … to increase chromatin accessibility to increase gene expression
CREB
CREB signaling in liver
Fasting: glucagon rules
> Glucagon binds
> cAMP and PKA singaling
> PKA phosphorylates CREB (CRE binding protein)
> HAT (CREB bound after activation) acetylates histone and opens up the CRE (cAMP response element)
> CREB binds CRE
> transcription CRE regulated genes
CRE activated genes
Gluconeogenic genes
> PEPCK
> G6P > for glucose-6-phosphatase
In which gluconeogenesis signalling is HDAC involved?
FoxO1
FoxO1 signalling when insulin rules (high glucose)
Insulin binds receptor
> activation PKB/Akt through signal transduction
> Akt/PKB phosphorylates FoxO1 and bound HDACs in cytosol
> FoxO1 and HDACs dissociate after FoxO1 is acetylated > no transcription
Response element
DNA element that can be bound by TFs
mainly enhancers
FoxO1 signaling when no insulin
Inactive acetylated FoxO1 is deacetylated by active HDACs (dephosphorylated) and FoxO1 becomes active and promotes gene expression of gluconeogenic genes like PEPCK and G6P
Transcription factor families
-Leucine zippers
-Helix turn helix
-Zinc fingers
Transcription factors are the smart bullets which need co-activators. Explain.
TFs are the only molecules who know where to bind DNA
> Example co-activator is the Mediator
> co-activator are regulated by phsophorylation
Mediator function
Bind TFs and RNA pol II, forming a ‘bridge’
> binding of mediator to TFs helps recruit RNA pol II
Most disease SNPs are in
Enhancers
Enhancer sequences
DNA sequences without promoter activity but can increase transcription even when located far away (1000s)
> serve as binding sites for signal/tissue specific transcription factors
> DNA binding proteins influence transcription initiation by altering local chromatin structure to expose gene or its regulatory sites (co-activators)
Islet-1
TF with important role in developing nervous system
> assay with reporter construct which connects with GFP
Transcription initiation regulation
-Folding of DNA brings TFs at enhancer into contact with transcription initiation complex
> transcription of human genes regulated by collaborating TFs which integrate all signals
TF SREBP2 promotes
Cholesterol biosynthesis and cholesterol uptake
TF LXR promotes
Cholesterol efflux
Eukaryotic TFs have multiple … and … domains
DNA binding and activation
> required to activate expression of most genes
> most TFs bind enhancers, some promoters
> bind through specific motifs in DNA
SREBP2 pathway
Cells are deprived of cholesterol
> Scap escorts SREBP from ER to Golgi
> In Golgi: S1P and S2P cleave TFs from transmembrane domain and release to nucleus to bind SRE and activate gene expression
Cells high cholesterol
> Trigger binding of Scap to Insig in the ER, transport SREBP to Golgi is blocked, no transcription
Which genes are activated by SREBP2
LDLR and HMGCR (HMG CoA reductase) genes, cholesterol uptake and synthesis
Insulin accepts another SREBP. Which one and what is the response?
SREBP1c
> in fed state
> Insulin releases Scap-SREBP from Insig
> SREBP1c activates FA synthesis genes ACC and FAS (Acetyl-CoA synthetase and Fatty acid synthase)
LXR pathway
High cholesterol in cell
> LXR + RXR already bound to response element, but bound by co-repressors at first
> Activated by oxysterols or synthetic ligands: binding co-activators after that
> transcription of genes like ABCA1 which is needed in cholesterol effluc
Which activators bind RXR and LXR
RXR bound by 9-cis retinoic acid
LXR bound by oxysterols or synthetic ligands
RNA processing control of Apolipoprotein B
ApoB100 > synthesized in liver for VLDL, full length protein
ApoB48: in intestines for chylomicrons
In ApoB48: the translation is stopped earlier
> a deaminase in the intestine converts the cytosine of codon 2153 int uracil, to make stop codon premature.
HC29: How large is the family of nuclear hormone receptors? How do they differ?
48 nuclear hormone receptors
> different substrate binding sites and DNA binding sites
Nuclear hormone receptor general principle
-Respond to binding of hormones in cytosol (steroid hormones which can pass PM)
-Activation and translocation to nucleus to bind response elements
Why can steroid hormones pass the PM?
They are amphipathic, can wiggle through PM
What if a hormone X is hydrophobic completely, can it pass the PM?
No, it would get stuck.
GRE
Glucocorticoid response element
> an enhancer
> activate program of genes through transcription initiation complex and DNA folding to the promoter and RNA pol II binding etc.
Human steroid hormones
-Estradiol (an estrogen)
-Progesterone
-Testosterone
-Aldosterone
-Calcitriol (active vitD)
-Cortisol
Most steroid hormones are …
amphipathic 3-oxo-sterols
All steroid hormones are derived from…
Cholesterol
> OH group cholesterol is hydrophilic and the rest hydrophobic inclusive carbon tail
-In steroid hormones, sides switched, carbon tail becomes hydrophilic and the rings with the now ketone (=O, was OH) group is hydrophobic.
Zinc finger TFs stick into…
the major groove of DNA
Estrogen receptor activation
-Binding estrogens like estradiol
-Zinc finger motifs bind DNA (zinc based domains)
-Ligand binding domain in Helix-12
-Binds as dimer
Conformational change estrogen receptor after binding ligand
Before: Helix-12 points outward, bound to inhibitory protein
after: points inward > activation > binding co-activator protein and DNA binding domain can bind DNA (the response element)
Tamoxifen treatment
Against breast cancer > the tumor cells express estrogen receptor and depend on pathway for growth.
> Tamoxifen inhibits transcription of genes regulated by estrogen and reduces secretion growth factors
> Tamoxifen is antagonist of estrogen receptor
Mechanism of action of Tamoxifen
In estrogen receptor-tamoxifen complex, tamoxifen extends from hydrophobic (ligand binding) pocket, preventing the co-activator from binding and inhibiting gene expression
> antagonist estrogen receptor
Tamoxifen is a prodrug, how is it activated
CYP2D6 and CYP3A4 metabolize tamoxifen into the active 4-hydroxy-tamoxifen
> hydroxylate: add OH group
> make drug more water soluble because then recognized as foreign for excretion, but activates the antagonist
Anabolic steroids and the androgen receptor
Steroid hormones (androgens) bind androgen receptor
> binding receptor enhances gene expresion for development of muscle mass
> Anabolic steroids are agonists of androgen receptors which resemble androgens
Glucocorticoid receptors activate a gene … by binding the activating ….
gene program by binding the steroid
Cortisol and the glucocorticoid receptor
Chronic stress hormone (works slower than adrenline, through gene expression)
> released upon fasting and chronic stress by adrenal gland cortex (adrenaline in medulla)
> less peaks during day
Glucocorticoid receptor ligands incl drugs
-Hydrocortisone (cortisol) works as anti-inflammatory and immunosuppressive drug to control inflammation in dermatitis and rhematism
-Prednisone is converted in liver to active drug prednisolone
-Prednisolone is 4 times more patient than hydrocortisone
-Dexamethasone is 30 times more potent than hydrocortisone
Glucocorticoids bind the glucocorticoid receptor (GR) > act slow via gene expression > which genes ?
Causes precursors of gluconeogenesis (glycerol and amino acids) to move o liver, where they are converted to glucose and stored to glycogen.
> similar effect as glucagon / adrenaline (but glucagon induces glycogen breakdown)
Side effects glucocorticoids
Osteoporosis
Effects cortisol
-Lipolysis in adipose tissue stimulated
-Protein degradation in muscle stimulated and synthesis inhibited
-Gluconeogenesis stimulated
-Glucose levels increase
-Beta oxidation stimulated
-Suppression immune response
-Promote glycogen formation (glycogen breakdown is then stimulated by adrenaline)
> fasted state responses
Long term treatment with glucocorticoids (GCs)
-Like untreated T1DM, but with them the glucagon is extremely high because insulin is absent
-State of starvation
> Proteolysis up
> Gluconeogenesis up
> blood glucose up
> insulin in plasma up
» glycogen synthesis up
» fat synthesis up
(breakdown arm and leg muscles for gluconeogenesis)
> lose muscle, gain fat
PPAR nuclear hormone receptors work with another nuclear receptor to form heterodimers. Which?
RXR (retinoid X receptors)
Activation PPAR
PPAR has ligand binding/dimerization domain, DNA binding domain and transactivation domain
> 9-cis retinoic acid (made from vitamin A) activates RXR
Vitamin A forms
-Retinol (COOH group)
-Retinal
-Retinoic acid > activates RXR
Function PPAR-alpha and PPAR-gamma
PPAR-alpha (liver) > burn fat
PPAR-gamma (adipocytes) > store fat
Ligands of PPAR-alpha nuclear receptors
-Free fatty acids (FFAs)
-Eicosanoids (C20)
-Fibrates (drugs)
Fibrates
Lipid lowering drugs for hypertriglyceridemia
> prodrug: ester bond first hydrolysed (OH hydroxyl end left)
> analog of FAs
Target genes PPAR-alpha
Upregulation of genes encoding
> CPT-1
> Very-long/medium chain acyl-CoA dehydrogenase (VLCAD/MCAD)
> HMG-CoA synthase
-So beta oxidation upregulated (and more ketone bodies)
> TAG levels in plasma down
> LPL up > VLDL and TAG in plasma down
> Apo-A1 > HDL up (made in liver and takes up cholesterol in periphery)
Ligands PPAR-gamma receptors (adipocytes)
-FFA unsaturated
-Eicosanoids (C20)
-Thiazolidinediones (TZDs)
TZD (glitazones)
Drugs for T2DM
> pioglitazone and rosiglitazone used today
> Thi (S)-axolidine (ring with NH in it) -dione (two ketone groups to carbons) > functional group
Target genes PPAR-gamma
-LPL: lipoprotein lipase
-FATP, CD36: FA transporters
-Acyl-CoA synthetase: FA esterification to prevent efflux
Effects PPAR-gamma activation
-Adipocyte differentiation
-Lipid synthesis (lipogenesis)
-TAG levels in plasma and liver down
-Anti-inflammatory
> Insulin resistance down
Side effects PPAR-gamma
-adipocyte differentiation, lipogenesis and less TAG in plasma and liver induces obesity
> change of diet needed
TZDs cause lipid redistribution from … to …
Abdominal to subcutaneous adipose tissue
Cirrhosis in liver disease characterized by ectopic fat deposition
Steatosis (fatty liver) > hepatitis (inflammation) > fibrosis (scarring) > cirrhosis (liver damage)
-Cells die because of too much fat > inflammation and scarring
Translational control of iron metabolism
IRP (iron responsive element binding protein) can bind either ferric iron in cell (Fe3+) or the IRE (stem loops structure) in the mRNA of both ferritin and transferrin receptor
Much iron
> IRP binds Fe3+
> IRP does not bind the IRE in 5’-UTR of ferritin mRNA, it is not blocked from translation: expression
> IRP does not bind IRE in 3’-UTR of transferrin receptor mRNA, not stabilized, degradation mRNA, no translation
Depletion Iron
> IRP binds IRE: blocks ferritin mRNA translation and stabilizes transferrin receptor mRNA molecule and translation (more iron uptake)
Iron as a double-edge sword
-It is essential for the function of hemoglobin and many enzymes like the electron transport chain complexes with Fe-S clusters and CYP enzymes
-Too much is toxic: Fenton reaction generates ROS which initiates ferroptosis (cell death)
Why translational control of iron regulating proteins?
Quick response, ready to go mRNA
> needs to be finetuned precisely without much fluctuation
Iron in body
-65% bound to hemoglobin in erythrocytes (reversible binding O2)
-Myoglobin in muscle (reversible binding O2)
-Storage of iron by ferritin in most cells (30%)
-Iron transport by transferrin in blood (0.7%)
How is iron-bound transferrin taken up by cells expressing the transferrin receptor
Receptor-mediated endocytosis
> lumen of endosome acidifies by influx H+
> Iron released and transported to cytosol via transporter
> Tranferrin releases receptor
> Exocytosis: receptor back on surface and transferrin recycled to blood to bind iron
Why storage iron by ferritin
Free iron is toxic and needs to be stored safely
> 24 ferritin proteins in big spherical shell
> in cytosol
> in equilibrium with low concentration of free iron
How is the regulation of ferritin and transferrin receptor in the cell by IRP called?
Reciprocal regulation
> one regulatory protein controls translation of two different mRNA in reciprocal manner
Nuclear receptor to transcription
NR - ligand binding - co-activator - HAT - DNA unwinding - transcription
PPAR-alpha vs PPAR-gamma in drugs
fibrates vs TZDs
Burn fat vs store fat
