Hormonal Regulation (Lecture 34)

Question 1

Genetic Evidence of the importance of TFs

Answer 1

There are diseases linked to mutations in transcription factors (TFs) proving their importance.
ex/ Form of diabetes - MODY (maturity onset diabetes of the young

By looking at the mutations one can learn the mechanism of action of TFs

Question 2

centra zone

Answer 2

The central zone is the zone of convergence of the DBD, LBD and the hinge region in one location. The receptors use this zone to communicate with each other (such as a domain effect when a ligand binds).

Question 3

explain the activity of NR HNF-4 alpha

Answer 3

PRMT1 (methyl transferase) is a coactivator of HNF-4𝛼 , it adds a methyl residue of an Arginine residue (R91). This solidifies the interaction between all of the units. In the diagram it touches the LBD of both subunits which helps the dimer to form and interact with DNA. This is how a coactivator can solidify the act of one transcription factor. = ‘Accelerator’

The ‘break’ is the phosphorylation of residue S78 of the LBD of one subunit in the same region by PKC (protein Kinase C) which creates a clash with Y319 of the other subunit’s LBD. Diagram C = it interferes with the connection of the two subunits and leads to loss of dimerization and loss of DNA binding.

Thus, even if the amino acids come
from far away, they integrate into a
single domain which is important for
the function of the receptors. Even
though the receptor is linear, the
protein is not. The folded structure
creates the convergence zone to help the reaction of the receptor.

Question 4

MODY Mutation

Answer 4

There are many mutations in the LBD, which causes interference with ligand binding

It is unusual that the majority of mutations are in the hinge region. These mutations cause
hyperinsulinemic hypoglycemia.

The mutation in the hinge region is in R127W and D126Y which missaligns the two DBDs
(DNA binding domain) and prevents a good interaction with AGGTCA half-site

Another set of mutations in 1314F and R324H, which fold back near the hinge region, reduce the
activity of the region and the receptor.

The wild type receptor functions well, but the mutations reduce the receptor activity, leading to
the MODY disease. Complete knockdown of the receptor HNF-4𝛼 results in death.

This is a disease that demonstrates the significance of transcription in metabolic control.

Question 5

NR control of Bile Acids is controlled by which receptor

Answer 5

FXR

Question 6

Bile Acids

Answer 6

Bile acids are storage acids that form micelles that help with the transport and absorption
of lipids. They are synthesized from cholesterol in the liver and circulate in the intestine. The lipids are brought back to the liver to be distributed or further metabolized. They cannot diffuse through membranes freely and require a transporter.

Question 7

how are bile acids synthesized?

Answer 7

BAs are made from cholesterol in a series of oxidation reactions.

The rate limiting enzyme is CYP7A1, which is in the first step of metabolizing cholesterol to
7α-hydroxycholesterol. 7α-hydroxycholesterol goes on to a neutral pathway to produce colic acid (the main BA) and CDCA.

These are terminal metabolites that are conjugated with Glycine or Taurine. The BAs cannot cross membranes because they are hydrophobic and hydrophilic. They must form micelles to be able to be transported.

7α-hydroxycholesterol is the ligand for nuclear receptor LXR.

FXR is a nuclear receptor that is the BA intracellular sensor: When BA levels are high, FXR
represses their synthesis.

Question 8

FXR ligands

Answer 8

CDCA and cholic acid

Question 9

removal of FXR in mice causes:

Answer 9

high levels of BA, cholesterol, Triglycerides and fatty liver as well as cardiovascular diseases arise form this

Question 10

FXR working in the intestines

Answer 10

The intestine has BAs which activate FXR that produces the growth factor FGF15.

Question 11

FXR working in the liver

Answer 11

The liver BAs activate FXR which activates another nuclear receptor called SHP, which through signaling through FGF15 inhibits the expression of CYP7A1 (rate limiting step in Bile Acid synthesis)

Question 12

what is the mechanism for FXR when BA are high

Answer 12

FXR does not directly control CYP7A1 expression

FXR forms a heterodimer with RXR. In the liver when CDCA activates FXR, FXR induces the expression of SHP. Then SHP works with nuclear receptor LRH, and this complex acquires corepressors to express the repression of Asbt and CYP7A1.

Asbt is the major BA transport system in enterocytes. Asbt deficiency causes major BA absorption disease.

FXR is the BA sensor which reacts to a high or low level of BAs to control their synthesis and transport

Question 13

what is the mechanism for FXR when BA are high

Answer 13

SHP and FGF15 are not expressed. LRH forms a heterodimer with RXR instead of SHP, and LRH-RXR complex works with coactivators to induce the expression of CYP7A1 and Asbt.

Question 14

effects of fructose on humans

Answer 14

giving fructose directly to humans increases
intra-abdominal adipose mass. This diet also increases postprandial plasma triglyceride (TG) concentrations because of a rapid increase in de novo lipogenesis because of the generation of triose phosphates (GA3P).

Question 15

what biochemical pathways is initiated by fructose, and how does this occur?

Answer 15

Ingesting fructose leads to lipid synthesis because of the increase
of lipogenic precursors.

Enzymes that are crucial for the synthesis of lipids are under the
control of the lipogenic precursors SREBP 1c, ChREBP which works with PGC-1β. They induce the other enzymes to increase lipogenesis in response to fructose.

The molecular pathway of upregulating the lipogenic enzymes by SREBP 1c, ChREBP, PGC-1β is unknown.

Question 16

TFs involved in the Control of Hepatic Glucose Metabolism

Answer 16

CREB (a leucine zipper factor) and GR (glucocorticoid receptor) as initiators

Question 17

what occurs when fasting

Answer 17

When fasting: Initially = Glycogenolysis. Second = Gluconeogenesis. Last = Ketogenesis.

Gluconeogenesis relies on amino acids, derived from muscle protein, and glycerol from fat is also used. Ketogenesis relies on lipolysis in fat.

All three pathways are regulated by transcription through the concerted action of many TFs, all subjected to different signals.

Question 18

TF cascade when fed

Answer 18

glucose > makes lipids through activation of ChREBP, LXR is involved in BA
synthesis which regulates CYP7A1. Insulin activates SREBP to stimulate lipogenesis.

Insulin also blocks the activity of FoxO1, inhibiting glycogenolysis, gluconeogenesis, and
ketogenesis.

Question 19

TF cascade when fasting

Answer 19

First response is glucagon
which activates CREB and glucocorticoids which activate GR. CREB and GR activate all the other factors (shown in figure).

There is a single dependent regulation of TFs already present in the cell (CREB and GR) which induce other factors to send more signals = cascade

Question 20

circadian cycle metabolic processes include:

Answer 20

In the morning, cortisol is released. This activates the GR and blood pressure increases. When it nears nighttime, melatonin is secreted. Then during sleep, blood pressure goes down. This is regulated by the ‘central clock’ in the brain and the ‘peripheral clock’ in organs.

Question 21

central clock

Answer 21

regulates sleep and waking and feeding. Feeding releases metabolic hormones through the various organs, and goes back to the central clock.

This ensures a synchronized life during day and night. Disruption = disease.

Question 22

Transcriptional Control of Circadian Metabolism

Answer 22

Master clock is in the suprachiasmatic nuclei (SCN) which synchronizes the peripheral clocks by:

1. Controlling rhythmic signals such as hormones, temperature
2. Imposing feeding/fasting rhythms
   - The signals are interpreted by immediate early genes which convey information to central clock controlled genes / output genes (CCC-OG). CCC regulates the expression of local clock controlled genes (LCC-OG).
   - There is a cascade where immediate early genes control the clock genes that control factors that can impose rhythms in the metabolic processes.

Question 23

core clock genes

Answer 23

Bmal and Reverb𝛼 (nuclear receptor)

Bmal are the activators and Reverb𝛼 are the repressors, they work in a negative transcriptional feedback loop

The Bmal/Clock activates Cry/Per and Reverb𝛼 which then inhibit Bmal1/Clock = cycle

Question 24

Nuclear receptors control the adaptive clock because

Answer 24

they can sense metabolites and hormones.

Question 25

Regulation of the Clock by NAD+

Answer 25

SIRT1

NAMPT is the rate limiting enzyme for the synthesis of NAD+. It is highly rhythmic and controlled by the core clock genes. The cofactor SIRT1 is an HDAC, and removes the acetyl group from PER, Bmal, PGC1 which activates them and their various processes.

The amount of NAD+ is regulated by the core clock and food intake and exercise.