Carbohydrates, Lipids, and Lactose Intolerance

Overview

• Lecture connects carbohydrate and lipid chemistry to lactose intolerance, emphasizing clinical and genetic perspectives.
• Exam: two multiple-choice questions specifically on lactose intolerance; other topics include blood coagulation, GPCR signaling, and basic nomenclature for lipids and carbohydrates.
• No lab this week due to safety concerns about students leaving late at night. The in‑person exam is still planned for Wednesday, with some in‑class review time.

Carbohydrates vs Lipids (Energy and Structure Review)

• In the comparison figure:

  • Molecule B has many nonpolar covalent bonds where electrons are shared equally.
  • Nonpolar covalent C–H bonds store more chemical potential energy than polar bonds.
  • Therefore, Molecule B has more potential chemical energy than Molecule A.

• Molecule B:

  • Classified as a fatty acid, not yet a fat.
  • A fat (triacylglycerol) = 3‑carbon glycerol backbone (each carbon with a hydroxyl group) + three fatty acids esterified to those carbons.
  • If only two fatty acids are attached to glycerol and the third carbon carries a phosphate plus other groups, the molecule is typically a phospholipid.

• Molecule A:

  • A six‑carbon carbohydrate (a hexose), likely similar to glucose based on the number of carbons and hydroxyl groups, though the exact isomer depends on OH orientation.
  • Carbohydrates store chemical potential energy but, carbon for carbon, less than fatty acids dominated by nonpolar C–H bonds.

• Aldose vs ketose classification:

  • These terms apply specifically to carbohydrates.
  • Aldose: carbonyl (C=O) at the end of the carbon chain (an aldehyde).
  • Ketose: carbonyl in the middle of the chain (a ketone).

• Linear vs cyclic carbohydrate forms:

  • Simple sugars such as glucose can interconvert between a linear and a cyclic form.
  • The cyclic (ring) form is more stable and is the predominant form in the body.
  • Some lab tests distinguish these forms, but for this course you only need to know that both exist and that the cyclic form is more common.

Lactose and Lactase: Biochemistry and Physiology

• Composition:

  • Lactose is a disaccharide composed of one glucose and one galactose molecule.
  • Glucose and galactose are monosaccharides that differ only in the orientation of one hydroxyl group.
  • The two monosaccharides are joined by a glycosidic bond (a specific type of covalent bond between sugars).

• Enzyme–substrate roles:

  • Lactose = substrate (disaccharide); it does not contain an active site.
  • Lactase = enzyme; it has an active site that binds lactose and catalyzes hydrolysis of the glycosidic bond.
  • Hydrolysis of lactose yields glucose + galactose.

• Intestinal context:

  • The small intestine is represented as a tube; its epithelial cells line the lumen.
  • When lactase is present on or in these cells:
    • Lactose from milk or dairy is hydrolyzed to glucose and galactose.
    • Glucose is transported efficiently into intestinal cells by glucose transporters and then into the bloodstream.
    • Galactose can also be taken up and metabolized.
  • When lactase is absent (lactose intolerance):
    • Lactose remains intact in the small intestine and passes into the large intestine.
    • The intestinal epithelium has few or no specific lactose transporters, so intact lactose does not readily cross into the blood.

• Microbiome fermentation and symptoms:

  • The large intestine contains a dense microbiome of bacterial species.
  • Under normal conditions, these bacteria do not encounter much lactose.
  • In lactase deficiency, lactose reaches the large intestine and certain bacteria ferment it vigorously:
    • Bacterial metabolism of lactose produces gas and other by‑products.
    • Symptoms include bloating, abdominal cramps, flatulence, and foul‑smelling gas.

• Osmosis and diarrhea:

  • Osmosis: water moves from regions of low solute concentration to regions of high solute concentration across a semipermeable membrane.
  • Undigested lactose in the intestinal lumen increases solute concentration there.
  • Water moves from body tissues into the gut lumen to balance the concentration, leading to excess water in the intestines.
  • This can cause diarrhea, which may be severe and dangerous, especially in malnourished or dehydrated individuals (for example, in famine conditions).

Microbiome, Nutrition, and Health

• The large‑intestinal microbiome:

  • Consists of diverse bacteria that help metabolize components of our diet that we cannot digest fully.
  • These bacteria can synthesize certain vitamins and nutrients that are important for human health.

• Broader physiological links:

  • There are emerging connections between the microbiome and aspects of neurophysiology, including mental health conditions like depression.
  • Thus, changes in what reaches the large intestine (e.g., undigested lactose) can influence both gastrointestinal function and possibly broader systemic health.

Clinical Testing for Lactose Persistence vs Intolerance

• What the test measures:

  • A standard lactose tolerance test measures blood glucose levels, not lactose.
  • Reason: lactose itself does not easily cross the intestinal epithelium into the bloodstream, whereas glucose does.
  • If lactase is active, lactose is cleaved to glucose, which then enters the blood and can be measured from a simple finger prick.

• Test procedure and interpretation:

  • Step 1: Measure baseline blood glucose level.
  • Step 2: The subject ingests a lactose load, usually via a large volume of milk.
  • Step 3: Blood glucose is measured at set intervals after ingestion.
  • Outcomes:
    • Significant rise in blood glucose after ingestion → lactase enzyme is active → individual is lactase persistent (lactose tolerant).
    • Little or no change in blood glucose → lactose not hydrolyzed effectively → individual is lactose intolerant.

• Enzymatic glucose detection:

  • The assay uses immobilized glucose oxidase on a strip or in a reaction well:
    • Glucose oxidase oxidizes glucose, transferring electrons to FAD within the enzyme and producing gluconolactone.
    • Reduced FADH₂ then donates electrons to oxygen, generating hydrogen peroxide (H₂O₂).
    • H₂O₂ reacts with a dye in the presence of the enzyme horseradish peroxidase; the dye changes color (e.g., yellow to pink).
  • This principle is similar to detection methods in western blots or ELISAs, where a secondary antibody carries an enzyme (often horseradish peroxidase) that generates a detectable signal.

Genetics and Regulation of the Lactase Gene

• Basic gene architecture:

  • Each gene can be divided into:
    • Regulatory (non‑coding) region: controls whether and how strongly the gene is expressed.
    • Coding region: contains codons that are transcribed into mRNA and translated into a protein (e.g., lactase).

• Regulatory region components:

  • Promoter: DNA segment where transcription factors bind and recruit RNA polymerase to start transcription.
  • Operator: binding site for repressor proteins; often located overlapping or near the promoter.
  • Additional elements include:
    • Up elements and distant enhancers that can be far from the promoter but interact with it when DNA loops, allowing “molecular kisses” that enhance or fine‑tune transcription.

• Coding region:

  • Transcribed into RNA after which codons direct amino acid sequence during translation.
  • For lactase, this region encodes the lactase protein that hydrolyzes lactose.

• Developmental regulation:

  • Almost all mammals express lactase as infants to digest milk.
  • Around weaning (for humans, around age two in many individuals), lactase expression is typically downregulated.
  • Downregulation relies on regulatory proteins:
    • A separate gene encodes a repressor protein.
    • When that repressor is produced, it binds to sites in the regulatory region (promoter/operator) of the lactase gene.
    • Repressor binding blocks transcription factor access and prevents RNA polymerase from initiating transcription.
    • Result: lactase levels fall, and the individual becomes lactose intolerant.

Molecular Basis of Lactase Persistence

• Key population genetics findings:

  • Researchers compared DNA from lactase‑persistent and non‑persistent individuals.
  • The coding region of the lactase gene showed no consistent differences correlated with the trait.
  • Differences were instead found in the non‑coding region near the lactase gene on chromosome 2.

• Regulatory mutations:

  • In many Europeans, a single base substitution (a T instead of a C at a specific non‑coding position) is associated with lactase persistence.
  • This mutation lies in a regulatory “switch” region, not in the protein‑coding sequence.
  • Other lactase‑persistent populations (e.g., in some African or Asian groups) have distinct mutations, but these also cluster in regulatory DNA rather than in the coding exons.

• Mechanism of persistence vs intolerance:

  • Lactose intolerant (non‑persistent):

    • Repressor protein can bind properly to the regulatory region (promoter/operator) of the lactase gene.
    • Binding prevents transcription factor access and blocks recruitment of RNA polymerase.
    • Transcription of lactase ceases in later childhood → no enzyme production in adulthood.

  • Lactase persistent (tolerant):

    • A regulatory mutation in the non‑coding region interferes with repressor binding to the lactase promoter.
    • Repressor is still produced by its own gene, but it cannot effectively bind the mutated promoter/operator region.
    • Transcription factors can bind and recruit RNA polymerase even in adults.
    • Lactase continues to be expressed beyond childhood, allowing digestion of lactose throughout life.

• Evolutionary context:

  • Historically, the ancestral human condition was likely to stop producing lactase after early childhood.
  • In populations experiencing famine or food shortage, domesticating animals and consuming milk provided a new nutrient source.
  • Individuals carrying regulatory mutations that maintained lactase expression could utilize milk without severe diarrhea and dehydration.
  • This offered a survival advantage under those environmental conditions and led to positive selection of lactase‑persistence alleles in certain populations.

Linking Lactase Regulation to the Lac System and IPTG

• Structural analogy:

  • IPTG (often mis‑spoken as IPG in lecture) is a structural analog of lactose.
  • It resembles lactose closely enough to be recognized by lac repressor but contains a bond configuration that prevents its cleavage.
  • Consequently, IPTG remains intact and continues to interact with the regulatory system without being broken down.

• Conceptual parallel in E. coli plasmid systems:

  • In bacteria and recombinant plasmid constructs:
    • The gene of interest (protein of interest) is placed under control of a lac promoter/operator.
    • A lac repressor gene is also encoded (often on the same plasmid) and expressed in the host cell.
    • Lac repressor binds the operator near the promoter, blocking transcription of the gene of interest by RNA polymerase.
  • This setup intentionally keeps the foreign protein (e.g., GFP) turned off during early bacterial growth:
    • High‑level expression of foreign proteins can stress or even kill E. coli.
    • Stressed cells may misfold the protein, causing it to aggregate into insoluble pellets.

• Induction by IPTG:

  • When the culture reaches an appropriate density and health, IPTG is added:
    • IPTG binds to lac repressor and prevents it from binding the operator.
    • The promoter becomes accessible; transcription factors and RNA polymerase can initiate transcription.
    • The gene of interest is then expressed at high levels.
  • This mirrors the natural concept that enzyme synthesis should respond to substrate availability:
    • In normal lactose metabolism, cells would not synthesize lactose‑metabolizing enzymes unless lactose were present.
    • IPTG mimics the “lactose present” signal but, because it cannot be degraded, provides a stable and controllable on‑switch in lab settings.

• Conceptual connection to human lactase:

  • In humans, lactose presence does not directly induce expression of the lactase gene as simply as in the bacterial lac operon.
  • However, both systems embody the principle that gene expression is regulated by combinations of promoters, operators, repressors, and small molecules that indicate nutrient availability.

Gene Expression Review in This Context

• Two major steps:

  • Transcription: DNA → RNA via RNA polymerase.
  • Translation: RNA → protein via the ribosome and tRNAs.

• Roles of regulatory elements:

  • Promoter:
    • Primary docking site for transcription factors.
    • Transcription factors bind and recruit RNA polymerase to begin transcription.
  • Operator:
    • Binding site for repressor proteins.
    • When a repressor is bound, it blocks access of transcription factors or directly blocks RNA polymerase binding at the promoter.
  • Up elements and enhancers:
    • Can be distant from the promoter but brought close by DNA bending.
    • These interactions can strengthen or fine‑tune transcription initiation.

• Repressors vs transcription factors:

  • Transcription factors: generally facilitate binding of RNA polymerase and activate or enhance transcription.
  • Repressors: bind operators (sometimes overlapping the promoter) and prevent transcription factor binding or RNA polymerase recruitment.

• Lactase persistence mutation in this framework:

  • The critical change is in the regulatory (non‑coding) region of the lactase gene.
  • The mutation prevents effective binding of the repressor to the promoter/operator.
  • Without repressor binding, transcription remains possible and lactase continues to be produced in adult cells.

GPCR Signaling Clarifications (Review Connection)

• G‑protein‑coupled receptors (GPCRs) reviewed in context of epinephrine and vasopressin:

  • Ligand binding:

    • When a ligand (e.g., epinephrine or vasopressin) binds its GPCR, the receptor activates a heterotrimeric G protein.
    • Gα dissociates from the Gβγ subunits in both cases.

  • Different Gα proteins and effects:

    • Epinephrine binding β‑adrenergic receptors:
      • Activated Gα typically stimulates adenylate cyclase (AC).
      • This increases cAMP levels and activates downstream signaling.
    • Vasopressin binding certain GPCR subtypes:
      • Activated Gα can inhibit adenylate cyclase.
      • This decreases cAMP, producing a different physiological response.

• Definitions in signaling pharmacology:

  • Agonist:
    • A ligand that activates a receptor, leading to Gα release and downstream signaling.
    • The resulting effect can be either stimulatory or inhibitory on a given effector (e.g., AC); what matters is that signaling is engaged.
    • Epinephrine and vasopressin are both agonists for their respective GPCRs.
  • Antagonist:
    • A ligand that binds to the receptor but does not cause Gα release.
    • It blocks or dampens signaling by preventing agonists from activating the receptor.

Key Terms and Concepts (Lactose Intolerance Focus)

Study Focus and Exam Preparation

• Carbohydrates and lipids:

  • Be able to classify molecules as fatty acids, fats (triacylglycerols), and phospholipids based on structure.
  • Review aldehyde vs ketone sugars (aldoses vs ketoses) and linear vs cyclic forms.

• Lactose and lactase:

  • Memorize that lactose = glucose + galactose.
  • Distinguish clearly between lactose (substrate, disaccharide) and lactase (enzyme, active site).
  • Understand how undigested lactose leads to gas, bloating, and osmotic diarrhea.
  • Remember that lactose tolerance tests measure blood glucose.

• Genetics of lactase persistence:

  • Identify regulatory vs coding regions.
  • Know that persistence is due to regulatory (non‑coding) mutations that prevent repressor binding to the lactase promoter/operator.
  • Be able to connect this to transcriptional control (promoter, operator, repressors, transcription factors, RNA polymerase).

• IPGT/IPTG and lac system:

  • Understand IPTG as a lactose analog used to relieve lac repressor binding in plasmid systems, turning on expression of a protein of interest.

• GPCRs and signaling:

  • Recall that both epinephrine and vasopressin activate GPCRs and release Gα.
  • Different Gα types can stimulate or inhibit adenylate cyclase.
  • Distinguish agonists from antagonists in terms of Gα release.

• Logistics:

  • Exam will be multiple choice, with no short answer.
  • Content roughly split between: (1) lipids, carbohydrates, lactose intolerance and genetics; and (2) previously covered topics such as GPCRs and blood coagulation.