Download Understanding the molecular biologist

Putting Things into Perspective:
Glucose Transport, from Whole Body Physiology to Molecular Genetics
Luc Tappy
Institut de physiologie, Université de Lausanne, CH-1005 Lausanne, Switzerland
phone +41-21-692-5552, fax + 41-21-692-5595, email: [email protected]
Learning Objectives
 To identify the nature of informations relevant to glucose uptake and metabolism which
can be obtained with molecular biology (gene mutations, gene polymorphism, quantitation of specific tissue mRNA levels, microarray technologies, transgenic animals)
 To list the key steps controlling glucose uptake in specific cell types and tissues (glucose transporter proteins and hexokinase isoforms)
 To critically evaluate how molecular biology data can be used in an integrated perspective (tissue/whole body glucose metabolism)
Introduction
Molecular biology can be defined as the science devoted to the study the chemistry and
function of genes. Its boundaries remain however poorly defined. The genome codes for
all proteins of our organism, and hence for all enzymes. It therefore plays a key role in
metabolic regulations.
The expression of specific genes can also be modulated by several factors, such as hormones and substrates. This leads to variable specific mRNA and protein synthesis. In this
lecture, we will review how gene mutations and gene expression can be monitored with
the powerful tools of molecular biology. We will critically assess whether and how these
data relate to alterations of metabolic pathways activities.
In addition, molecular biology, by artificially altering genes in mice, can provide transgenic
models relevent for the study of human genetic diseases and for the understanding of
physiological metabolic regulations.
The aim of this lecture will not be to describe in details the techniques of molecular biology. Instead, it will focus on how molecular biology can improve our understanding of the
physiology and pathophysiology. As an exemple of gene mutation, we will focus on the
metabolic alterations present in patients with glucokinase gene mutations. In order to illustrate how tissue mRNA levels and identification of gene networks can be interpreted in an
integrated perspective, we will also focus on the control of hepatic de novo lipogenesis.
Key-steps in Glucose Uptake and Metabolism
For all cell types, glucose has first to be transported from the extracellular to the intracellular space, and then to be phosphorylated into glucose-6-phosphate to be further metabolized. Subsequent metabolism may correspond to glycolysis/oxidation, glycogen synthesis,
hexose monophosphate pathway, or de novo lipogenesis (fatty acid synthesis). Various
isoforms of glucose transporter proteins exists. The isoform expressed predominantly
varies according to the cell type.
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GLUT 1 and 3 have a low Km for glucose, and are expressed in cells which continuously rely on glucose metabolism, such as CNS cells.
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GLUT 2 is present in pancreatic beta- and alpha-cells, liver cells, and some cells of the
CNS. It has a high Km for glucose.
GLUT 4 is expressed in major insulin-sensitive tissues. It also has a low Km, but has
the peculiarity to be present inside the cell (microsomial fraction) in resting cells, and to
be translocated to the surface of the cell in response to insulin. This translocation, by
increasing several-fold the number of transporter proteins expressed at the surface of
the cell, is responsible for the stimulation of glucose transport by insulin.
All these GLUTs allow cell glucose uptake by facilitated diffusion. An other type of glucose
transporter protein allows an active glucose transport coupled with sodium transport and is
present in enterocytes (SGLT1) and kidney cells (SGLT2).
After glucose has been transported into the cell, it is converted into glucose-6-P under the
action of the enzyme hexokinase. Four isoforms of this enzyme are known. Most cell types
express hexokinase I, II, or III, which are characterized by a low Km for glucose and a
rnegative feedback of glucose-6-P on the enzyme activity. Hexokinase IV, or glucokinase
is characterized by a high Km for glucose, and no feedback of enzyme activity by glucose6-P. It is expressed in pancreatic beta- and alpha cells, liver cells, and specialized
hypothalamic cells.
From a functional perspective, it is important to recognize that both GLUT2 and glucokinase are expressed in cell types in which glucose metabolism has to vary according to
extracellular glucose concentration (glucose sensors). The high Km for glucose of both
proteins, and the absence of retrocontrol by glucose-6-phosphate, ensure that glucose
uptake and phosphorylation in these cells is proportional to extracellular glucose concentration throughout the physiological range of glycaemia.
Gene Mutations
Gene mutation is a relatively frequent phenomenon, with the consequence that several
variants of virtually each gene are encountered in the general population. Gene mutations
can be documented by restriction fragment gene polymorphism (RFLP) or direct sequencing of specific genes. Based on epidemiological data, it is recognized that several human
diseases have a genetic origin. Identification and characterization of gene mutations can
be attained by candidate gene approaches (ie the study of RFLP or the sequencing of
genes which are hypothetically involved in the pathogenesis of the disease) or by genomewide scanning. These techniques have proved quite efficient to identify single gene mutations as the origin of inherited diseases. Many diseases however are thought to be multigenic (i.e. mutation of two or more genes interact to cause the disease) and/or multifactorial (i.e. the coexistence of both genetic end environemental factors cause the diseases).
Identification of genes involved in such multigenic diseases has to date remained elusive.
Identification of a mutation in a candidate gene by itself is not sufficient to conclude that it
causes a disease. This conclusion should be based on:
1. an association between the presence of the mutation and the occurrence of the disease; this association may, however, be difficult to assess if the penetrance of the mutated gene is low (i.e. if the gene mutation does not affect the phenotype in a large
number of subjects)
2. mutagenesis experiments documenting that the identified mutation leads to altered
synthesis or function of the corresponding protein.
Mutation of the Glucokinase Gene:
Maturity Onset Diabetes of the Young 2 (MODY2)
MODY is a relatively rare type of familial diabetes, characterized by an autosomal dominant mode of inheritance. It is caused by the mutation of a single gene. Several gene mutations have been identified as being at the origin of different subtypes of MODY (MODY
1-5).
MODY2 is secondary to a mutation in the glucokinase gene. In the homozygous state,
such mutations are lethal. In the heterozygeous state, they cause a mild form of diabetes.
Several different mutations were identified in different families. The causal role of the gene
mutation in the pathogenesis of the disease was demonstrated by
1. characterization of the mutated proteins, which showed that the mutations significantly
increased th Km for glucose, and/or decreased the Vmax of the enzyme.
2. the production of transgenic mice expressing mutated glucokinase in pancreatic beta
cells or in the liver, showing that the presence of the mutation actually altered the phenotype.
Clinical studies performed in humans with MODY2 have extensively documented the consequences of glucokinase gene mutations. Patients have an impaired glucose-induced
stimulation of insulin secretion, impaired post-prandial suppression of hepatic glucose production, decreased post-prandial glycogen synthesis, and stimulation of glucagon secretion and of glucose production during hypoglycemia occuring at a higher than normal glucose threshold.
These alterations are consistant with a role of glucokinase as the major glucose sensor in
pancreatic beta cells, liver cells, and hypothalamic cells involved in glucoregulation and the
defence against hypoglycemia.
Gene Expression
Synthesis of specific mRNA from gene DNA template is the first step in protein synthesis.
The transcription of a given gene into its correponding mRNA varies according to the physiological and environmental conditions. This modulation of gene transcription is due to the
actions of intracellular substances (transcription factors, hormones, substrates,…) which
act on promotor or repressor regions of the gene. Monitoring of the level of a given mRNA
in a tissue can be evaluated by Northern blotting, or by reverse transcriptase polymerase
chain reaction (competitive RT-PCR).
These approaches have proven very useful to evaluate the expression, or level of transcription, of genes in various conditions. In particular, these methods allowed to elucidate
how major hormones and nutrients alter the expression of genes coding for key regulatory
enzymes.
It is important to keep in mind that the level of mRNA coding for a regulatory enzyme does
not necessarily correlate with the activity of the metabolic pathway. In many instances, an
increase in the mRNA level does not result in an increased level of the corresponding
protein. In addition, enzymes may be activated/deactivated, under the action of hormones,
or be allosterically regulated by intermediate substrates, and the control of enzyme activity
may override the importance of enzyme concentration.
Finally, the concentrations of intermediary substrates (i.e. the precursor and product of the
biochemical reaction catalyzed by the enzyme of interest) may be the most important factor in determining the activity of this metabolic pathway. These points will be illustrated by
some data available regarding the regulation of de novo lipogenesis .
De novo Lipogenesis and its Regulation
Fatty acids (initially palmitate) can be synthesized from acetyl-CoA in the process of de
novo lipogenesis. Carbohydrate and amino acids may be the source of acetyl-CoA for this
process. The initial steps in this pathway are the transfer of acetyl-CoA from the mitochondria to the cytoplasm, its conversion into malonyl-CoA under the action of the enzyme
acetyl-CoA carboxylase, and elongation of malonyl-CoA under the action of the enzyme
fatty acid synthase. Overall, the pathway is essentially stimulated by insulin, which activates acetyl-CoA carboxylase and increases the expression of acetyl-CoA carboxylase and
fatty acid synthase genes.
Hepatic de novo lipogenesis can be monitored by isotopic methods in vivo. It is very low
under normal conditions, but can be increased several-fold during isoenergetic feeding
when simple carbohydrates form a major portion of total calories, or during hyperenergetic
feeding. It is increased in insulin-resistant obese and type 2 diabetic patients. It is also increased in critically ill patients. In these patients, de novo lipogenesis increases when carbohydrate intake increases, even under conditions of isoenergetic conditions; in contrast,
inhibition of gluconeogenesis, another insulin-sensitive glucose pathway, is severly impaired.
Role of Specific Genes in the Regulation of de novo Lipogenesis
Several transgenic models facilitated the identification of key-genes in the control of de
novo lipogenesis. Hepatic glucose metabolism involves glucose transport into hepatocytes
followed by phosphorylation by glucokinase. These steps appear important in the control
of de novo lipogenesis, since mice overexpressing the glucokinase gene in hepatocytes
develop liver steatosis.
SREBP 1c is a key transcription factor involved in the control of hepatic glucose metabolism. It is stimulated by insulin, and activates the expression of both glucokinase and fatty
acid synthase genes in hepatocytes. Transgenic animals overexpressing SREBP 1c develop hepatic steatosis.
Recent work has unravelled the mechanisms by which liver cells become resistant to insulin’s actions on gluconeogenesis, while de novo lipogenesis remains fully insulin-responsive. The transduction system used for insulin signalling differ for these two pathways.
Suppression of gluconeogenesis involves activation of insulin receptor substrate 2 (IRS2).
This protein is downregulated by hyperinsulinemia. Hence the impaired suppression of
gluconeogenesis in insulin resistant hyperinsulinemic animals. In contrast, stimulation of
de novo lipogenesis involves the activation by insulin of SREBP 1c, which in turn activates
the fatty acid synthase gene. This pathway remains fully active in insulin resistant, hyperinsulinemic animals.
These in-vitro data suggest a major role for insulin in the control of lipogenic genes. In vivo
studies however indicate that not only insulin, but also, and possibly more important, the
amount of lipogenic substrates reaching the liver, are involved in the control of de novo
lipogenesis. This illustrates the key role played by substrate concentrations in metabolic
control.
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