The Scientist http://www.the-scientist.com/2005/2/28/16/1   Volume 19 | Issue 4 | 16 | Feb. 28, 2005
By Nicole Johnston

Timing is everything, even with regard to metabolism. To test a role for the molecular clock in glucose homeostasis, Garrett FitzGerald and colleagues at the University of Pennsylvania recently studied mice with impaired Bmal1 and Clock, the core genes behind circadian rhythm.1 These genes encode transcription factors known to play an important role in recovering from insulin-induced hypoglycemia. The studies may implicate dysregulated clock functions in metabolic syndrome, a condition believed to affect as many as 47% of the US population. Features of metabolic syndrome include obesity, high triglyceride levels, insulin resistance, and hypertension.

"Ten percent of genes in the transcriptome tend to oscillate in a circadian fashion," says FitzGerald. "The cassettes of genes that oscillate tended to be those involved in glucose metabolism, lipid metabolism, response to vascular injury, and adipocyte maturation." With congruence of those cassettes, he says, " [it] strikes you that these are the functions dysregulated in the metabolic syndrome. As a first step, we decided to focus on glucose metabolism."

IMPLICATING THE CLOCK

The master molecular clock, located in a region of the brain known as the suprachiasmatic nucleus (SCN), regulates circadian rhythm. It is thought that the clock controls the rate of neuronal firing in the SCN, thus influencing behavior and metabolism.

Removal of the SCN is known to impair glucose homeostasis, but what isn't known is whether disruption of satiety centers neighboring the SCN may also be involved. Until now, no direct evidence has suggested a role for the molecular clock in glucose homeostasis or insulin sensitivity.

Genes related to the molecular clock are also expressed in tissues throughout the body, suggesting a role for peripheral clocks as well. Just how these distant clocks communicate with the master clock isn't known, however. "The major problem with [understanding] circadian rhythm is that a lot is known about gene expression, but not a lot is known about circadian physiology," says Ueli Schibler, a molecular biologist at the University of Geneva, Switzerland. Circadian physiology is still in its relative infancy because it is difficult to study it in mammals, which hinders the ability to draw a direct correlation between circadian rhythm and physiology. FitzGerald says his studies are the first to address function.

"What [FitzGerald] has done is determined several parameters of energy homeostasis or sugar metabolism," says Schibler. "It is important to know these parameters and he has shown that they are circadian."  The researchers showed that glucose levels peak early in the day in mice carrying normal copies of both genes. In mutant mice, however, this circadian regulation disappeared. Moreover, a high-fat diet further affected carbohydrate metabolism by amplifying the circadian variation with respect to glucose tolerance and insulin sensitivity.

EXTERNAL CUES

"The big surprise was that when we knocked out Bmal1, the core element of the molecular clock, there was no recovery from insulin-induced hypoglycemia," says FitzGerald. Together, these findings led the authors to conclude that the molecular clock, along with dietary cues, may directly influence glucose homeostasis: What you eat and when you eat may both influence your insulin response. "If you superimpose environmental cues, you're likely to get an amplified response," explains FitzGerald. "Circadian rhythm recedes into the background, and environmental cues tend to drive things to a greater degree."

"Now, diurnal changes are not just driven by the light/dark cycle, but appear to be the output of the circadian clock, as shown by a genetic approach," says Steve Kay, a molecular biologist at the Scripps Research Institute in La Jolla, Calif. "These are real bona fide clock outputs and that's a very significant advance."

"This paper is a good example of probably what's an important role of the clock in providing a temporal filter for acute or stochastic perturbations or signals, i.e., stress," says Kay. "What may be an important role for the clock is gating your sensitivity to these signals."

"The consequences of circadian rhythm and their impact [go] beyond just when you're awake. It influences how you handle food intake," says Chris Bradfield, University of Wisconsin at Madison. "Biological clocks probably impinge on most aspects of normal physiology. This is just one of the [first] and most modern examples of this idea."

The findings raise another interesting question, says Bradfield. "What isn't clear is: What is the role of the central clock versus the peripheral clocks? Because in this case they're all disrupted." He adds that tissue-specific deletions can address this question.

References
1. RD Rudic et al, "Bmal1 and CLOCK, two essential components of the circadian clock, are involved in glucose homeostasis," PLoS Biol 2: e377. Nov. 2, 2004

From Sugar to Fat, A Link Uncovered

Carbohydrate metabolism was thought to be completely understood for twenty years. But Uyeda says that one question in particular kept gnawing at him, namely, how carbohydrates induce fat synthesis and stimulate the conversion to storage of fat, independently from insulin.

ChREBP was first identified by his group in 2001, by its ability to bind the carbohydrate response element of the liver pyruvate kinase gene.1 Earlier this year, they showed that lipogenic-enzyme expression was significantly reduced in mice lacking ChREBP expression.2 Just recently, they showed direct evidence for ChREBP in the glucose regulation of acetyl-CoA carboxylase and fatty acid synthase, two key enzymes involved in fat formation.

The breakthrough came through their development of a new method for rapid extraction of a stable factor from nuclei and its subsequent purification. "It was an extraordinarily difficult task to purify and identify the transcription factor to initiate this entire process," says Uyeda. "That is the reason the factor was known to exist for over 15 years but not identified."

He believes that this factor and its regulation explains how ingested carbohydrates are converted into acetylCoA, a substrate required for fat synthesis, via glycolysis. The other important finding, he says, is that ChREBP induces all fat-synthesizing enzyme genes, thereby coordinating carbohydrate metabolism and its conversion to stored fat, completely independent of insulin.

"Since carbohydrate is the major source of energy and fat, if it is not used for [immediate] energy needs then it is converted to fat for future energy needs," he explains. "Liver is the major tissue responsible for the conversion, and ChREBP in liver plays a major role in lipogenesis."

"It took heroic protein chemistry to get that thing out. It's really elegant work" says Richard Veech of the National Institute on Alcohol Abuse and Alcoholism, Bethesda, Md. "Then [Uyeda] went on to show how it was controlled by a simple substrate. Not only was this a new transcription factor that controls steps in glycolysis, fatty-acid synthesis, and the hexose monophosphate shunt, but it was all integrated together by a single metabolite: xylulose 5-phosphate."3

"To me, what it really points out is that nutrients in our diet – glucose and fructose, which are really abundant in the modern diet – are there for more than to provide fuel," says Howard Towle, a molecular biologist at the University of Minnesota, Twin Cities. "They are also signals that turn on biochemical pathways that affect how our metabolism is controlled. The obvious link is with obesity and type II diabetes."

"If you eat lots of carbs, you're going to increase the pathways that increase fat synthesis," says Veech. "It's very interesting to speculate that that mechanism is contributing in a low-carb diet," says study coauthor, Bonnie Miller, a biochemist also at UT Southwestern. "I think a large number of people have not been aware of glucose's regulatory effects. They pretty much always thought in terms of insulin," she says.

Uyeda says it is not surprising that reducing carbohydrates in one's diet aids in reducing weight, but points out that most proteins and fat are ultimately converted to glucose in the liver. "Total caloric intake needs to be reduced," he says. But that's not all. "Any excess pyruvate generated from carbohydrate metabolism, which is not oxidized in mitochondria, will be stored as fat via acetyl CoA. Therefore, exercise to reduce this excess pyruvate is also essential."

References:

1. H Yamashita et al, "A glucose-responsive transcription factor that regulates carbohydrate metabolism in the liver," Proc Natl Acad Sci 2001, 98: 9116-21.

2. S Ishii et al, "Carbohydrate response element binding protein directly promotes lipogenic enzyme gene transcription," Proc Natl Acad Sci 101: 15597-602. Nov. 2, 2004

3. RL Veech "A humble hexose monophosphate pathway metabolite regulates short- and long-term control of lipogenesis," Proc Natl Acad Sci 2003, 100: 5578-80.

4. K Iizuka et al, "Deficiency of carbohydrate response element-binding protein (ChREBP) reduces lipogenesis as well as glycolysis," Proc Natl Acad Sci 2004, 101: 7281-6.

The liver provides for long-term energy needs of the body by converting excess carbohydrate into fat for storage. Insulin is one factor that promotes hepatic lipogenesis, but there is increasing evidence that glucose also contributes to the coordinated regulation of carbohydrate and fat metabolism in liver by mechanisms that are independent of insulin. In this study, we show that the transcription factor, carbohydrate response element-binding protein (ChREBP), is required both for basal and carbohydrate-induced expression of several liver enzymes essential for coordinated control of glucose metabolism, fatty acid, and the synthesis of fatty acids and triglycerides in vivo.

How Carbohydrates Make Fats

Dr. Mae-Wan Ho traces the tangled paths of how diet affects metabolism affects gene transcription affects metabolism…

Sources for this report are available in the ISIS members site.

It has long been known that a high-carbohydrate diet stimulates the synthesis of fatty acids and induces the transcription and expression not only of all the enzymes needed to make fatty acids, but also the enzymes breaking down glucose into the necessary building blocks for making all kinds of fat.

It appears that two distinct transcription factors are involved in providing the signals for making fats. Transcription factors bind to promoters of genes to boost transcription and hence gene expression. One transcription factor, SREBP-1c (Sterol Response Element Binding Protein), is stimulated by insulin, and binds to the SRE (Sterol Response Element) in the promoter region of genes encoding key enzymes that make cholesterol.

A second element, the carbohydrate response element, ChoRE, is involved in the transcription of fat-making enzymes after stimulation by high glucose in the absence of insulin. ChoRE sits in the promoters of enzymes involved in making other fats.

Two years ago, the research team headed by Kosaku Uyeda in the Dallas Veterans Affairs Medical Centre and Department of biochemistry, University of Texas in the United States, purified the protein that binds to the ChoRE in the promoter of the gene encoding liver pyruvate kinase from the livers of 800 rats that had been fasted and then refed a high-carbohydrate diet. This ChoRE binding protein (ChREBP) contains amino-acids in certain positions of the polypeptide chain that can be phosphorylated (accepting a phosphate group) by protein kinase A. Adding a phosphate group to serine in position 196 inhibits the protein from entering the nucleus, and adding a phosphate group to the threonine in position 666 inhibits its binding to the liver pyruvate kinase promoter site, both of which prevent transcription of the genes involved.

It has been known for a long time that cholesterol in the diet suppresses cholesterol synthesis in the body, mediated through feedback inhibition of SREBP production.

Feeding fat also inhibits carbohydrate metabolism, and the chain of biochemical events have been worked out by Uyeda’s group. Fatty acids are activated by ATP (adenosine triphosphate, the major energy intermediate in biochemical reactions) in a reaction that produces AMP (adenosine monophosphate). Thus, an increase in fatty acids boosts the level of AMP. AMP stimulates a protein kinase to phosphorylate ChREBP thereby inhibiting it from binding to its promoter site, preventing gene transcription.

Feeding high carbohydrate diet has the opposite effect on ChREBP, in that it activates the protein to enter the nucleus and to bind to its promoter site, thus enhancing transcription. Uyeda’s group has published new findings on how this is achieved, via the sugar phosphate, xylulose 5-phosphate, an obscure terminal player in the hexose monophosphate shunt, a side branch from the main glycolytic pathway that breaks down glucose.

The enzyme phosphofructokinase (PFK) sits at the intersection of the glycolytic pathway and the hexose monophosate shunt. Its activity is controlled in liver by the concentration of the metabolic molecule fructose-2,6-diphosphate, which stimulates PFK to proceed along the glycolytic pathway that eventually supplies all the building blocks for making fats.

Fructose-2,6 diphosphate is produced and destroyed by the same enzyme that catalyses both the forward and reverse reactions. The kinase activity, which makes fructose-2,6-diphosphate from fructose-6-phosphate by adding a phosphate group, is inhibited, while the phosphatase activity, which removes phosphate to regenerate fructose-6-phosphate, is activated by a cyclic-AMP dependent protein kinase that donates a phosphate group to the enzyme itself.

(Phosphate groups coming on and off small molecules and especially so, big molecules like enzymes and transcription factors, is the most common way to change their activities, as biochemists have been finding out for some decades now.)

A high-carbohydrate diet stimulates the kinase activity of this enzyme via a specific protein phosphatase (PP2A) that removes a phosphate group from the enzyme. PP2A itself is activated by, yes, xylulose-5-phosphate.

In the latest report from Uyeda’s group, PP2A and others in the same family, turn out to be agents that also activates ChREBP (by removing phosphate from it), so that it can enter the nucleus and bind to its promoter sites. Though their action on ChREBP, PP2A and family members are involved in promoting the transcription of a host of genes that make fats out of carbohydrates.

This must be one of the most heroic and sustained feats of scientific sleuth in our time. The group has hunted down all the culprits responsible for integrating the major metabolic pathways and gene transcription, showing how changing one’s diet appropriately can make metabolic sense. It is definitely not all in the genes.

Genes don’t determine our fate. Metabolic intervention can do wonders, for genes are at least as much the servants as masters of experience.

Am J Physiol Endocrinol Metab 287: E424-E430, 2004. First published April 20, 2004; doi:10.1152/ajpendo.00568.2003 0193-1849/04 $5.00

http://ajpendo.physiology.org/cgi/content/abstract/287/3/E424

Modulation of carbohydrate response element-binding protein gene expression in 3T3-L1 adipocytes            and rat adipose tissue

Zhibin He,1 Tao Jiang,1 Zhuowei Wang,1 Moshe Levi,1,2 and Jinping Li1

1Division of Renal Diseases and Hypertension, Department of Medicine, and 2Department of Physiology and Biophysics, University of Colorado Health Science Center, Denver, Colorado 80262

Submitted 12 December 2003 ; accepted in final form 10 April 2004

Carbohydrate response element-binding protein (ChREBP) is a rat homolog of human Williams-Beuren syndrome region 14 and a member of the basic helix-loop-helix leucine zipper transcription factor family. Its activation was found to be inducible by carbohydrate in the liver nuclear extracts from rats fed a high-sucrose diet. ChREBP is able to bind to the carbohydrate response element on the promoter of L-type pyruvate kinase and initiate the gene transcription. The detailed expression profile and transcriptional regulation of the ChREBP gene in adipocytes have not been characterized. In the present study, we provide evidence showing that 1) the ChREBP gene is expressed in differentiated 3T3-L1 adipocytes and rat adipose tissue; 2) insulin, glucose, and the antidiabetic agent troglitazone can significantly upregulate the gene expression of ChREBP in 3T3-L1 adipocytes, whereas free fatty acids suppress its expression in this cell type; 3) fasting followed by refeeding with a high-carbohydrate diet resulted in a 10-fold increase of ChREBP mRNA level in rat adipose tissue; and 4) ChREBP expression in adipose tissue is not significantly affected by the diabetic state. Taken together, the results we present are consistent with the idea that ChREBP is an important modulator of adipocyte biology and that its expression in adipose tissue is subject to combined regulation by glucose and insulin in vivo. The induction of ChREBP may serve as a novel pharmacological pathway for troglitazone-mediated hypoglycemic effects in vivo. adipogenesis; lipogenesis

Address for reprint requests and other correspondence:
J. Li, 4200 East 9th Ave., C281, Denver, CO 80262 (E-mail: jinping.li@uchsc.edu ) and
Dr. Moshe Levi, 4200 East 9th Ave., C281, Denver, CO 80262 (E-mail: moshe.levi@uchsc.edu ).

Another Great Article in PDF format:

Carbohydrate Responsive Element-binding Protein (ChREBP): A Key ...

File Format: PDF/Adobe Acrobat

... new specific transcription factor termed “carbohydrate responsive element binding protein, ChREBP” in ... dependent mechanism in response to glucagon ...

Abstracts/UyedaChREBP NIH talk.pdf

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