Beating the Clock for a Better Understanding of Metabolism

Timing is everything, even with regard to metabolism.

Nicole Johnston(njohnston@the-scientist.com)
Feb 27, 2005
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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...

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.

From Sugar to Fat, A Link Uncovered

Contrary to diet-industry hype implicating insulin as the key player influencing fat formation, Kosaku Uyeda and colleagues at the University of Texas (UT) South-western Medical Center, Dallas, have discovered how sugar can directly lead to fat formation, bypassing regulatory hormones. They identified a transcription factor called carbohydrate response element-binding protein (ChREBP) that triggers liver enzymes to convert sugar directly into fat. The effect was more pronounced in mice fed a high-carbohydrate diet.

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."

"A glucose-responsive transcription factor that regulates carbohydrate metabolism in the liver," Yamashita H, Proc Natl Acad Sci , 2001 Vol 98, 9116-21"Carbohydrate response element binding protein directly promotes lipogenic enzyme gene transcription," Ishii S, Proc Natl Acad Sci Vol 101, 15597-602 Nov. 2, 2004"A humble hexose monophosphate pathway metabolite regulates short- and long-term control of lipogenesis," Veech RL, Proc Natl Acad Sci , 2003 Vol 100, 5578-80"Deficiency of carbohydrate response element-binding protein (ChREBP) reduces lipogenesis as well as glycolysis," Iizuka K, Proc Natl Acad Sci , 2004 Vol 101, 7281-6