Tuesday, August 16

ChREBP: The forgotten factor

The discussion between Taubes and Guyenet has revived the long-life debate about the influence of carbohydrates/insulin in the pathogenesis of obesity and MetSyn. I think both hypotheses (carbohydrate excess and food reward) are complementary to each other. The problems associated with Taubes hypothesis are obvious and had been reviewed elsewhere. The food reward hypothesis, on the contrary, is very well documented in the literature and most researchers have shown consistent results. The question is, can food reward be a dominant factor in obesity in the absence of sugar? Generally, people get obese consuming high energy diets both high in sugar and fat. If we exclude sugar from the equation, would then the same people get obese? The food reward associated with junk food would be the same in the absence of sugar (assuming that there is sugar-free junk food)? Is high sugar (and probably veggie oils) the ultimate cause of leptin resistance and food reward? Is palatability only relevant when talking about high fat-high sugar foods? 

I think this is why maybe the two hypotheses are complementary. I have yet to see any evidence of someone getting obese eating a low carbohydrate high fat diet. Sure, you can gain weight with a low carbohydrate diet, and you can regain the weight lost after a period of calorie restriction eating a low carbohydrate diet. But can you get obese in the absence of sugar? We will never see any formal evidence of this theory, but I think is a valid speculation.

Anyways, on to the topic of the post. As a nerd as I am, I like to look at physiological processes deep inside. When discussing about the role of sugar/carbohydrates in obesity, the transcription factor carbohydrate response element-binding protein (ChREBP) is hardly mentioned. This transcription factor binds to the carbohydrate response element (ChoRE) located in the promoter of target genes and stimulates transcription. ChREBP is a member of the basic helix-loop-helix/leucine zipper (bHLH/ZIP) family of transcription factors and its expression is ubiquitous, being most abundant in lipogenic organs such as liver, brown and white adipose tissues, small intestine, kidney and muscle. 

Insulin and glucose both coordinate the transcription of key enzymes involved in de novo lipogenesis and glycolysis, the former by activation of SREBP1c and LXR. Both regulate different pathways which are integrated in the response to a high carbohydrate load (in this case, in the hepatocyte):

Copyright © 2011, The American Society for Clinical Investigation.

At low blood glucose concentrations, ChREBP is located in the cytoplasm, phosphorylated at the Ser196 residue. When blood glucose levels rise, glucose enters the hepatocyte and is phosphorylated by glucokinase and then converted to xylulose-5-phosphate (Xu-5-P) in the hexose monophosphate shunt (HMS). Xu-5-P activates protein phosphatase 2A delta (PP2A delta) and dephosphorylates ChREBP*, which can then enter the nucleus and stimulate transcription by dimerizing with Mlx. On the other hand, insulin stimulates transcription of both ChREBP and SREBP1c. Although Xu-5-P has been proposed as the key regulator of ChREBP, glucose itself can activate ChREBP through its GRACE (glucose response activation conserved element) domain, by unkown mechanisms. ChREBP activity seems to be controlled mainly at the post-transcriptional level. 

Inhibition of ChREBP is mediated by phosphorylation of Ser196 (inactivating nuclear import) and Thr666 (preventing DNA binding) by PKA and AMPK. 

Target enzymes regulated by ChREBP include Liver Type Pyruvate Kinase (L-PK), the NADPH supply system (glucose-6-phosphate dehydrogenase, transketolase, malic enzyme, etc.), glucose 6 phosphatase (G6P), Acetyl CoA Carboxylase (ACC) and Fatty Acid Synthase (FAS). 

Studies with ChREBP knockout mice

Iizuka et al (1) tested the importance of ChREBP for induction of several enzymes implicated in glycolysis, fatty acid synthesis and de novo lipogenesis. ChREBP -/- mice had slightly elevated glucose and insulin levels, deposited a large amount of glycogen in liver (but not in muscle), had almost half the plasma FFA of wild type (WT) mice and less adipose tissue, when fed a standard diet. Compared to WT mice, the level of LPK mRNA in ChREBP -/- mice was only 27% of that measured in age-matched WT. ACL, ACC1 and FAS mRNA levels were also lower in KO mice. Levels of mRNA for malic enzyme showed the greatest reduction (59%). When mutant mice were fed a high-sucrose diet, plasma FFA were markedly reduced and mice experienced progressive hypothermia, culminating in death in less than 1 week (>50% of the ChREBP -/- mice). When fed a high-fructose diet, they became moribund in a few days. This last observation was explained by low levels of fructokinase and triose kinase. 

To induce glycolysis and lipogenesis, they fed the mice a high starch diet. This diet increased levels of blood glucose compared to the standard diet, in both WT and KO mice. Plasma insulin in ChREBP -/- mice fed the high starch diet was significantly higher than any other group. They were moderately insulin-resistant (assessed by glucose tolerance tests) and had 40% greater liver weights than WT (from increased glycogen storage). Despite having increased glucose and insulin levels, ChREBP -/- mice fed the high starch diet showed reductions in liver mRNA for ACL, ACC1, FAS, malic enzyme, SCD-1 and LCE. This resulted in hepatic fatty acid synthesis rates that were 65% lower compared to WT. LPK remained lower in ChREBP -/- mice even when fed the high starch diet, and Glut-2 mRNA was <10% of that measured in WT. Glucose 6-Pase and PEPCK were also reduced**. 

Inhibition of ChREBP expression in ob/ob mice in vivo using a RNA-interference technique improves hepatic steatosis by decreasing lipogenic rates, leading to decreased levels of triglycerides and NEFA, and improving insulin signaling in liver, skeletal muscle and white adipose tissue (2). Using a double mutant model (ob/ob ChREBP -/-) (leptin deficient-ChREBP deficient), Iizuka et al (3) observed that inactivation of ChREBP expression reduced fat synthesis and obesity (body weight was very similar between ChREBP -/-, WT and ob/ob ChREBP -/-), and improved glucose tolerance and appetite control in ob/ob mice. Thus, deletion of ChREBP was able to override some phenotypic characteristics of leptin-deficient mice. According to this, it has been suggested that reducing ChREBP might protect against beta-cell dysfunction in type 2 diabetes because it inhibits the expression of Pdx-1, MafA, GcK and insulin (4), as well as PPARa (5).

Pleiotropic properties of ChREBP

Although ChREBP is a key regulator of glycolysis, fatty acid synthesis, gluconeogenesis and de novo lipogenesis, it seems that it has other important roles. Yun-Seung et al (6) tried to identify ChREBP target genes and gene expression patterns using ChIP-seq. They treated HepG2 cells with 25mM glucose for 8 hours. They found 783 target genes involved in different pathways. The most enriched pathway was lipid metabolism, followed by gluconeogenesis, as suspected.  Nevertheless, there were other target genes that are associated with diverse functions, such as protein dimerization, embryonic development, among others. 

One very intesting study was published by Tong et al (7). They investigated the role of ChREBP in cancer cell proliferation and metabolism using HCT116 colorectal cancer cells and HepG2 hebatoblastoma cells. It was seen that these cells require ChREBP to maintain their proliferative state and is rapidly upregulated upon growth factor stimulation. Moreover, comparison between HCT116 cells transfected with ChREBP siRNA and without transfection showed that inhibition of ChREBP caused a reduction in glucose uptake and lactate production, and increased oxygen consumption rates. This reflects increased mitochondrial respiration and decreased aerobic glycolysis. Transfected HCT116 cells also showed a reduction in glucose flux through the pentose phosphate pathway and de novo lipid biosynthesis. These observations were confirmed by 13C NMR. RNA microarray analysis of transfected cells showed an increase in the expression of p21, MDM2 and TIGAR, all of which are p53-dependent targets. Although the level of total p53 was the same in ChREBP deficient and non-deficient cells, the level of p53 that was phosphorylated in Ser-15 increased as ChREBP expression declined, effect that was explained by increased ROS concentrations. Supression of ChREBP resulted also in G1 and G2/M arrest. The authors finally showed that when injected to nude mice, ChREBP knockdown cells formed smaller tumors in vivo compared with control cells. 

Relevance to humans

Using animal studies to propose novel hypotheses can be fun. However, we cannot extrapolate findings directly. This is important when discussing metabolic pathways and the effect of different diets. For instance, mice have a basal metabolic rate that is 7 times greater than humans, so a 40% calorie restriction in mice mimics a therapeutic fasting in humans (8).There is evidence that mRNA levels of several lipogenic enzymes are different between rats and humans. In general, the lipogenic capacity of adipose tissue is lower in humans than rats, which may be explained by decreased ChREBP mRNA expression and lower abundance of SREBP1c (9). But it is interesting to note that expression of ChREBP differs in adipose tissue and liver. In obese subjects, hepatic ChREBP expression increases while decreasing in adipose tissue, and there is a significant increase of ChREBP mRNA and protein levels during preadipocyte differentiation (10). Differences between lean and obese subjects are shown below (A) in liver, omental and subcutaneous fat, as well as expression versus lean liver (considered as 1) (B).

We can see that lean people have less levels of ChREBP mRNA in liver, both have higher levels in both omental and subcutaneous adipose tissue. Because we know that we cant make conclusive statements only with mRNA levels (see here), we must look at protein levels to see the full picture:

Western Blot analysis of hepatic tissues of both lean and obese subjects (A) revealed the pressence of ChREBP (95 kDa) in obese but not in lean samples. Comparison to b-actin is shown in B. Protein concentration could not be detected in adipose tissue samples (indicating low absolute values and correlation between ChREBP mRNA and protein abundance). If there is a relevance for ChREBP and obesity-MetSyn in humans, we must look then at the liver, which is central to the disease. 

We have seen in studies mentioned above that inhibition of ChREBP reduces hepatic steatosis, confirming the role of lipogenesis and de novo lipogenesis in the process. It is often stated that de novo lipogenesis is very low in humans and has no physiological relevance. I differ. It might be not relevant to adiposity directly, but indirectly by promoting hepatic insulin resistance and steatosis (11). Maybe this is why very low carbohydrate diets work very well treating NAFLD (12,13), as hepatic DNL is increased with a high carbohydrate-low fat diet (14). This should also amielorate hepatic insulin resistance and subsequently hyperglycemia, not only by decreasing dietary glucose, but by restoring hepatic insulin signaling. A high fat diet should also reduce ChREBP liver activity, thereby reducing hepatic steatosis (15).

Summing up

There is not much human research with ChREBP but until now, the evidence supports its importance in the pathogenesis of obesity and MetSyn. For me, it seems that a chronic high sugar diet could influence development of obesity and MetSyn by chronic stimulation of ChREBP. This creates an obsogenic environment in which lipids begin to accumulate in the liver, leading to loss of hepatic insulin signaling. ChREBP overexpression in beta-cells could also contribute to insulin resistance and diabetes onset. These effects could be further exacerbated by excessive fructose consumption, as fructose has been shown to increase ChREBP DNA binding (16). This might explain why some high carbohydrate diets (ie. paleoish) do not cause obesity or MetSyn, compared with high sugar diets. Gut flora's effect on lipid metabolism and obesity might also include upregulating hepatic ChREBP, as shown with conventionalization of germ-free mice with normal microbiota from conventionally raised animals (17).

In conclusion, ChREBP might be the connection between high carbohydrate/sugar diets and obesity/MetSyn, as it promotes lipogenesis. This transcription factor directly links glucose/fructose to metabolic dysregulation and probably other "kind" of diseases, like cancer. The ability to improve the metabolic state of ob/ob mice suggests that a dietary treatment which reduces ChREBP activity should be the default treatment (at least initially) to MetSyn and leptin resistance. Especially when fructose seems to be the bioactive compound behind leptin resistance in a Western-type diet (18). Moreover, this could explain why maybe a high carbohydrate diet can predispose to obesity. But as always, more research is needed. 

* Recent research points out to G6P as the relevant molecule for ChREBP activation.

** Which suggests that increased liver glycogen was not by gluconeogenesis. 

ResearchBlogging.orgDel Pozo CH, Vesperinas-García G, Rubio MA, Corripio-Sánchez R, Torres-García AJ, Obregon MJ, & Calvo RM (2011). ChREBP expression in the liver, adipose tissue and differentiated preadipocytes in human obesity. Biochimica et biophysica acta PMID: 21840420

Iizuka K, & Horikawa Y (2008). ChREBP: a glucose-activated transcription factor involved in the development of metabolic syndrome. Endocrine journal, 55 (4), 617-24 PMID: 18490833


  1. "For me, it seems that a chronic high sugar diet could influence development of obesity and MetSyn by chronic stimulation of ChREBP"

    Lucas why did you use the word sugar, and not carbohydrate? Did you mean sucrose/HFCS ?

    so once again we see that carbohydrate ( glucose ) is a fundamental part for driving the biochemistry that promotes an obesogenic environment in the body.

    Anyway great post Lucas, found it very informative, look forward to more of your work.

  2. Hi Kindke,

    I dont think high carbohydrate diets per se are inherently obsogenic. This is shown by different cultures with a high carb diet but no obesity. But, if we look at the typical SAD, I think carbohydrates are the main problem and not fat, especially sugar.

  3. I like to believe that the two main foods that contribute to weight gain and obesity are sugar and refined wheat.

    I think they are also among the most rewarding foods in the diet, as well as those foods that stimulate the greatest amount of insulin release.

    So, if Taubes is right, cutting out sugar and refined wheat should lead to weight loss. If Stephan is right, it should too.

    Personally I believe that both of them are right. Taubes is viewing this at the level of the adipocyte, while Stephan is focused on the brain's control over the whole thing.

    I'm sure there are a lot of different factors that are contributing to the obesity epidemic, and both insulin and food reward being major players.

  4. Why is food availability not mentioned.Many animals eat in excess by instinct to allow the accumulation of fat for survival.Humans seem the same way in that we have large appetites.Having large appetites and ease of finding high calorie foods that its kinda obvious that the obesity epidemic is due to food abundance mainly.Take for example me and my tendency to lose control of my eating.I ate my dinner at noon and do not eat till 4am again.So I sit and watch TV and surf the net knowing that right under my building is a 7eleven with these killer nachos smothered in cheese and chili.I do not eat it just by willpower but my mind wants it since I believe we are all geared for as much fatgain as possible....survival is where its at.

  5. Hi Again Lucas,

    Could you have a look at this study, its fairly recent and seems to suggest that disruption of the bodies Glucose-sensing neurons via ATP-sensitive potassium ion channels produces a rodent with an extremely lean phenotype, that is incredibly resistant to obesity from a high-fat high calorie diet, ( the mouse also over-eats ),

    the mouse is also quite lazy and shows reduced endurance to exercise.

    My conclusion from this study was that when your body has a reduced capacity to react to hyperglycemia, energy expenditure shoots up dramatically.

    Would be great if you could give me a second opinion on this.

  6. Wolfstriked,

    Food availability per se doesn't seem to be a problem, as evidenced by Japan, France, Kitava, former USA, etc. 7 elevens don't pop up everywhere and then hope for people to come--people want them because they're addicted to their crap. What if we replaced every fast food/convenient store with a butcher shop?

  7. Good point John.Could be just an American thing where we eat big....

  8. Hi Kindke,

    Interesting study indeed. I think this study shows first, the importance of glucose sensing in the brain for energy homeostasis. The lean phenotype cannot be fully explained by this study, as the KO of the gene deletes all of KATP channels, not only those in the hypothalamus. Nevertheless, this deletion in skeletal muscle increases energy expenditure.

    What can we take out from this study? The only thing that could be extrapolated is that glucose sensing in the brain is important for food intake homeostasis. If your body doesnt sense glucose, you are "starving" so orexigenic hormones go up. We dont know if this is "good", because the effects could be due to: a.hypothalamic KO, b. muscle KO, c.muscle + hypothalamic KO, etc. It seems that the lean phenotype can be attributed to increased energy expenditure in skeletal muscle as it has become energy inefficient, seen also by the poor performance during exercise. This may override the effect of hyperphagia on weight gain.

    This has nothing to do with hyperglycemia per se. I would expect that if a hypothalamic exclusive KO KATP model is used, mice would be extremely obese and with all the characteristics of MetSyn.

  9. The genetic component of obesity is equal to that of height. Voluntary factors have extremely limited ability to affect body weight over the very long term.

    Dr. Douglas Coleman, Dr. Rudolph Leibel, Dr. Michaeal Rosenberg and Dr. Jeffrey Friedman have all watched data accumulate which shows that body weight , in both humans and animals, is involuntarily regulated.

    The Internet people are all missing one of the biggest components of obesity- GENES. Obesity is more heritable than NAY other medical condition studied with the possible exception of height alone- and only by the tiniest smidgen.

    With regrad to obesity, all of the Internet people are asking "what cheese is the moon made of?"

    Dr. Leibel's extensive work shows that when it comes to body weight- our bodes have a mind of their own.

    1. Hi RAZZ,

      "The genetic component of obesity is equal to that of height. Voluntary factors have extremely limited ability to affect body weight over the very long term."

      Yes and no. Monogenic obesity is rare (which means that is not very prevalent or that we havent been able to trace that gene). I do think genetic factors are very important, but epigenetic, which are (to some extent), modifiable.

  10. Hi Lucas, :)

    The formal way to phrase it is "80 to 90 percent of the variance in obesity can be ascribed to genetic factors"

    Another fact:

    15 % of morbidly obese people are that way solely because they have mutations in one of these 4 genes: NPY-R's, MC4R , NPY- LEPRb, aMSH-LEPRb.

    Dr. Friedman and Dr. Liebel believe this number will grow significantly greater over the years as we discover more. It certainly has so far. Back in 2004, Friedman's quote was 5 to 10 %

    Here is Dr. Jeffrey Friedman talking about it:

    Since the link may not show up on your Blog if I type the address just Google search "Dr. Jeffrey Friedman Sokol Science Lecture Series"

    All of ther above information is contained in it. However, it is a long lecture.

    Best wishes,

  11. Hi Lucas,

    I forgot to mention:

    Unfortunately, the body has a mind of its own regarding our weight- independet of what we are doing. This is unpleasant to learn, but it has been studied by Dr. Jeffrey Friedman, Dr. Michael Rosenbaum and Dr. Rudopl,h Liebel back and forth and sideways six different times in their own words.

    It's true. But, it is recently gained scientific knowledge. Lose even 10 % of body weight and the body aggressively responds by lowering energy expenditure by 25%- even after being corrected for that you;'re lighter.This coupled with increased hunger, increased muscular efficiency. This effect does not go away EVEN after 6 years.

    This is bad news.