- Aug 2019
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Given that APC−/− tumors can efficiently transport both glucose and fructose, we sought to determine the metabolic fate of glucose and fructose using 13C isotopic tracing. We isolated tumors from APC−/− mice and exposed them to four different labeling conditions for 10 min ex vivo: 13C-glucose (labeled at all six carbons), 13C-fructose (labeled at all six carbons), 13C-glucose + unlabeled fructose, and 13C-fructose + unlabeled glucose.
To study glucose and fructose metabolism in tumors the they traced the breakdown of the molecules.
- The scientists labeled glucose and fructose with a radioactive atom that can be traced even as the molecule is broken down.
- They incubated tumor tissues with labeled-glucose, labeled-fructose or a mix of labeled-glucose + fructose, or a mix of labeled-fructose + glucose. These tumor tissues absorb the sugars and metabolize them. Note: Adding a mixture of sugars to the tumor allows the scientists to determine how the metabolic pathways are related.
- The different components after metabolism are then determined in lab to trace the metabolic pathway of how tumors break down sugars.
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Despite the relatively well-studied pathway of fructose metabolism in the liver and in the small intestine, the role of fructose metabolism in tumors is mostly unknown.
This paragraph details the pathway of fructose and glucose metabolism within cells. In normal cells this is how glucose and fructose are broken down and converted into energy. It is unknown how tumors metabolize these molecules and so the scientists move on to explore this in their next series of experiments.
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product inhibition
This is a mechanism to control production in biological settings. This means when something is being made (ex. protein) and reaches a certain concentration then the production is stopped. This can also be called a negative-feedback loop.
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In aggregate, these results indicate that intestinal tumors can transport fructose directly from the intestinal lumen, where the fructose concentration is high after oral administration of HFCS.
Summarizing findings from the experiments up till this point: 1) fructose often reaches the colon at high concentrations when passive transporters in the upper intestines are saturated. 2) tumors in the lower intestines and colon absorb fructose efficiently, as demonstrated by decreased fructose in the liver in tumor-bearing mice 3) colorectal tumors are able to absorb more fructose than healthy intestinal cells because they have more fructose transporters
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Similar results have been reported for other fructose-metabolizing enzymes, ketohexokinase (KHK) and aldolase B in human colon tumors (22, 23).
Colorectal tumor fructose metabolism has been looked at in the past. In these studies the enzymes required to break down fructose were studied, rather than looking at fructose transporters as this paper did. The past research found similar results where tumors had higher levels of enzymes that break down fructose than normal human tissues did.
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on a tumor tissue microarray containing 25 cases of human colon tumors ranging from early-stage adenomas to metastatic carcinoma (fig. S5B)
In order to investigate tumor metabolism in human tissues scientists used a tissue microarray where tiny samples of human tumors or tissues were cultured and studied. They compared metabolism between 25 different tumors, of varying severity, to normal human intestinal cells.
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Further supporting our hypothesis, we found that GLUT5 was expressed at higher levels in APC−/−tumors as compared to IECs (fig. S5A), and in human colon tumors as compared to adjacent normal IECs
Investigating tumor uptake of fructose further, the scientists looked at sugar uptake proteins in colorectal tumors of both mice and human tissues. They found that colorectal tumor cells expressed more of the passive fructose transporter than normal intestinal cells do.
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Furthermore, the amount of fructose reaching the liver and serum was reduced in tumor-bearing APC−/− mice compared to WT mice (Fig. 2A), implying that fructose is trapped by the tumors instead of being transported to the liver and blood
The scientists also looked at how much fructose and was reaching the liver and blood to test metabolism of the sugar. They found that in tumor-bearing mice there was less fructose metabolized in the liver and less reached the blood. This suggests that the tumors consumed more of the fructose.
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we confirmed that APC−/− tumors efficiently transported both fructose and glucose following a bolus of HFCS (fig. S4B).
After administering high fructose corn syrup with the labeled carbons on glucose and fructose to a mouse with colorectal tumors the scientists found that the tumor took up and metabolized both glucose and fructose.
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Given these findings, we hypothesized that fructose in the intestinal lumen might be efficiently transported and metabolized by tumors located in the distal small intestine and colon.
The authors wanted to test whether tumors near the end (distal) of the intestines or in the colon consume fructose since it can be found in much higher concentrations in the colon than glucose can. Approach: They marked glucose and fructose molecules with C14 (radioactive carbon) which can be traced as the sugars get broken down and metabolized. This way if they find C14 from fructose and glucose in a tumor they can conclude that it metabolizes both sugars.
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Indeed, we found that fructose concentration was significantly increased in the colonic lumen (4.4 mM at peak 30 min) in WT mice after an oral bolus of HFCS (fig. S4A), consistent with impaired fructose uptake in the small intestine.
The scientists repeated experiments from previous work to validate their methods. They fed mice (oral bolus) high fructose corn syrup and then 30 minutes later (to allow for digestion) measured fructose levels in the colon. Similar to previous studies, they found elevated fructose in the colon suggesting the passive transporters in the intestine were saturated and allowed fructose to pass through undigested.
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Relatedly, a recent study in mice showed that fructose doses greater than 1 g/kg (~1% of daily calorie intake) overwhelm fructose absorption in the small intestine, resulting in a higher concentration of fructose in the colon (20)
The same results seen in human studies looking at gastrointestinal discomfort after fructose consumption were recapitulated in mouse studies. In animal models scientists are able to more closely control variables and more inversely monitor outcomes. In this case they were able to directly measure fructose concentrations in the colon rather than relying on gastrointestinal symptoms. They found high concentrations of fructose in the colon after oral delivery, suggesting that the passive transporters in the intestine were unable to uptake fructose at levels higher than 1g per kg of weight.
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The consumption of as little as 5 g of fructose can lead to the saturation of GLUT5 in the small intestine (i.e., malabsorption), resulting in an increased concentration of fructose in the lumen of the colon (large intestine) of healthy humans (17–19)
A past study administered various amounts of fructose to people after fasting. They then assessed gastrointestinal symptoms that stem from fructose passing through into the intestines and colon undigested (malabsorption). They found that even 5g of fructose can lead to the saturation of passive transporters in the intestine. Ingestion of any amount of fructose over 5g will result in high levels of fructose in the colon.
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passive transporter (GLUT5)
Fructose is only absorbed through diffusion into a cell, this means it relies on there being a lower concentration of fructose in a cell compared to the intestine. Passive absorption often leads to a saturation of the channels and so not as much fructose can be absorbed.
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sodium-coupled glucose transporters (SGLTs)
Sodium-coupled glucose transporters are found in the intestine. They use energy gathered from sodium ion transport into the bloodstream to generate energy to import glucose into a cell. Using energy to import a molecule up a concentration gradient (there is more glucose inside the cell than outside so it costs energy to import more) is termed active transport.
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These results suggest that the chronic intake of modest amounts of HFCS in liquid form facilitates tumor growth in the setting of APC deficiency independent of obesity and the metabolic syndrome.
Summarizing their results: In APC deficient mice (where colorectal tumors readily develop) consumption of even small amounts of high fructose corn syrup increases tumor growth.
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We observed similar results in a study of another mouse model, CDX2P-CreERT2; APCflox/flox (fig. S3, D to H), where intestinal tumors develop mainly in the colon instead of the small intestine (15).
Previous study of another genetically modified mouse model (where tumors were induced to grow mostly in the colon) demonstrated similar results. This suggests that this mouse model or genetic manipulation (APC deletion) itself is not somehow more sensitive to high fructose corn syrup than other mutations that lead to colorectal cancer. Comparing previous results increases confidence that any colorectal cancer tumors will respond to high fructose corn syrup in a similar fashion.
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Although the total number of tumors was similar (fig. S3, A and B), HFCS treatment significantly increased the number of large adenomas (>3 mm in diameter) and high-grade tumors in the HFCS group compared to the Con group (Fig. 1, C to F, and fig. S3C).
Results from experiment Part 2: Comparing mice fed with different amounts of high fructose corn syrup did not change the number of tumors formed. However, the size of the tumors, and the aggressiveness of the cancer differed between the mice fed a small amount of high fructose corn syrup and the control group with no high fructose corn syrup. This demonstrates that even a small amount of high fructose corn syrup is increasing tumor growth.
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Chronic treatment of HFCS using this strategy did not induce obesity or metabolic dysfunction in APC−/− mice (Fig. 1, A and B, and fig S2).
Result from experiment Part 1: Mice given a small amount of high fructose corn syrup did not become obese (as intended) and they did not develop metabolic syndrome. This suggests that high fructose corn syrup itself is not causing metabolic syndrome, but rather obesity is causing these health complications.
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To uncouple the metabolic effects caused directly by HFCS from those caused by HFCS-induced obesity, we treated APC−/− mice with a restricted amount (400 μl of 25% HFCS) of HFCS daily via oral gavage starting the day after tamoxifen injection (referred to as the HFCS group).
Here they did a two-part experiment: 1) They tested whether high fructose corn syrup itself induced metabolic dysfunction by giving mice a limited about of high fructose corn syrup so that they did not become obese.
2) To test the effects of high fructose corn syrup on colorectal tumor formation and growth the authors compared tumor characteristics between mice fed with different amounts of high fructose corn syrup. First, they treated all mice with tamoxifen to activate tumor formation. Then they broke them down into three groups: HFCS- mice that were treated with a limited amount of high fructose corn syrup to prevent obesity, WB- mice that had high fructose corn syrup mixed in with water so they consume a lot of it, and Con- a control group with no high fructose corn syrup administered. They then compared the formation of tumors and their characteristics to look at the effects of high fructose corn syrup.
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The consumption of HFCS in this manner led to obesity in both WT and APC−/− mice (fig. S1), and to metabolic dysfunction in WT mice (fig. S2) over an 8-week period.
In response to consumption of high fructose corn syrup mice in both groups gained a lot of weight and began displaying symptoms of metabolic dysfunction (high blood pressure, abnormal cholesterol etc.). It was unclear as to whether high fructose corn syrup consumption or obesity led to the development of metabolic dysfunction so the authors went on to test this next.
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We first determined the physiological effects of HFCS administered to APC−/− and wild-type (WT) mice
The scientists were first interested in looking at how high fructose corn syrup affects an entire mouse, and compare the effects on normal mice and their genetically modified mouse (APC -/-). They did this by mixing high fructose corn syrup into their water and allowing them to drink as much as they wanted (ad libitum). They monitored the mice's weight over time.
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tumor suppressor that is frequently mutated (75 to 80%) in the early stages of CRC development (13).
Scientists have found that in a high majority of colorectal cancers the APC gene is defective. This high correlation suggests that the APC gene is a major cause for the development of colorectal cancer and tumors.
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Wnt signaling
Wnt signaling is group of a pathways that regulate gene transcription and growth. Normally APC controls and limits growth that Wnt stimulates but when APC is deleted or mutated Wnt signaling is uncontrolled and leads to cancer formation.
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APC,
Adenomatous Polyposis Coli is a tumor suppresor gene meaning that when it is functional, APC controls cell growth and prevents tumor formation. When it becomes mutated or deleted (as in the mouse models), uncontrolled cell growth leads to tumor formation.
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To untangle the link between sugar consumption, obesity, and cancer, we mimicked SSB consumption in a genetically engineered mouse model of intestinal tumorigenesis. In this model, the adenomatous polyposis coli (APC) gene is deleted in Lgr5+ intestinal stem cells upon systemic tamoxifen injection (Lgr5-EGFP-CreERT2; APCflox/flox, hereafter APC−/− mice) (11, 12).
The scientists need a mouse model that will develop intestinal tumors so that they can study the effects of sugar-sweetened beverages on the tumor. They manipulated the mouse genes so that after injecting a drug (Tamoxifen) a gene in the intestine is deleted and tumors begin to form. With this genetically engineered mouse the scientists can induce tumor formation of the mouse, then track tumor size and metabolism to look at the effects of high fructose corn syrup in a diet.
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endocrine systems
A system of glands in the body that produce hormones that regulate metabolism, mood, sleep, development etc.
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metabolic syndrome
A cluster of factors such as high blood pressure, high blood sugar, excess body fat and abnormal cholesterol which contribute to diseases such as diabetes, heart-disease and strokes.
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confounders
Multiple factors at play which can affect an outcome or result. In this case it is impossible to separate the variables of obesity, which causes a host of complications such as high blood pressure, and abnormal cholesterol, from the direct effects of sugar-sweetened beverages.
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Indeed, studies have shown that excessive consumption of SSBs causes obesity and that being obese increases the risk of CRC, especially in men
Taking big data sets such as questionnaires or patient data, computational biologists can run statistical tests to look at correlations between variables. In this case data has shown that people who drink a lot of sugar-sweetened beverages (such as soda) tend to be more overweight and in men there was a significant correlation to developing colorectal cancer.
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serum
The fluid component of blood left after cells and clotting factors are removed.
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intestinal lumen
Lumen; The inside space of a tubular structure. The intestine is a long digestive organ that contains a tube of cells which absorb nutrients of food that is passing through the inside of the tube, which is called the intestinal lumen.
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tumor grade i
A scale on which tumors are judged by abnormality and the cells' likelihood to spread.
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high-fructose corn syrup
A sweetener made from corn starch. It contains a mixture of glucose and fructose molecules which taste the same and have the same calories, though they are processed differently in the body.
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tumorigenesis
formation of cancerous clusters of cells (tumors), where cell growth is uncontrolled
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AMP deaminase (AMPD2)
define
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Notably, there was almost no labeling of downstream metabolites of F1P from 13C-fructose when unlabeled glucose was added to the medium (Fig. 2D and fig. S6A), suggesting that the presence of glucose saturates aldolase and prevents fructose from being cleaved into three carbon units in this time frame. Because KHK produces F1P much faster than aldolase cleaves it, F1P accumulates (fig. S6B). This results in an acute drop in cytosolic ATP in tumors from APC−/− mice that had received HFCS as compared to Con tumors following a bolus (and Fig. 2E).
ADDING EMPHASIS
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As expected, F1P was predominantly 13C-labeled at all six positions (M+6) in tumors treated with 13C-fructose or 13C-fructose + unlabeled glucose (47.1 and 67.1%, respectively), as assessed by the percentage of labeling (Fig. 2C); these findings confirm the activity and presence of KHK in the tumors.
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tandem mass spectrometry (LC-MS/MS)
Tandem means having two in a row. and mass spectrometry is a method used to analyze samples to look at chemical makeup by looking at charge-to-mass signatures of individual atoms in a sample. Putting two mass spectrometers in a row increases the sensitivity of this method so that ions that are close in mass can be told apart. A great analogy and explanation can be found in this Youtube video.
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distal
adjective; far from the center. The end-most part of the intestines.
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radiolabeled
define
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ad libitum
adverb; as much as desired. The authors put high-fructose corn syrup in water for the mice to drink as much as they wanted.
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myriad
Noun; large and diverse amount of
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Define APC
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