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High-risk diet

Responsible for weight gain, the blocking of insulin begins in the brain and increases predisposition to cardiovascular diseases and cancer

Standing by a metal container reminiscent of a windowless train carriage, Andressa Coope prepares a yellowish paste that is rich in pork fat. The white rats that will be fed this diet, are kept there in stacked up cages, drawing our attention not only for being fat, but also because they have a small tube like an antenna implanted on the top of their heads. It is through this tube that the biologist will inject the substances that are expected to show the effects of a fatty diet on the body and further strengthen the recent conclusion of the State University of Campinas/Unicamp teams of which she is a member: long-term consumption of fat-rich diets such as those in western countries like Brazil and the USA, besides making one gain weight, can be fatal for the body.

The excess of cream-filled sweets, breads, fried foods and fatty meats hinders the proper functioning of the hormone insulin, which carries glucose to the inside of the cells in different organs and tissues, where this sugar is transformed into the energy that is essential for life. 15 years of work were required for the teams of Mário José Abdalla Saad, José Barreto Carvalheira and Lício Velloso at the Unicamp School of Medical Sciences to show that this biochemical maladjustment known as insulin resistance begins in the body and in the muscles. It later has repercussions throughout the entire body, reducing the use of the energy that comes from foods and increasing hunger. Consequently, obesity, diabetes, hypertension, cardiovascular diseases and even cancer – in sum, the world’s current killer health problems – develop more easily.

A rare example of the integration between the phenomena that occur within cells and other more global phenomena that regulate the workings of organs and tissues, the almost 200 papers published by the Unicamp teams now show, precisely, where, how and when insulin resistance arises. Insulin resistance is the first step in the development of 90% of the cases of diabetes, which affect 180 million people worldwide. Promising alternatives for treating these problems are also arising out of these studies.

It was by keeping the animals’ bowls always full that Saad’s team found out that the hypothalamus and muscle cells are the first to become insulin-resistant, ten days after the start of a fat-rich diet. In the second stage, this hormone stops working properly in the liver and blood vessel cells. Only after five months does the problem become manifest in the adipose tissue, formed by cells that specialize in accumulating fat.

This sequence of the problem’s development enables us to understand better why people who develop insulin resistance generally become obese – although it does not explain all obesity cases, a problem which may also be of genetic origin or due to other types of hormonal dysfunctions. The chief reason is that everything starts in the hypothalamus, a region at the center of the brain that is responsible  for both hunger control and for energy expenditure. A few minutes after the first bites are taken out of a sandwich, the blood sugar level increases, stimulating the pancreas to release insulin. The hypothalamus detects the higher rates of this hormone and, in turn, reduces the production of two others: orexin, responsible for the sensation of hunger, and the melanine concentrating hormone (MCH), which controls the metabolism in addition to hunger.

Velloso’s team recently demonstrated part of this mechanism and the link between obesity and diabetes by regulating the production of MCH in the hypothalamus. Obese rats had high amounts of this hormone in their blood and spent less energy, whereas the thin ones had less MCH and burnt calories faster. The animals that were given extra doses of this hormone became insulin resistant, obese and diabetic. Velloso tells us that because it makes the body save energy, by reducing body temperature imperceptibly, MCH has become a good target for the pharmaceutical industry where the treatment of obesity is concerned: blocking the action of this hormone might reduce hunger and increase energy expenditure, making the body’s temperature rise slightly.

When insulin is no longer able to carry blood glucose into cells, however, this entire biochemical mechanism is derailed. The high levels of blood sugar continue to induce the pancreas to make insulin, but even these higher doses are not recognized by the hypothalamus, which increases the release of the two hormones that increase hunger and reduce energy expenditure, as if the body were facing a long fast. As a result, one enters a vicious circle in which the amount of insulin and blood sugar are always high, damaging the cells of the liver, blood vessels and nerves.

As if this were not bad enough, in the first five months of insulin resistance the   adipose tissue cells continue to absorb glucose and to turn it into fat, increasing the “love handles’ or “spare tire” around the waist. “This sequence suggests that a very old survival mechanism may be at play to this day”, comments Saad: by becoming insulin resistant, the brain allows one to eat freely and accumulate energy, as if food were going to become scarce shortly afterwards.

The contribution of the  Saad, Carvalheira and Velloso teams led to our understanding of how  the rise in insulin resistance  is not limited to  interaction between the body’s tissues and organs. The Campinas groups’ studies, coupled with those from other research centers abroad, also helped to identify what happens at the cellular and molecular levels. When the body is running well, insulin approaches the cells carrying a glucose molecule and it fits into proteins on the surface of the cells called insulin receptors. The cell then opens and allows the glucose to enter. The glucose is then involved in a number of successive chemical reactions until it is transformed into energy or is stored as an energy reserve as fat in adipose tissue, or as glycogen in the liver and muscles.

Successive Pantagruelian meals break this routine, changing the functioning of the enzymes that would normally allow the glucose to enter the cell and follow its habitual path. Saad’s team described two new mechanisms whereby cell confusion and insatiable hunger are established. In one of these, which Marco Carvalho-Filho described in 2005 in Diabetes, an enzyme called iNOS (inducible nitrous oxide synthase) blocks the action of the molecules to which insulin binds itself on the surface of the cells. When he discovered these connections, Saad imagined an action strategy: reducing insulin resistance by blocking the action of iNOS, a path that appears to be promising according to preliminary laboratory studies.

Another insulin resistance mechanism brings to the scene two other enzymes, known by the abbreviations JNK and IKK-beta. Activated by the consumption of high fat diets, these enzymes also keep insulin from connecting to cells and carrying glucose into them, as shown by Patrícia Oliveira Prada, from Saad’s team, in an article published in 2005 in the journal Endocrinology. This time the damage is large, because these cell surface molecules are not only used by insulin. They are also crucial for other hormones to work, such as those that regulate hunger and blood pressure. The blocking of these cell surface molecules, highlights Saad, is one of the common origins of obesity, diabetes and hypertension.

The biochemical mechanisms whereby illnesses may be related appear deep within cells. In 1995, while doing post-doctoral work under Saad’s supervision, Velloso began working on the connection between insulin, which controls the amount of glucose circulating in the body, and angiotensin II, which controls blood pressure. This might explain a phenomenon that has been known for a long time: diabetics often suffer from hypertension as well.

Published in 1995 and 1996, the earliest results showed how angiotensin opposes the action of insulin and  has also inspired new treatment strategies. It was Carla Carvalho, in her post-doctoral work with Saad, who found that anti-hypertension drugs that block the action of angiotensin also help to treat diabetes and obesity, as they reduce insulin resistance in vein and artery cells. Currently a researcher at the Biomedical Sciences Institute of the University of São Paulo (USP), Carla has also shown that the integrated action of an excess of insulin and of LH (luteinizing hormone), which helps to control the menstrual cycle, may contribute to forming polycystic ovaries, which are common among obese young women. In this way,  she explained why weight loss was a way of normalizing the functioning of the ovaries.

In  pursuit of even more in-depth explanations for these connections, Saad suspected that the three enzymes, iNOS, JNK and IKK-beta that keep insulin from working might have a common origin. As he showed after a great deal of work, the three can be activated by proteins called TLR-4 found on cell membranes, that are toll-like receptors. Mice with a genetic mutation that switches off this protein made better use of glucose, gained less weight and did not develop insulin resistance, even when submitted to a high fat diet.

For Saad, these results suggest that TLR-4 may very well be the missing link between the consumption of fatty diets and the development of insulin resistance. By connecting with this cell-surface receptor, fats might activate one of the three enzymes that block the action of insulin, keeping the body from making use of the glucose. By deactivating the TLR-4 receptor in the mice’s cells, Saad also observed a reduction in one of the types of the blood’s defense cells. This might indicate a connection between obesity and a very slight inflamation of the entire body, which doctors do not always notice and that is generally found among people whose weight is well above the level regarded as healthy – people with a body mass index (obtained by dividing the weight by the height squared) above 30: a person who is 1.7 m tall is obese if he/she weighs more that 87 kg.

However, what activates TLR-4″ Possibly, a type of fat found mainly in red meats, according to a study conducted by Velloso’s team, currently in its publication phase. “It all starts because we eat too much animal fat”, says Velloso. “Hunger and the epidemics caused by infectious diseases, which were the two main reasons for death among our ancestors, may have selected genes that favor energy storage and fast response to infections”, he comments. The very buildup of fat can be seen as a defense mechanism in the event of food scarcity, as was the case back in the time when humans lived in caves. However the body maintains this instruction to eat a lot to this day, even when food is not scarce.

Over the course of these many years of work, the Unicamp teams also found that those who are highly overweight also run a higher risk of developing cancer. “Excess insulin fosters tumor growth”, says Carvalheira, a physician who coordinates one of the three Campinas teams that are showing the links between these diseases. Carvalheira found this association between insulin resistance and a greater propensity to developing cancer in an experiment with two groups of mice. The two groups were given cancer cell injections; one consumed a diet rich in fats, while the other was given a more balanced diet. At the end, those who stuffed themselves with fat were shown to be 50% more prone to developing tumors, and their tumors where 1.5 times bigger. A study published in August in the New England Journal of Medicine proved this relation, but inversely, by comparing the cause of death of almost eight thousand obese people who had had stomach reducing surgery and who had started eating in moderation  versus another eight thousand who did not undergo surgery. Among the first group, death from diabetes dropped by 92% and from cancer by 60%, although mortality due to accidents and suicide was 58% higher.

“The link between obesity and cancer seems to be clear”, says Carvalheira. He relied on this knowledge to create a way to fight the lack of appetite that usually goes with cancer. In another experiment, he found that metformin, a diabetes treatment drug, might increase the consumption of food by a factor of two and the survival period by 30%. This is a new application for a known drug, although its use in these cases will have to undergo further testing, until it is shown to be truly safe.

This is not the only alternative found recently for fighting insulin resistance. “We bring into the lab those issues that arise during our work at the hospital”, comments Saad. Attuned to the possibility of applying the knowledge that resulted from the study of rodents, Saad, Velloso and Carvalheira have found some ways to reduce the blocking of insulin. One of them consists of DNA fragments called oligonucleotides, which block the action of a protein called PGC-1 and leave insulin freer to deliver sugar to the cells. Results with their experiments with mice encouraged a domestic pharmaceutical company to go ahead with the development of a drug based on these DNA fragments, which may become available to those who need it within ten to fifteen years – if everything works out and if the international criteria for drug development are followed.

Paty Karol, from Saad’s team, described in her doctoral thesis an oligonucleotide (DNA fragment) that undid the insulin blocking in the hypothalamus of rats and reduced it in muscles and in the liver. As a result, the animals ate less. However, this compound is still far from reaching drugstore shelves.

Until then, perhaps a simpler way to avoid excess insulin may really be physical exercise. Marcelo Flores, under Carvalheira’s guidance, showed that prolonged exercise of medium to high intensity reduced rats’ appetite by increasing the sensitivity of the hypothalamus to two hormones that control hunger: insulin and leptin. However, the exercise must be taken regularly and in an ongoing basis. In a Unicamp experiment, a group of rats had to swim for an hour a day for eight weeks, whereas another group was sedentary. Afterwards all the animals were allowed to stuff themselves with fatty foods, sweets and highly caloric drinks. Surprisingly, those that had been exercising developed higher insulin resistance than the sedentary ones. Those who had undergone the swimming regime gained more weight, reproducing one of the most visible changes that Argentine soccer player Maradona experienced after retirement from soccer. Conclusion: although a sedentary life may be subject to criticism, exercising regularly in order to lose weight and then stopping abruptly may prove to be disappointing.

The Project
Molecular insulin resistance mechanisms in the hypothalamus and peripheral tissues (01/03176-5); Modality: Thematic Project; Coordinator: Mário José Abdalla Saad – Unicamp Investment; Investment: R$ 1,146,794.71 (FAPESP)