9UNP0465SD_Henry_Lardy_Wordpress

- Henry Lardy: It's a pleasure to participate in this symposium that is honoring Gobind. I have to tell you my first relationship with Gobind, which happened many years ago when he was at the University of British Columbia. At that time I was a member of the Scientific Panel of the Life Insurance Medical Research Fund. And at one of our annual meetings, there was an application from Gobind Khorana to support his research. The application had been assigned to two medical doctors who reported to the committee that the application looked like good chemistry, but it was pure chemistry and they didn't see its relevance to heart disease. So I interceded to say that the compounds that Dr. Khorana was synthesizing were phosphorylated nucleotides and they were the same type of compounds that supplied the energy for heart muscle contraction. The result was that the grant was approved. Then some years later, the newspapers announced that Gobind Khorana had won the Nobel Prize. And the next day, the Life Insurance Medical Research Fund was in the newspapers reporting that they had supported Professor Khorana's work since time immemorial. 

So I was glad to see that I had something useful to do on the committee. I don't think considering any other grants was anywhere nearly as important. I'm going to show the first slide, which doesn't seem to have to do anything with topic that I'm talking about, but we'll bring in the relevance later on. Mammalian spermatozoa use fatty acids from their phospholipids to drive motility. And they need oxygen, of course, to do that. If lactate is supplied, there is a great increase in oxygen consumption, and a great increase in in the motility as we rated it from 1 to 4+. Glucose supplies energy both by glycolysis and respiration, so oxygen uptake is not increased as much as it is with lactate. But it supports excellent motility. A very unexpected finding was that beta-hydroxybutyrate, both in epidermal sperm and ejaculated sperm, inhibited the low rate of respiration, but it supported an excellent degree of motility. So we stated that obviously, oxidation of beta-hydroxybutyrate was very efficient in generating high energy phosphate for motility. 

Now that's a far cry from the steroids that I'm going to talk about, but I'll come back to that slide. When I became an Emeritus Professor in 1988, the university allowed me to retain one laboratory with a single bench. One can do chemistry on a single bench, but you cannot do the type of metabolic studies that we had done in the previous 43 years. So when searching for a chemistry problem, I chose to work on steroids, because that offered the greatest opportunity to learn. I knew practically nothing about steroids, but the newspapers had been carrying stories about a new compound called DHEA, which is Dehydroepiandrosterone. And that steroid seemed to have magical properties. It caused weight loss in obese animals, without any depression of their food intake. It didn't cause weight loss in non-obese individuals. It was an effective treatment for some kinds of cancers, and it improved memory in mice, old mice. It enhanced the effectiveness of the immune system. 

But large doses, about half of 1% of the animal's diet, were required to obtain there responses. DHEA is the most abundant steroid in the human body. There is more DHEA than all other steroids combined. We make about 15-25mg a day. It's all made in the adrenalin glands, but it is transformed to other compounds in a variety of tissues. The formation of DHEA decreases in people as they age. The result is that an 80-year-old has about 10% DHEA in his blood stream as compared to his concentration at 20-30 years of age when it is maximum. All steroids are derived from cholestrol. And the pathways of the derivation are shown here. In the upper left hand corner, the cholestrol is converted to pregnalanone. 

And in the middle column, the 2-carbon side chain of pregnalanone is cleaved to yield DHEA on the far right side. The DHEA has been known to be converted to several other compounds, but the relationship of DHEA to their synthetic pathway and to their function have never been elucidated. Now I'm going to switch to mitochondrial electron transport. The next slide describes the fact that when puridine nucleotides such as NADH are oxidized, there is a coupling of phosphorylation at three sites to make three equivalents of adenosine tryphosphates from inorganic phosphate and ADP. In 1958, two research groups independently reported that insect muscle oxidized NADH forming glycolysis by a pathway that skipped the first phosphorylation step. And that pathway is shown here. Puridine nucleotides that are generated by the oxidation of substrates yield NADH. And the NADH reduces dihydroxyacetone phosphate to yield the glycerol 3-phosphate. There is a glycerol 3-phosphate dehydrogenase in the mitochondria that oxidizes that product and transfers electrons to ubiquinone, and thus it skips the first phosphorylation site. Naturally, the energy that's not captured as ATP at that first site would generate heat, and therefore we investigated the most highly thermogenic hormone known, namely that of a thyroid hormone. 

When 2% dessicated thyroid powder is fed to rats for a week, the liver glycerol phosphate dehydrogenises increases some 20-fold. It increases the activity in all tissues that we studied, except for brain, which does not respond. It has been shown that all of the tissues that I show here as responding with an increase of a glycerol 3-phosphate dehydrogenase also show an increased oxygen uptake when excised from a hyperthyroid animals. Thyrodectomy shown in the righthand column reduces the enzyme in all the tissues, but it does not affect the enzyme in brain. So in the cellular oxidation of glucose, the cytosolic NADH is generated by removal of two of the electrons from glucose metabolism, which represents one-sixth of entire electron transfer in the metabolism of glucose completely. And since it decreases efficiency by one-third, you can multiply one-sixth by one-third and you can get 5.4% which is the fraction of the glucose energy that is lost as heat. That is much less than is measured in hyperthyroid human subjects. Earlier workers, Barbara McClintock especially in 1930s, demonstrated that human subjects who were hyperthyroid consumed 30-35% more oxygen to do a given amount of work, as compared to normal subjects. And other investigators had shown that hypothyroid subjects would perform a given piece of work with less increase of oxygen uptake than normal subjects. So with the glycerol phosphate shuttle acting on glycolytically generated NADH, we know that it doesn't account for the decreased work efficiency in the hyperthyroid state, because 5.4% doesn't measure up to the 30-35% more oxygen that is required for a given piece of work in hyperthyroid subjects. 

So I searched for evidence that the electron removal in the tri-carboxylic acit cycle might be involved. And in 1965 I found that reducing equivalents generated in the mitochondria tri-carboxylic cycle were transferred to the cytosol in form of malic acid and some other reduced acids. Cytosolic malic enzyme converts malic and NADP to pyruvate and NADPH. The enzyme that reduces dihydroxy acetone phosphate to glycerol 3-phosphate uses either of the puridine nucleotides, NADH or NADPH. The pyruvate will return to the mitochondria, where pyruvate carboxylate converts it to oxyl-acetate, and the reduced puridine nucleotide generated in the tri-carboxylic cycle will reduce the oxyl acetate to malate. All the reducing equivalents generated in the carbohydrate metabolism are available for the glycerol phosphate shuttle, and the heat liberated maintains normal body temperature. We have found that the glycerol phosphate shuttle is inhibited by beta-hydroxybutyrate, which accounts for decrease of oxygen consumption, and an increased motility of sperm shown earlier, because it eliminates the inefficient metabolism that occurs through the glycerol phosphate dehydrogenase. We think that beta-hydroxybutyrate is also responsible for the drop of body temperature in hibernating animals. Hibernating animals eat well and accumulate body fat before they seek their winter quarters. Our Q-measurements show that hibernating animals are living completely on the fat oxidation which yields ketone bodies, acetacylic acid, and beta-hydroxybutyrate. 

The latter inhibits the glycerol phosphate shuttle and thus shuts down body heat production. That accounts for the inhibition of its normal body temperature and drop of temperature to 1 or 2 degrees above that of environment. In 1977, Terry -- reported that mice genetically destined to become obese, gained much less weight when fed Dehydroepiandrosterone despite eating as much as their obese litter matess. I thought immediately that they must be wasting energy, and that glycerol phosphate shuttle might be activated by DHEA. So, we examined that question. And as you can see here, like the thyroid hormone, DHEA increased the glycerol phosphate dehydrogenase, an effect that is not only additive of the thyroid hormone, it was synergistic. The enzyme response to DHEA is proportional to the amount of DHEA into the diet. As you can see, there's a steady increase in the percent of control of glycerol phosphate dehydrogenase and the malic enzyme and the percentage of DHEA in the diet is increased. In addition to this effect on weight loss and on the glycerol phosphate shuttle, DHEA improves memory in old mice. It enhances the synthesis and secretion of insulin, and it increases the amount of mitochondria in liver. 

But as I mentioned earlier, it must be fed in relatively large amounts to obtain those responses. Previous chemists had found and characterized several derivatives of the DHEA in the tissues and excreta of cancer patients. We wondered whether these derivatives might be more active than their precursor, DHEA. So we undertook a study to determine how the derivatives were formed. And we show here, that when DHEA is incubated with tissues it is converted to a variety of compounds. The major compound is a reduction at the 17 position to form estradiol. It is also hydroxylated at 7 position to yield 7 alpha hydroxy DHEA, which is next oxidized to 7 oxo-DHEA. And that in turn is reduced at the 7 position to form 7 beta dihydroxy DHEA. Earlier investigators thought that 7 beta dihydroxy DHEA was formed by direct hydroxylation of DHEA. But we couls demonstrate that no 7 beta dihydroxy DHEA was formed until 7 oxo DHEA had accumulated in the incubation medium. 

The final product that we have found thus far is hydroxylated at the 16 alpha hydroxy position to yield a 7 beta 16 alpha dihydroxy DHEA. As we go through this sequence of metabolic change, the compounds become more active as inducers of enzymes, and also they're more active in stimulating memory of old mice. Two physicians and psychiatrists at Columbia University Medical School were aware of our paper showing that 7 oxo DHEA enhanced memory in old mice. And knowing that the compound worked in brain, they tested it on five women who had been abused as children, and who had suffered post dramatic stress syndrome for many years. They had been treated with many different drugs, all of which had been ineffective. These five ladies showed some improvement the first week when they were given 250mg of 7 oxo DHEA daily. And all were cured and went back to work within a month, when the dose was doubled to 500mg daily. I must say, that that was a very successful and dramatic finding, but we were disappointed when we learned some months later that they had applied for a use patent on our compound 7 oxo DHEA for the treatment of depression. Another interesting compound that's produced is the androstenediol shown in the upper right hand corner of this chart. It has been known for many years that androstenediol is a weak estrogen. 

However in 1996, we demonstrated that it is a full-blown androgen without any further alteration. Most importantly, the androgen activity of androstenediol was not inhibited by the drugs that are currently used to treat prostate cancer. When prostate cancer patients are treated with Casodex or Hydroxyflutamide, drugs that inhibit the function of the androgen receptor, the cancer goes into remission for some months, and then it resumes growth, and it is not influenced by anti-testosterone agents. Physicians term that "androgen independent" cancer growth. We postulated that androstenediol was supporting the androgen independent growth, and Japanese investigators have confirmed our hypothesis by finding high concentrations of androstenediol in the so-called androgen independent gland of prostate cancer patients. My colleague Dr. Pagma Marwa synthesized some 60 new steroids in the search for an effective inhibitor of anadoil. And we found that beta hydroxy acetoxy androst 1-5 dien 17 ethelyn ketel was effective against diol, and it also inhibited the function of testosterone. We studied the tolerance and the pharmaco-kinetics of androstenediol in rats. We found that the only effect of this compound adic, as we abbreviate it, was to shrink the prostate and the seminal vesicles. 

It did not cause histological changes in any other tissues. Obviously, it should be tested in prostate cancer patients. So we obtained the patent, which the university assigned to Holiseeden Corporation in San Diego, because they had supported our work on the compound. And our university policy is that individual companies that support research have a right to develop the findings. They were pleased to work on our patent, but in recent times we have found that they've been busy synthesizing analogues of our compound in the hope that they can find a substance that will escape the patent stipulations, and they will therefore escape paying royalties to the University of Wisconsin. Well, we're now trying to recover our rights to that patent, but I'm afraid it will take a great deal of money to develop it in clinical trials, if we ever do recover the patent rights. I wish to emphasize that the studies of the function of the steroids emphasize that they stimulate the production of the glycerol phosphate dehydrogenates that participates in the struggle. And that allows oxidization to go on without coupling to energy capture. And that was responsible for heat generation, maintaining our normal body temperature, and we soon will find out whether it has anything to do with the drop in body temperature in hibernating animals. Thank you. 

(applause)