Date of Award
5-1969
Document Type
Dissertation - Restricted
Degree Name
Doctor of Philosophy (PhD)
Department
Biological Sciences
First Advisor
James J. Smith
Second Advisor
Alvin F. Rieck
Third Advisor
David W. Glenister
Fourth Advisor
Robert W. Rasch
Fifth Advisor
Joseph Barboriak
Abstract
Adipose tissue physiology as it existed some 30 years ago can only be reconstructed from the meager reports in the literature. Adipose tissue was considered to be a connective tissue filled with droplets of fat. The fat stores were believed to be purely passive in nature and not in any way involved in the general energy metabolism of the body. Its main task was believed to be that of insulating the body against heat loss and of providing mechanical support for certain organs and tissues.
Today the fat cell is considered not merely a connective tissue cell filled at random with fat droplets, but a cell belonging to a specific organ differentiated even in the embryological state and representing an active center of energy metabolism. It should be recalled that some 30% of the dietary carbohydrate is converted to fat before it is further metabolized. In vitro experiments have shown that adipose tissue is the major site of conversion of carbohydrates into fat.
This tissue has also been shown to be the main source of free fatty acids in serum. Although they comprise only about 5% of the total fatty acids, free fatty acids have an extremely rapid turnover and so constitute an important link between the release and the transport (mostly on albumin molecules) from fat depots to other tissues. It is now recognized that they provide a source of energy for the working cell and are in fat metabolism what glucose is in carbohydrate metabolism.
The feature common to several classes of lipids (phospholipids, neutral lipids and steroid esters) is a fatty acid with a variable number of carbon atoms extending up to fifty. It is the fatty acid that goes to the heart of lipid function, be it as a constituent of membranes or a fuel. How this fatty acid is constructed from smaller building blocks has posed one of the classical problems of biochemistry.
In 1920, Osborne and Mendel concluded in a review on the indispensability of fats in the diets that "if true fats (i.e. compounds soluble in ether) are essential for nutrition during growth, the minimum necessary must be exceedingly small." It was probably not realized at that time, however, how difficult it is to prepare experimental diets completely free of fat.
Therefore, it was a great step forward when Evans and Burr began to use carefully extracted casein, and sucrose in place of starch (starch from wheat, corn and rice contains about 1% of lipids which can only be partly extracted by the classical lipid solvents) in their highly purified diets.
It was soon found that the fatty acid fraction of the lipid molecule played a vital role in the nutritional status of animals. Burr and Burr recognized that a deficiency syndrome developed in animals subjected to their fat free diets. The symptoms, occurring after 70 to 90 days on the diet, were described as scaliness of the skin on the back of the animals, followed by a cessation of growth. The term "essential fatty acid" was introduced by the same authors for linoleic acid since it was found effective in curing fat-deficient rats. Other fatty acids were subsequently identified as having essential fatty acid activity.
Essential fatty acids (EFA) are those fatty acids that either cannot be biosynthesized or are synthesized in inadequate amounts by animals that require these nutrients for growth, maintenance, and proper functioning of many physiological processes. A requirement for essential fatty acids has been established for many species of animals. Undesireable consequences of EFA deficiency have been reported in the rat, mouse, chicken, guinea pig, hamster, dog, calf, pig, lamb, goat, some insects and microorganisms, and humans.
It is now recognized that there are at least three essential fatty acids, linoleic (delta 9,12-octadecadienoic), linolenic (delta 9, 12, 15-octadecatrienoic), and arachidonic (delta 5, 8, 11, 14-eicosatetraenoic) that vary in activity in alleviating symptoms of EFA deficiency. Metazoans cannot de-novo synthesize them so they must be provided in the diet. Multicellular organisms can synthesize other unsaturated fatty acids from small precursors derived from carbohydrates. For example, oleic acid which is formed from acetyl-CoA can be further dehydrogenated to yield polyunsaturated fatty acids. However, these fatty acids are nonessential. Fulco and Mead identified in tissues of rats fed a fat-free diet a trienoic acid that accumulates a delta 5, 8, 11-eicosatrienoic acid arising from oleic acid. This trienoic acid is found in significant amounts in the heart, liver, adipose tissue, and erythrocytes of animals fed diets low in essential fatty acids, and constitute the most characteristic feature of the fatty acid composition of tissues from EFA deficient animals.
Besides the decrease in growth and the skin changes, an increased metabolic rate and decrease in food efficiency are observed in EFA-deficient animals.
Since essential fatty acids enter the structure of membranes, it would be expected that in EFA-deficiency, structural changes would take place in these membranes. In EFA-deficient mice it was observed that liver mitochondria were markedly enlarged and spherical in shape with an increase in the number and length of cristae. Further
studies suggest that mitochondria are the most sensitive indicators of EFA depletion.
Studies on isolated mitochondria from the livers of EFA-deficient rats showed that their mitochondria had an increased tendency to swell, were more fragile, and had a lower phosphorous-to-oxygen (P/0) ratio than normal hepatic mitochondria, indicating an uncoupling of oxidative phosphorylation and interference with normal membrane structure. Mitochondria from EFA-deficient rats also exhibited an increased oxygen consumption, a faster oxidation of Krebs cycle intermediates, greater succinic acid dehydrogenase activity, and extensive structural damage.
The thyroid activity of fat-deficient rats was studied specifically by Morris et al. They concluded that the recorded increase in oxygen consumption in deficient animals is not due to an altered thyroid function. Macmillan and Sinclair gave injections of radioactive iodine to fat-deficient mice, and found no hyperactivity of the thyroid gland. Thus, hyperthyroidism appears to be ruled out as an explanation of the metabolic and structural alterations described in EFA-deficiency.
This later observation is pertinent since thyroid hormones when given in large pharmacological amounts retard the growth of young animals and cause a loss in body weight of adults. They greatly alter heat production and increase oxygen consumption. Thus, the administration of a single large dose of thyroxine causes a rise in basal metabolic rate which begins after a latent period of a few hours. The activity of some of the Krebs cycle enzymes is directly or indirectly enhanced by thyroid hormones. It has been repeatedly demonstrated that thyroid hormone administration lowers the phosphorylation efficiency of mitochondria and uncouples oxidative phosphorylation.
Recent experimental results have suggested that the thyroid hormones effect the enzymes for oxidative phosphorylation indirectly by altering the structure of the mitochondria. Tapley in a study of the water content of isolated rat liver mitochondria suspended in a sucrose medium, where oxidation or phosphorylation are excluded, found that such uncoupling agents as thyroxine and calcium ions caused rapid swelling of the mitochondria, whereas other uncoupling agents such as Dinitrophenol and "Dicumarol" inhibited the usual slight spontaneous swelling. Thus the swelling phenomenon is not a consequence of the process of oxidative phosphorylation, and the mitochondrial structure, especially its membrane, may be the primary site of action of the thyroid hormones.
Higher swelling rates of EFA-deficient mitochondria were observed in vitro by Hayashida et al.
There is also a third condition which seems to share some fundamental aspects with the essential fatty acid syndrome and hyperthyroidism. This condition is the Hutchinson-Gilford Syndrome (Progeria). No more than a few cases have been reported in the literature. This rare disease is characterized by hypermetabolsim (sic), cessation of weight gain, decreased food efficiency and early death from complications due to extensive arteriosclerotic lesions. In spite of the striking metabolic similarities with hyperthyroidism, the thyroid function has been shown to be normal in progeria.
In view of the above observations it was decided to investigate the possibility that a common metabolic abnormality is present in essential fatty acid syndrome, hyperthyroidism and progeria. As a first step, the in vivo physiological effect of thyroxine on the elongation and desaturation of fatty acids of rat liver mitochondria will be explored and compared with the metabolic situation seen in the essential fatty acid syndrome.