Five years earlier, Bernard had shown that gastric digestion of cane sugar and starch resulted in the formation of readily-absorbable glucose. Now in 1848, he decided to explore where that glucose was distributed in the body - the économie animale as he called it23,46. Using a sensitive analytical (copper reduction) method developed by BarreswillTMB47,p130,190, he was surprised to find glucose in blood samples - from animals and man - that were eating a diet completely free of carbohydrate; indeed, even if they had been fasting for several days. Could this mean that some glucose was being synthesized in the body itself?
To clarify the issue and to identify the possible source of the glucose, he took blood from abdominal blood vessels of fasting animals. He soon discovered particularly large amounts in the hepatic vein leaving the liver. There was also some glucose in the portal vein just as it entered the liverTMB47,p123. That puzzled him, since during fasting there should have been no nutrient in the portal vein tributaries draining the intestine. He therefore reasoned that the liver itself had to be the source of that glucose, entering the portal vein by reverse flow. His theory was supported by finding that the portal vein glucose level was still high after he placed a ligature around that vein between intestine and liver.
He logically moved to an analysis of liver tissue samples and to his most important discovery: in every liver he examined - and from every species of mammal, bird, reptile and fish - he found copious quantities of glucose23,24. There was no glucose in any other organ. Initially, he could not believe that the liver could possibly synthesize glucose. That organ's function was known to be the secretion of bile - and had not the famous Xavier Bichat and so many before him insisted that each organ had only one function? Furthermore, there was a widespread belief that animals could not synthesize nutrients. The influential chemists Jean-Baptiste Dumas and Jean-Baptiste Boussingault were adamant that plants - and only plants - could do that. Accordingly, Bernard was obliged to at least consider that rather than synthesize glucose, the liver simply stored it until it was needed by the body. He would need to address that issue at some point.
Meanwhile, his immediate priority was to identify what was controlling the release of glucose into the hepatic veins leaving the liver. With his belief that chemical functions of the body were often under the control of the nervous system, he decided to see what would happen if he cut the pneumogastric nerves (the vagus nerves as they are now called). It was a logical experiment since those nerves, part of the parasympathetic nervous system possess nerve branches which terminate in the liver. When he cut the vagus nerves, it did indeed result in less glucose leaving the liver through the hepatic veinsTMB20volI:p288,360,366. However, when he stimulated the vagus nerves electrically (his famous counterproof principle) glucose release from the liver did not increase. He therefore argued that it could not be the vagus nerves which were responsible.
In 1849 he explored further and higher, into the brain. Using a needle, he stimulated the floor of the fourth brain ventricle, from where the vagus (as well as other) nerve fibres were known to originate. This time, blood glucose did rise impressively34. Bernard called this interesting phenomenon piqûre diabetes. However, it was only a temporary glucose rise, never lasting more than one day. It could therefore hardly be called diabetes. Furthermore, the blood glucose rise was not solely under the control of the vagus nerves, since if he cut them before doing his piqûre, it did not prevent the glucose rise: another failed counterproof38,TMB25,vol2:p431.
Somewhat later, and determined to explore every avenue, he cut the spinal cord just above the exit of its splanchnic nerves which carry sympathetic nerve fibres - belonging to the other part of the autonomic nervous system. This time the piqûre phenomenon was indeed blockedTMB47p368. Yet Bernard was mistaken in thinking that the sympathetic nerves had a direct effect on the liver. Only many decades later, it was shown that sympathetic stimulation results in release of adrenaline from its nerve endings. It is this which secondarily promotes glucose discharge from the liver.
One morning, Bernard discovered an animal's liver that his assistant had forgotten to clear away from the laboratory bench following the previous day's experiment, in which they had together been analysing its glucose content. Rather than throwing away that liver, Bernard impulsively decided to take further samples from it. To his surprise, he found that even more glucose was present than on the previous day134. This was the first hint that the liver was indeed capable of manufacturing glucose (rather than just storing it). It led him to quickly devise washout experiments that he hoped would provide absolute proof.
He injected water forcibly into the portal vein as it entered the liver. At the same time he took repeated samples from the hepatic vein leaving the liver, until he could no longer detect any glucose in them. One day later, he repeated this washout procedure on the same liver. Glucose again appeared in the hepatic veins, and in even greater amounts than before. He now had his proof. Glucose was not just stored in the liver, but had actually been synthesized there62,TMB27vol2p110,TMB47p181,300,568.
Not only was this an important step forward in understanding glucose metabolism, but in one stroke it had demolished the age-old theory of 'one-organ one-function'. It had also destroyed the equally entrenched belief that animals were incapable of synthesizing nutrients. Furthermore, glucose was apparently produced in one organ, secreted into the circulation and then acted elsewhere. Bernard saw this as a model for a considerably wider concept: that other organs such as the thyroid, spleen, suprarenal and even the thymus gland might in future be shown to be 'glands of internal secretion'. Even though glucose is certainly not a hormone, Bernard's concept of internal secretion was the first step in defining the endocrine systemTMB20p96,TMB27vol2p412,TMB31p59.
He was now intent on identifying the chemical precursor of glucose in the liver; yet without knowing exactly what substance he was looking for, it was at that time difficult for anyone to provide an answer. While he continued to think and experiment, he gave the still-unknown precursor the descriptive name glycogène (glycogen). Meanwhile, many publications and presentations resulted from his glucose studies: in 1849 to the Society of Biology37,39,41,46,60 (which he himself had helped to establish), and in 1850 to the Academy of Sciences34 who the very next year awarded Bernard, for the third time, its prize in Experimental Physiology62. In 1853 he was also awarded a Doctorat in Natural Sciences for his discoveriesTMB10.
Bernard returned to his work on curare. Again, he showed that its effect was exclusively on motor nerves: the sensory nerves were left perfectly intact. Then he disovered that if the animal could be kept alive by artificial respiration, the curare effect would wear off, and muscle function would fully recover61,65. This led to the use of curare as a muscle relaxant in tetanus143,145 and in severe epilepsy; and then also for abdominal surgery. It also prompted Bernard to propose that poisons might be used more systematically to "...analyze the most delicate phenomena of the living mechanism" - a sort of physiological dissection. He accordingly experimented on strychnine, as well as on other poisons26,TMB24. Yet it was curare which continued to fascinate him, and he researched and wrote about it for a further twenty years168,169,205,228. It also led him to study asphyxia and the actions of anaestheticsTMB41. Interestingly, he also discovered that administering either curare or strychnine induced a temporary rise of blood glucose: a phenomenon which he assumed was again due to nerve stimulation. As always, his ideas and results found their way into an array of notebooks. Of these, the cahier de notes (cahier rouge) is in many ways his most revealing document. Compiled between 1850 and 1860, this unique and now famous notebook contains all manner of observations and ideas: and not just about his research. Reading it is essential to understanding his flow of thoughtTMB57,TMB59.
Perhaps spurred on by its relevance to diabetes and glucose metabolism, Bernard switched his research effort to the physiology of sympathetic nerves themselves. In 1727, Pourfour de Petit had described a dilatation of the pupil of the eye (mydriasis) in a man whose side of the neck had been severely damaged by a gunshot wound. Petit had shown the reverse phenomenon (miosis) when he cut the sympathetic nerve on one side of the neck. In 1851, Bernard repeated Petit's experiment and found that in addition to the pupillary constriction, the eyelid drooped (ptosis), and there was recession of the eye in the orbit (enophthalmos). He also observed that skin temperature on that side of the head had become higher70,73,TMB25,156, a phenomenon which he showed was due to an increased blood flow. It was much later in 1869, that the Swiss physician Johann Horner additionally observed reduced sweating in a woman with a tumour invading the sympathetic nerve in the neck. The complete clinical syndrome is widely called Horner's Syndrome. In France it is more correctly referred to as the Syndrome de Claude Bernard-Horner.
As part of his counterproof concept, Bernard electrically stimulated the sympathetic: the animal's pupil dilated, the eyelid retracted and skin temperature fell, accompanied by reduced blood flow to that side of the head75,76,80. The rare clinical syndrome which corresponds to this counterproof experiment in animals88 is referred to as the Pourfour de Petit Syndrome or the Claude Bernard Syndrome. Through his observations, Bernard proposed that the sympathetic nervous system controlled blood flow throughout the body, and was thus probably a principle regulator of body heat. This was the debut of his many researches on that subject. Although there was a flurry of challenges from other researchers concerning the originality of his findings and conclusions - especially from Budge and Waller - the Academy of Sciences eventually awarded him, for the fourth time its experimental physiology prize.
Bernard was becoming weary from his research, and from the repeated challenges to his results and conclusions, all of which he had to defend in public. His defence was mostly successful, but the conflicts were now taking their toll. His relationship with Fanny was also going from bad to worse, and he really needed a break. In 1853, the PLM (Paris-Lyon-Marseilles) railway line was finally completed. With this, his annual visit back to St Julien to see his mother and help supervise the grape harvest became quicker and less arduous. When he arrived that year, his mother told him that she had decided (partly to minimize inheritance tax) to hand over the family vineyards to him, and the cottage to his sister. Coming home would now become even more of a pleasure for him: yet Fanny would only rarely share these visits. She really did not get along with Madame Bernard.
Solutions and ideas...