Come for the Cholera, Stay for the Knowledge

In terms of diseases, ones that cause epidemics and pandemics often take the spotlight due to their ability to cause widespread panic. Cholera, from the Vibrio cholerae bacterium, is one of the oldest diseases that still causes the deaths of thousands every year. It does so by severely dehydrating the diseased person which quickly leads to acute renal and organ failure. Throughout history there have been countless epidemics and we are currently in the seventh pandemic that has gone global. Although we know the way the disease is transmitted (feces contamination) and the mechanism behind it, there is still much work to be done. New vaccinations to fend off cholera are crucial to preventing outbreaks that can happen at any time, especially in areas where war and natural disasters are common. It should be mentioned that while cholera is often thought of as a disease that only affects developing nations, developed nations are no less susceptible to this deadly disease given its ability to pass through undercooked seafood and contaminated food. One of the most interesting and important things to understand about cholera is that its treatment is relatively simple, intravenous replenishing of fluids and antibiotics; however, this isn’t an option for those who don’t have reliable access to hospitals. If left untreated, there is a 50-70% chance that the person will die within a day due to the dehydration that leads to organ failure. The reason why cholera is still an important area of research is because it can cause large outbreaks relatively quickly and they are often difficult to contain. In addition, new strains and mutations of cholera have been found that make them particularly aggressive. By developing better, more effective vaccinations and increasing education on ways to prevent its spread, we might put an end to this disease.

Three Fat Mice

Obesity continues to be a rising epidemic in many countries and has been linked to various metabolic disorders and metabolic dysregulation. Although there has been much research about the effects of metabolic disorders and dysregulation of cells in relation to obesity,  little is known about the metabolic changes that occur in obese adipose tissue. Given that there is a significant mass of adipose tissue in obese people, understanding the metabolic dysregulation that occurs is important. It has been known that adipose tissues produce and secrete various biologically active molecules that are collectively called adipocytokines/adipokines. The dysregulation of adipocytokines has been shown to have a key role in the pathophysiology of metabolic disorders and atherosclerosis in obesity, one of the leading causes of death around the world.  Nagao et al. uses both static and in vivo analyses to evaluate the metabolic dynamics in obese mice; they found that glutamate and other metabolites of the tricarboxylic acid cycle (TCA) was increased only in white adipose tissue (WAT) of obese mice but not in the liver or skeletal muscles((1)). In addition, in vivo analyses show that glucose derived metabolites were dynamically and specifically produced in obese WAT when compared to lean WAT. It was also found that high levels of one of the metabolites, glutamate, could potentially be associated with adipocyte dysfunction in obesity.

Previous work has shown that hypertrophied adipocytes from obese adipose tissue have increase lipid catabolism given the large excess of free fatty acids and glycerol present. Other work by Nagao et al. reports that adipose tissue secrets uric acid and overproduction of uric acid may contribute to enhanced purine metabolism. It has also be shown that obese subjects and animal models demonstrate a change in the amino acid profiles of their plasma. This means that the metabolic dysregulation of adipose tissue extends beyond what is already known about glucose and fatty acid metabolism and can lead to problems throughout the body . In addition, most research focuses on a single aspect of metabolic dysregulation when it is known that there is an arguably significant overlap between the various metabolic pathways in adipose tissue. Nagao et al. gives a novel report of the metabolic dynamics in in vivo tissues of obese mouse models.

The purpose of this study was to determine the static and dynamic and static metabolic changes in obese adipose tissue when compared to lean adipose tissue and show the association between metabolic changes and what is already known about hypertrophied adipocytes. Nagao et al. focused on the actions of adiponectin, a molecule important in glucose regulation and fatty acid catabolic and insulin, an highly important signaling molecule.

In order to get a baseline of the effect of obesity on adipose tissue metabolic, static metabolic analysis was conducted using LC/MS-MS and GC/MS on epididymal white adipose tissue (Epi WAT). The authors were looking for the relative levels of many metabolites involved in various pathways including glycolysis, pentose phosphate pathway (PPP), TCA cycle, and amino acids. The authors used three types of mice; ob/ob mice that were genetically mutated to eat excessively and were predisposed to obesity, C57 lean control mice, and diet induced obesity (DIO) mice that were fed a high fat/high sucrose diet. When comparing the levels of metabolites in ob/ob and C57 mice the authors found that there were increased levels of glycolytic and TCA metabolites in ob/ob mice. However, there were no significant changes in liver tissues or skeletal muscles indicating that it the metabolic changes occur specifically in adipose tissue.  Amino acid quantification was done using the same methods and found that there were higher concentrations of alanine, and glutamate in ob/ob Epi WAT mice. Next, the authors looked at the DIO mice in order to determine what happens to the metabolite levels when a subject becomes obese. They found that the differences were similar to the comparison between DIO and ob/ob mice. This suggests high levels of glutamate and other TCA associated metabolites in adipose tissues.

Figure 1. ob/ob mouse on left, normal mouse on right

Nagao proposed that either the rapid turnover of the TCA cycle due to excess fatty acids and glycerol present results in high levels of glutamate or that stopping the TCA cycle causes an accumulation of glutamate. In order to investigate and analyze adipose tissues in vivo, Nagao et al. used a combination of C13 isotopic glucose injections and a high-resolution metabolome analysis on ob/ob, C57, and DIO mice. This allowed them to take timed samples to find out where the glucose was, and what it was being broken down into. They determined that the rise in glutamate levels was due to a cataplerotic TCA flux in the adipose tissues in both ob/ob and DIO mice. Figure 2 shows that there was an excess of TCA metabolites across the board in addition to a higher plasma glucose levels in adipose tissue. Figure 3 shows that metabolic turnover in liver and skeletal muscle was not markedly different.  This suggests that the adipose tissues are using the excess glucose to create excess TCA metabolites which in turn may reduce the activity of the TCA cycle. In addition, the fact that increased metabolites were only in adipose tissue, it may reflect a need for rapid turnover and the expansion of adipose cells.

Figure 2. Metabolic turnover analysis of Epi WAT in DIO mice
Figure 3. Metabolic turnover analysis of the liver and skeletal muscle in DIO mice

In vivo analysis suggests the TCA cycle metabolites that are derived from glucose may induce a rise in glutamate in obese adipose tissue to to an abundance of glycogen. These high glutamate levels have negative downstream effects that are implicated in obesity and atherosclerosis. Given our many discussions on how we shouldn’t think of metabolic pathways as isolate events, this work nicely highlights the ways in which many metabolic pathways intersect and are affected by one another.

An interesting thing to look at would be whether or not the metabolic dysregulation can be reverted back to normal activity if the subject were to lose weight and whether or not the metabolic activity is forever changed.

 

  1. Nagao et al. January 2017. Increased Dynamics of Tricarboxylic Acid Cycle and Glutamate Synthesis in Obese Adipose Tissue In Vivo Metabolic Turnover Analysis. J Bio Chm. 292:4469-4483

Super Old, Still Relevant

I’ve chosen cholera as my topic because it is one of the epidemics that is still plaguing many of the world’s developing nations despite its well understood mode of transmission. Although it is relatively easy to treat with the advent of vaccines, it is important to note that epidemics are difficult to control. I’ve divided my findings into genetics, disease mechanism, public health, and research on treatments. The major themes of research in cholera have been discovering its bacterial nature, assessing its mode of transmission, and establishing healthcare protocols in case of epidemics. Recent focus has been on genetics of cholera because new strains have been appearing that are resistant to antibacterials. In addition, new vaccines that are both more effective, less costly, and more easily reproduced. In addition weighing the benefits and downsides of producing vaccines that protect for a long amount of time but require more upkeep rather than less coverage but single dose vaccines are a hot topic in cholera treatments. Although we know how the bacteria get into the system, the mechanisms that allow its survival are not all understood. New findings suggest that the bacteria are able to extract what they need from the host cell that allow them to turn on their toxicity. Overall, the trends are about containment of cholera epidemics, trying to spread public knowledge about its mode of transmission, and finding new ways to attack and prevent it.

Blame the Biota

The role of gut microbiota in relation to the human body has grown in importance due to its implication with many diseases that are prevalent in the western world such as diabetes, inflammatory bowl disease, and obesity. Although obesity is on the rise, there are many strategies available for people to lose weight, however, one of the main problems is maintaining the weight loss; instead, almost 80% of individuals find themselves cycling between weight loss and weight gain. Gut microbiota has become a new target for investigating the causes of obesity, however most experiments that have been done quantify and identify the types of microbiota in the human body while the underlying metabolic pathways that drive post-diet weight gain remain unknown. Thaiss et al. has found that there were intestinal microbiome signatures that persisted even after successful dieting in obese mice that contributed to faster weight-regain and metabolic changes after re-exposure to obesity-promoting conditions.

Changes in metabolic homeostasis and the subsequent increase in risk for obesity have more recently been linked to changes in the gut microbiome. By using mouse models of weight loss and regain to mimic recurring obesity, the authors try to show that although obese mice can lose the weight, there is a change in the gut microbiota that persists over time and increases post-weight loss weight gain. In addition, the find that metabolites produced by the gut microbiota may be viable targets for treatment to prevent regain of weight.

The study used mouse models to study weight loss and recurring obesity to try and understand the mechanisms involved in repeated cycles of weight loss and gain. Mice were cycled on high-fat diets (HFD) and normal chow (NC), which promoted a cycle of weight loss and weight gain that would resemble a typical human’s weight loss and gain. Control mice were fed either all NC or HFD. They observed that the weight-loss, weight-gain cycle led mice to be susceptible to increased weight gain the second time, even if they returned to the original weight. In addition to increased weight gain, they found that there were metabolic difficulties such as enhanced glucose intolerance, and elevated levels of leptin and low-density lipoprotein (LDL) but not high-density lipoprotein (HDL). All three of which have been correlated to various inflammatory diseases. Second and third cycles of HFD-induced obesity further increased the weight regain. The authors suggest that repeated cycles of weight loss and gain may lead to accelerated weight regain, in addition to other metabolic damage.

Enhanced recurrent weight gain after treatment of obesity.

Figure 1. Shows the weight gain over time of control HFD (blue) and NC (black) mice along with mice subjected to weight cycling (red) and mice given a HFD once (green).

From these findings, the authors proposed that it was the initial obese state in the mice that caused the abnormalities that persisted, predisposing them to the metabolic disorders after the cycling occurred. They found that this was not the case but that it was the composition of the gut microbiota that changed after the first obesity state that never reverted back to its pre-obese state. Instead, it was in between the obese and pre-obese states. This is significant because although shifts in diet have the ability to change microbiota within days of beginning, the findings show that there are microbial changes that have long-lasting effects.

To further understand the long-term effects of obesity on gut microbiota, the authors performed a genomic sequencing on the gut microbiota and identified 733 bacterial genes whose abundance was altered by the HFD and did not return to the NC levels even after dieting. They also found that the microbiota composition only reverted back to the pre-obese state after 21 weeks, indicating that weight loss maintenance must be prolonged enough that the gut microbiota can revert back and even then, it is does not ensure the prevention of weight regain. Fecal transplants from previously obese and control mice to germ-free mice were also performed and mice were fed either HFD or NC diets. They found that NC fed mice who had not gone under the weight gain/loss cycle were similar to normal NC fed mice which the authors indicate means that the post-obese microbiome itself does not have obesogenic properties. However, when mice were fed HFD, there was enhanced weight gain and glucose intolerance even if the mice were not previous obese. This suggests that it is likely that the post-obese microbiota increases the likelihood of metabolic complications upon re-exposure to obesity-causing conditions.

The authors also suggested that the post-obese microbiome configuration might give a possible prediction as to how much weight would be regained upon introducing the mice to a HFD again. By using a DNA-based prediction, they found that it was the composition of the gut microbiota as a whole that may change the post-obese microbiome, rather than specific species. This means that targeting the entire biome may be a more effective treatment to prevent weight-regain than single-species targeting.

Although reversion of the post-obese microbiome took longer than expected, the authors found that modulating the microbiome during the post weight-loss period could prevent some of the secondary weight gain the associated metabolic complications. The authors tested this by subjecting weight-cycling mice to daily fecal transfers from mice that had either never gone under weigh-cycling or mice that were in a post-diet phase. Those that received the transplant from non-cycling found that there was less regain of weight once subjected to an obesity-inducing environment.

Given the increasing rates of obesity in the western world, finding and preventing the weight regain that is extremely common in obese people is one of the keys to stopping the cycle of weight gain and regain. Although the underlying metabolic pathways are not yet known exactly, the authors found that alterations in gut microbiota have lasting effects on subjects regardless of whether they return to their original weight or not. This provides for a new target that can be highly specified towards an individual which may promote decreased weight regain.

Reference

Thaiss, Christopher A., Itav, Shlomik., Rothschild, Daphna., et al. Persistent microbiome alterations modulate the rate of post-dieting weight regain. Nature. February 2016, 544-551.

 

Rodents, and Cows, and Shrimp, Oh My

People’s fascination with diseases goes back to the ancient times and has not subsided since. Thankfully, we’ve come a long way from diagnosing people with unbalanced humors and treatments consisting of ingesting various animal parts and light mutilation. As someone who is also interested in history, I thought it may be interesting to discuss diseases that have drastically altered the course of history in ways that people may not realize.

  1. Bubonic plague-carried around by flea infested rodents, the plague spread throughout much of Asia around 1336. Making its way along the Silk Road, it spread to India and surrounding countries by 1346. By October of 1437, the plague had reached Europe. Current research has indicated that between 40-50% of Europe, and 75-80% of Spain perished. This caused massive social and economic change that would shape the future of Europe. A weakened feudal system allowed peasants more control and social movement that they would not quickly forget when their landowners tried to regain control. A historical nightmare to be sure yet new cases arise every year.
  2. Smallpox-one of the few diseases that scientists have managed to eradicate, smallpox was once rampant among much of the world. Thought to have originated somewhere in Egypt, it made its way to India, China, Japan, and finally Europe where it would cause the death of millions. Brought to America by colonizers, it decimated native populations and the shaped the path for the 13 colonies.
  3. Cholera-likely to have originated in the India subcontinent and spread throughout the world, it was the cause of seven pandemics and still persists today as an epidemic in many developing nations. It became the first reportable disease in America due to its prevalence in the population. Although seemingly easy to prevent with the advent of proper waste disposal and clean water, it remains a serious problem in places without these amenities. Due to its prevalence, many new scientific advances have come from finding treatments and vaccines.

Achy Breaky Heart (Blog Spotlight Practice)

Angiogenesis is an important area of research because the proliferation of blood vessels is crucial to cardiovascular health in the body. As the authors state, angiogenesis relies on the proliferation and migration of endothelial cells which are important to the growth of the vascular network. Although regulatory molecules have been identified, the importance of metabolism has not. Fatty acid oxidation (FAO) has been linked to ATP production but its importance to angiogenesis has not been defined. CPT1 was chosen as the molecule of focus in FAO because it is the rate limiting step of FAO and is likely to have the largest impact when modified. The way the importance CPT1A, the most abundant isoform of CPT1, and CPT1C, a less abundant isoform was detected, was by silencing it. They found that there was a decreased vessel sprout length and number due to decreased endothelial cells (EC). The next step was to study the effects in vivo, using mice that lacked CPT1A which was found to show the same results as the in vitro tests indicating that impairment of angiogenesis was due to EC proliferation defects.

Investigating the mechanism by with FAO regulates EC proliferation was another point of interest for the authors because it helps define its importance to angiogenesis. They highlight several different approaches, each with varying levels of success but provided new information as to the role of FAO in angiogenesis. It was determined that although FAO is involved in ATP production, decreased levels of ATP or energy stress that were causing a lack of sprouting. The authors then suggested a novel role for FAO in EC proliferation given that it is used for de novo synthesis of nucleotides. They found that although impaired ability to synthesize proteins, it did not prevent prevent CPT1A silencing. The most novel thing the group found was that FAO was involved in the synthesis of DNTp in angiogenesis which was unexpected given that most of the DNTp in the body was thought to come from glycolysis and glucose.

The novelty of this article is in the authors’ ability to place the importance of FAO in angiogenesis which had not been done before. In addition, it affected proliferation, not migration, of EC proliferation which is why it can be seen that there is a marked decrease in proliferation, however placement remains somewhat the same. They also discovered a previously unknown role of fatty-acid-derived carbons in de novo deoxyribonucleotide synthesis which provides for carbons for the production of aspartate and glutamate. Although not replaceable for DNA synthesis, they were for protein and RNA synthesis indicated that knock-out CPT1A ECs produced their own rNTPs. These are important discoveries from a human perspective because the proliferation of blood vessels is very important in long-term maintenance of the body in people with cardiovascular problems. It also helps place FAO into a new context and a possible target for drugs.

Although we’re only learning the basics of lipid metabolism, it is important to remember that we only know a fraction of what there is still to discover. There is a lot we still don’t know about the pathways we rely on every day and this is just one tiny piece of a larger puzzle.

Why do Biochemistry?

I think to study biochemistry means to take biological systems and processes and break them down to the molecular scale to understand how and why they happen. It gives biological relevance to many of the molecules and chemical reactions we know exist and can help develop new ones that have real world application.

I chose to study biochemistry because for me, it’s the best of both worlds. I like being able to give chemical reactions relevance in a biological system. I think biochemistry is necessary to study in the sense that it is interdisciplinary. Learning how to build connections and share information with other groups within a given field is important regardless of the field of study.

As a (hopefully) future surgeon, I might not have to explain metabolic pathways, but learning how to communicate science with non-science people is very important and a skill that I think is lacking in the scientific and medical community.