Weaponized insulin: how the venom of a sea-dwelling predator could treat diabetes

Written by: Padmini S. Pillai

Original Article: Safavi-Hemami et al. PNAS 2015
The Gist of it:
The history of medicine is filled with tales of converting poisons into potions. Researchers have now discovered that the venomous cocktail released by deadly sea-dwelling snails could potentially lead to a fast-acting diabetes drug.
Cone snails are native to coral reefs found in the warm waters of the Indo-Pacific. These predators hide in tiny crevices to stalk schools of small fish and paralyze them with neurotoxin-laden harpoons. A subset of cone snails called net-hunters trap their meals by first secreting a venom called nirvana cabal to disorient potential victims. Next, they extend a false mouth to engulf their now delirious prey. An injection of paralytic toxins then serves as the final blow. Scientists knew nirvana cabal was responsible for the rapid deceleration and dazed stupor of these fish, but what ingredient in this noxious cocktail was causing fish to fall prey to the snails’ deadly embrace?
A study led by Dr. Baldomero Olivera found that insulin is the major component of nirvana cabal. Produced by pancreatic cells in humans, insulin lowers blood sugar levels in the body. In cone snails, the nervous system makes insulin to control sugar metabolism. In contrast, the weaponized insulin net-hunters use on prey is made in the venom gland. When fish take in the venom through their gills, it enters their bloodstream, causing hypoglycemia, or low blood sugar, rapidly slowing their movement. This allows the snail to seize its prey.
Olivera’s team analyzed DNA from the venom glands of two cone snail species and found a sequence that looked like that of the insulin gene of fish, not the insulin gene of the predator itself. They injected the molecule encoded by that DNA, called Con-Ins G1, into zebrafish and observed a significant drop in blood sugar. Similarly, when added to a fish tank, Con-Ins G1 caused fish to slow down and swim less. Likewise, worm-eating cone snails produce worm-specific insulin in their venom glands instead of the fish-specific insulin. This suggests that the type of weaponized insulin released by the net-hunting cone snail depends on its diet.
The conclusions of this study are not entirely morbid. Studies into the structure of cone snail venom insulin and a comparison with human insulin could possibly lead to better diabetes medications with fewer side effects. It turns out that this venomous, picky eater’s need to slow down its prey could eventually pave the way for a new generation of life-saving drugs.
24_doodle

Cone snail venom contains a fast-acting insulin to slow down prey.

The Nitty Gritty:
The researchers performed next-generation sequencing on the transcriptome of the Conus geopraphus venom gland and identified a transcript, Con-Ins G1, with high sequence similarity to fish insulin. Con-Ins G1 was most highly expressed and almost exclusively found in the distal region of the venom gland, closest to the injection apparatus. C. tulipa, another cone snail that uses the net-hunting strategy to capture prey, expressed similar insulin transcripts. Analysis of mollusc- and worm-hunting cone snails revealed that the type of insulin expressed in the venom gland correlated with its diet. Sequencing of the Con-Ins G1 protein by mass spectrometry revealed novel post-translational modifications. Chemical synthesis of the molecule was performed using selenocysteines. To study the effects of synthetic Con-Ins G1 (sCon-Ins G1), streptozotocin was injected intraperitoneally into zebrafish to induce hyperglycemia. Administration of sCon-Ins G1significantly lowered blood glucose, which was similar to the effect of human insulin. When sCon-Ins G1 was applied to the water column of a fish tank containing zebrafish larvae, researchers observed reduced locomotor activity, as measured by time spent swimming and movement frequency.
Original Research Article: Safavi-Hemami, H., et al. “Specialized insulin is used for chemical warfare by fish-hunting cone snails.” Proceedings of the National Academy of Sciences of the United States of America 201423857 (2015). doi:10.1073/pnas.1423857112

Insect-powered tweezers and other things you didn’t know you needed

Written By: Ritu Raman

Original Article: Akiyama et al. Lab on a Chip 2013

The Gist of It

Biological materials (such as our bodies) have many desirable qualities that engineers have been unable to copy in synthetic (man-made) materials. For example, when you work out, you get stronger. When you fall and hurt yourself, you heal. But what if we could make machines that adapted to their surroundings in the same way? This is exactly what Akiyama and colleagues investigated. Because muscle is more flexible, self-repairing, and energy efficient than synthetic alternatives, they decided to use muscle dissected from an insect to make a moving machine. Muscle contracts inside the body when it receives a trigger from the nervous system. The authors of this study replicated this trigger by stimulating the dissected muscle with short pulses of electricity. They then wrapped the muscle around a pair of tweezers and showed that they could make the tweezers close every time the muscle contracted in response to the electrical pulse. The insect-powered tweezers were able to work in air and at room temperature, demonstrating that a muscle-powered machine could function in a real-world environment. Now maybe you have no use for insect-powered tweezers (and I don’t blame you; who does?), but that doesn’t make this study any less impactful! It serves as a proof-of-concept that biological tissue can be used to power a synthetic machine. This sets the stage for a whole host of moving, walking, gripping, swimming machines that could be useful in our daily lives. And, like other biological systems, these machines may be able to do things like exercise to get stronger and heal when they’re damaged. Personally, I can’t wait for the day that becomes a reality!
23_doodle

Electrically stimulated contraction of insect muscle controls the opening and closing of a tweezer.

The Nitty Gritty
Akiyama et al. dissected insect dorsal vessels (DV) from final stage larvae of Thysanoplusia intermixta (i.e. inchworms). The muscle tissue was preserved in cell culture medium supplemented with fetal bovine serum, penicillin, and streptomycin. Microtweezers fabricated via photolithography were sterilized, and the DV tissue was placed around the tweezers and embedded in notches to ensure stable tethering. Light microscopy was used to track the tweezers’ deformation in response to electrically stimulated contraction of the muscle. Knowing the dimensions and material properties of the microtweezers, the authors were able to calculate the force produced by the muscle that resulted in the measured deformation. Calculations were verified via finite element analysis simulations. The authors used a paraffin coating on top of the culture medium reservoir to prevent medium evaporation, ensuring stable operation of the devices in air for up to five days.
Original Research Article: Akiyama, et al. “Atmospheric-operable bioactuator powered by insect muscle packaged with medium.” Lab on a Chip 13.24 (2013): 4870-4880.

Bad fats in the brain: Loss of fat-modifying enzyme is the cause of a childhood brain disease

Written By: Kathleen Cunningham

Original Article: Pant et al. Journal of Clinical Investigation 2019
The Gist of It:
In your brain and spinal cord, neurons have a fatty covering that protects them, similar to the plastic coating around your cell phone charge cord. This covering is made of myelin. Myelin is made by cells in your brain called oligodendrocytes and it has many roles, including helping to maintain the proper electrical signals in the neurons in your brain. However, in patients with leukodystrophies (LD), a group of rare disorders that affect the nervous system, myelin is not made or broken down abnormally Patients with LD develop the disorder in infancy and can have severe symptoms, such as poor motor function, involuntary muscle contractions, seizures, and death. Using genetic sequencing, Pant and colleagues found a change in the gene DEGS1 that caused LD in 19 patients from 13 different families. The DEGS1 gene makes a protein that can change specific types of fats in your cells from a saturated form to a desaturated form, an important step in making myelin. The authors measured the amount of the saturated lipid and found that it is increased in patient cells. To test whether changes in DEGS1 may really cause LD, the authors created a zebrafish model by decreasing the amount of DEGS1. Zebrafish larvae with less DEGS1 had unusual shape during development, as well as abnormal movement and fewer oligodendrocytes making myelin. These changes are consistent with what is observed in patients. Excitingly, the authors tested a drug (FTY720) that is known to affect fat saturation levels and block the accumulation of this saturated fat that was previously approved by the Food and Drug Administration (FDA) as a treatment for multiple sclerosis. This drug was able to reduce the fat accumulation and improve the number of oligodendrocytes and the movements of the drug-treated zebrafish. The success of this drug in zebrafish opens the door to the potential of finding a treatment for this rare but deadly childhood disease.
22_doodle

Zebrafish were used to test whether DEGS1 mutations identified in leukodystrophy patients can cause features similar to disease in humans and to test possible treatments

The Nitty Gritty:
Pant et al. first conducted whole exome sequencing in a series of patients with undiagnosed leukodystrophies. They found a frameshift mutation in the delta-4 desaturase, sphingolipid 1 (DEGS1) gene in the proband patient which was not present in multiple genomic databases in healthy individuals. They next used GeneMatcher and communications with the Reference Center for Leukodystrophies to find an additional 18 patients with candidate causative mutations in the DEGS1 gene. All of the patients were homozygous or compound heterozygotes for DEGS1 mutations.  The researchers also categorized the specific common features of these LD patients and found that 79% of patients shared poor psychomotor development, dystonia, and spasticity. The patients also presented with eye movement defects and seizures. All patients were confirmed by MRI to have white matter lesions and hypomyelinating LD, with a median patient age of 3.6 years. Interestingly, patients with the mutations predicted in silico to confer the greatest loss of function had the most severe disease, all dying before the age of 7. Using fibroblasts collected from patients, the researchers next observed possible consequences of the loss of DEGS1 function; they showed a dramatic accumulation of the DEGS1 lipid substrate Dihydroceramide (DhCer). As DhCer accumulation can cause a buildup of excess reactive oxygen species (ROS), Pant et. al next measured ROS levels and showed that patients’ fibroblasts have increased levels. Further, when treated with exogenous DhCer, control fibroblasts showed an increase in ROS levels, while patient fibroblast ROS levels were saturated. To further confirm that DEGS1 was important in key myelinating cell types in vivo, the researchers turned to zebrafish. Zebrafish are one of the smallest and easiest laboratory models that preserve key components of myelinated axons. Indeed, zebrafish DEGS1 was expressed in myelinating oligodendrocytes in zebrafish larvae. The researchers designed a morpholino to knock down DEGS1 in zebrafish. This resulted in accumulation of DhCer, loss of myelination and myelinating cells, and locomotor disability. Since the enzymatic pathway for DEGS1 is known, the authors examined an FDA-approved drug known to target the ceramide synthesis pathway, FTY720. FTY720 inhibits the enzyme that synthesizes DhCer prior to the DEGS1 step in the pathway. Excitingly, the drug rescued DhCer levels as well as myelination and motor function in zebrafish; it also rescued DhCer and ROS levels in patient fibroblasts. Therefore, the authors have identified an exciting potential treatment for a rare but devastating childhood disease.

 

Original Research Article: Devesh C. Pant, et al. “Loss of Sphingolipid Desaturase DEGS1 causes hypomyelinating leukodystrophy” The Journal of Clinical Investigation 10.1172 (2019): e123959.

Bacteria from fermented soybean paste decrease obesity symptoms

Written by: Ken Estrellas

Original Article: Kim et al, PLoS ONE 2018
The Gist of It:
Lots of recent attention has been focused on the “gut microbiome,” the collection of microorganisms that live in our intestines. These bugs have been linked to everything from digestion and the immune system to communication with the brain. While probiotic foods (or foods that contain live microorganisms that are good for you) such as yogurt, kombucha, and sauerkraut are often associated with changing and improving the gut microbiome, the bacteria present in these foods may also have positive effects on our overall health without even directly affecting the gut microbiome! In a recent study published in PLoS ONE, Kim and colleagues studied the way certain Bacillus bacteria can affect metabolism, inflammation, and obesity. First, mice were given a high-fat diet to cause obesity. Then, they were either left untreated or treated with one of two bacterial cocktails: a mixture of five Bacillus strains from fermented soybean paste or a mixture of other types of bacteria. Obese mice treated with the Bacillus mixture gained weight less quickly, had lower levels of blood glucose, and had significantly reduced fat accumulation in some areas than the other mice. This was potentially caused by decreased expression of genes associated with fat storage. Bacillus-treated mice also showed lower expression of genes associated with inflammation in the liver, epididymal fat tissue, and skeletal muscle. Gene expression and protein levels of occludin, a key protein which helps prevent leakage in several tissues, were both higher in Bacillus-treated obese mice compared to other obese mice. Interestingly, when the gut bacterial populations of the Bacillus-treated mice were compared with populations from other mice, there were no differences in the amounts of different gut bacteria present. This shows that the health improvements in the Bacillus-treated mice were not due to changes in the microbiome itself but due to other effects on the body. Overall, this study helps shed light on the ways certain organisms like Bacillus can affect our overall health and wellness and explain why certain people might benefit from an extra helping of soy!
21_doodle.jpg

Bacteria found in soybeans can potentially have beneficial effects on your health.

The Nitty Gritty:
In order to elucidate the mechanisms by which certain Bacillus strains may be protective against metabolic disorders, Kim et al. tested the effects of various bacterial regimens on wild-type C57BL/6 mice induced to obesity with a high-fat diet (HFD). Non-HFD and HFD-only controls were compared to HFD + “VSL#3” (4 Lactobacillus strains + 3 Bifidobacteria strains + 1 Streptococcus thermophilus strain; HFD + V) and HFD + “Bacillus” (5 different strains of Bacillus isolated from Doenjang, a long-term fermented soybean paste; HFD + B). HFD + B mice demonstrated significantly decreased levels of body weight accumulation and blood glucose over 13 weeks of study. Decreased subcutaneous and mesenteric adipose tissue were observed in HFD + B mice, potentially due in part to locally decreased levels of lipid uptake genes (Cd36, Ldlr) and lipogenic genes (Srebp1c, Acc, Fas, Scd1) in both areas. Phospho-Akt S473 levels in skeletal muscle and epididymal adipose tissue (EAT) were significantly increased in HFD + B mice compared to other treatment groups, a potential indicator of enhanced insulin functionality. Relative mRNA levels associated with the inflammatory markers TNFα, IFNγ, MCP-1, and IL-1b were elevated in obese mouse liver, EAT, and skeletal muscle vs the negative control. These levels were all decreased in HFD + B mice vs HFD alone, and there was a significant decrease in IL-6 observed in EAT and decreases in IL-12 observed in liver and EAT. Adiponectin expression was elevated in both the serum and EAT of HFD + B mice, and increased intestinal barrier function was demonstrated via decreased endogenous serum LPS and increased ileal occludin expression; these data are potentially suggestive of increased insulin sensitivity. Improved hepatic steatosis and increased fatty acid oxidation (as demonstrated by Acox1 and Cpt1 expression and PGC1α protein levels) were also observed in HFD + B mice. Treatment of HFD mice with each of the five Bacillus strains individually revealed different effects on body weight, glucose tolerance, and hepatic triglyceride levels, suggesting a combinatorial effect with these strains. Finally, fecal sampling of the gut microbiota revealed some alterations in Firmicutes / Bacteroidetes and Bacteroides / Prevotella ratios in HFD + B mice vs other treatment groups; however, these differences were not statistically significant and suggest that the Bacillus mixture confers its benefits through mechanisms other than direct alterations of gut microbiome composition. This study represents an in-depth characterization of the biological mechanisms that underlie potentially beneficial effects of food-derived Bacillus strains and may inform future studies of how certain foods and microbiota affect metabolic disorders.
Original Research Article: Kim, Bobae, et al. “Protective effects of Bacillus probiotics against high-fat diet-induced metabolic disorders in mice.” PLoS ONE 13(12): e0210120.