Source: University of Utah submitted to NRP
ISARIA SINCLAIRII & CORDYCEPS EXTRACTS FOR THE TREATMENT OF INSULIN RESISTANCE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1018623
Grant No.
2019-67018-29250
Cumulative Award Amt.
$200,000.00
Proposal No.
2018-07907
Multistate No.
(N/A)
Project Start Date
Mar 1, 2019
Project End Date
Feb 28, 2021
Grant Year
2019
Program Code
[A1341]- Food Safety, Nutrition, and Health: Function and Efficacy of Nutrients
Recipient Organization
University of Utah
201 S President Circle RM 408
Salt Lake City,UT 84112-9023
Performing Department
Nutrition and Integrative Phys
Non Technical Summary
Ceramides (a lipid) generated by inflammatory cytokines have important regulatory roles in development of insulin resistance. Ceramides, which accrues under obesity, may be the most toxic metabolites that accumulates in peripheral tissues. Blocking ceramide production in rodents using ceramide synthesis inhibitor myriocin improves features of diabetes and its cardiovascular complications.The proposed Seed Grant, which aligns withFunction and Efficacy of NutrientsProgram Area Priority (A1341), aims to investigate if the extacts ofIsaria sinclairii(IS)andCordycepsfungi,containing bioactive componentmyriocin,prevent the developments of pathologies associated with chronic inflammation.Myriocin was originally isolated from an extract of the fruiting bodies of the fungusIS. ThoughISand relatedCordycepsspecies have been long-used in traditional Chinese medicine as a treatment for multiple conditions, it has not been approved for clinical trials in people. When given to mice or rats,ISorCordycepsextracts improve insulin sensitivity and glucose tolerance and ameliorate hypertension. WehypothesizethatISorCordycepsextracts will prove to be an effective nutrition capable of reducing tissue ceramide levels and warding off features of metabolic disease.We will test this hypothesis through the followingAims:1) Evaluate the myriocin content of a variety ofIS/Cordycepsspecies;2) Assess the effects ofIS/Cordycepsextracts on glucose homeostasis and nutrient metabolism in mice pre-disposed to diabetes; and3) Probe the biochemical mechanism underlyingCordycepsandISactions. Study findings may identify an immediately available nutritional treatment to be used to improve human health and prevent inflamation.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
70114701010100%
Knowledge Area
701 - Nutrient Composition of Food;

Subject Of Investigation
1470 - Mushrooms and other edible fungi;

Field Of Science
1010 - Nutrition and metabolism;
Goals / Objectives
Aim 1: Evaluate the myriocin content of a variety ofIsaria Sinclairii/Cordycepsspecies.Aim 2: Assess the tissue-specific effects ofCordycepsand Isaria Sinclairiiextracts on glucose homeostasis and nutrient metabolism in mice pre-disposed to diabetes.Aim 3: Probe the biochemical mechanism underlyingCordycepsandIsaria Sinclairiiactions.
Project Methods
Administration of Fungal Extracts:PowderedIsaria sinclairiiorCordycepsobtained will be mixed into chow and high fat diets as previously described and C57Bl6/J animals fed over a course of 12 weeks.Metabolic Phenotyping:Our laboratory has established comprehensive phenotyping protocols for assessing metabolic changes in mice challenged with high fat diets (Diet D12452, Research Diets, Inc., New Brunswick, NJ; 60% kCal from fat).The dosing regimen chosen will attempt to mimic that recommended for human consumption on a per/kg basis.Indirect Calorimetry and Body Composition:We will assess energy expenditure and thermogenesis twice during the study protocol using non-invasive mouse metabolic chambers (Columbus Instruments) that directly measure O2consumption, CO2production, food intake, water intake, and activity. Body composition will be assessed by MRI as above.Glucose Homeostasis:At 10- and 12-weeksglucose utilization in the fasting and fed state will be assessed both before and after insulin/glucose challenge. Where warranted, insulin sensitivity will be measured by euglycemic clamp using3H-glucose and14C-deoxyglucose for glucose kinetics, tissue-specific glucose uptake, and hepatic glucose production.Serum and Tissue Analysis:Glucose levels will be measured using a Beckman glucose analyzer. Cytokine (tumor necrosis factor-alpha, interleukin-6, and interleukin-1) levels will be quantified using the Multiplex system from Luminex according to manufacturer instructions. Levels of free fatty acids, organic acids, amino acids, and sphingolipids will be assessed by either GC or LC-mass spectroscopy by the University of Utah Metabolomics Core. Other blood chemistry determinations (e.g., serum cholesterol, alanine aminotranseferase, alkaline phosphatase, lactate, C-reactive peptide, etc.) will be assessed using a Clinical Vitros 250 Chemistry Analyzer according to the manufacturer's instructions. Tissue histology of adipose and liver will be as we previously described.Evaluating Effects on Insulin Signaling:Following the end of the hyperinsulinemic-euglycemic clamp, animals will be sacrificed with a lethal dose of sodium pentobarbitol and insulin-sensitive tissues (e.g., liver, heart, white adipose tissue, soleus, epitrochlearis, and red and white gastrocnemius muscles) will be excised. Insulin signaling will be assessed by Western blotting with phospho-specific (vs. wild-type control) antibodies recognizing specific insulin signaling intermediates (e.g. phosphorylated Akt/PKB, GSK3b, mTOR, p70 S6K, etc.). Moreover, we will conduct real time-PCR to measure transcripts of genes involved in anabolic or catabolic processes.Assessing Mitochondrial Function:Using the methods, described previously we will assess respiratory rates (basal, uncoupled, maximal, etc.), electron transport chain complex activity, citrate synthase activity, mitochondrial membrane potential, ATP generation, and reactive oxygen species generation. Metabolomic profiling will be used to obtain acylcarnitine profiles and to determine the levels of organic acids from the citric acid cycle, as well as pyruvate, and lactate.Characterization ofCordycepsandIsaria sinclairiiMyriocin Content:Myriocin content in theCordycepsandISwill be analyzed at the Metabolomics Core at the University of Utah. Moreover, the Core will also be instrumental in determining the efficacy of these medicinal preparations by analyzing the sphingolipids and ceramide content in serums and tissues post completion ofin vivostudy.Statistical Considerations:For clamp studies, cohorts of 6-8 are sufficient to detect a difference of 20% with ana-error level of 5% and a b-error level of 50%.

Progress 03/01/19 to 02/28/21

Outputs
Target Audience:The target audience comprises scientists and medical doctors with the sole focus on understanding and finding better treatment options for metabolic diseases such as diabetes and heart diseases. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The undergraduate, graduate students, and post-doctoral fellows received training in various techniques related to this project. This includes but not limited to metabolite extraction for mass-spectrometry, measuring blood glucose, conducting glucose and insulin tolerance tests, assessing body composition using NMR, analyzing metabolic chamber (Comprehensive Laboratory Animal Monitoring System) data, and analyzing the data using GraphPad Prism software. How have the results been disseminated to communities of interest?We had planned to present these findings at the Keystone Conference on Integrating Metabolism and Immunity, 2020 and Annual Nutrition Science Meeting, 2020 but both of these events were canceled due to the pandemic. We now plan to present these data in the next coming months at the virtual version of the above-mentioned conferences. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? During the term of the funding, we have successfully completed Aim 1. We acquired 8 different commercial preparations of Cordyceps either grown locally in the United States or those grown in various Asian countries such as in China, Taiwan, and Singapore and marketed either as a powder in a capsule, bottled liquid extract, or raw material that needed to be further processed for human consumption. We used 5 mg of these commercial preparations and applied mass spectroscopy to quantify the amount of myriocin.11 Interestingly, we identified two commercially available Cordyceps sample 1 and 8 that contained the highest amount of myriocin. We also found that one of the samples, sample 6 contained no myriocin, and could be used as a perfect negative control for our studies. Since sample 1 is locally grown in the United States, exhibits a comparable amount of myriocin to that of sample 8 obtained from Singapore, and is relatively cheaper and available as capsules unlike sample 8 that requires further processing, we decided to test the efficacy of sample 1 to improve metabolic function in diet-induced obese (DIO) mice as listed in Aim 2. With the achievement of several significant metabolic improvements like lowering body weight, increasing energy expenditure from Aim 2, we investigated the mechanism of Cordyceps to improve insulin signaling and improve adipocyte metabolism in Aim 3. These studies indicated that sample 1 improved hepatic insulin signaling and elicited a broad spectrum of metabolic improvements in adipose tissue such as reduced adipocyte size and browning/beiging of adipocytes. To test the efficacy of identified Cordyceps sample 1 in improving glucose homeostasis and nutrient metabolism, powdered sample 1 at two doses low (19.7mg/kg) and high (39.5mg/kg) were mixed into the high-fat diet (Diet D12452, Research Diets, Inc., New Brunswick, NJ; 60% kCal from fat) as previously described.3 These doses were calculated on the basis of the pharmacological dose of sample 1 that was recommended by the manufacturer for human consumption. As controls, we used a high-fat diet that either contained an equivalent amount of starch derived from rice or Cordyceps sample 6 (39.5mg/kg) that did not contain any traces of myriocin. Feeding 4-weeks old C57Bl6/J mice with a high-fat diet supplemented with Cordyceps extracts from sample 1 for 12-weeks reduced ceramides and other complex sphingolipids in several tissues such as liver and intestine as well as in the serum in a dose-dependent manner. These data are consistent with our hypothesis that Cordyceps extracts that contain traces of myriocin inhibit ceramide synthesis. The mice that were fed Cordyceps extracts from sample 1 exhibited robust phenotypes. They were protected from obesity and exhibited reduced total fat mass. Significant increases in lean mass were only observed in the high dose group, although there was a trend in the low dose group. The animals consuming a diet supplemented with Cordyceps extracts from sample 1 exhibited increased energy expenditure compared to animals fed with starch supplemented diet. In comparison, animals fed with Cordyceps extracts from sample 6 that did not contain any traces of myriocin exhibited worsening of all the above-stated phenotypes. Mice in sample 1 supplementation groups displayed reduced circulating blood glucose levels after 9 weeks of dietary regimen and modest improvements in glucose and insulin tolerance after 4 weeks and resolved hepatic steatosis in the end or 12 weeks of the treatment regimen. In contrast, animals fed with Cordyceps extracts from sample 6 exhibited elevated blood glucose throughout the treatment. These improvements observed following sample 1 treatment were due to enhanced activation of Akt/PKB in the liver, the anabolic enzyme that is activated in response to insulin and inhibits gluconeogenesis in the liver, and increases glucose uptake in adipose tissue and muscle. Histological analysis of the adipose tissue revealed that animals fed Cordyceps extracts from sample 1 had reduced (~50%) cross-sectional area of the adipocytes from epididymal, subcutaneous white adipose tissue, and brown adipose tissue. Since we previously have found that pharmacological treatment of myriocin in obese mice promotes browning/beiging of subcutaneous fat and increases the thermogenic activity of the brown fat, we reasoned these metabolic transformations could account for the improvements ensuing the treatment with Cordyceps extracts from sample 1. Indeed, Cordyceps extracts from sample 1elevated the expression of uncoupled protein-1 (UCP-1) in the subcutaneous and brown fat depots. Given the previous finding of the gut toxicity of myriocin administration, we determined the pathology of the small intestine and colon. From the H&E staining section, there was no detectable inflammation or other pathologic symptoms been found in both the small intestine and colon from the low dose sample 1 group. There was minor to mild inflammation in the colon from the high dose group. These findings pave the way for the clinical trial of IS/Cordyceps products as well as oral consumption of low-dose myriocin as a therapy for insulin resistance and other metabolic diseases.

Publications

  • Type: Journal Articles Status: Under Review Year Published: 2021 Citation: Ying L*, Chaurasia B*#, Kaddai V, Wilkerson JL, Maschek JA, Cox J, Wei P, Bensard C, Meikle JP, Clevers H, Shayman AJ, Hirabayashi Y, Holland WL, Rutter J, Summers SA (2021). Serine Palmitoyltransferase Controls Stemness of Intestinal Progenitors (Biorixv, the preprint server biology) *co-first and # Co-corresponding author.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Talbot CL, Chaurasia B, (2021). Ceramides: A Key Lipotoxic Modulator of Adipocyte Metabolism. Frontiers in Physiology (Invited review). # Corresponding author
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Chaurasia B and Summers SA (2021). Ceramides in Metabolism: Key lipotoxic players. Annual Review of Physiology. February; (10): 83:303-330. # Shared Corresponding author
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Chaurasia B#, Ying, L, Talbot CL, Maschek JA, Cox J, Schuchman HE, Hirabayashi Y, Holland LW, Summers SA (2021). Ceramides are necessary and Sufficient for Diet-Induced Impairment of Brown Adipose Tissue. Molecular Metabolism. March; (19): 45:101145. # Corresponding author
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Chaurasia B#, Talbot CL and Summers SA (2020). Adipocyte CeramidesThe Nexus of Inflammation and Metabolic Disease. Frontiers in Immunology. September; (11): 576347. # Corresponding author
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Ying L, Talbot CL and Chaurasia B# (2020). Ceramides in Adipose Tissue. Frontiers in Endocrinology. June; (11): 407.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Charron MJ, Williams L, Seki Y, Du XQ, Chaurasia B, Saghatelian A, Summers SA, Katz EB, Vuguin PM and Reznik SE (2020). The Molecular Mechanism of Antioxidant Effects of N-acetylcysteine in Mice Programmed for Metabolic Disease. Diabetes. May 2020, db191129.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Summers SA, Chaurasia B and Holland WL (2019). Metabolic Messengers: Ceramides. Nature Metabolism. October 24; doi:10.1038/s42255019-0134-8
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Buffolo M, Pires KM, Ferhat M, Llkun O, Makaju A, Achenbach A, Bowman F, Atkinson DL, Holland WL, Amri EZ, Chaurasia B, Franklin S and Boudina S (2019). Identification of a Paracrine Signaling Mechanism Linking CD34high Progenitors to the Regulation of Visceral Fat Expansion and Remodeling. Cell Reports. October; 8(29): 270-282
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Karanth S, Chaurasia B, Bowman FM, Tippetts T, Holland WL, Summers SA and Schlegel A (2019). FOXN3 controls liver glucose metabolism by regulating gluconeogenic substrate selection. Physiological Reports. September; 7(18) e14238
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Chaurasia B, Tippetts T, Monibas RM, Liu J, Li Y, Wang L, Wilkerson JL, Sweeney CR, Pereira RF, Sumida DH, Maschek JA, Cox JE, Kaddai V, Lancaster GI, Siddique MM, Poss A, Pearson M, Satapati S, Zhou H, McLaren DG, Previs SF, Chen Y, Qian Y, Petrov A, Wu M, Shen X, Yao J, Nunes CN, Howard AD, Wang L, Erion MD, Rutter J, Holland WL, Kelley DE, Summers SA. (2019). Targeting a Ceramide Double-Bond Improves Insulin Resistance and Steatohepatitis. Science. July; 365(6451): 386-392.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Ying L, Tippetts TS and Chaurasia B# (2019). Ceramide dependent lipotoxicity in metabolic diseases. Nutrition& Healthy aging. 5: 1-12.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Kiser PD, Kloesnikov AV, Kiser JZ, Dong Z, Chaurasia B, Wang L, Summers SA, Hoang T, Blackshaw S, Peachey NS, Kefalov VJ, Palczewski K, (2019). Conditional deletion of Des1 in the mouse retina does not impair the visual cycles in cones. FASEB Journal. January 15:fj201802493R
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Perspective: Kusminski CM and Scherer PE (2019). Lowering ceramides to overcome diabetes. Science. July; 365(6451): 319-320
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Commentary: Samuel VT and Shulman GIS (2019). Nonalcoholic fatty liver disease, insulin resistance, and ceramides. The New England Journal of Medicine. November 7; 381:1866-1869.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Commentary: J�rvinen HY (2020). Ceramides-a cause of insulin resistance in NAFLD in both murine models and humans. Hepatology.doi:10.1002/hep.31095.


Progress 03/01/19 to 02/29/20

Outputs
Target Audience:The target audience comprises of scientist and medical doctors with the sole focus on understanding and finding better treatment options for metabolic diseases such as diabetes and heart diseases Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The undergraduate, graduate students and post-doctoral fellows received training in various techniques related to this project. This includes but not limited to metabolite extraction for mass-spectrometry, measuring blood glucose, conducting glucose and insulin tolerance tests, assessing body composition using NMR, analyzing metabolic chamber (Comprehensive Laboratory Animal Monitoring System) data, and analyzing the data using GraphPad Prism software. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?The first aim in the remaining period of this funding will be to complete the ongoing study and perform hyperinsulinemic/euglycemic clamps with glucose tracers to determine insulin sensitivity in muscle, liver and white adipose tissue. In addition to this we plan to probe the biochemical mechanism that underliesCordycepsaction. We will achieve this by performingmechanistic studies of the tissue changes, including an assessment of alterations in insulin signaling, mitochondrial function, and lipid metabolism in muscle, liver, and adipose tissue. A lipidomic/metabolomic profile will be obtained to quantify sphingolipids, glycerolipids, and other nutrient metabolites in these tissues and serum.

Impacts
What was accomplished under these goals? During the term of the funding thus far we have successfully completed theAim 1.We acquired8 differentcommercial preparations ofCordycepseither grown locally in United Sates or those grown in various Asian countries such as in China, Taiwan and Singapore, and marketed either as a capsule or to be further processed to be consumed as soup for humans. We used 5 mg of these commercial preparations and appliedmass spectroscopy to quantify the amount of myriocin.Interestingly, we identified two commercially availableCordycepssample 1 and 8 that contained the highest amount of myriocin. Since, sample 1 is locally grown in United States, exhibits comparable amount of myriocin to that of sample 8 obtained from Singapore, is relatively cheaper and available as capsule unlike sample 8 that requires further processing, we decided to test the efficacy of sample 1 to improve glucose homeostasis and nutrient metabolism in mice pre-disposed to diabetes as listed inAim 2. To test the efficacy of identifiedCordycepssample 1in improving glucose homeostasis and nutrient metabolism, powdered sample 1 at two doses low (19.7mg/kg) and high (39.5mg/kg) were mixed into the high fat diet(Diet D12452, Research Diets, Inc., New Brunswick, NJ; 60% kCal from fat)as previously described.1These doses were calculated on the basis of pharmacological dose of sample 1 that was recommended by the manufacturer for human consumption. As controls we used high fat diet that either contained equivalent amount of starch derived from rice orCordycepssample 6 (39.5mg/kg) that did not contain any traces of myriocin. C57Bl6/J mice are currently being fed with these modified high fat diets. The preliminary data obtained after 4-6 weeks of feeding from these diets suggest that animals fed with high fat diets that containCordycepssamples 1, are refractory to weight gain an effect mediated by reduced accumulation of fat mass without alteration in lean mass, exhibits reduction in fed blood glucose levels, and improves insulin sensitivity. As proposed in the initial proposal, we aim to continue to feed these mice with above mentioned diets for additional 6 weeks for a total of 12 weeks.

Publications