Graham Barlow (Graduate Student, Bollyky/Nolan labs, Immunology) – “Deep Profiling of Autoimmunity in the Pancreas of Type 1 Diabetes Patients Using Highly Multiplexed Microscopy”
Type 1 Diabetes (T1D) is an autoimmune disease characterized by dysfunctional interplay between cells of the immune system. Understanding these interactions with more granularity will lead to more targeted and strategic immunotherapies but research has been stymied by the technical inability to observe these interactions directly. CO-Detection by indEXing (CODEX) is a multiplexed microscopy platform developed by the Nolan lab that is uniquely poised to illuminate pancreatic autoimmunity. We applied CODEX to tissue sections of cadaveric pancreata from human donors with and without T1D. We employed an antibody panel targeting 55 pancreatic and immune cell antigens and developed computational approaches for extracting the spatial relationships between cells. This is allowing us to characterize the immune infiltrate, explore its spatial distribution, and infer key transitions in disease progression.
Kaisha Benjamin (Graduate Student, Endy Lab, Bioengineering) – “Engineering a Live Bacterial Therapeutic for Type 1 Diabetes”
Type 1 Diabetes (T1D), also known as insulin-dependent diabetes, is a chronic endocrine disorder that is caused by the autoimmune destruction of the insulin-producing β-cells in the pancreas. T1D is characterized by elevated blood glucose levels and is one of the most common endocrine and metabolic conditions occurring in childhood. There is no cure for T1D and, as a result, T1D patients depend on exogenous sources of insulin for their survival. Inadequate glycemic control results in serious microvascular and macrovascular complications such as stroke, neuropathy, and kidney failure. As an alternative to current insulin therapy, I aim to develop a synthetic live bacterial therapeutic for T1D using the skin commensal bacterium Staphylococcus epidermidis. Specifically, I will design and install into S. epidermidis a synthetic integrated genetic system that senses blood glucose levels (BGL) and releases a biologically active single-chain insulin analog in response to the appropriate BGL (i.e., >130 mg/dl glucose). I will achieve this through three specific aims: (1) the development of a biosensor, to detect elevated BGL; (2) the integration of the glucose sensor with an insulin actuator to create a biological insulin control system; and (3) the installation of the integrated genetic system into S. epidermidis to create a bacterial β-cell. Together, my proposed aims will facilitate the development of a novel approach to T1D treatment.
Romina Bevacqua (Post-doctoral fellow, Kim lab, Developmental Biology) – “Molecular and Functional Studies of SIX2 and SIX3 Transcription Factors in Human Pseudoislets”
Knowledge about the mechanisms regulating human islet cell maturation and function is essential for advancing efforts to regenerate and replace β-cells in diabetes. Recent studies (Arda et al 2016) identified the transcription factors SIX2 and SIX3 as new candidate regulators of human β-cell function, neither of which is expressed in mouse islets. Both factors are encoded at loci previously linked by GWAS to impaired fasting glucose (Kim et al. 2011). Gain-of-function studies suggest that SIX3 and SIX2 are sufficient to enhance insulin content and secretion in immature β-cells (Arda et al. 2016), but the function of these factors in adult β-cells remains unknown. Here, we used loss-of-function genetic approaches in human ‘pseudoislets’ to investigate roles of SIX2 and SIX3. In vitro glucose-stimulated insulin secretion (GSIS) assays normalized to total insulin content showed that insulin secretion was blunted after SIX2 or SIX3 knockdown (p<0.05). By contrast, glucagon secretion was increased (p<0.05). Ongoing high-throughput RNA sequencing (RNA-Seq) and chromatin immunoprecipitation sequencing (ChiP-Seq) analysis should elucidate molecular mechanisms underlying these findings. Expanding our understanding of the roles of SIX3 and SIX2 in human β-cells could accelerate progress in multiple strategies being used to replace or regenerate functional human β-cells.
Ivan Carcamo-Oribe (Research Scientist, Knowles lab, Cardiovascular Medicine) – “Co-Expression and Predictive Network Based Key Driver Analysis of Insulin Resistance in Human iPSC Lines”
Insulin resistance (IR) precedes the development of type 2 diabetes (T2D) and increases cardiovascular disease risk. Although genome wide association studies (GWAS) have uncovered new loci associated with T2D, their contribution to explain the mechanisms leading to decreased insulin sensitivity has been very limited. Thus, new approaches are necessary to explore the genetic architecture of insulin resistance. To that end, we generated an iPSC library across the spectrum of insulin sensitivity in humans. RNA-seq based analysis of 310 induced pluripotent stem cell (iPSC) clones derived from 100 individuals allowed us to identify differentially expressed genes between insulin resistant and sensitive iPSC lines. Analysis of the co-expression architecture uncovered several insulin sensitivity-relevant gene sub-networks, and predictive network modeling identified a set of key driver genes that regulate these co-expression modules. Functional validation in human adipocytes and skeletal muscle cells (SKMCs) confirmed the relevance of the key driver candidate genes for insulin responsiveness.
Nicolas Cuttriss (Clinical Assistant Professor, Pediatrics) – “Democratizing Type 1 Diabetes (T1D) Knowledge in Rural and Underserved Communities: Project ECHO T1D”
T1D care for many patients in both the pediatric and adult populations in the U.S. falls on primary care providers (PCPs). Persistently suboptimal outcomes for people with T1D and a lack of access to subspecialty care mandates the development of innovative healthcare delivery models. Using the Project ECHO (Extension for Community Healthcare Outcomes) model, Stanford University and the University of Florida partnered to develop and pilot a “ECHO T1D” teleECHO™ clinic. The goal of the pilot was to demonstrate the feasibility of an ECHO model for T1D and improve the ability of PCPs to manage patients with T1D. Utilizing a “Hub‑and‑Spoke” model, we recruited and partnered with PCPs at non‑specialty diabetes practices across Florida and California and held weekly hour teleECHO sessions consisting of didactic presentations and case reviews. Precision recruitment methods to target clinics in remote and medically underserved regions included: (1) geocoding to identify high‑need areas, (2) claims data to identify PCPs treating T1D, (3) survey data from PCPs. The 18‑month pilot program is ongoing and enrollment demonstrated extremely high interest amongst PCPs as the pilot program filled beyond capacity. In California, 11 Spoke sites enrolled with 37 clinics serving roughly 900 adult and pediatric patients with T1D. In Florida, 12 Spoke sites enrolled with 67 clinics who serve roughly 1,300 patients with T1D. PCPs and their clinic staff have become local experts in T1D, providing a more informed level of care for the many patients who lack access to endocrinologists.
Eric Jay Daza, Marily Oppezzo (both from Stanford Prevention Research Center) and Katarzyna Wac (University of Copenhagen) – “Effects of Sleep Deprivation on Blood Glucose, Food Cravings, and Mood in Non-Diabetics: An N-of-1 Randomized Trial Pilot Study”
Sleep deprivation is a prevalent and rising health concern, one with known effects on blood glucose (BG) levels, mood, and calorie consumption. However, the mechanism by which sleep deprivation affects calorie consumption (e.g., measured via self-reported types craved food) is unclear, and may be highly idiographic or individual-specific. Single-case or “n-of-1” randomized trials are useful in exploring such idiographic effects by exposing each subject to both treatment and control conditions, thereby characterizing effects specific to that individual. Our primary goals were twofold: 1) To generate individual-specific hypotheses of the effect of sleep deprivation on next-day BG level, mood, and food cravings in non-diabetic individuals. The observed effects on BG level and mood of our study participants would be compared to the average effects from the literature. 2) To assess the utility and feasibility of our n-of-1 studies for generating such idiographic hypotheses for personalized management of sleep behavior.
Laya Ekhlaspour (Instructor, Pediatrics)– “Impact of Fat Content on Postprandial Glucose Excursions While in a Hybrid Closed-Loop System”
Currently with most hybrid closed-loop insulin delivery systems, the meal bolus is based solely on the carbohydrate content. We compared the glycemic response to meals with both low and high fat content for 8 hours following dinners with subjects using a hybrid closed-loop (HCL) system. Forty-three subjects aged 6-49 years old completed supervised outpatient clinical trials of a HCL system. The carbohydrate, protein and fat content of each meal was recorded by the medical staff, using food labels, and information from chain restaurants. We divided 36 dinners into two categories based on their fat intake. We considered a meal low fat (LF) if it had < 16 grams of fat, n=19 and high fat (HF) if it had >30 grams of fat, n=17. We calculated the area under the curve (AUC) above 140 mg/dl for 8 hours following the start of the meals following a standard premeal insulin bolus. There was a significant difference in AUC above 140 mg/dl between high fat and low-fat meals. HF = 7036 mg/dl. min ± 17064 and LF = -4478 ± 13554 (p=0.03). There was a significant increase in glucose between 120 and 240 minutes following a HF comparing to a LF dinner (p< 0.0001). Meals with HF content can significantly increase the CGM values 2-4 hours following meal onset and the effect of delayed gastric emptying and insulin resistance can persist for hours even with closed-loop control. Further studies to determine optimal insulin delivery based on nutritional inputs may improve overall glycemic control. "
Mohsen Fathzadeh (Post-doctoral fellow, Knowles lab, Cardiovascular Medicine) – “FAM13A Affects Body Fat Distribution and Function”
Insulin resistance (IR) is a major driver of T2D and results in increased cardiovascular risk partly due to IR-associated dyslipidemia (e.g. high plasma triglyceride and low plasma HDL-cholesterol levels). Increases in indices of body fat distribution (e.g. waist-hip-ratio adjusted for BMI, WHRadjBMI) are correlated with IR. Large genome-wide association studies (GWAS) of fasting insulin adjusted for BMI (FIadjBMI), a proxy for intravenously assessed IR, and WHRadjBMI showed a significant positive association at the FAM13Alocus. These findings suggest that FAM13Amight regulate insulin and fat distribution even in normal-weight individuals, a “lipodystrophy-like” phenotype. We characterized the role of FAM13Aacross human tissues by integrating GWAS data with tissue-specific gene expression, and analyzed tissue specific gene expression from Fam13a knockout (KO) mice. Humans:We performed colocalization analysis of FAM13Agene expression in subcutaneous adipose (SAT), visceral adipose (VAT), liver and skeletal muscle from the Genotype Tissue Expression project (v7) with 7 IR-related GWAS traits using eCAVIAR. Using a modified colocalization posterior probability (mCLPP) computed in eCAVIAR, we observed colocalizations (mCLPP > 0.8) between SAT FAM13A expression and FIadjBMI, WHRadjBMI, triglycerides and HDL-cholesterol. We did not observe similar colocalizations in the other tissues, suggesting that FAM13A plays an active and differential role in SAT. Mice:To evaluate the biological effect of Fam13a, we performed differential expression (DE) analysis in VAT and SAT tissue between wildtype and KO male mice on both chow and high fat diet (total n = 32 tissue samples). After adjustment for the effects of diet and type of adipose tissue, we observed 255 differentially expressed genes at 5% FDR. Pathway analysis showed a strong enrichment of oxidative phosphorylation and a depletion of immunodeficiency pathways in KOs. To understand the differential impact of Fam13a KO across tissues, we performed a DE analysis stratified by adipose tissue type adjusted for sex and diet. We observed a much more pervasive effect of the KO on the transcriptome in SAT (96 DE genes) compared to VAT (0 DE genes) at 5% FDR. In conclusion, our results establish FAM13A as the causal gene in the locus; indicate that the primary effect is in SAT; and highlight oxidative phosphorylation and immunodeficiency as key biological pathways underlying the association with IR.
Christopher Gardner (Rehnborg Farquhar Professor, Medicine) – “The DIETFITS (Diet Intervention Examining The Factors Interacting with Treatment Success) Study”
The DIETFITS (Diet Intervention Examining The Factors Interacting with Treatment Success) study was a randomized clinical trial to determine the effect of healthy low-fat (HLF) diet vs a healthy low-carbohydrate (HLC) diet on weight change among 609 overweight/obese, non-diabetic adults. Main findings showed no difference in amount of weight lost between the 2 diet groups, but substantial variability within diet groups (~80 lb range of weight change) as hypothesized. The original hypothesis was that insulin resistance (IR) would predict differential weight loss success by diet group (i.e., effect modification). INS-30 values from baseline OGTTs were used as a proxy measure of IR to test the hypothesis; the main hypothesis was not supported. However, the rich study data yielded other potential measures of IR beyond INS-30. We hypothesize there may be subsets of the population that benefit differentially from an HLF vs HLC diet, and that some measures of IR are likely to be more sensitive than others at detecting this. The objective of this study is to conduct secondary analysis of DIETFITS to more thoroughly explore whether the subset of individuals with IR would benefit more from HLF vs HLC diet in terms of weight loss and CVDRFs. We will evaluate several measures of IR including fasting insulin, HOMA-IR, Matsuda index, insulin area-under-the curve (AUC) during OGTT and triglyceride/HDL-C ratio. Analyses will be completed prior to the Frontiers in Diabetes Research Symposium.
Keren Hilgendorf (Research Scientist, Jackson lab, Microbiology and Immunology) – “The w-3 Fatty Acid Receptor Ffar4/Gpr120 Triggers Camp-Dependent Adipogenesis via Primary Cilia”
Adipocyte hypertrophy and de novo adipogenesis mediate WAT expansion. Hypertrophy in excess is linked to insulin resistance. Adipogenesis in vitro is induced by insulin, glucocorticoids, and IBMX. However, the physiological signals that activate adipogenesis are poorly defined. Quiescent preadipocytes are uniformly ciliated in vitro, and cilia are important for adipogenesis. Patients with dysfunctional cilia are diabetic and obese. However, the function of ciliary signaling in preadipocytes is unknown. Here, we use a mouse model to show that the primary cilium is an in vivo marker of preadipocytes and critical for initiating adipogenesis. Specifically, 30% of all perivascular cells in fat pads are ciliated, and these ciliated cells are activated in response to HFD. Further, Tulp3, critical for trafficking GPCRs to the primary cilium, is required for adipogenesis. We discovered that Ffar4/Gpr120 is localized to preadipocyte cilia in vitro and in vivo. Addition of Ffar4 ligand, the ω3 fatty acid DHA, activates ciliary Ffar4 and promotes adipogenesis of both 3T3-L1 and primary preadipocytes. Ffar4 activation acutely raises cAMP in the cilium. This in turn activates EPAC, promotes chromatin remodeling, and induces expression of Pparγ. Thus, DHA is a physiologically relevant ligand that replaces the pharmacological agent IBMX to promote adipogenesis, and this requires the primary cilium. These data provide a molecular rationale for the anti-diabetic effects of DHA and explain how ciliary dysfunction causes obesity and diabetes.
Owen Jiang & Yunshin Jung (Research Scientist & Post-doctoral fellow, Svensson lab, Pathology) – “Isthmin-1 Is an Endocrine Activator of the pi3k Pathway That Improves Glucose Homeostasis”
The metabolic benefit of thermogenic fat cells, including brown and beige adipocytes, is thought to arise from both their classical heat generating functions and also their ability to secrete adipokines. Previous work has indicated that thermogenic adipose tissue can crosstalk to liver and other organs, but the specific molecules involved in this crosstalk have not been fully explored. Here, we have identified Isthmin-1 ISM1 as a secreted protein that is highly inducible during adipocyte differentiation. In humans, higher gene expression levels of ISM1 in adipose tissue correlates with increased BMI. Administration of ISM1 protein to or overexpression in obese mice improves insulin sensitivity without causing hypoglycemia, and demonstrate liver and subcutaneous adipose tissue as dominant target organs of ISM1. Phospho-kinase screening identifies that ISM1 is activating the classical PI3K signaling pathway in a similar manner to insulin, but unlike insulin, ISM1 has integrated suppressive actions on both expression of gluconeogenic enzymes and lipogenic genes in liver and hepatocytes. Thus, ISM1 shares the common PI3K/AKT pathway with insulin but can uncouple lipogenesis from gluconeogenesis. Our data identify a new hormone signaling pathway that prevents the development of diabetes and hepatic steatosis with therapeutic potential for the treatment of metabolic diseases.
Carl Johnson (Graduate Student, Jackson lab, Stem Cell Biology and Regenerative Medicine) – “Preadipocyte Cell Cycle Control: Hypertrophy or Hyperplasia”
Organisms can store newly acquired fat within existing adipocytes (hypertrophy) or within newly created adipocytes (hyperplasia). These two pathways are linked to distinct health outcomes with strong associations between hypertrophy and metabolic disorders, type 2 diabetes, and poorly vascularized hypoxic fat pads. Disordered fat pads have been shown to antagonize preadipocyte function, compounding a bias for hypertrophy, creating a feedback loop for worsening metabolic health.
The physiological signals that trigger the mitotic entry and differentiation remain incompletely known we undertook an unbiased genome-wide CRISPR KO screen in the mouse preadipocyte cell line 3T3-L1 to determine the broad genetic requirements for adipogenesis. 3T3-L1 cells are contact inhibited before they undergo two rounds of mitoses after the addition of the differentiation cocktail, but before any lipid accrual, allowing us to interrogate the transition from quiescence to pre-differentiative mitosis. Using the same sgRNA library, genes involved in the cell cycle were counter-screened in a proliferation assay to remove hits that were generally toxic or lethal. The remaining cell cycle genetic hits appear to control the pre-differentiative mitoses while leaving the proliferative mitoses demonstrably unencumbered. When cell cycle inhibitors were added they inhibited both mitosis and 3T3-L1 differentiation. Overall, a better understanding of the mechanisms by which preadipocytes transition to their pre-differentiative mitoses could lead to therapies that directly target the underlying cause(s) of hypertrophic fat and the metabolic diseases that follow.
Seokho Kim (Post-doctoral fellow, Kim lab, Developmental Biology) – “Discovering Mechanisms Regulating Islet Development and Maturation in Pigs”
Replacement of insulin-producing β cells represents a major goal for the treatment of diabetes. The use of human stem cells to derive functional β cells has shown promise in recent years based on advances in understanding of the genetic and signaling basis of pancreatic organogenesis. However, due to inaccessibility of human tissues at key developmental stages, most of this understanding reflects studies of rodents, whose islet morphology and regulation appear distinct from humans. Although human pancreatic diseases like monogenic diabetes and pancreatitis have been modeled effectively in pigs, porcine pancreas developmental biology is relatively understudied. To address this knowledge gap, we have investigated porcine islet cells throughout fetal and neonatal development using histological and molecular approaches. Fluorescence-activated cell sorting (FACS) followed by high-throughput RNA profiling (RNA-Seq) provided comprehensive gene expression profiles of porcine β and α cells throughout fetal and neonatal development. Detailed morphometric analysis revealed a dynamically regulated positive endocrine cell allocation and islet architecture over time. Gene expression analysis with RNA-seq libraries clearly identified cell- and stage- specific gene expression patterns in sorted porcine β and α cells. Expression of genes involved in cell proliferation, including MKI67, declined as porcine β and α cell development advanced and these findings were supported by morphometry. In addition, expression of PDX1, MAFA and NKX6.1 were restricted to pig β cells. Gene Ontology analysis identified enriched pathways like glucose or insulin response in β and α cells, hormone processing, and intracellular signal transduction. Over 50 genes previously linked to diabetes in human genome wide association studies, including SLC30A8 and SLC2A2, were found to be developmentally regulated in the pig. Further genome-wide comparison of porcine transcriptome profiles to extant human and mouse RNA-seq datasets revealed that porcine β cell gene expression was closer to that of human islets than to mouse islets at neonatal stages. Our studies also showed that the cell specific expression patterns of PDX1, NKX6.1, MAFB, and other factors more closely resemble that in humans than mice. data. Taken together, this study strongly supports the pig as a relevant model for the study of pancreatic and endocrine organogenesis which may allow for detection of transient gene expression signatures not previously appreciated due to a lack of granularity in human fetal and post-natal sample procurement. Delineating how porcine pancreatic cells mature at relevant fetal and post-natal stages will enhance our understanding of the signaling, morphogenetic and transcriptional programs that generate functional β and α cells, and accelerate progress in producing functional replacement human islet cells for diabetes.
Kyle Kovary (Graduate Student, Teruel lab, Chemical and Systems Biology) – An Integrated Screening Approach to Identify Drug Repurposing Targets for Treating Insulin Resistance”
Obesity and insulin resistance are reaching epidemic levels in the United States and the developed world. This trend will lead a to public health crisis due to the increased risks for cardiovascular and metabolic diseases such as type 2 diabetes. To combat these diseases, it is important to restore proper function to adipose tissue given the central role it plays in metabolic and endocrinological homeostasis. The transcription factor PPARg is the master regulator of the development, maintenance, and function of adipose tissue. Efforts to therapeutically target PPARg to re-sensitize adipose tissue to insulin to combat type 2 diabetes has yielded several drugs, such as thiazolidinediones (TZDs). While TZDs are able to re-sensitize adipocytes to insulin in type 2 diabetes patients, they have dangerous cardiac side effects and are no longer widely used therapeutically. Targeting peripheral regulators of PPARg is an attractive strategy to reactivate PPARg activity that may avoid the harmful side effects of direct and potent PPARg activators such as TZDs. In order to expand our understanding of the PPARg regulatory network, we are utilizing an integrated approach that combines CRISPR knock-out screening, RNA-sequencing, and deep proteomics to gain a more comprehensive understanding of the PPARg regulation network. With this integrated approach, we aim to identify PPARg regulators that can be targeted with existing drugs that have known clinical profiles, utilizing existing drug repurposing databases. We hope that this strategy will yield druggable targets that can be translated from bench to bedside in a more accelerated fashion.
Caitlin Maikawa (Graduate Student, Appel lab, Bioengineering) – “Supramolecular CB-PEG Designer Excipients for Improved Insulin Formulations”
Insulin has been the focus of diabetes treatment for over 80 years, yet treatment with insulin and amylin analogues is more effective than insulin alone and amylin replacement is necessary to restore meal-time glucagon suppression. However, amylin analogues (e.g., pramlintide) are incompatible with insulin analogues in standard formulations. The burdensome requirement for separate administrations has limited pramlintide adoption to less than 1.5% of rapid-acting insulin users. Furthermore, the minimal overlap in the pharmacokinetics of these two hormones following subcutaneous administration limits their potential synergistic effects. In this presentation we will show how simultaneous supramolecular PEGylation of insulin and pramlintide enables a stable insulin-pramlintide co-formulation. Our approach exploits host-guest interactions of cucurbituril-PEG with end-terminal aromatic amino acids on the proteins. This dual-hormone co-formulation can be stabilized for over 100 hours under stressed conditions, compared to only 10 hours for commercial insulin. Using rat and pig models of insulin-deficient diabetes, we demonstrate that co-formulation simultaneously modifies the pharmacokinetic profiles of insulin and pramlintide, and restores meal-time glucagon suppression. This approach to insulin-pramlintide co-formulation more closely mimics endogenous co-secretion of insulin and amylin and shows promise as a dual-hormone replacement therapy for diabetes that enhances treatment efficacy and reduces patient burden.
Latha Palaniappan (Professor, Medicine) – “Strength Training Regimen fOr Normal weiGht Diabetics (STRONG-D) Study”
Prevalent among Asian populations and the elderly, normal-weight diabetics (NWD) represent approximately 1 in 5 individuals with Type II Diabetes Mellitus (T2DM). Although NWD face higher mortality rates than overweight diabetics, current literature lacks data on effective lifestyle recommendations for this population. Standard T2DM treatment emphasizes aerobic exercise for weight loss; however, given the increased mortality associated with low weight and diabetic individuals’ low lean muscle mass, strength training may be more beneficial in helping NWD achieve glycemic control. The Strength Training Regimen for Normal Weight Diabetics (STRONG-D) Study aims to determine the best exercise regimen for NWD. Modeled after the design of the Health Benefits of Aerobic and Resistance Training in T2DM patients (HART-D) study, STRONG-D is a three-arm randomized controlled trial that compares the clinical effectiveness of structured strength training only, aerobic training only, and combination (strength + aerobic) training. The three arms will have a combined total of 282 randomized participants that follow a 9-month exercise intervention. The primary measured outcome is HbA1c levels, while secondary endpoints include physical fitness, body composition as well as leg strength and endurance. Through this research model, the STRONG-D Study will compare the efficacy of different exercise regimens in improving diabetes management and health outcomes for NWD, with the goal of developing evidence-based exercise guidelines for these individuals.
Latha Palaniappan (Professor, Medicine) – “Initiate and Maintain Physical Activity in Clinics (IMPACT) Study”
The American Diabetes Association (ADA) recommends 150 minutes of aerobic exercise per week and resistance training twice per week for patients with Type II diabetes mellitus (T2DM); however, 38% of patients do not exercise at recommended levels and 31% do not exercise at all. Structured exercise interventions have been shown to be effective in reducing hemoglobin A1c (HbA1c) levels in T2DM patients, but novel approaches are needed to translate these findings into practice. The Initiate and Maintain Physical Activity in Clinics (IMPACT) Study will determine the optimal level and frequency of structured regimen needed for patients to initiate and maintain physical activity long-term. IMPACT is a three-arm randomized controlled trial evaluating patient-centered outcomes, clinical effectiveness, and cost-effectiveness of a specialized physical activity treatment for T2DM patients. Participants are tracked over 30 months with 6 months of intervention and 24 months of follow-up. Participants are randomized into 1x per week exercise, 3x per week exercise, or usual care. Primary study outcomes include absolute change in HbA1c levels, anthropometry, patient satisfaction, self-reported quality of life, and the incremental cost-effectiveness ratio for each study arm. These findings will enable the implementation of structured exercise regimens, designed specifically to address the ADA’s guidelines and improve patient outcomes, as part of the recommended lifestyle modifications for patients with T2DM.
Juri Park (Visiting Scholar, Endocrinology) – “Predictors of Incident Type 2 Diabetes in Normal Weight Individuals with Normoglycemia: The Korean Genome and Epidemiology Study (KoGES)”
The population with type 2 diabetes mellitus (T2DM) is heterogeneous; yet, focus has mostly centered on obese individuals to understand the pathogenesis of T2DM. To broaden understanding of T2DM mechanisms, we conducted a retrospective study of normal weight individuals in the KoGES study to understand risk for incident T2DM in 1467 normal-weight (NW, <23 kg/m2) vs 2536 overweight (OW, ≥23 kg/m2) individuals with normoglycemia at baseline followed from 2001 to 2014. Incidence of T2DM was similar in NW vs OW individuals (10.2 vs 11.2 per 1000 person-years). Risk factors for T2DM, however, differed between the 2 populations. In OW individuals, measures of insulin resistance were predictors of T2DM. In NW individuals, measures of beta-cell dysfunction and current smoking were significant and not insulin resistance. In both populations, hypertension was a significant risk factor. Risk factors for incident T2DM differ between NW and OW individuals which need further investigation to more precisely define and treat subsets of individuals within T2DM.
Atefeh Rabiee – “Dynamic Reversal of Insulin Sensitivity during Adipogenesis by the Timing and Order of CEBPB and CEBPA Levels”
Dysfunctional adipose tissue is a hallmark of insulin resistance (IR), a disease that is highly correlated with diabetes and cardiovascular disease. The development of healthy functioning adipocytes (fat cells) requires that, early in adipogenesis, the expression of the transcription factor C/EBPβ increases, resulting in an upregulation in the expression of its downstream target PPARγ, the master regulator of adipogenesis. Under normal conditions, C/EBPβ expression subsequently decreases as adipogenesis progresses. However, under inflammatory (IR) conditions, expression of C/EBPβ remains high, and reports suggest that this high C/EBPβ contributes to the inflammatory state. Understanding how C/EBPβ switches from being adipogenic (promoting insulin sensitivity) to inflammatory (promoting IR) could be a promising new way to treat IR, but the mechanisms underlying this switch are poorly understood. Here, we present evidence that high C/EBPβ in adipocytes under inflammatory conditions leads to IR by blocking access of C/EBP onto the promoters of adipogenic genes. Here we show that the timing of expression of C/EBPβ and C/EBP needs to be precisely controlled for adipogenesis to correctly progress and disruption of this timing leads to IR.
Mehdi Razavi & Rosita Primavera (Post-doctoral fellows, Thakor lab, Radiology)– “Dexamethasone-Loaded Microplates Improve the Outcome of Islets Transplanted in PDMS Bioscaffolds”
Bioscaffolds for the transplantation of pancreatic islets have been studied for the treatment of type-1 diabetes. However, several studies have shown that islets survival is limited in vivo, due to an extensive inflammatory reaction. Recently, we developed novel biodegradable microparticle (µPLs), which can release dexamethasone (DEX), an anti-inflammatory drug, in a sustained manner. Our DEX-µPLs are square in shape with a size of 20x20x10µm, and significantly reduced pro-inflammatory cytokines expression on LPS-stimulated primary macrophages. Hence, we hypothesized that supplementing islets transplanted in bioscaffolds with our DEX-µPLs can enhance islet survival by attenuating any host mediated inflammatory response. In this work, we synthesized a bioscaffolds made of PDMS, coated with collagen and loaded with DEX-µPLs. When islets were transplanted into the epididymal fat pad of diabetic mice, our bioscaffolds significantly reduced blood glucose values compared to islets alone (average over the first month post-transplantation: 534±65 vs 295±46 mg/dL; P<0.05). Furthermore, mice transplanted with PDMS-Collagen-DEX-µPLs bioscaffolds showed faster responses to i.p. glucose injections (glucose clearance rate: 2.59±0.04 vs 2.02±0.23 mg/dL/min P<0.05). Collectively these results show that localized administration and release of DEX can improve the outcome of islet transplantation."
Krissie Tellez (Graduate Student, Kim lab, Developmental Biology) – “Gluca-Gone: Exploring in Vivo Mechanisms Regulating Glucagon Secretion in a Glucagon Null Mouse”
Endocrine cells within the pancreatic islets of langerhans react to changes in blood glucose levels by secreting hormones that act on target tissues. Glucagon is produced by islet α cells and is secreted in response to physiological settings of low blood glucose, like nocturnal fasting. While glucagon secretion is impaired in pathological settings like diabetes, little is known about mechanisms regulating glucagon secretion. This is in part due to the inability to distinguish human from endogenous glucagon in transplantation models, like immunocompromised NSG mice. To promote genetic and physiological studies to elucidate mechanisms underlying glucagon secretion, we generated mice lacking endogenous glucagon using CRISPR/Cas9 in an NSG background (NSG-GKO). Specifically, we targeted exon 3, which encodes the amino acids comprising mature processed glucagon peptide. We recovered mice with “in-frame” deletions of glucagon that eliminated mature glucagon but maintained production of incretins like Glucagon-like-peptide-1 (GLP-1) produced from preproglucagon processing. NSG-GKO mice displayed known phenotypes of glucagon signaling loss including hypoglycemia, hyper aminoacidemia, hypoinsulinemia, and alpha cell hyperplasia. Human islet transplantation reconstituted circulating glucagon in GKO-NSG mice, which rescued glucagon null phenotypes. Importantly, transplanted human islets showed regulated glucagon secretion in response to stimulants like hypoglycemia. Therefore, the GKO-NSG mouse is a novel model enabling physiological and genetic studies of transplanted human α cells. Using this novel transplantation model, we are using loss-of-function and gain-of-function genetics to identify candidate regulators of glucagon secretion in human α cells.
Stefan Tholen (Post-doctoral fellow, Teruel lab, Chemical and Systems Biology) – “Circadian Glucocorticoid Oscillations Are Required to Maintain Functional Brown Adipose Tissue”
In healthy mammals glucocorticoids (GC) are secreted in circadian oscillations. Disruption of this circadian rhythm promotes obesity and development of obesity-related disorders. As a therapeutic treatment expansion of brown adipose tissue (BAT) or boosting its respiratory activity gained significant attention. However, how BAT is controlled by circadian GC oscillations has hardly been investigated and could impact therapeutic strategy. Here we show that flattening of daily GC oscillations in mice not only results in body weight gain mainly due to increase of white fat depots, but also leads to lipid accumulation in BAT. Lipid accumulation in BAT precedes expansion of white fat depots and is characterized by an initial phase of increased lipid uptake, and less energy expenditure due to downregulation of factors playing a key role in the transcriptional cascade regulating brown adipocyte differentiation including UCP-1. Finally, early lipid uptake results in misbalance of triglyceride synthesis and breakdown leaving the tissue dysfunctional. Interestingly, mice in which GC levels were raised in the evening phase displayed normal BAT morphology. This provides evidence that as long GCs increase in the correct circadian time frame there is no lipid accumulation in BAT and that it is not the total GC concentration but rather losing the off-periods that result in dysfunction of BAT. These novel findings indicate an important regulatory role of GCs in BAT biology."
Robert Whitener (Post-doctoral fellow, Kim lab, Developmental Biology) – “Leveraging the Flexibility of the mAbCAR System to Target Tregs to Human Islets”
Replacement of insulin producing β cells through allotransplantation of islets isolated from cadaveric donors shows great promise as a highly effective treatment for type 1 diabetes (T1D) patients. However, these patients require chronic immunosuppression to prevent graft rejection. Leveraging the immunomodulatory role of regulatory T cells (Tregs) represents an attractive strategy for inducing durable allotolerance to grafted islets and could allow for the reduction or elimination of immunosuppressive. Previously published in vivo results indicate that engineered mAbCAR-Tregs traffic to MHC mis-matched islet grafts and promote allotolerance in mice. We now report additional studies to screen candidate target molecules for mAbCAR-Treg targeting to human islets.In an in vitro transwell migration assay, SDF1α directed mAbCAR-Tregs responded to soluble SDF1α in a dose dependent manner. Using this platform, we have begun pilot experiments utilizing islets as a source for soluble targets and as direct targets to screen islet cell surface antigens, including the recently published β cell antigen NTPDase3. In parallel experiments, homing of directed mAbCAR-Tregs to human islets transplanted into the right kidney capsule of NSG recipient mice is monitored longitudinally via BLI technology. Identification of islet antigens which can be used to drive localization of Tregs to transplanted islets is a key factor towards broadening the applicability of islet transplant therapy. The flexibility of the mAbCAR-Treg system has been demonstrated to be a viable strategy for inducing allotolerance to transplanted islets in mice and is highly flexible, enabling rapid validation of candidate antigens for downstream clinical studies.
Si Wu (Post-doctoral fellow, Snyder lab, Genetics) – “Systematic Investigation of Overweight and Normal Weight Type Ii Diabetes by Applying Metabolomics”
The majority of people living with Type 2 Diabetes Mellitus (T2DM) are overweight or obese due to the additional burden of applying insulin to properly control blood sugar levels. However, some people, especially Asian populations, develop T2DM with a low BMI (normal weight). This discrepancy may be resulted from differences in body fat and muscle mass for a given BMI. In order to investigate the influences of body weight on the onset of T2DM, we collected plasma samples from 9 over-weight and 17 normal-weight diabetic patients as a pilot study, then applied untargeted metabolomics profiling on these plasma samples. By combining Hydrophilic Interaction (HILIC) and Reverse-Phase Liquid Chromatography (RPLC)-Mass Spectrometry, we comprehensively detected and relatively quantified 5,469 metabolites. We then employed various pattern recognition algorithms (principal component analysis, hierarchical clustering, Welsh t-test, etc), clearly separated over-weight and normal-weight diabetic groups based on their plasma metabolite profiling. Furthermore, we statistically identified 52 significantly different metabolites between these two groups, mainly involved in lipid metabolism. These results not only demonstrate that untargeted metabolomics is able to serve as an efficient diagnostic tool to distinguish over-weight and normal-weight diabetic patients, but also provide meaningful insights into molecular mechanisms about tight relationships between diabetes and obesity, further facilitate biomarker discovery for early diagnosis and differentiation of normal/over weight diabetes.
Meng Zhao (Post-doctoral fellow, Svensson lab, Pathology) – “Identification of c16orf89 as a New Metabolic Hormone Produced by Thyroid Gland”
Thyroid gland plays a critical role in regulating thermogenesis and metabolism in mammals. In humans, disorders of thyroid function have been associated with metabolic diseases, such as obesity and type 2 diabetes. However, the mechanisms underlying the interactions between thyroid, thyroid hormones, and the development of metabolic diseases are still not well understood. We have identified c16orf89 as a new polypeptide hormone secreted from thyroid gland. c16orf89 is highly expressed in thyroid in both humans and mice, and the expression is suppressed in diet-induced obese mice. Pharmacological administration of recombinant c16orf89 protein normalizes blood glucose in diabetic mice within hours of a single injection. Consistent with the pharmacological treatment, whole-body c16orf89 ablation results in an increase of blood glucose levels independently of body weight. Mechanistically, c16orf89 stimulates the phosphorylation of AMPKα at Thr 172, suggesting that c16orf89 re-wires the nutrient status of the cell. These findings reveal a previously unstudied thyroid-secreted metabolic factor, which enables the potential development of new protein therapeutics for type 2 diabetes and also links thyroid function in metabolic disease through a new endocrine pathway.