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Title: Diabetic Conditions Confer Metabolic and Structural Modifications to Tissue-Engineered Skeletal Muscle
Abstract Skeletal muscle is a tissue that is directly involved in the progression and persistence of type 2 diabetes (T2D), a disease that is becoming increasingly common. Gaining better insight into the mechanisms that are affecting skeletal muscle dysfunction in the context of T2D has the potential to lead to novel treatments for a large number of patients. Through its ability to emulate skeletal muscle architecture while also incorporating aspects of disease, tissue-engineered skeletal muscle (TE-SkM) has the potential to provide a means for rapid high-throughput discovery of therapies to treat skeletal muscle dysfunction, to include that which occurs with T2D. Muscle precursor cells isolated from lean or obese male Zucker diabetic fatty rats were used to generate TE-SkM constructs. Some constructs were treated with adipogenic induction media to accentuate the presence of adipocytes that is a characteristic feature of T2D skeletal muscle. The maturity (compaction and creatine kinase activity), mechanical integrity (Young's modulus), organization (myotube orientation), and metabolic capacity (insulin-stimulated glucose uptake) were all reduced by diabetes. Treating constructs with adipogenic induction media increased the quantity of lipid within the diabetic TE-SkM constructs, and caused changes in construct compaction, cell orientation, and insulin-stimulated glucose uptake in both lean and diabetic more » samples. Collectively, the findings herein suggest that the recapitulation of structural and metabolic aspects of T2D can be accomplished by engineering skeletal muscle in vitro. Impact Statement The tissue engineering of skeletal muscle to model disease and injury has great promise to provide a tool to develop and/or improve therapeutic approaches for improved health care. A tissue-engineered skeletal muscle model of one of the most common and debilitating diseases, type 2 diabetes, has been developed in vitro as evidenced by the structural and metabolic alterations that are consistent with the disease phenotype in vivo. « less
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Tissue Engineering Part A
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National Science Foundation
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  1. Key points

    Previous studies in fetuses with intrauterine growth restriction (IUGR) have shown that adrenergic dysregulation was associated with low insulin concentrations and greater insulin sensitivity.

    Although whole‐body glucose clearance is normal, 1‐month‐old lambs with IUGR at birth have higher rates of hindlimb glucose uptake, which may compensate for myocyte deficiencies in glucose oxidation.

    Impaired glucose‐stimulated insulin secretion in IUGR lambs is due to lower intra‐islet insulin availability and not from glucose sensing.

    We investigated adrenergic receptor (ADR) β2 desensitization by administering oral ADRβ modifiers for the first month after birth to activate ADRβ2 and antagonize ADRβ1/3. In IUGR lambs ADRβ2 activation increased whole‐body glucose utilization rates and insulin sensitivity but had no effect on isolated islet or myocyte deficiencies.

    IUGR establishes risk for developing diabetes. In IUGR lambs we identified disparities in key aspects of glucose‐stimulated insulin secretion and insulin‐stimulated glucose oxidation, providing new insights into potential mechanisms for this risk.


    Placental insufficiency causes intrauterine growth restriction (IUGR) and disturbances in glucose homeostasis with associated β adrenergic receptor (ADRβ) desensitization. Our objectives were to measure insulin‐sensitive glucose metabolism in neonatal lambs with IUGR and to determine whether daily treatment with ADRβ2 agonist and ADRβ1/β3 antagonists for 1 month normalizes their glucose metabolism. Growth, glucose‐stimulatedmore »insulin secretion (GSIS) and glucose utilization rates (GURs) were measured in control lambs, IUGR lambs and IUGR lambs treated with adrenergic receptor modifiers: clenbuterol atenolol and SR59230A (IUGR‐AR). In IUGR lambs, islet insulin content and GSIS were less than in controls; however, insulin sensitivity and whole‐body GUR were not different from controls. Of importance, ADRβ2 stimulation with β1/β3 inhibition increases both insulin sensitivity and whole‐body glucose utilization in IUGR lambs. In IUGR and IUGR‐AR lambs, hindlimb GURs were greater but fractional glucose oxidation rates andex vivoskeletal muscle glucose oxidation rates were lower than controls. Glucose transporter 4 (GLUT4) was lower in IUGR and IUGR‐AR skeletal muscle than in controls but GLUT1 was greater in IUGR‐AR. ADRβ2, insulin receptor, glycogen content and citrate synthase activity were similar among groups. In IUGR and IUGR‐AR lambs heart rates were greater, which was independent of cardiac ADRβ1 activation. We conclude that targeted ADRβ2 stimulation improved whole‐body insulin sensitivity but minimally affected defects in GSIS and skeletal muscle glucose oxidation. We show that risk factors for developing diabetes are independent of postnatal catch‐up growth in IUGR lambs as early as 1 month of age and are inherent to the islets and myocytes.

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  2. Introduction Blood sugar homeostasis relies largely on the action of pancreatic islet hormones, particularly insulin and glucagon. In a prototypical fashion, glucagon is released upon hypoglycemia to elevate glucose by acting on the liver while elevated glucose induces the secretion of insulin which leads to sugar uptake by peripheral tissues. This simplified view of glucagon and insulin does not consider the paracrine roles of the two hormones modulating the response to glucose of α- and β-cells. In particular, glucose-stimulated glucagon secretion by isolated α-cells exhibits a Hill-function pattern, while experiments with intact pancreatic islets suggest a ‘U’-shaped response. Methods To this end, a framework was developed based on first principles and coupled to experimental studies capturing the glucose-induced response of pancreatic α- and β-cells influenced by the two hormones. The model predicts both the transient and steady-state profiles of secreted insulin and glucagon, including the typical biphasic response of normal β-cells to hyperglycemia. Results and discussion The results underscore insulin activity as a differentiating factor of the glucagon secretion from whole islets vs . isolated α-cells, and highlight the importance of experimental conditions in interpreting the behavior of islet cells in vitro . The model also reproduces the hyperglucagonemia, whichmore »is experienced by diabetes patients, and it is linked to a failure of insulin to inhibit α-cell activity. The framework described here is amenable to the inclusion of additional islet cell types and extrapancreatic tissue cells simulating multi-organ systems. The study expands our understanding of the interplay of insulin and glucagon for pancreas function in normal and pathological conditions.« less
  3. Introduction: Myocardial fibrosis and dysfunction is one of the major cardiac complications of long-term diabetes. Prolonged hyperglycemia is known to induce myocardial dysfunction often leading up to heart failure. Hypothesis: The objective of this study was to investigate the cardioprotective effect of glycyrrhizin (GLC) on myocardial damage in engineered in-vitro human cardiac tissues. Engineered 3D tissue chips present an ideal microenvironment via therapeutically relevant interfaces to study molecular- and cellular-level events and mimic human-specific disease states, and identify new therapeutic targets in vitro. Methods: AC16 human cardiomyocyte cells were used to 3D bioprint cardiac tissue chips based on prior published work. In our study, the 3D bioprinted cardiac tissue chips (CTC) were cultured using normo- (5mM) and hyper-glycemic (25mM) conditions for up to 48 hrs. For the GLC treatment group, a subset of CTC cultured using hyperglycemic conditions were treated with 50 mM of GLC for 24 hours. Results: CTC cultured under hyperglycemic conditions demonstrated altered levels of connexin-43 (CX43) and Troponin-I implying cardiomyocyte injury. Exposure to hyperglycemia revealed changes in epigenetic markers: histone methylation marker (H3K9me)-1, Sirtuin-1, and Histone Deacetylase (HDAC)-2 as well as in inflammatory and stress related mediators such as heat shock protein (HSP)-60, receptor for advancedmore »glycation end products (RAGE), toll like receptor (TLR)-4, high mobility group box (HMGB)-1 and CXC chemokine receptor (CXCR)-4. CTC exposed to 25mM glucose for 24 hours resulted in the downregulation of HSP60 and Sirtuin-1. Prolonged exposure to hyperglycemia led to decrease in the expression of CX43 and CXCR4; thereby adversely affecting cardiomyocyte function. Upregulated expression of DNA-binding nuclear protein HMGB1 along with changes in H3K9me1 indicated long-term hyperglycemia-induced damage to cardiomyocytes. GLC treated CTC exhibited a decrease in the expression of RAGE, TLR4 and also demonstrated altered expression of CX43, CXCR4, and troponin I. Conclusions: This study suggests that GLC possesses cardioprotective effects in human cardiomyocytes exposed to prolonged hyperglycemia.« less
  4. Under healthy conditions, the pancreas responds to a glucose challenge by releasing insulin. Insulin suppresses lipolysis in adipose tissue, thereby decreasing plasma glycerol concentration, and it regulates plasma glucose concentration through action in muscle and liver. Insulin resistance (IR) occurs when more insulin is required to achieve the same effects, and IR may be tissue-specific. IR emerges during puberty as a result of high concentrations of growth hormone and is worsened by youth-onset obesity. Adipose, liver, and muscle tissue exhibit distinct dose-dependent responses to insulin in multi-phase hyperinsulinemic-euglycemic (HE) clamps, but the HE clamp protocol does not address potential differences in the dynamics of tissue-specific insulin responses. Changes to the dynamics of insulin responses would alter glycemic control in response to a glucose challenge. To investigate the dynamics of insulin acting on adipose tissue, we developed a novel differential-equations based model that describes the coupled dynamics of glycerol concentrations and insulin action during an oral glucose tolerance test in female adolescents with obesity and IR. We compared these dynamics to the dynamics of insulin acting on muscle and liver as assessed with the oral minimal model applied to glucose and insulin data collected under the same protocol. We found thatmore »the action of insulin on glycerol peaks approximately 67 min earlier ( p < 0.001) and follows the dynamics of plasma insulin more closely compared to insulin action on glucose as assessed by the parameters representing the time constants for insulin action on glucose and glycerol ( p < 0.001). These findings suggest that the dynamics of insulin action show tissue-specific differences in our IR adolescent population, with adipose tissue responding to insulin more quickly compared to muscle and liver. Improved understanding of the tissue-specific dynamics of insulin action may provide novel insights into the progression of metabolic disease in patient populations with diverse metabolic phenotypes.« less
  5. Abstract

    Type 1 diabetes mellitus (T1DM) is an autoimmune disease characterized by insulin deficiency, and patients with diabetes have an increased risk of bone fracture and significantly impaired fracture healing. Proinflammatory cytokine tumor necrosis factor‐alpha is significantly upregulated in diabetic fractures and is believed to underlie delayed fracture healing commonly observed in diabetes. Our previous genetic screen for the binding partners of progranulin (PGRN), a growth factor‐like molecule that induces chondrogenesis, led to the identification of tumor necrosis factor receptors (TNFRs) as the PGRN‐binding receptors. In this study, we employed severalin vivomodels to ascertain whether PGRN has therapeutic effects in diabetic fracture healing. Here, we report that deletion of PGRN significantly delayed bone fracture healing and aggravated inflammation in the fracture models of mice with T1DM. In contrast, recombinant PGRN effectively promoted diabetic fracture healing by inhibiting inflammation and enhancing chondrogenesis. In addition, both TNFR1 proinflammatory and TNFR2 anti‐inflammatory signaling pathways are involved in PGRN‐stimulated diabetic fracture healing. Collectively, these findings illuminate a novel understanding concerning the role of PGRN in diabetic fracture healing and may have an application in the development of novel therapeutic intervention strategies for diabetic and other types of impaired fracture healing.