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Ultrastructural 3D Visualization of Insulin Degludec Multi-Hexamers Upon Subcutaneous Injection in the Pig
Abstracts / Can J Diabetes 40 (2016) S27–S74
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162 Caspase 8 Plays a Pivotal Role in Adipose Tissue Inflammatory Signalling and Glucose Homeostasis CYNTHIA T. LUK*, SALLY Y. SHI, MANSA KRISHNAMURTHY, STEPHANIE A. SCHROER, MINNA WOO Toronto, ON
161 Ultrastructural 3D Visualization of Insulin Degludec Multi-Hexamers Upon Subcutaneous Injection in the Pig TORBEN SEESTED*,†, ANTHONY BURGESS, CHARLES PYKE†, ERICA NISHIMURA† Måløv, Denmark The long action of insulin degludec (IDeg) is attributed to the selfassembly of IDeg di-hexamers into soluble multi-hexamers. Using immuno-gold electron microscopy (iTEM), we have previously identified the formation of IDeg multi-hexamers in 2D in the extracellular matrix (ECM), after subcutaneous injection in pigs. In this study, correlative iTEM and focused ion beam scanning electron microscopy (FIB-SEM) were used to visualize the ultrastructural distribution of IDeg multi-hexamers in 3D. For comparison, insulin glargine (IGlar) was also visualized. Tissue biopsies were obtained from the neck of a pig 10 min after subcutaneous injection with 20 IU of IDeg U100 or the U300 formulation of IGlar. The tissue was then embedded into epoxy resin and anti-insulin iTEM was performed on ultrathin sections. Morphological markers close to immuno-gold positive structures were correlated to SEM micrographs obtained from the entire resin blockface. Finally, FIB-SEM tomography was performed, resulting in volumes generated from 5 to 7 nm thin slices. IDeg was visualized in the ECM as long structures measuring ~10 nm in width. FIB-SEM visualization confirmed that, in 3D, these structures connected into long multi-hexamers that distributed into an intricate network of densely packed, yarn-like clusters. In comparison, the morphology of IGlar U300 appeared to consist of amorphous micro-precipitates that, in 2D or 3D, appeared heterogeneous in both shape (spherical and cylindrical) and size. In summary, distinct ultrastructural 3D morphologies of IDeg and IGlar were visualized and were consistent with the different strategies for protraction: micro-precipitation of IGlar, and assembly of IDeg into multi-hexamers.
Chronic inflammation is a major contributor to obesity-associated insulin resistance and diabetes. Caspase 8 is best recognized for its critical role in the extrinsic apoptosis pathway but recent data suggests that this pathway may also interact with inflammatory NF-κB signalling. The objective of this study is to determine the role of caspase 8 in regulating adipose tissue inflammation and glucose homeostasis. Previously, we found that with obesity and insulin resistance, caspase 8 increased in adipose tissue of both mice and humans. We treated differentiated 3T3-L1 adipocytes with the caspase 8 inhibitor ZIETDFMK and found decreased activation of both apoptosis and NF-κB signalling. Using adipose tissue-specific caspase 8 knockout mice, we found that disruption of caspase 8 also decreased apoptosis and inflammation in vivo. Under both basal and high fat diet-fed conditions, knockout mice had improved glucose homeostasis compared to littermate controls. To determine if caspase 8 regulates glucose homeostasis via NF-κB signalling, we generated adipocyte-specific caspase 8 knockout mice with inducible expression of activated Ikkβ, resulting in adipocyte-specific concomitant activation of NF-κB signalling. The improved glucose homeostasis in caspase 8-deficient mice was partially abolished in double mutant mice, indicating that caspase 8 regulation of NF-κB signalling plays an important role in adipose tissue inflammation and glucose homeostasis. Together, these data show that caspase 8 plays an important role in cell death and inflammation in adipocytes at least partially via its regulation of NF-κB signalling. This represents a novel signalling cascade in adipose tissue contributing to obesity-associated insulin resistance.
163 Hyperinsulinemia Causes Age-Dependent Insulin Resistance and Reduces Lifespan JAMES D. JOHNSON, NICOLE M. TEMPLEMAN Vancouver, BC Hyperinsulinemia is commonly considered to be a downstream consequence of insulin resistance and obesity, but newer models where insulin production can be directly modulated are challenging the prevailing paradigm. Here, we examined insulin levels, glucose tolerance, insulin sensitivity, body composition and lifespan in large cohorts of male and female Ins1−/−:Ins2+/− experimental mice and their Ins1−/−:Ins2+/+ control littermates that were fed 1 of 2 distinct diets. At 2 years of age, female Ins1−/−:Ins2+/− experimental mice had ~20% reduced circulating insulin regardless of diet when compared with Ins1−/−:Ins2+/+ control littermates. Rather than having negative repercussions on glucose homeostasis, decreasing insulin led to fasting glucose that was 1 mM lower, with a significantly reduced HOMA-IR, and ~15% improved insulin sensitivity in insulin tolerance tests in aged, experimental mice across both diets. Body mass and fat mass were reduced by 10% across both diets, while fat-free mass and grip strength were unchanged in mice with modestly reduced insulin. We also find that a modest reduction in circulating insulin was sufficient to impart significant lifespan extension across both diets, independently of IGF-1 levels. RNA-sequencing of liver samples from 30-week-old mice demonstrated both pandiet and diet-specific changes in several key growth, metabolism and longevity-related pathways. Our results demonstrate that a
S59
162 Caspase 8 Plays a Pivotal Role in Adipose Tissue Inflammatory Signalling and Glucose Homeostasis CYNTHIA T. LUK*, SALLY Y. SHI, MANSA KRISHNAMURTHY, STEPHANIE A. SCHROER, MINNA WOO Toronto, ON
161 Ultrastructural 3D Visualization of Insulin Degludec Multi-Hexamers Upon Subcutaneous Injection in the Pig TORBEN SEESTED*,†, ANTHONY BURGESS, CHARLES PYKE†, ERICA NISHIMURA† Måløv, Denmark The long action of insulin degludec (IDeg) is attributed to the selfassembly of IDeg di-hexamers into soluble multi-hexamers. Using immuno-gold electron microscopy (iTEM), we have previously identified the formation of IDeg multi-hexamers in 2D in the extracellular matrix (ECM), after subcutaneous injection in pigs. In this study, correlative iTEM and focused ion beam scanning electron microscopy (FIB-SEM) were used to visualize the ultrastructural distribution of IDeg multi-hexamers in 3D. For comparison, insulin glargine (IGlar) was also visualized. Tissue biopsies were obtained from the neck of a pig 10 min after subcutaneous injection with 20 IU of IDeg U100 or the U300 formulation of IGlar. The tissue was then embedded into epoxy resin and anti-insulin iTEM was performed on ultrathin sections. Morphological markers close to immuno-gold positive structures were correlated to SEM micrographs obtained from the entire resin blockface. Finally, FIB-SEM tomography was performed, resulting in volumes generated from 5 to 7 nm thin slices. IDeg was visualized in the ECM as long structures measuring ~10 nm in width. FIB-SEM visualization confirmed that, in 3D, these structures connected into long multi-hexamers that distributed into an intricate network of densely packed, yarn-like clusters. In comparison, the morphology of IGlar U300 appeared to consist of amorphous micro-precipitates that, in 2D or 3D, appeared heterogeneous in both shape (spherical and cylindrical) and size. In summary, distinct ultrastructural 3D morphologies of IDeg and IGlar were visualized and were consistent with the different strategies for protraction: micro-precipitation of IGlar, and assembly of IDeg into multi-hexamers.
Chronic inflammation is a major contributor to obesity-associated insulin resistance and diabetes. Caspase 8 is best recognized for its critical role in the extrinsic apoptosis pathway but recent data suggests that this pathway may also interact with inflammatory NF-κB signalling. The objective of this study is to determine the role of caspase 8 in regulating adipose tissue inflammation and glucose homeostasis. Previously, we found that with obesity and insulin resistance, caspase 8 increased in adipose tissue of both mice and humans. We treated differentiated 3T3-L1 adipocytes with the caspase 8 inhibitor ZIETDFMK and found decreased activation of both apoptosis and NF-κB signalling. Using adipose tissue-specific caspase 8 knockout mice, we found that disruption of caspase 8 also decreased apoptosis and inflammation in vivo. Under both basal and high fat diet-fed conditions, knockout mice had improved glucose homeostasis compared to littermate controls. To determine if caspase 8 regulates glucose homeostasis via NF-κB signalling, we generated adipocyte-specific caspase 8 knockout mice with inducible expression of activated Ikkβ, resulting in adipocyte-specific concomitant activation of NF-κB signalling. The improved glucose homeostasis in caspase 8-deficient mice was partially abolished in double mutant mice, indicating that caspase 8 regulation of NF-κB signalling plays an important role in adipose tissue inflammation and glucose homeostasis. Together, these data show that caspase 8 plays an important role in cell death and inflammation in adipocytes at least partially via its regulation of NF-κB signalling. This represents a novel signalling cascade in adipose tissue contributing to obesity-associated insulin resistance.
163 Hyperinsulinemia Causes Age-Dependent Insulin Resistance and Reduces Lifespan JAMES D. JOHNSON, NICOLE M. TEMPLEMAN Vancouver, BC Hyperinsulinemia is commonly considered to be a downstream consequence of insulin resistance and obesity, but newer models where insulin production can be directly modulated are challenging the prevailing paradigm. Here, we examined insulin levels, glucose tolerance, insulin sensitivity, body composition and lifespan in large cohorts of male and female Ins1−/−:Ins2+/− experimental mice and their Ins1−/−:Ins2+/+ control littermates that were fed 1 of 2 distinct diets. At 2 years of age, female Ins1−/−:Ins2+/− experimental mice had ~20% reduced circulating insulin regardless of diet when compared with Ins1−/−:Ins2+/+ control littermates. Rather than having negative repercussions on glucose homeostasis, decreasing insulin led to fasting glucose that was 1 mM lower, with a significantly reduced HOMA-IR, and ~15% improved insulin sensitivity in insulin tolerance tests in aged, experimental mice across both diets. Body mass and fat mass were reduced by 10% across both diets, while fat-free mass and grip strength were unchanged in mice with modestly reduced insulin. We also find that a modest reduction in circulating insulin was sufficient to impart significant lifespan extension across both diets, independently of IGF-1 levels. RNA-sequencing of liver samples from 30-week-old mice demonstrated both pandiet and diet-specific changes in several key growth, metabolism and longevity-related pathways. Our results demonstrate that a