Physiological Levels Of Insulin Research Articles

Localized In Vivo Electro Gene Therapy (LiveGT)-Mediated Skeletal Muscle Protein Factory Reprogramming

Gene electrotransfer (GET) has gained significant momentum as a non-viral gene delivery method for various clinical applications, primarily in the cancer immunotherapy and vaccine development space. Preclinical studies have demonstrated exogenous gene delivery and expression in various tissues, including the liver, skin, cardiac muscle, and skeletal muscle. However, protein replacement applications of this technology have yet to be fully actuated. Plasmid DNA skeletal muscle delivery has been shown to maintain expression for up to 18 months. In the current study, we evaluated localized skeletal muscle delivery for protein replacement applications. We developed localized in vivo electro gene therapy (liveGT) protocols utilizing mono- and biphasic pulse sequences for localized pulse delivery directly to skeletal muscle with a custom monopolar platinum electrode. Plasmid DNA encoding human insulin and human glucokinase were chosen for this study to evaluate the liveGT platform for protein replacement potential. Initial in vitro GET was performed in mouse myoblasts to evaluate human insulin and glucokinase co-delivery. This was followed by liveGT-mediated reporter gene delivery in the skeletal muscle of Sprague–Dawley rats for pulse sequence selection. Protein replacement potential was evaluated in healthy (non-diabetic) rats with liveGT-mediated human insulin and glucokinase co-delivery to skeletal muscle. Human and rat insulin levels were measured via ELISA over the course of 3 months. Fed-state blood glucose measurements were monitored in correlation with serum human insulin levels. LiveGT-mediated skeletal muscle reprogramming successfully produced physiological levels of human insulin in serum over the course of 3 months. Hypo- and hyperglycemic events were not observed. Therefore, liveGT is a safe and viable platform for potential protein replacement therapies.

12440 Exploring The Potential Protective Role Of The Bacterial-derived Metabolite Indole-3-propionic Acid Against Obesogenic Insults In Placenta Cell Lines

Abstract Disclosure: J. Ernst: None. L. Conlon: None. R. Gupta: None. L. Kang: None. M.E. Heard-Lipsmeyer: None. Obesity during pregnancy is associated with detrimental changes in placental function that lead to the development of complications such as gestational diabetes and preeclampsia as well as altered metabolism in the offspring. With the incidence of obesity reaching epidemic levels world-wide, it is imperative to find mechanisms to mitigate these risks during pregnancy. The gut-microbiome is a diverse system that regulates numerous body functions through the secretion of specific metabolites. Gut dysbiosis has recently emerged as one mechanism that contributes to placental dysfunction and clear associations between obesity and gut dysbiosis are evident. Indole-3-propionic acid (IPA) is a gut microbiome-derived metabolite that has anti-inflammatory, anti-oxidative stress and glucose regulatory functions, all of which are dysregulated in obesity. Previous studies have found diminished levels of IPA in serum of women with overweight/obesity. However, it remains unknown what the physiological functions of IPA are in the placenta and if it could have potential protective effects against perturbations caused by obesity. The objective of this study is to determine what physiological role IPA plays in the placenta and if IPA can protect against obesogenic insults that contribute to placental pathology. For this study, we developed an in vitro model of obesity where two human placental cell lines, BeWo (choriocarcinoma) and HTR8 (normal immortalized trophoblasts) were cultured in media conditioned by mature human adipocytes (ACM). RNA sequencing (RNAseq) analysis was used to determine acute effects of IPA treatment after 24 hours in the presence and absence of ACM. We further examined the influence of IPA on insulin signaling with physiological and hyperinsulinemia levels of insulin at 24 and 72 hours utilizing QPCR and western blot analyses. RNAseq analysis revealed treatment with physiological levels of IPA induced changes in genes associated with normal placental function, autophagy and inflammatory mediated processes and other pathways. Our RNAseq analysis offers initial insight into the molecular mechanisms of IPA in placental cells. Future analyses will investigate the potential role of insulin signaling, glucose homeostasis and oxidative stress. Collectively, this study highlights the importance of a healthy gut microbiome and potentially serves as a window of opportunity to mitigate the onset or severity of pregnancy-related disorders such as gestational diabetes by enhancing the gut microbiome prior to pregnancy. Presentation: 6/1/2024

12440 Exploring The Potential Protective Role Of The Bacterial-derived Metabolite Indole-3-propionic Acid Against Obesogenic Insults In Placenta Cell Lines

Abstract Disclosure: J. Ernst: None. L. Conlon: None. R. Gupta: None. L. Kang: None. M.E. Heard-Lipsmeyer: None. Obesity during pregnancy is associated with detrimental changes in placental function that lead to the development of complications such as gestational diabetes and preeclampsia as well as altered metabolism in the offspring. With the incidence of obesity reaching epidemic levels world-wide, it is imperative to find mechanisms to mitigate these risks during pregnancy. The gut-microbiome is a diverse system that regulates numerous body functions through the secretion of specific metabolites. Gut dysbiosis has recently emerged as one mechanism that contributes to placental dysfunction and clear associations between obesity and gut dysbiosis are evident. Indole-3-propionic acid (IPA) is a gut microbiome-derived metabolite that has anti-inflammatory, anti-oxidative stress and glucose regulatory functions, all of which are dysregulated in obesity. Previous studies have found diminished levels of IPA in serum of women with overweight/obesity. However, it remains unknown what the physiological functions of IPA are in the placenta and if it could have potential protective effects against perturbations caused by obesity. The objective of this study is to determine what physiological role IPA plays in the placenta and if IPA can protect against obesogenic insults that contribute to placental pathology. For this study, we developed an in vitro model of obesity where two human placental cell lines, BeWo (choriocarcinoma) and HTR8 (normal immortalized trophoblasts) were cultured in media conditioned by mature human adipocytes (ACM). RNA sequencing (RNAseq) analysis was used to determine acute effects of IPA treatment after 24 hours in the presence and absence of ACM. We further examined the influence of IPA on insulin signaling with physiological and hyperinsulinemia levels of insulin at 24 and 72 hours utilizing QPCR and western blot analyses. RNAseq analysis revealed treatment with physiological levels of IPA induced changes in genes associated with normal placental function, autophagy and inflammatory mediated processes and other pathways. Our RNAseq analysis offers initial insight into the molecular mechanisms of IPA in placental cells. Future analyses will investigate the potential role of insulin signaling, glucose homeostasis and oxidative stress. Collectively, this study highlights the importance of a healthy gut microbiome and potentially serves as a window of opportunity to mitigate the onset or severity of pregnancy-related disorders such as gestational diabetes by enhancing the gut microbiome prior to pregnancy. Presentation: 6/1/2024

Serum osteoglycin is stable during various glycemic challenges in healthy men

PurposeOsteoglycin is hypothesized to be metabolically active and may enhance insulin action. We hypothesized that osteoglycin levels increase during hyperglycemia as a physiological response to enhance the effects of insulin.MethodsEight healthy males were included in a cross-over study consisting of three study days following an 8 h fast. First, we performed an oral glucose tolerance test (OGTT); second, an isoglycemic intravenous glucose infusion (IIGI); and third, a control period consisting of a three hour fast. We analyzed blood samples for circulating osteoglycin levels during the study days. Repeated measures ANOVA was performed to compare levels of s-osteoglycin between OGTT, IIGI, and the fasting control.ResultsThere were no differences in baseline osteoglycin levels among study days (p > 0.05). We observed no significant changes neither in absolute s-osteoglycin levels by time (p = 0.14) nor over time by study day (p = 0.99). Likewise, we observed no significant changes in percentage s-osteoglycin levels neither by time (p = 0.11) nor over time by study day (p = 0.89).ConclusionWe found that s-osteoglycin levels were stable for three hours during OGTT, IIGI, and fasting in healthy males. Based on the present study, circulating s-osteoglycin levels may be measured independently of fasting or non-fasting conditions. Furthermore, circulating physiological levels of glucose and insulin did not affect s-osteoglycin levels.

Insulin resistance and metabolic diseases - a review

Introduction: This review paper aims to discuss the effects of insulin resistance, and its association with various metabolic diseases. Insulin resistance (IR), characterized by diminished tissue responsiveness to physiological insulin levels, is a key component in various metabolic diseases, including type 2 diabetes, cardiovascular disorders (CVDs), obesity, and non-alcoholic fatty liver disease (NAFLD).
 State of knowledge The consequences of IR are profound; in type 2 diabetes, it hinders glucose utilization by muscle cells, leading to hyperglycemia, muscle damage, and loss of mass and strength. IR also plays a central role in NAFLD, promoting lipid accumulation, hepatic inflammation, fibrosis, and cell death.
 Materials and methods: A review of chosen literature in the PubMed and Google Scholar databases was conducted, using the following key words: „Insulin resistane”, „Insulin resistance in obesity”, „Obesity”, „Insulin resistance NAFLD”, „Diebetes”, „Insulin resistance CVD”
 Summary: The consequences of insulin resistance are far-reaching. In the context of type 2 diabetes, it impedes glucose utilization by muscle cells, resulting in hyperglycemia and subsequent muscle cell damage, contributing to loss of mass and strength. Insulin resistance is also a central player in the pathogenesis of non-alcoholic fatty liver disease (NAFLD), fostering lipid accumulation, hepatic inflammation, and, ultimately, fibrosis and cell death.
 Conclusions: In conclusion, understanding insulin resistance is paramount in addressing the rising prevalence of metabolic diseases globally Controlling insulin resistance emerges as a crucial aspect of managing these metabolic disorders and their complications. Further research into the mechanisms of IR formation and effective intervention strategies is imperative for improving public health outcomes.

Nutrient Uptake by the Hindlimb of Growing Pigs Treated with Porcine Somatotropin and Insulin

The shift in nutrient partitioning induced by porcine somatotropin (pST) is accompanied by a decrease in insulin sensitivity for whole-body glucose uptake. The relative contribution of metabolic changes in the hindlimb was investigated in eight pigs (55 kg) that had received recombinant pST (120 μg/kg) or excipient (control) for 7 d. Uptake of metabolites by the hindlimb was measured under basal conditions and during hyperinsulinemic-euglycemic clamps at low [14 ng/(kg·min)] and high [360 ng/(kg·min)] insulin infusion rates. Dextrose infusion rate required to maintain euglycemia was used as an index of whole-body glucose uptake in response to exogenous insulin. Effects of pST on hindlimb and whole-body glucose uptake were evident only at physiological levels of insulin (basal and low insulin infusion rate). During the low rate of insulin infusion, dextrose infusion rate was 79% lower for pST-treated pigs and glucose uptake by the hindlimb was 59% lower compared with control pigs. The decrease in glucose uptake by the hindlimb was entirely accounted for by the estimated reduction in glucose utilization by adipose tissue of the hindlimb. Glucose:oxygen quotients were reduced during basal (57%) and low insulin infusion (63%) with pST treatment, indicating a change in the pattern of substrate utilization. This is consistent with the concept that pST directs nutrients away from adipose and towards muscle growth by altering the response of tissues to homeostatic signals such as insulin.

Insulin Resistance: From Mechanisms to Therapeutic Strategies.

Insulin resistance is the pivotal pathogenic component of many metabolic diseases, including type 2 diabetes mellitus, and is defined as a state of reduced responsiveness of insulin-targeting tissues to physiological levels of insulin. Although the underlying mechanism of insulin resistance is not fully understood, several credible theories have been proposed. In this review, we summarize the functions of insulin in glucose metabolism in typical metabolic tissues and describe the mechanisms proposed to underlie insulin resistance, that is, ectopic lipid accumulation in liver and skeletal muscle, endoplasmic reticulum stress, and inflammation. In addition, we suggest potential therapeutic strategies for addressing insulin resistance.

Inflammatory status of the pancreas in NOD mice that do not develop overt diabetes.

Type 1 diabetes (T1D) is an autoimmune disease in which immune cells target the pancreatic islets and destroy the β-cells, resulting in hyperglycemia and decreased plasmatic insulin levels. The non-obese diabetic (NOD) mouse is the most used animal model for studying diabetes because it spontaneously develops T1D and shares similarities with the human disease. A hallmark feature of this model is the appearance of insulitis, defined as an inflammatory cell infiltration of the pancreatic islets. However, a small percentage of NOD mice do not develop overt diabetes even after 28–35 weeks of age. Thus, we questioned the status of the pancreatic islets in these non-diabetic NOD mice, with particular focus on islet inflammation and plasmatic insulin levels, in comparison to pre-diabetic (11 weeks old) and new-onset diabetic mice. Diabetes progression was evaluated by assessing blood glucose and pancreas histology. The inflammatory score was determined on Hematoxylin–Eosin (HE)-stained sections of pancreas. Plasma insulin was detected by enzyme-linked immunosorbent assay (ELISA). The results showed that inflammation increased in an age-dependent manner in all mice, irrespective of their diabetic status. Mostly affected within the analyzed groups were the 28 weeks old non-diabetic NOD mice, in which insulin production was reduced and inversely correlated with the inflammatory status. We conclude that in NOD mice, pancreatic inflammation progresses independently of diabetes onset and clinical signs of disease. Most likely, the NOD females that do not develop overt diabetes preserve a small mass of functional β-cells, which is able to provide the physiological insulin levels and avoid diabetes onset.

The use of D-chiro-Inositol in clinical practice.

D-chiro-Inositol has been widely used in clinical practice to induce ovulation in women with polycystic ovary syndrome. Only recent evidence established that this molecule acts through two different mechanisms, with potentially different outcomes. On the one hand, under a metabolic perspective, D-chiro-Inositol improves insulin signaling, thus restoring physiological insulin levels in resistant subjects. On the other hand, at a cellular level, it downregulates the expression of steroidogenic enzyme aromatase, which is responsible for the conversion of androgens to estrogens. We reviewed current literature in different databases, searching for D-chiro-Inositol in relation with one of the following keywords: myo-inositol, PCOS, infertility, insulin resistance, aromatase, androgen and inositol, testosterone, estrogen and inositol, estradiol, hypogonadotropic hypogonadism, fat tissue, estrogens and cancer, anovulation, uterine myoma, endometriosis, endometrial hyperplasia. D-Chiro-Inositol treatment may be helpful in restoring physiological hormonal levels in various clinical disorders. However, D-Chiro-Inositol intervention should be carefully designed to avoid possible undesired side effects stemming from its multiple mechanisms of action. We evaluated the optimal D Chiro-Inositol administration for different pathologies, defining dosages and timing. Even though further studies are required to validate our preliminary results, this paper is primarily intended to guide researchers through some of the pathways of D-Chiro-Inositol.

Insulin resistance and type 2 diabetes in children

Type 2 diabetes (T2D) is an emerging health risk in obese children and adolescents. Both environmental (lack of physical activity, excess nutritional intake, sedentary lifestyle) and genetic factors contribute to this global epidemic. The growing prevalence of T2D in youth is also associated with a consistently increased incidence of metabolic and cardiovascular complications. Insulin resistance (IR), i.e., whole-body decreased glucose uptake in response to physiological insulin levels, determines impaired glucose homeostasis and it is recognized as cardinal trigger of T2D and cardiovascular disease in both adults and children. In particular, IR and beta-cell dysfunction lead to the persistent hyperglycemia which characterizes T2D. Indeed, both pathological states influence each other and presumably play a crucial, synergistic role in the pathogenesis of T2D, although the precise mechanisms are not completely understood. However, beta-cell dysfunction and IR induce impaired glucose metabolism, thus leading to the progression to T2D. Therefore, understanding the mechanisms correlated with the decline of beta-cell function and IR is crucial in order to control, prevent, and treat T2D in youth. This review focuses on the current knowledge regarding IR and T2D in children and adolescents and showcases interesting opportunities and stimulating challenges for the development of new preventative approaches and therapeutic strategies for young patients with T2D.

Physiologically inspired culture medium prolongs the lifetime and insulin sensitivity of human hepatocytes in micropatterned co-cultures

Given significant species-specific differences in liver functions, cultures of primary human hepatocytes (PHHs) are useful for assessing drug metabolism and to mitigate the risk of drug-induced hepatotoxicity in humans. While significant advances have been made to keep PHHs highly functional for 2–4 weeks in vitro, especially upon co-culture with both liver- and non-liver-derived non-parenchymal cells (NPCs), the functional lifespan of PHHs is 200–400 days in vivo. Therefore, it is desirable to determine culture conditions that can further prolong PHHs functions in vitro for modeling chronic drug exposure, disease pathogenesis, and to provide flexibility to the end-user for staggering drug incubations across multiple culture batches. Most PHH culture platforms utilize supraphysiologic levels of glucose and insulin and bovine-derived serum when including NPCs, which can alter PHH functions. Therefore, here we developed a culture medium containing physiologic levels of glucose (5 mM), insulin (500 pM), and human serum (10 % v/v) and tested its effects on micropatterned co-cultures (MPCCs) in which PHHs are organized onto collagen domains of empirically optimized dimensions and surrounded by 3T3-J2 murine fibroblasts that express liver-like molecules and induce higher PHH functions than liver-derived NPCs. Our physiologically-inspired culture medium allowed better retention of PHH morphology, polarity, and functions (albumin and urea, cytochrome-P450 activities, and sensitivity to insulin-mediated inhibition of gluconeogenesis) for up to 10 weeks relative to the traditional medium. Finally, PHHs in the physiologic medium displayed clinically-relevant responses to prototypical drugs for hepatoxicity and cytochrome-P450 induction. Ultimately, our physiologic culture medium could find broader utility for the continued development of PHH-NPC co-cultures for drug development, investigating the effects of patient-derived sera on PHH functions and disease phenotypes, and for use in cell-based therapies.

1741-P: Immune Regulation of Postprandial Glycemia and Whole-Body Insulin Sensitivity through Myeloid Production of IL-10

As feeding increases signals that potently induce gene expression in immune cells, we aimed to elucidate the role of myeloid cells in normal postprandial metabolism. Feeding increased serum levels of LPS, which is known to induce multiple genes in immune cells. Notably, LPS together with insulin potently induced IL-10 in macrophages, which could also be detected in the postprandial portal vein in vivo. In cultured hepatocytes, physiological levels of insulin alone did not suppress gluconeogenic gene expression, although when added together with medium from macrophages stimulated with LPS and insulin, they were markedly suppressed. This suppression was not seen in medium from macrophages of IL-10 knockout mice, showing this to be an IL-10 dependent process. Plasma glucose concentration ad libitum was elevated in mice injected with adenovirus encoding shRNA targeting the IL-10 receptor, and the normal postprandial suppression of gluconeogenic genes in liver was compromised, showing the importance of the IL-10 signal in postprandial glucose metabolism. Furthermore, myeloid-specific knockout of Akt1 and Akt2 resulted in similar phenotypes that were rescued by incrementing mTOR signaling, pointing to an Akt-mTOR dependent mechanism. As whole-body deficiency of IL-10 has been reported to decrease body weight and increase insulin sensitivity, we assessed the effect of myeloid deficiency of IL-10 by conducting bone marrow transfer from IL-10 knockout mice to wild type mice. While these phenotypes were recapitulated, postprandial glucose production was not improved despite markedly increased whole-body insulin sensitivity. These results pointed to the differential actions of myeloid IL-10 where baseline production maintains normal body weight, while postprandial increases in the hepatic-portal system suppresses hepatic gluconeogenic gene expression, collectively showing the need for context-dependent administration of IL-10 based therapies. Disclosure G. Toda: None. K. Soeda: None. N. Arakawa: None. Y. Masuda: None. N. Kobayashi: None. H. Suwanai: None. Y. Izumida: None. T. Yamauchi: Research Support; Self; AeroSwitch, Asahi Mutual Life Insurance Company, Astellas Pharma Inc., AstraZeneca K.K., Boehringer Ingelheim International GmbH, Daiichi Sankyo Company, Limited, Kowa Pharmaceutical company,limited., Kyowa Hakko Kirin Co., Ltd., Merck Sharp & Dohme Corp., Mitsubishi Corporation Life Sciences Limited, Mitsubishi Tanabe Pharma Corporation, Novartis Pharma K.K., Novo Nordisk Inc., NTT Docomo Inc., Ono Pharmaceutical Co., Ltd., Sanofi K.K., Sanwa Kagaku Kenkyusho, Shionogi & Co., Ltd., Sumitomo Dainippon Pharma Co., Ltd., Taisho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Company Limited, TOSOH CORPORATION. Other Relationship; Self; Covidien Japan Inc. (Medtronic Japan Co., Ltd.), Eli Lilly Japan K.K., Johnson & Johnson, Kissei Pharmaceutical Co., Ltd. T. Kadowaki: Research Support; Self; Astellas Pharma Inc., Daiichi Sankyo, Mitsubishi Tanabe Pharma Corporation, MSD Corporation, Novartis Pharma K.K., Novo Nordisk Pharma Ltd., Ono Pharmaceutical Co., Ltd., Sanofi, Sumitomo Dainippon Pharma Co., Ltd., Takeda Pharmaceutical Company Limited. Speaker’s Bureau; Self; Abbott, Astellas Pharma Inc., AstraZeneca K.K., Boehringer Ingelheim Pharmaceuticals, Inc., Cosmic Corporation, Daiichi Sankyo, Eli Lilly Japan K.K., FUJIFILM, Kowa Company, Ltd., Kyowa Hakko Kirin Co., Ltd., Medscape Education, Medtronic, Mitsubishi Tanabe Pharma Corporation, MSD Corporation, NIPRO Medical Corporation, Novartis Pharma K.K., Novo Nordisk Pharma Ltd., Ono Pharmaceutical Co., Ltd., Sanofi, Sanwa Kagaku Kenkyusho, Sumitomo Dainippon Pharma Co., Ltd., Taisho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Company Limited, Terumo Medical Corporation. Other Relationship; Self; Asahi Mutual Life Insurance. K. Ueki: None. Funding Japan Society for the Promotion of Science (16K15488, 17K16141, 19K17954)

SUN-649 Metabolic and Functional Regulation of T Cells by Insulin and Insulin like Growth Factor 1

Obesity leads to altered immunity characterized by increased risk of autoimmunity, poor response to infection, and impaired vaccine response. T cells play an important role in this obesity-associated immune response; however, the mechanisms by which T cells are altered in obesity remain unknown. Our goal is to identify nutritionally regulated hormones and cytokines that link whole body nutrition and immunity, and to understand the mechanisms by which such factors can alter T cell response in obesity. To that end, we have identified the hormones insulin and insulin-like growth factor-1 (IGF-1) as potential links between nutritional status and T cell metabolism and function. Insulin is secreted from pancreatic beta cells in response to increasing blood glucose levels, and circulating insulin levels are elevated in obesity due to insulin resistance in metabolic tissues. IGF-1 levels are influenced by protein intake and nutrition status, and free (bioactive) levels of IGF-1 are elevated in obesity. To study the role of insulin and IGF-1 on T cell function and metabolism, we treated activated CD4 T cells with physiologic levels of insulin or IGF-1 in vitro for 24 hours. Treatment of CD4 T cells with insulin or IGF-1 increased glucose uptake, glycolytic metabolism, and mitochondrial metabolism while altering inflammatory cytokine production. In particular, both insulin and IGF-1 decreased IFN-γ production, whereas IGF-1 specifically increased IL-17 production from both bulk activated CD4 T cells and T cells skewed toward a T helper 17 (Th17) phenotype. Using a T cell-specific insulin receptor (IR) conditional knockout mouse, we found that loss of IR signaling decreased glucose uptake and mitochondrial metabolism and increased IFN-γ production by activated T cells. Moreover, IR appears to be required for both insulin and IGF-1 effects on T cells. Lastly, we investigated the CD4 T cell subset-specific expression of both IR and IGF-1 receptor (IGF-1R). We found that each CD4 T cell subset had its own unique expression of both IR and IGF-1R; however Th17 cells had a striking increase in IGF-1R expression compared to the other T cell subsets, indicating a specific role for IGF-1 in promoting inflammation. These findings underscore the ability of the nutritionally-regulated hormones insulin and IGF-1 to modulate CD4 T cell metabolism and function and thereby alter T cell immunity, which has direct clinical relevance in both normal physiology and in obesity.

Effects of β-hydroxybutyrate treatment on glycogen repletion and its related signaling cascades in epitrochlearis muscle during 120 min of postexercise recovery.

We investigated the effects of β-hydroxybutyrate (β-HB), the most abundant type of ketone body in mammals, on postexercise glycogen recovery in skeletal muscle by using an in vitro experimental model. Male ICR mice swam for 60 min and then their epitrochlearis muscles were removed and incubated with either physiological levels of glucose (8 mmol/L) and insulin (60 μU/mL) or glucose and insulin plus 1, 2, or 4 mmol/L of sodium β-HB. Four millimoles per liter β-HB had a significant positive effect on glycogen repletion in epitrochlearis muscle at 120 min after exercise (p < 0.01), while 2 mmol/L of β-HB showed a tendency to increase the glycogen level (p < 0.09), and 1 mmol/L of β-HB had no significant effect. We further investigated the effect of 4 mmol/L β-HB treatment on the signaling cascade related to glycogen repletion in the epitrochlearis muscles throughout a 120-min recovery period. After incubating the muscles in 4 mmol/L of β-HB for 15 min postexercise, the Akt substrate of 160 kDa Thr642 (p < 0.05) and Akt Thr308 (p < 0.05) phosphorylations were significantly increased compared with the control treatment. At the same time point, 5'-AMP-activated protein kinase and acetyl-coenzyme A carboxylase phosphorylations were significantly lower (p < 0.05) in the epitrochlearis muscle incubated with 4 mmol/L of β-HB than in the control muscle. Our results demonstrate that postexercise 4 mmol/L β-HB administration enhanced glycogen repletion in epitrochlearis muscle. Four millimoles per liter β-HB treatment was associated with alternation of the phosphorylated status of several proteins involved in glucose uptake and metabolic/energy homeostasis at the early stage of postexercise.

Multi-dimensional Transcriptional Remodeling by Physiological Insulin In Vivo

Regulation of gene expression is an important aspect of insulin action but invivo is intertwined with changing levels of glucose and counter-regulatory hormones. Here we demonstrate that under euglycemic clamp conditions, physiological levels of insulin regulate interrelated networks of more than 1,000 transcripts in muscle and liver. These include expected pathways related to glucose and lipid utilization, mitochondrial function, and autophagy, as well as unexpected pathways, such as chromatin remodeling, mRNA splicing, and Notch signaling. These acutely regulated pathways extend beyond those dysregulated in mice with chronic insulin deficiency or insulin resistance and involve a broad network oftranscription factors. More than 150 non-coding RNAs were regulated by insulin, many of which alsoresponded to fasting and refeeding. Pathway analysis and RNAi knockdown revealed a role for lncRNA Gm15441 in regulating fatty acid oxidation in hepatocytes. Altogether, these changes in coding and non-coding RNAs provide an integrated transcriptional network underlying the complexity of insulin action.

Model-Based Tool for Personalized Adjustment of Basal Insulin Supply in Patients With Intensified Conventional Insulin Therapy.

The decisive factor in successful intensive insulin therapy is the ability to deliver need-based-adjusted nutrition-independent insulin dosages at the closest possible approximation to the physiological insulin level. Because this basal insulin requirement is strongly influenced by the patient's lifestyle, its subtlety is of great importance. This challenge is very different between patients with type 1 diabetes and those with insulin-dependent type 2 diabetes. Furthermore, it is more difficult to finetune a basal insulin dosage with intensified conventional insulin therapy (ICT), due to delayed insulin delivery, compared to insulin pump therapy, which provides continuous delivery of small doses of exclusively short-acting insulin. In all cases, the goal is to achieve an optimal basal delivery rate. We hypothesized that this goal could be achieved with a modeling tool that determined the optimal basal insulin supply based on the patient's anamnestic data and monitored glucose values. This type of modeling tool has been used in health insurance programs in Germany to improve insulin control in patients that receive ICT. Our retrospective data analysis showed that this modeling tool provided a significant improvement in metabolic control, significant reductions in HbA1c and Q scores, and improved time-in-range values, with reduced daily insulin levels. The model-based basal rate test could provide additional data of the actual effect of the basal insulin adjustment in intensified insulin treated diabetes to the physician or treatment team.

Peripheral Insulin Extraction in Non-Diabetic Subjects and Type 2 Diabetes Mellitus Patients.

Insulin has to be transported across the capillary endothelium to stimulate muscle glucose uptake. We investigated insulin uptake from the peripheral circulation in non-diabetic (ND) individuals and in type 2 diabetes (T2D) patients. Single-center cross-sectional study involving 40 ND (age 65±11 years) and 30 T2D patients (age 67±8 years). Thirty-six participants were studied in the fasted state (22 ND subjects and 14 T2D patients termed NDF and T2DF) and 34 participants 1-h following a glucose challenge (18 ND subjects and 16 T2D patients indicated as NDG and T2DG). Main outcome measure was fractional extraction (FE) of insulin (FEins) and glucose using the forearm balance method. In NDF, FEins was 18 (10-26) % at lower insulin levels (63 51-80] pmol/l), while in NDG at higher insulin levels (776 [543-1176] pmol/l), FEins was 9 (4-16) % (p = 0.01 vs. NDF). In NDF only, a negative correlation was observed between FEins and arterial plasma insulin load (rho = - 0.575;p = 0.006) and fasting plasma glucose levels (rho = - 0.551;p = 0.01). In T2DF FEins was 6 (1-19) % and not different from FEins in T2DG (10 2-14) %), and was not associated to fasting glucose. FEins tended to be higher in NDF compared to T2DF (p = 0.07). We propose that in ND individuals, besides passive diffusion, an active high-affinity pathway with limited capacity around lower physiologic insulin levels exists for insulin transendothelial transport, contributing to glycemic control. In T2D patients, this mechanism of peripheral insulin uptake is diminished or even absent. Modulation of insulin extraction from the circulation may be a novel target to improve glucose metabolism in T2D.

Dissection of Insulin-Dependent Pathways in Skeletal Muscle under Physiological and Diabetic Conditions

Insulin signaling regulates growth and metabolism, in part, by regulation of gene expression. To gain more insight into transcriptional pathways activated by physiological levels of insulin in vivo we performed euglycemic insulin clamp in mice at low (4 mU.kg-1.min-1) and high (12 mU.kg-1.min-1) concentrations of insulin and analyzed changes in gene expression in skeletal muscle by RNA-sequencing. At 3 h, low insulin regulated 451 genes while high insulin regulated 1771 genes by at least 1.5-fold (FDR&amp;lt;0.25). Interestingly, the most up regulated pathways in muscle were potential targets of estrogen-related receptor alpha (Esrra) and were related to TCA cycle and oxidative phosphorylation, while the most suppressed genes were targets of FoxO1 involved in autophagy. Insulin also inhibited the expression of over 40 genes involved in mRNA processing/splicing, mostly within the U2 ribonucleoprotein complex, which regulates the assembly and catalytic activity of the spliceosome. To determine how these insulin-dependent pathways might be altered in diabetes, we intersected the clamp gene expression data with gene expression data in streptozotocin (STZ) or high fat diet (HFD) obesity-induced diabetes focusing on genes oppositely regulated by insulin and diabetes. This approach revealed 93 insulin-regulated genes that were dysregulated in STZ and HFD muscle. Most related to TCA cycle and oxidative phosphorylation, however, changes in mRNAs involved in RNA processing/splicing also overlapped in HFD samples, thus indicating potential insulin resistance-specific gene expression signatures. Together these data demonstrate that insulin is a potent regulator of gene expression in vivo altering mRNA diversity through both changes in expression and dysregulation of the spliceosome, with the latter being most prominent in states of insulin resistance. These differences provide unique targets for therapeutic approaches to type 1 and type 2 diabetes involving muscle. Disclosure T.M. Batista: None. R. Garcia Martin: None. W. Cai: None. S. Suk: None. H. Noh: None. J.K. Kim: None. C. Kahn: Advisory Panel; Self; CohBar, ERX Therapeutics, AntriaBio, Inc.. Board Member; Self; Kaleio Biosciences.

Loss of CEACAM1 in Endothelial Cells Contributes to the Development of Cardiac Fibrosis

Carcinoembryonic Antigen-related Cell Adhesion Molecule 1 (CEACAM1) has been implicated in various physiological processes in the vascular systems, including blood vessel formation, and endothelial barrier function. Consistent with a role for hepatic CEACAM1 in promoting insulin sensitivity by mediating insulin clearance and maintaining physiologic plasma insulin levels, we have recently identified a role for hepatic CEACAM1 in preventing cardiac hypertrophy and dysfunction. The aim of this study was to elucidate the contribution of endothelial cell CEACAM1 in cardiac function and fibrosis that has remained elusive. To this end, we generated an endothelial cell-specific Ceacam1 knockout mouse line (VECadCc1−/−). Gomori Trichrome Blue Staining revealed a significant perivascular and interstitial fibrosis in the heart of VECadCc1−/− mice compared to their control littermates. Consistently, the mRNA levels of markers of fibrosis [collagen 1A1, α-SMA and fibroblast-specific protein1 (FSP1)] were elevated with a reciprocal decrease of endothelial cell makers (CD31), indicating endothelial mesenchymal transition (EndMT) in these mice. Cardiac fibrosis was associated with increased activation of the TGF-β1-Smad2/3 signaling pathway. Immunofluorescence staining co-localized CD31 with α-SMA in the myocardial perivascular section of VECadCc1−/− hearts. Moreover, mRNA level of α-SMA, FSP1, SNAIL and SLUG were up-regulated while that of CD31 was down-regulated in mouse heart endothelial cells (MHEC) isolated from VECadCc1−/− mice. The Akt/eNOS pathway was basally inactivated, leading to lower nitric oxide (NO) level in MHEC isolated from VECadCc1−/− mice. Moreover, loss of endothelial CEACAM1 caused reduced association with SHP-2 phosphatase and consequently, reduction in insulin-stimulated IRS-1/Akt/eNOS activation.These studies identified an important role for the loss of CEACAM1 in endothelial cells as a mechanism underlying cardiac fibrosis. Disclosure H.T. Muturi: None. H.E. Ghadieh: None. S.S. Khuder: None. S. Jash: None. R. Abu Helal: None. N.A. Daniels: None. J. Liu: None. V. Puri: None. G. Vazquez: None. R. Gupta: None. S.M. Najjar: None.

Drosophila insulin release is triggered by adipose Stunted ligand to brain Methuselah receptor.

Animals adapt their growth rate and body size to available nutrients by a general modulation of insulin-insulin-like growth factor signaling. In Drosophila, dietary amino acids promote the release in the hemolymph of brain insulin-like peptides (Dilps), which in turn activate systemic organ growth. Dilp secretion by insulin-producing cells involves a relay through unknown cytokines produced by fat cells. Here, we identify Methuselah (Mth) as a secretin-incretin receptor subfamily member required in the insulin-producing cells for proper nutrient coupling. We further show, using genetic and ex vivo organ culture experiments, that the Mth ligand Stunted (Sun) is a circulating insulinotropic peptide produced by fat cells. Therefore, Sun and Mth define a new cross-organ circuitry that modulates physiological insulin levels in response to nutrients.