No description available.

Obesity, a disease characterized by excess adipose tissue (AT), is a growing worldwide epidemic. The Centers for Disease Control and Prevention (CDC), in 2017-2018, reported the prevalence of obesity in adults in the United States was 42.4% . Obesity increases the risk for many other serious health conditions such as type 2 diabetes, cardiovascular diseases, stroke, and some cancers. In individuals with obesity, the hypertrophic expansion of adipocytes, the main cell type within AT, is not matched by new vessel formation, leading to AT hypoxia. As a result, hypoxia inducible factor-1⍺ (HIF-1⍺) accumulates in adipocytes inducing a transcriptional program that upregulates profibrotic genes and biosynthetic enzymes such as lysyl oxidase (LOX) synthesis. This excess synthesis and crosslinking of extracellular matrix (ECM) components cause AT fibrosis. Although fibrosis is a hallmark of obese AT, the role of fibroblasts, cells known to regulate fibrosis in other fibrosis-prone tissues, is not well studied. Adipocytes are mechanoresponsive and affected by different microenvironmental cues, including hypoxia and mechanical (un)loading. Yet, no study has focused on the role of the aforementioned factors on the adipocyte mechanical response, including actin cytoskeletal remodeling. This dissertation aims to develop an in vitro model of healthy/diseased AT to explore the effect of microenvironmental cues on adipocyte function and actin cytoskeletal remodeling. The first aim is to study (1) the crosstalk between fibroblasts and adipocytes in a co-culture model and (2) the effect of hypoxia on the ras homolog gene family member A (RhoA)/Rho-associated coiled-coil kinases (ROCK) mechanical pathway and actin cytoskeletal remodeling in adipocytes. We confirmed that hypoxia creates a diseased phenotype by inhibiting adipocyte maturation and inducing actin stress fiber formation facilitated by myocardin-related transcription factor A (MRTF-A/MKL1) nuclear translocation. The second aim explores the effects of mechanical unloading (simulated microgravity) on key adipocyte functions and actin cytoskeletal remodeling. This study demonstrated that mechanical unloading enhances adipocyte maturation via increased lipogenesis and lipolysis and cortical actin remodeling, which together further enhanced glucose uptake. However, disrupting cortical actin remodeling by using inhibitors or exposure to a high concentration of free fatty acids (FFAs) diminished enhanced adipocyte functions observed in simulated microgravity. Overall, the results of these studies support the importance of microenvironmental cues on adipocyte actin cytoskeletal remodeling. Therefore, targeting mechanical pathways that regulate actin cytoskeletal remodeling can be used to improve adipocyte function and AT metabolism and possibly treat related diseases such as type 2 diabetes and obesity. / Bioengineering

INTRODUCTION: Exercise increases insulin stimulated glucose uptake (insulin action) if expended energy (kcal) is withheld following exercise, but the effect is blunted when expended energy is replaced as carbohydrate. Restricting carbohydrate and replacing expended energy as fat maintains increased insulin action in rodents; however, this effect has not been evaluated in humans. In humans, restricting carbohydrate intake following exercise may be a useful strategy to maximize the effect of individual exercise bouts on insulin action and promote gains in metabolic health over time. Therefore, the purpose of this study was to determine if carbohydrate restriction following exercise (carbohydrate deficit) increased insulin action in sedentary, overweight adults as hypothesized. METHODS: Ten healthy, sedentary, men and women, aged 21±2 years, body fat 37.3±3.1%, and VO2peak 34.6±1.2ml×kg-1×min-1 completed three, two-day experimental conditions in random order: 1) a no-exercise baseline condition (BASE), 2) exercise followed by a high-carbohydrate meal (HIGH-CHO= 76.3±2.5% CHO), and 3) exercise followed by a low-carbohydrate meal (LOW-CHO=17.8±0.1% CHO). On DAY 1, subjects came to the laboratory (early evening) and expended 30% of total daily energy expenditure on a cycle ergometer at 70% of VO2peak. Following exercise, an isocaloric meal (HIGH-CHO or LOW-CHO) was consumed to refeed the expended energy during exercise and venous blood samples were taken to record the insulin and glucose responses to the meals. Twelve hours later (Day 2), whole-body insulin action (steady-state glucose uptake per unit insulin) was measured using a continuous infusion of glucose with stable isotope tracers. A paired t-test was used to detect differences between exercise bouts and the glucose and insulin responses to the post-exercise meals. A one-way repeated measures ANOVA was performed to evaluate the effect of experimental condition on insulin action (p<0.05, for all tests). RESULTS: Intensity (VO2peak), duration (minutes) and energy expenditure (kcal) were similar between exercise bouts. After exercise, plasma glucose and insulin concentrations were significantly higher following the HIGH-CHO meal compared to the LOW-CHO meal (p<0.001, respectively). The next morning, insulin action was similar between experimental conditions (p=0.30). Non-oxidative glucose disposal was increased during the glucose infusion in Low-CHO compared to BASE (27.2±3.2 vs. 16.9±3.5µM×kg-1×min-1, p<0.05). Carbohydrate oxidation was reduced in Low-CHO (8.6±1.3µM×kg-1×min-1) compared to High-CHO (12.2±1.2µM×kg-1×min-1), and to BASE (17.1 ± 2.2 µM×kg-1×min-1), p<0.05 respectively. Resting fat oxidation was increased in Low-CHO compared to BASE (109.8 ± 10.5 mg×min-1 vs. 80.7 ± 9.6 mg×min-1, p<0.05) and remained elevated during the glucose infusion. CONCLUSION: Limiting carbohydrate, but not energy intake after exercise (carbohydrate deficit) resulted in increased non-oxidative glucose disposal, decreased carbohydrate oxidation and increased fat oxidation during the glucose infusion, compared to baseline, indicating a favorable shift in energy metabolism. Creating a carbohydrate deficit, by withholding expended carbohydrate but not energy following exercise may be a sensible strategy to promote favorable gains in insulin action that requires further evaluation.

Inactivity reduces insulin action. Energy surplus causes similar reductions to insulin action. Unless energy intake is reduced to match low energy expenditure during inactivity, a concurrent energy surplus may account for the lower insulin action. This study evaluated the effect of inactivity (sitting) with and without energy surplus on insulin action. Fourteen young (26.1 ± 4.5 years (M ± SD)), lean (23.7 ± 7.1% fat), fit (VO 2peak = 49.1 ± 3.3 ml*kg -1 *min -1 ) men (n=7) and women (n=7) completed each of 3, 24-hour conditions: an active condition (i.e. high energy expenditure with energy intake matched to expenditure) = ACTIV; (2) reduced energy expenditure (inactivity) with no reduction in energy intake (i.e. energy surplus) = INACTIV; (3) inactivity with energy intake reduced to match low energy expenditure = INACTIV LO-CAL. Insulin action was measured during a glucose infusion the following morning. Data were analyzed using linear mixed-effects models with planned contrasts. Compared to ACTIV, insulin action, defined as whole-body rate of glucose disappearance ( R d ) scaled to steady-state plasma insulin, was reduced 39% in INACTIV ( p < 0.001) and by 18% in INACTIV LO-CAL ( p = 0.07). Insulin action was also higher in INACTIV LO-CAL compared to INACTIV ( p =0.04). These results suggest that 1 day of sitting elicits large reductions in insulin action. Energy surplus accounts for half of the decline in insulin action, suggesting other factors are involved in the metabolic response to inactivity.

No description available.

No description available.

Adrenergic signalling is part of the sympathetic nervous system and is activated upon stimulation by the catecholamines epinephrine and norepinephrine. This regulates heart rate, energy mobilization, digestion and helps to divert blood flow to important organs. Insulin is released to regulate metabolism of carbohydrates, fats and proteins, mainly by taking up glucose from the blood. The insulin and the catecholamine hormone systems are normally working as opposing metabolic regulators and are therefore thought to antagonize each other. One of the major regulators involved in insulin signalling is the mechanistic target of rapamycin (mTOR). There are two different complexes of mTOR; mTORC1 and mTORC2, and they are essential in the control of cell growth, metabolism and energy homeostasis. Since mTOR is one of the major signalling nodes for anabolic actions of insulin it was thought that catecholamines might oppose this action by inhibiting the complexes. However, lately there are studies demonstrating that this may not be the case. mTOR is for instance part of the adrenergic signalling pathway resulting in hypertrophy of cardiac and skeletal muscle cells and inhibition of smooth muscle relaxation and helps to regulate browning in white adipose tissue and thermogenesis in brown adipose tissue (BAT). In this thesis I show that β-adrenergic signalling leading to glucose uptake occurs independently of insulin in skeletal muscle and BAT, and does not activate either Akt or mTORC1, but that the master regulator of this pathway is mTORC2. Further, my co-workers and I demonstrates that β-adrenergic stimulation in skeletal muscle and BAT utilizes different glucose transporters. In skeletal muscle, GLUT4 is translocated to the plasma membrane upon stimulation. However, in BAT, β-adrenergic stimulation results in glucose uptake through translocation of GLUT1. Importantly, in both skeletal muscle and BAT, the role of mTORC2 in β-adrenergic stimulated glucose uptake is to regulate GLUT-translocation. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.</p>

Ovine placental lactogen (oPL) is postulated to be involved in the repartitioning of maternal nutrients during pregnancy, through its effect on insulin metabolism. Ovine pancreatic insulin responses to exogenous glucose are depressed during pregnancy and this depression becomes more pronounced as gestation advances. In addition, under the hormonal environment of rising oPL and growth hormone (oGH) concentrations, maternal whole body glucose irreversible loss (GIL) increases. The percentage of GIL accounted for by uterine glucose uptake also increases with advancing gestation and increasing litter size. Regression analysis of oPL concentration with glucose uterine uptake as a percentage of GIL, accounts for 39% of variation. Maternal oPL concentrations which increase with gestational age, were significantly greater in multiple bearing ewes and ewes subjected to reduced metabolisable energy (ME) intakes. It is postulated that through actions on pancreatic sensitivity, oPL plays a major role as a homeorhetic control during pregnancy. Elevated oPL concentrations were strongly associated with continually depressed pancreatic insulin secretory ability. The reduction in pancreatic sensitivity to glucose was not as a result of elevation in GH or non-esterified fatty acid (NEFA) concentrations. Muscle insulin receptor number and affinity were found to increase with increasing litter size, suggesting that pregnancy associated insulin resistance occurs predominantly in adipose tissue. During ovine pregnancy there is a specific stimulation of maternal gluconeogenesis. As gestation advances, an increasingly greater proportion of this glucose is partitioned to the gravid uterus. The development of insulin resistance, together with the suppression of pancreatic activity, ensures the preferential uptake of glucose by non-insulin dependent tissues over insulin dependent tissues. These activities favour uterine glucose uptake, decrease adipose glucose uptake, and also promote adipose mobilisation and hepatic gluconeogenesis, so as to meet the increasing energy requirement of pregnancy. It is postulated that through these effects on insulin secretion and associated adipose tissue mobilisation factors, oPL plays a major role in homeorhesis during pregnancy. / Doctor of Philosophy (PhD)

The aim of the present thesis was to further increase our understanding of mechanisms contributing to and maintaining cellular insulin resistance in type 2 diabetes (T2D). For this reason, the effects of high glucose and insulin levels on glucose transport capacity and insulin signaling, with emphasis on insulin receptor substrate 1 (IRS-1) were assessed in fat cells. Altered levels of IRS-1 have previously been observed in adipose tissue from insulin-resistant and T2D subjects. A high glucose level (≥15 mM) for 24 h exerted only a minor impairment on glucose transport capacity in human adipocytes, as opposed to rat adipocytes. However, when combined with a high insulin level (104 µU/ml), basal and insulin-stimulated glucose transport was significantly impaired in both human and rat adipocytes. This was associated with a depletion of IRS-1 and IRS-2 protein levels in rat adipocytes, as a result of post-translational changes and altered gene transcription, respectively. In human adipocytes was only IRS-1 protein levels reduced. The high glucose/high insulin setting achieved maximal impairment of glucose transport within 6 h. Subsequent incubations of rat adipocytes under physiological conditions could partially restore insulin sensitivity. Interestingly, in both human and rat fat cells, decreased levels of IRSs occurred after the establishment of impaired glucose transport, suggesting that the observed depletion of IRSs is a consequence rather than a cause of insulin resistance. Nonetheless, IRS depletion is likely to further aggravate insulin resistance. Tyrosine phosphorylation of IRS-1 upon insulin stimulation activates the signaling pathway that mediates glucose transport. Pre-treatment of human adipocytes with high glucose and insulin levels was not associated with any alterations in the total IRS-1 Tyr612 phosphorylation following 10 min insulin stimulation. However, a significant increase in basal Tyr612 phosphorylation was observed. Furthermore, a rise in basal IRS-1 Ser312 phosphorylation was found. This is associated with reduced IRS-1 function and is considered to target IRS-1 to degradation pathways, and thus could potentially explain the observed decrease in IRS-1 protein levels. Our results imply an enhanced activation of insulin’s negative-feedback control mechanism that inhibit IRS-1 function. This could potentially have contributed to the observed impairment of insulin action on glucose transport in these cells. Accordingly, we have also shown that the downstream activation of protein kinase B upon insulin-stimulation is significantly impaired in human adipocytes exposed to the high glucose/high insulin setting, indicating a defect in the signaling pathway mediating glucose transport. We also investigated whether there are humoral factors in the circulation of T2D patients that contribute to peripheral insulin resistance. Human adipocytes cultured for 24 h in medium supplemented with 25% serum from T2D subjects, as compared to serum from non-diabetic subjects, displayed significantly reduced insulin-stimulated glucose uptake capacity. The effect could neither be attributed to glucose, insulin, FFA, TNF-α or IL-6 levels in the serum, but other circulating factor(s) seem to be of importance. In conclusion, chronic conditions of elevated glucose and/or insulin levels all impair insulin action on glucose turnover, but to different extents. A clear distinction between rat and human fat cells in the response to these different milieus was also observed. Alterations in the function of the key insulin signaling protein IRS-1 might be involved in the mechanisms underlying the impaired glucose uptake capacity. IRS-1 reduction however, occurs after but probably aggravates the existing insulin resistance. The effects of high glucose and/or insulin levels may be of importance in T2D, but additional novel factors present in the circulation of T2D patients seem to contribute to cellular insulin resistance.

An der Spitze der Morbiditäts - und Mortalitätsstatistik steht weltweit das Metabolische Syndrom, bestehend aus androider Adipositas, pathologischer Glukosetoleranz, Dyslipidämie und arterieller Hypertonie, verbunden mit einer erhöhten Inzidenz atherosklerotischer Gefäßerkrankungen. Der Replikationsinitiator 1 (Repin1) wurde kürzlich als mögliches Kandidatengen für Adipositas sowie damit verbundene metabolische Funktionsstörungen in kongenen sowie subkongenen Rattenstämmen identifiziert. Ziel der Arbeit war es, den Einfluss von Repin1 auf den Fettzellstoffwechsel zu untersuchen. Hierfür wurde die Expression von Repin1 in 3T3– L1 Präadipozyten und differenzierten 3T3-L1 Adipozyten mittels siRNA Technologie stark vermindert, um so auf mögliche Funktionen des Proteins schließen zu können. Nachfolgend wurden Veränderungen des Zellstoffwechsels mittels Glukosetransport, Palmitataufnahme sowie Triglyceridgehalt der Adipozyten untersucht. Repin1 wird in der 3T3-L1 Zelllinie exprimiert und zeigt eine steigende Expression während der Adipogenese. Der Knockdown von Repin1 resultierte in kleineren Fettzellen mit geringerer basaler, jedoch verstärkter insulinstimulierter Glukoseaufnahme. Auch der Fettstoffwechsel zeigte sich alteriert: Neben einer reduzierten Palmitataufnahme war die Expression verschiedener Schlüsselgene der Fetttropfenfusion, des Glukose-sowie des Fetttransportes verändert. Fazit: Repin1 reguliert die Expression von Genen, die eine Rolle bei der Festlegung der Fettzellgröße und des basalen und Insulin-stimulierten Glukosetransports in Adipozyten spielen.