Indiana University-Purdue University Indianapolis (IUPUI) / Insulin is a hormone that plays a central role in the regulation of human metabolism, and as a drug, is used in the treatment of diabetes mellitus. Hyperglycemia characterizes this condition due to a range of reasons from impaired insulin production by pancreatic beta cells to abnormalities resulting in resistance to insulin action. Depending on time and mechanism of action, the main types of insulin analogs are basal and prandial. Basal insulin analogs are slow-acting insulins that maintain a continuous basal level of insulin in the bloodstream. Prandial insulin analogs are fast-acting and their therapeutic goal is to avoid immediate and late post-prandial hyperglycemia. Most analogs face the problem of chemical degradation and amyloid-like fibril formation (fibrillation) in delivery devices. Thus, many modifications have been made to insulin in the effort to make it more stable and faster-acting. This thesis aims to study the effects of modifications that could be used to design an insulin analog with improved chemical and physical properties, while maintaining biological activity. We studied six amino-acid substitutions to native human insulin in different combinations: desB1 , AB2 , EB3, EA8 , EA14, and EB29. Analogs of the protein were chemically synthesized. Then, fibrillation and circular dichroism assays were performed using purified proteins. The results suggested that EB3 and EA14 are stabilizing modifications that prevent fibril formation, whereas EA8 and EA14 increase the structural stability of an analog. Our findings also suggested that certain modifications in isolation may not impact overall stability, but when combined with others, may show detectable effects, which is why EA8 and EA14 became the focus of further experiments. Cell-based activity assays indicated that all the analogs had similar biological activities. Future work will assess chemical degradation, solubility, amide proton exchange (as monitored by NMR), and mitogenicity.

Introduktion: Diabetes typ 1(T1DM) är en kronisk autoimmun sjukdom som är dödlig om den går obehandlad.  Immunförsvaret attackerar och förstör insulinproducerande β-cellerna som finns i de Langerhanska cellöarna i pankreas vilket leder till hel eller delvis insulinbrist. Symptom som polyuri, onormal trötthet, viktnedgång och polydipsi är det första tecknet på sjukdomen och uppkommer inte förrän 70 till 80% av β-cellerna är förstörda. Egenvården är komplex och innehåller blodsockerkontroller och insulintillförsel dygnet runt för resten av livet. Misskötsel av sjukdomen kan leda till hypoglykemi, hyperglykemi eller ketoacidos. Neuropati, diabetesretinopati och nefropati är komplikationer som kan uppstå vid långvarig T1DM. Sjukdomen behandlas med insulin-derivat som ges subkutant. Ett annat sätt att behandla T1DM är med teknosfär insulin som inhaleras. Exubera och Afrezza är två exempel på inhalationsinsulin. Exubera var den första inhalationsinsulinet på marknaden men drogs tillbaka av tillverkaren Pfizer två år efter lanseringen. Pfizer drog tillbaka läkemedlet på grund av låg försäljning vilket är ovanligt. Några år senare utvecklades Afrezza som en förbättrad version av Exubera och säljs enbart i USA och Brasilien. Afrezza ger hosta, hypoglykemi och en liten negativ påverkan på lungfunktionen.   Syfte: Syftet med arbetet var att undersöka om teknosfär insulin som inhaleras kan användas som ett alternativ för behandling mot T1DM. Metod: Informationen för det här litteraturarbetet hittades i databasen Pubmed, kurslitteraturen och internet. Clinicaltrials.gov användes för att få fram mer information och artiklar. Artiklarna valdes noggrant ut för att täcka helheten av teknosfär insulin. Resultat: Studien med Exubera gav inga definitiva slutsatser om behandlingseffekterna eftersom det var för litet antal händelser. Så en slutsats på att Exubera ger mer biverkningar kunde ej uteslutas. Artikel 2,3 och 4 gav någorlunda likartade resultat då en jämförelse mellan teknosfär insulin och subkutant insulin gjordes. Resultatet blev att teknosfär insulin som inhaleras har snabbare absorption och effekt men lägre verkningseffekt än subkutant insulin. Sista artikeln visade att okomplicerade akuta övre luftvägsinfektioner (URTI) inte har en påverkan på läkemedlet Afrezza eller på absorptionen av insulin. Diskussion: Exubera drogs från marknaden på grund av den låga försäljningen vilket kan bero på alla biverkningar som läkemedlet gav och även på inhalatorn. Priset på Exubera var en annan anledning till den dåliga försäljningen. Den prissattes dubbelt så högt jämfört med insulin som ges subkutant. Afrezza är en förbättrad version av Exubera där inhalatorn är mindre och är enbart godkänd i USA och Brasilien. Afrezza ger upphov till mindre hypoglykemihändelser men har en liten negativ påverkan på lungfunktionen. Teknosfär insulin är ett alternativ för patienter med nål- och sprutfobi. Slutsats: Teknosfär insulin är inte sämre läkemedel än insulin som ges subkutant. Det finns bra läkemedel ute på marknaden som fungerar bra och ger inga biverkningarna som Afrezza och Exubera. Detta kan vara en av faktorern som bidrar till mindre försäljning av läkemedlen.

Hormones are essential components in the body and their imbalance leads to pathological consequences. T2DM, insulin resistance and obesity are the most commonly occurring lifestyle diseases in the past decade. Also, an increased cancer incidence has been strongly associated with obese and T2DM patients. Therefore, our aim was to study the influence of high insulin levels in accumulating DNA damage in in vitro models and patients, through the induction of oxidative stress. The primary goal of this study was to analyze the genotoxicity induced by the combined action of two endogenous hormones (insulin and adrenaline) with in vitro models, through the induction of micronuclei and to see if they cause an additive increase in genomic damage. This is important for multifactorial diseases having high levels of more than one hormone, such as metabolic syndrome and conditions with multiple pathologies (e.g., T2DM along with high stress levels). Furthermore, the combination of insulin and the pharmacological inhibition of the tumor suppressor gene: PTEN, was to be tested in in vitro models for their genotoxic effect and oxidative stress inducing potential. As the tumor suppressor gene: PTEN is downregulated in PTEN associated syndromes and when presented along with T2DM and insulin resistance, this may increase the potential to accumulate genomic damage. The consequences of insulin action were to be further elucidated by following GFP-expressing cells in live cell-imaging to observe the ability of insulin, to induce micronuclei and replicative stress. Finally, the detrimental potential of high insulin levels in obese patients with hyperinsulinemia and pre-diabetes was to be studied by analyzing markers of oxidative stress and genomic damage. In summary, the intention of this work was to understand the effects of high insulin levels in in vitro and in patients to understand its relevance for the development of genomic instability and thus an elevated cancer risk. / In-vitro-Genotoxizitätsstudien mit hohen Konzentrationen von Insulin und die Kombination mit Adrenalin zeigten keinen additiven Anstieg der Mikrokernzahl. Der Insulinrezeptor und der AKT-Signalweg waren in den insulinvermittelten Genomschaden involviert. Die endogenen ROS-Quellen, Mitochondrien und NOX, waren an dem insulinvermittelten DNA-Schaden beteiligt. Hohe Konzentrationen von mitochondrialen ROS alleine, verursacht durch einen Komplex III Mitochondrien-Inhibitor, führten zu Zytotoxizität, aber nicht zu einer Zunahme des Genomschadens. Daher ist die durch das NOX-Enzym vermittelte ROS-Produktion wahrscheinlich der gemeinsame Faktor des genotoxischen Signalweges von Insulin und Adrenalin. Die Überstimulation des NOX-Enzyms führte zu einer Sättigung der zellulären biologischen Effekte und fehlender Additivität bei der Induktion von Genomschaden. Dies könnte jedoch unter physiologischen Bedingungen anders sein, da die Hormonspiegel niedriger sind und die ROS-Quellen nicht durch jedes einzelne der Hormone bereits maximal genutzt und daher erschöpft werden. Damit könnte die Möglichkeit eines additiven Genomschadens in vivo bestehen. Die Rolle des AKT-Signalwegs bei der Insulin-vermittelten genomischen Schädigung ist bereits etabliert und hier wurde nun die Funktion des negativen Regulatorproteins PTEN untersucht. Die Ergebnisse zeigten, dass die PTEN Inhibierung nicht nur zu einer erhöhten Genotoxizität durch MN-Induktion führte, sondern auch zur Beeinträchtigung der mitochondrialen Funktion. Obwohl kein Anstieg von ROS nach PTEN-Inhibierung beobachtet wurde, könnte die mitochondriale Dysfunktion zur metabolischen Imbalance sowie zur Zunahme des Genomschadens führen. Dies könnte insbesondere bei Patienten mit bestimmten PTEN-assoziierten Syndromen und Krebserkrankungen, die eine defekte PTEN-vermittelte Tumorsuppressorfunktion, DNA-Reparaturdefekte und kompromittierte antioxidative Abwehrmechanismen aufweisen, eine wichtige Rolle spielen. Wenn diese Patienten zusätzlich von Hyperinsulinämie betroffen sind, könnte eine Akkumulation von Genomschaden erfolgen und das Risiko zur Krebsentstehung wäre erhöht. Der Mechanismus der Genomschadensinduktion durch Insulin wurde bisher mit einer ROS-vermittelten DNA-Oxidation in Verbindung gebracht, aber noch nicht mit der mitogenen Signalgebung. Bei dieser beschleunigte das mitogene Potential des Insulins die Zellteilung und verursachte einen leichten replikativen Stress. Der milde replikative Stress könnte der Kontrolle durch die mitotischen Checkpoint-Proteine entgehen und zu Chromosomen-Fehlverteilungen und Chromosomenbrüchen führen. Dieser Effekt wurde in der Krebszelllinie Hela in Form von multipolaren Spindeln und Mikronuklei beobachtet und es ist nicht klar ob normale Zellen mit effizienterer Kontrolle dies verhindern könnten. Insgesamt könnte ein durch hohe Insulinspiegel vermittelter Schaden im Kontext anderer Komorbiditäten wie etwa PTEN Syndromen, metabolischem Syndrom oder Adipositas zu einer Akkumulation von DNA-Schäden führen. Schließlich zeigte die Analyse von Proben adipöser Patienten eine Zunahme von DNA-Schaden und oxidativem Stress im Vergleich zu den gesunden Kontrollen. Der Anstieg des DNA-Schadens war am höchsten in der Untergruppe der Patienten mit Insulinresistenz. Hoher Insulinspiegel bedeutet somit ein Risiko vom erhöhten oxidativen Stress und Genomschaden, insbesondere in Kombination mit Komorbiditäten. Erschwert wird das Verständnis dieser multifaktoriellen Zusammenhänge durch das komplexe Zusammenspiel von oxidativem Stress und seiner zellulären Regulation in vielen physiologischen sowie pathophysiologischen Prozessen. Daneben ist es eine Herausforderung, Genomschäden bei den geringen Wirkspiegeln hormoneller Effekte zu detektieren. Weitere Untersuchungen der komplexen Insulin-vermittelten Genomschadenswege werden notwendig sein, um mögliche Risiken der Hyperinsulinämie bei Erkrankungen wie Stoffwechselkrankheiten, Diabetes Typ 2 und Adipositas besser zu charakterisieren.

Abnormal regulation of growth hormone (GH) secretion has been reported in some patients with insulin-dependent diabetes (IDD). We compared the GH responses in 32 healthy subjects (age 25 ± 2 SE years) and in 23 IDD patients (28 ± 1.9 years old, diabetes duration 10.4 ± 2 years, and glycohemoglobin levels 9.3 ± 2.0%). During acute, severe hypoglycemia (glucose < 40 mg/dl), the mean GH levels were similar. When prolonged mild hypoglycemia was induced (58.0 ± 2.0 mg/dl in the controls and 54.0 ± 2.0 mg/dl in the IDD patients), the mean GH levels were similar, although the increase in GH was delayed in the latter group. During brief (30 min) exercise at 40-50% of VO2 max, GH rose comparably in both groups (IDD patients maintained euglycemia with basal insulin infusion). However, with more prolonged and intense exercise using a glucose clamp to maintain euglycemia, GH rose to 5.4 ± 2.2 ng/ml in controls and 26.4 ± 12.6 ng/ml in the diabetics (P < 0.05). When the combination of intense exercise and hypoglycemia (~ 55 mg/dl) was used, GH rose to a peak of 21.7 ± 2.7 ng/ml in the controls and to 33 ± 3.0 ng/ml in the diabetics (P = NS). Our data show that in insulin-infused IDD patients made euglycemic for these experiments: a) The GH response to acute, severe hypoglycemia was identical to that in the controls and the response to mild, prolonged hypoglycemia was delayed, but of similar magnitude compared with controls; b) Exercise-induced GH responses were observed in both groups, but exaggerated in the diabetics at a higher exercise intensity; c) Hypoglycemia during exercise produced an additive effect on GH secretion in the controls but not in the IDD patients. We conclude that the wide range of abnormal GH secretory responses in type I diabetes reflects a central, possibly hypothalamic, defect in GH regulation.

Recent studies have confirmed the presence of insulin receptors in the rat brain although their function has still not been well defined. The present study explores the possibility that insulin receptors in the brain can alter or contribute to central neurotransmission. Insulin caused a dose-dependent inhibition of hippocampal pyramidal neurons. The pattern of inhibition mirrored the binding kinetics of insulin in the hippocampus. Two related peptides, proinsulin and desoctapeptide insulin, had neuronal effects consistent with their binding to insulin receptors in the brain. Proinsulin was effective in doses 30-fold greater than insulin, whereas desoctapeptide insulin had little or no effect. These observations indicate that the inhibitory effect of insulin in this tissue may be insulin receptor-mediated and support a previously suggested functional role of insulin in the central nervous system.

Introduction: Metabolic syndrome and depression are considered to be important risk factors for the development of cardiovascular diseases. The prevalence of metabolic syndrome is estimated to be around 25% of the adult population in industrialized countries, including the population of Czech Republic. The prevalence of depression is estimated to be around 15% of the same adult population. It is not clear yet on the base of poor literature, which is so far available, whether there is a causal relationship between these factors or not. Objective: To try to find a relationship between metabolic syndrome and depression in a population sample using clinical and metabolic parameters. Methods: The prevalence of depressivity or other psychopathologies was evaluated with the use of self-report questionnaires in a randomly selected population sample of 259 people living in Pilsen. The questionnaires were mailed to the subjects. Those of them who responded were invited to the examination of anthropometric and laboratory parameters defining the metabolic syndrome and to the examination of some other parameters. The occurrence of risk factors of the metabolic syndrome of insulin resistance and the relationship between depression and metabolic syndrome was investigated. Metabolic syndrome of insulin resistance...

Commercial broiler chickens are withdrawn from feed prior to harvesting, transport and processing. This feed withdrawal period does not reduce carcass yield but is significant in reducing the potential for carcass contamination from undigested feed or fecal material that may remain in the digestive tract if feed is not withdrawn. However, withdrawing feed can result in an increase in bacterial contamination due to the decrease in physiological pH during fasting. Recently, consumers are more interested in how food animals are raised, prepared for, and processed. In response to these concerns, the feasibility of developing a “feed withdrawal” feed that could be provided to commercial broilers during the traditional feed withdrawal period was evaluated. The physiological effect of fasting during the feed withdrawal period is also not well understood. The focus of this study was to determine if feedstuffs that are readily accessible to commercial feed mills was evaluated to determine if body weight loss could be reduced and commercial broilers could be processed acceptably when allowed access to this feed during the traditional feed withdrawal period. In addition, the physiological response of commercial broilers at different ages to fasting was determined. The emphasis of this study was to determine the effect of fasting periods of commercial broilers on the hormones insulin and glucagon, circulating levels of glucose and body temperature compared to fulled birds. The results of this research suggests that a “feed withdrawal feed” that is available to commercial feed mills is feasible to allow commercial broilers access to feed during the traditional feed withdrawal period and still be processed with no contamination concerns. In addition, the impact of fasting on the hormones glucagon and insulin, circulating levels of glucose and body temperature were shown to be significantly changed during a fasting period.

This study examined effects of carbohydrate (C), carbohydrate-protein (CP), or placebo (P) beverages following eccentric resistance exercise on muscle damage by serum creatine kinase (CK), muscle protein breakdown by urinary 3-methylhistidine (3MH), muscle soreness, isokinetic muscle strength, muscle glycogen resynthesis, and serum hormones. Untrained males (N=26) underwent a 9-day controlled meat-free diet and 24 hr urine collections. To reduce glycogen, subjects cycled for 40 min at 70% of VO_2peakfollowed by 5 cycling sprints on day 4 evening. On day 5, fasted subjects performed 100 eccentric leg flexions at 120% of 1-RM and drank C (n=8, 1.25 g C/kg), CP (n=9, 0.875 g C/kg, 0.375 g protein/kg), or P (n=9) beverages immediate post-exercise (IPE) and 2 hr later. Muscle biopsies were taken IPE on day 5, and days 6 and 8 mornings. Blood was obtained days 4-10 fasted plus IPE, 3 hr, and 6 hr post-exercise on day 5. At 3 hr on day 5, insulin was higher for CP (24.6 ( 15.5 &amp;#181;IU/ml) and C (17.2 +/- 10.9 &amp;#181;IU/ml) than P (5.3 +/- 0.4 &amp;#181;IU/ml, p<.05). Glycogen was low on day 5, partially recovered on day 6, and normal by day 8 (p<.01) with no difference among groups. Isokinetic quadriceps peak torque at 60^o/s decreased 24% on day 6 and remained depressed by 21% on d 8 (p<.01) for all groups. Soreness peaked on day 7 and CK on day 8 (p<.01) with no differences by group. CK increased (p<.01) over day 5 (delta CP 24.6 +/- 19.1, delta P 39.2 +/- 71.6, delta C 70.8 +/- 60.4 U/L) and was highest for C (p<.05). On day 5, CP had lowest 3MH (193.0 +/- 13.8 &amp;#181;mol/d) versus C (251.1 +/- 22.5 &amp;#181;mol/d, p<.05). Testosterone at 3 hr on day 5 was lower for C (4.2 +/- 0.3 ng/ml) and CP (4.3 +/- 0.3 ng/ml) versus P (5.1 +/- 0.2 ng/ml, p<.05). In summary, glycogen, muscle strength and soreness were unaffected by beverage. However, a CP beverage may be beneficial for reducing muscle damage and protein breakdown on the day of eccentric resistance exercise. / Ph. D.

Cold exposure impacts intestinal remodelling and metabolism. Signaling by glucagon-like peptide 1 (GLP-1), GLP-2 and glucose-dependent insulinotropic polypeptide (GIP) are tightly linked to nutrient intake and absorption. However, these peptide hormones' necessity to mediate gut adaptation and metabolic alternations during cold exposure has been incompletely explored. We hypothesize that GLP-1, GIP, and GLP-2 are released in proportion to required energy needs during cold exposure to enable efficient nutrient absorption and gut adaptation and subsequently impact nutrient handling. We evaluated morphological changes in the intestinal in wildtype, Glp1r-/-Glp2r-/- and Glp1r-/-Gipr-/- mice exposed to chronic cold or thermoneutral conditions for four weeks. Food intake and gut hormone secretion were significantly increased in all mice housed at 4-6 ̊C compared to those housed at thermoneutrality. Concomitantly, we observed increased remodeling measured by crypt to villus height (increased villi length) and intestinal circumference (increased circumference) in cold-exposed wildtype and Glp1r-/-Gipr-/- mice housed. In contrast, intestinal morphology in Glp1r-/-Glp2r-/- mice was unchanged in response to cold. Associated with these morphometric changes, we observed significant increases in fasting concentrations of GLP-1. These data suggest that GLP-1 and GLP-2 are key signaling molecules secreted from the gut in response to chronic cold exposure to enable intestinal remodeling.

<p> This study was designed to investigate possible changes in insulin sensitivity (IS) with cessation of training. Six endurance trained athletes were studied at 12, 60 h and 7 days following cessation of training. In-vivo IS was established by a glucose clamp technique (Greenfield et al. Diabetes 30, 1981) and expressed as the metabolic clearance rate of glucose (MCR) in ml. plasma cleared kg-1. min-1. At 12 h after the last training session the mean MCR was 15.6+1.8 compared with 7.8+1.2(p<0.01) in age and weight matched sedentary controls. The MCR decreased to 10.1+1.0 after 60 h and decreased significantly to 8.5+0.5(p<0.05) after 7 days of detraining. In-vitro IS was measured by determining the insulin binding of fractionated young erythrocytes by the method of Polychronakos et al. (Clin. Inves. Med.4,14B,1981). Insulin binding was 10.4+0.9% at 12 h and decreased significantly to 8.1+0.7%/4xl0^9 cells after 60 h of detraining (p<0.001). In conclusion: 1) detraining of endurance athletes resulted in a rapid decrease in IS. After 7 days, glucose MCR reached values indistinguishable from sedentary controls. 2) changes in IS observed may be partially mediated by alterations in insulin binding to receptors. 3) since the high IS observed with endurance athletes on the initial test disappeared shortly after cessation of training, it is probably an acute effect of the last exercise bout rather than a chronic effect of training.</p> / Thesis / Master of Science (MSc)