Background: The augmented whole body glucose uptake response to insulin during the postprandial state is described as meal-induced sensitization (MIS). MIS occurs when the presence of food in the upper gastrointestinal tract (GIT) activates two feeding signals (activation of hepatic parasympathetic nerves and elevation of hepatic glutathione level), and causes insulin to release hepatic insulin sensitizing substance (HISS), which stimulates glucose uptake in peripheral tissues. The impairment of HISS release results in the absence of meal-induced insulin sensitization (AMIS), causing progression to a cluster of metabolic, vascular, and cardiac dysfunction, which we refer to as components of the AMIS syndrome. Objectives: The objective of my doctoral research was to study the manipulation of the HISS-pathway, in age- and diet-induced AMIS models, with exercise ± antioxidants. Also, in a separate project I studied the signaling pathways involved with the HISS action in skeletal muscle. Methods: The 7-day voluntary running was used as exercise intervention to manipulate the HISS pathway in healthy and prediabetic rats. The interaction of an antioxidant cocktail, SAMEC (S-adenosylmethionine + vitamin E + vitamin C), with the effects of exercise on postprandial insulin response was studied. Moreover, in the signaling studies the insulin and 5'-adenosine monophosphate activated protein kinase (AMPK) pathways were examined to test their possible involvement with the HISS action in skeletal muscle. Results: Voluntary running-wheel exercise for 7 days increases the postprandial glucose uptake response to insulin in health and in prediabetes through enhancement/restoration of HISS action. Supplementation with SAMEC during 7 days of exercise does not either harm or add benefits to the positive effects of exercise on insulin sensitivity. Finally, the signaling studies indicate that HISS increases the rate of glycogen synthesis in muscle through an insulin/AMPK-independent pathway.

Problem: Gestational obesity and insulin resistance pose a significant threat to the future health of our population. Mothers and children of these metabolically maladaptive pregnancies experience extensive morbidity and mortality. This study characterized postprandial insulin sensitivity in female Sprague Dawley rats at 5-days and 15-days gestation. Based on these findings, a model of gestational obesity was developed using 35% sucrose supplementation (SS). The efficacy of a preventative and a therapeutic intervention at modulating sucrose-induced gestational insulin resistance in Sprague Dawley rats was elucidated. Methods: Insulin sensitivity in the post-prandial state includes insulin-dependent and Hepatic Insulin Sensitizing Substance (HISS)-dependent components, and can be characterized with the Rapid Insulin Sensitivity Test (RIST). HISS is a putative hepatic factor released in the fed-state that selectively increases glucose uptake in skeletal muscle, kidney and heart. In the first phase of this study, the effects of insulin were assessed in pregnant animals (5 and 15-days gestation) and their virgin controls. Groups of 15-day gestation and virgin animals had SS for 8-weeks (with a 2-week recovery), 10-weeks or 22-weeks. Half of all of the 10-week SS animals were treated with either SAMEC (given chronically, containing S-adenosyl-methionine, vitamin C and vitamin E) or BENAC (given once the night before the acute study, containing bethanechol chloride and n-acetyl-l-cysteine). Body weight, weight gained over the gestational period, fat pad mass, post-prandial glycemia, plasma insulin and triglyceride concentrations were measured in all groups. Results: 5-days gestation was associated with preserved direct insulin action and increased HISS-dependent insulin action. 15-days gestation was associated with a mixed insulin resistance: both direct and HISS-dependent insulin action were reduced. SS in these pregnant and virgin rats eliminated HISS-dependent insulin action, associated with hyperinsulinemia, hypertriglyceridemia and obesity. In the SS group given 8-weeks of sucrose (then a 2-week recovery), virgins spontaneously partially recovered HISSdependent insulin action. At 15-days gestation, recovery was complete with reductions in plasma insulin and triglyceride concentrations, and normalization of body weight and fat pad mass. 10-week SS resulted in complete absence of HISS-dependent insulin action, and produced a model of gestational obesity. Prolonged (22-week) SS did not result in hyperglycemia or elevation of plasma insulin concentration above 10-week SS. SAMEC in 10-week SS 15-day pregnant and virgin rats prevented the loss of HISS-dependent insulin action, and normalized plasma insulin and triglyceride concentrations. BENAC given to 10-week SS virgin and 15-day pregnant rats normalized overall insulin responses secondary to restoration of HISS-dependent insulin action. This was accompanied by a reduction (for virgins) and normalization (at 15-days gestation) of plasma insulin and triglyceride concentrations. In 15-day pregnant controls (no sucrose), BENAC increased the HISS-dependent insulin action significantly above baseline and reduced plasma triglycerides and insulin below control levels. Conclusions: These results suggest an explanation for the insulin resistance occurring in pregnancy, whereby HISS may facilitate metabolic adaptation. HISS may represent a pathophysiological missing link in the insulin resistant disorders of pregnancy. These findings substantiate a series of unexplored treatments (including BENAC and SAMEC) for the epidemic of gestational obesity and diabetes in mothers-to-be and the deleterious metabolic programming occurring in the next generation.

Cathepsin S is a protease important in major histocompatibility complex (MHC) class II antigen presentation and also in degrading the extracellular matrix. Studies, most of them experimental, have shown that cathepsin S is involved in different pathological conditions such as obesity, inflammation, atherosclerosis, diabetes, and cancer. The overall hypothesis of this report is that high levels of circulating cathepsin S, is a biomarker that reflects pathology induced by inflammation and obesity. The overall aim of this report was to investigate possible associations between circulating cathepsin S, inflammation, glucometabolic disturbance, and its associated diseases in the community. As cathepsin S appears to be a novel risk marker for several pathological conditions, we also wanted to examine the effect of dietary intervention on circulating cathepsin S concentrations. This thesis is based on data from three community-based cohorts, the Uppsala longitudinal study of adult men (ULSAM), the prospective investigation of the vasculature in Uppsala seniors (PIVUS), and a post-hoc study from the randomized controlled NORDIET trial. In the first study, we identified a cross-sectional positive association between serum cathepsin S and two markers of cytokine-mediated inflammation, CRP and IL-6. These associations were similar in non-obese individuals. In longitudinal analyses, higher cathepsin S at baseline was associated with higher CRP and IL-6 levels after six years of follow-up. In the second study, we identified a cross-sectional association between increased serum levels of cathepsin S and reduced insulin sensitivity. These associations were similar in non-obese individuals. No significant association was observed between cathepsin S and insulin secretion. In longitudinal analysis, higher cathepsin S levels were associated with an increased risk of developing diabetes during the six-year follow-up. In the third study, we found that higher serum levels of cathepsin S were associated with increased mortality risk. Moreover, in the ULSAM cohort, serum cathepsin S was independently associated with cause-specific mortality from cardiovascular disease and cancer. In the fourth study, we identified that adherence to an ad libitum healthy Nordic diet for 6 weeks slightly decreased the levels of plasma cathepsin S in normal or marginally overweight individuals, relative to the control group. Changes in circulating cathepsin S concentrations were correlated with changes in body weight, LDL-C, and total cholesterol. Conclusion: This thesis shows that circulating cathepsin S is a biomarker that independently reflects inflammation, insulin resistance, the risk of developing diabetes, and mortality risk. Furthermore, a Nordic diet moderately reduced cathepsin S levels in normal-weight and overweight men and women. This effect may be partially mediated by diet-induced weight loss and possibly by reduced LDL-C concentrations.

Emerging evidence suggests that vitamin D may be associated with type 2 diabetes (T2DM), however current data are inconsistent regarding metabolic disorders underlying T2DM. The objectives of this thesis were to investigate the association of vitamin D with the primary pathophysiological disorders of type 2 diabetes: namely insulin resistance (IR) and beta (β)-cell dysfunction, and the metabolic syndrome (MetS). All studies included individuals participating in the PROspective Metabolism and ISlet cell Evaluation (PROMISE) cohort study, comprising 712 subjects 30 years and older, and at risk of T2DM at baseline. Serum 25-hydroxyvitamin D [25(OH)D] was measured to assess vitamin D nutritional status. Validated oral glucose tolerance test derived indices for IR and β-cell function were calculated. In the first cross-sectional study, multivariate linear regression analyses indicated a significant inverse association of serum 25(OH)D with IR (β=-0.003, p=0.007) and a significant positive association of 25(OH)D with β-cell function (β=0.004, p=0.03) at the baseline PROMISE clinic visit (n=712). In another cross-sectional study also conducted using data from the baseline PROMISE clinic visit, higher 25(OH)D was found to be significantly associated with a reduced presence of the MetS after multivariate adjustment (OR=0.76, 95% CI 0.62-0.93). Low serum 25(OH)D was also significantly associated with various MetS components. In light of the findings in the first cross-sectional study, the third study examined prospective associations of baseline 25(OH)D with 3-year follow-up IR and β-cell function (n=489). Although baseline 25(OH)D was not significantly associated with follow-up IR, a significant positive association of baseline 25(OH)D with β-cell function at follow-up was observed (β=0.005, p=0.015). Lastly, in a longitudinal substudy (n=127), seasonal changes in 25(OH)D over 2.5 years did not significantly affect changes in IR and β-cell function. In conclusion, results indicated that baseline serum 25(OH)D was cross-sectionally related to IR, β-cell function and the MetS, and was prospectively related to β-cell function at the 3-year follow-up. In addition, seasonal changes in 25(OH)D do not adversely affect IR and β-cell function over time. These findings suggest a potential role for higher 25(OH)D levels in reducing diabetes risk, although additional longitudinal studies are warranted.

Patients undergoing elective surgery typically fast for 8-12 hours before surgery. However, oral preoperative carbohydrate ingestion may increase postoperative insulin sensitivity and reduce complications. To determine the effects of carbohydrate supplementation prior to CABG or spinal surgery, 38 patients were randomized to receive a carbohydrate supplement or to fast for 12 hours preoperatively. Baseline and postoperative measurements of insulin sensitivity were completed using the short insulin tolerance test and homeostasis model assessment (HOMA). Patient discomfort was measured immediately before surgery. Insulin sensitivity was not significantly different between groups. However, the supplemented group experienced a significantly smaller rise in glucose levels following surgery (p=0.03) and had higher postoperative HOMA-β scores (p=0.02). Fasted patients were significantly more thirsty (p=0.01), hungry (p=0.04) and anxious (p=0.01) before surgery and experienced a significantly longer hospital stay (p=0.008). Carbohydrate supplementation improved outcomes, warranting re-evaluation of fasting practices prior to major surgery.

Central resistance to the actions of insulin and leptin is strongly associated with obesity and type 2 diabetes mellitus (T2DM). These anorexigenic hormones modulate one another’s actions at the neuronal level. To investigate the cellular events underlying the effect of insulin resistance on leptin modulation of hypothalamic neurons, a neuronal cell model was established. The rHypoE-19 cell line expresses the insulin and leptin receptors alongside a complement of signaling molecules rendering it an appropriate model to study the molecular events underlying leptin and insulin crosstalk. Hyperinsulinemia was used to induce insulin resistance and leptin regulation of the rHypoE-19 neurons was analyzed prior to and following the induction of insulin resistance. It was found that the attenuation of insulin signal transduction affects leptin signaling and transcriptional modulation of the rHypoE-19 neurons. These studies will ultimately lend itself to an improved understanding of the complex cellular events that accompany neuronal hormone resistance.

Emerging evidence suggests that vitamin D may be associated with type 2 diabetes (T2DM), however current data are inconsistent regarding metabolic disorders underlying T2DM. The objectives of this thesis were to investigate the association of vitamin D with the primary pathophysiological disorders of type 2 diabetes: namely insulin resistance (IR) and beta (β)-cell dysfunction, and the metabolic syndrome (MetS). All studies included individuals participating in the PROspective Metabolism and ISlet cell Evaluation (PROMISE) cohort study, comprising 712 subjects 30 years and older, and at risk of T2DM at baseline. Serum 25-hydroxyvitamin D [25(OH)D] was measured to assess vitamin D nutritional status. Validated oral glucose tolerance test derived indices for IR and β-cell function were calculated. In the first cross-sectional study, multivariate linear regression analyses indicated a significant inverse association of serum 25(OH)D with IR (β=-0.003, p=0.007) and a significant positive association of 25(OH)D with β-cell function (β=0.004, p=0.03) at the baseline PROMISE clinic visit (n=712). In another cross-sectional study also conducted using data from the baseline PROMISE clinic visit, higher 25(OH)D was found to be significantly associated with a reduced presence of the MetS after multivariate adjustment (OR=0.76, 95% CI 0.62-0.93). Low serum 25(OH)D was also significantly associated with various MetS components. In light of the findings in the first cross-sectional study, the third study examined prospective associations of baseline 25(OH)D with 3-year follow-up IR and β-cell function (n=489). Although baseline 25(OH)D was not significantly associated with follow-up IR, a significant positive association of baseline 25(OH)D with β-cell function at follow-up was observed (β=0.005, p=0.015). Lastly, in a longitudinal substudy (n=127), seasonal changes in 25(OH)D over 2.5 years did not significantly affect changes in IR and β-cell function. In conclusion, results indicated that baseline serum 25(OH)D was cross-sectionally related to IR, β-cell function and the MetS, and was prospectively related to β-cell function at the 3-year follow-up. In addition, seasonal changes in 25(OH)D do not adversely affect IR and β-cell function over time. These findings suggest a potential role for higher 25(OH)D levels in reducing diabetes risk, although additional longitudinal studies are warranted.

Patients undergoing elective surgery typically fast for 8-12 hours before surgery. However, oral preoperative carbohydrate ingestion may increase postoperative insulin sensitivity and reduce complications. To determine the effects of carbohydrate supplementation prior to CABG or spinal surgery, 38 patients were randomized to receive a carbohydrate supplement or to fast for 12 hours preoperatively. Baseline and postoperative measurements of insulin sensitivity were completed using the short insulin tolerance test and homeostasis model assessment (HOMA). Patient discomfort was measured immediately before surgery. Insulin sensitivity was not significantly different between groups. However, the supplemented group experienced a significantly smaller rise in glucose levels following surgery (p=0.03) and had higher postoperative HOMA-β scores (p=0.02). Fasted patients were significantly more thirsty (p=0.01), hungry (p=0.04) and anxious (p=0.01) before surgery and experienced a significantly longer hospital stay (p=0.008). Carbohydrate supplementation improved outcomes, warranting re-evaluation of fasting practices prior to major surgery.

Central resistance to the actions of insulin and leptin is strongly associated with obesity and type 2 diabetes mellitus (T2DM). These anorexigenic hormones modulate one another’s actions at the neuronal level. To investigate the cellular events underlying the effect of insulin resistance on leptin modulation of hypothalamic neurons, a neuronal cell model was established. The rHypoE-19 cell line expresses the insulin and leptin receptors alongside a complement of signaling molecules rendering it an appropriate model to study the molecular events underlying leptin and insulin crosstalk. Hyperinsulinemia was used to induce insulin resistance and leptin regulation of the rHypoE-19 neurons was analyzed prior to and following the induction of insulin resistance. It was found that the attenuation of insulin signal transduction affects leptin signaling and transcriptional modulation of the rHypoE-19 neurons. These studies will ultimately lend itself to an improved understanding of the complex cellular events that accompany neuronal hormone resistance.

Bradykinin (BK) acts through eNOS to reduce MAPK-mediated feedback inhibition of insulin signalling. Preliminary data suggest that the sGC-cGMP-PKG pathway, a prominent NO target, is involved. Our present study aimed to support the role of this pathway with atrial natriuretic peptide (ANP), which uses a receptor associated GC (NPR-A) to generate cGMP. We found that treating adipocytes with ANP mimicked BK effects on insulin-stimulated glucose uptake, Tyr-IRS-1 and Akt/PKB phosphorylation, as well as JNK and ERK1/2 inhibition. These outcomes depended on GC-cGMP-PKG signalling since A71915 (NPR-A antagonist), and KT-5823 (PKG inhibitor), completely abrogated them, while zaprinast (phosphodiesterase inhibitor), prolonged ANP actions. Furthermore, decreased MAPK phosphorylation was independent of upstream kinase activity, suggesting that MAPK phosphatases may be involved. These data indicate that BK and ANP act through the GC-cGMP-PKG pathway to potentiate insulin signalling via attenuated feedback inhibition. Stimulating the GC-cGMP-PKG pathway may, therefore, be a promising therapy for T2DM.