Browsing by Subject "Diabetes"

Aims: Measurement of glycated hemoglobin (HbA1c) is an important indicator of glucose control over time. Point-of-care (POC) devices allow for rapid and convenient measurement of HbA1c, greatly facilitating diabetes care. We assessed two POC analyzers in the Peruvian Amazon where laboratory-based HbA1c testing is not available.

Methods: Venous blood samples were collected from 203 individuals from six different Amazonian communities with a wide range of HbA1c, 4.4-9.0% (25-75 mmol/mol). The results of the Afinion AS100 and the DCA Vantage POC analyzers were compared to a central laboratory using the Premier Hb9210 high-performance liquid chromatography (HPLC) method. Imprecision was assessed by performing 14 successive tests of a single blood sample.

Results: The correlation coefficient r for POC and HPLC results was 0.92 for the Afinion and 0.93 for the DCA Vantage. The Afinion generated higher HbA1c results than the HPLC (mean difference = +0.56% [+6 mmol/mol]; p < 0.001), as did the DCA Vantage (mean difference = +0.32% [4 mmol/mol]). The bias observed between POC and HPLC did not vary by HbA1c level for the DCA Vantage (p = 0.190), but it did for the Afinion (p < 0.001). Imprecision results were: CV = 1.75% for the Afinion, CV = 4.01% for the DCA Vantage. Sensitivity was 100% for both devices, specificity was 48.3% for the Afinion and 85.1% for the DCA Vantage, positive predictive value (PPV) was 14.4% for the Afinion and 34.9% for the DCA Vantage, and negative predictive value (NPV) for both devices was 100%. The area under the receiver operating characteristic (ROC) curve was 0.966 for the Afinion and 0.982 for the DCA Vantage. Agreement between HPLC and POC in classifying diabetes and prediabetes status was slight for the Afinion (Kappa = 0.12) and significantly different (McNemar’s statistic = 89; p < 0.001), and moderate for the DCA Vantage (Kappa = 0.45) and significantly different (McNemar’s statistic = 28; p < 0.001).

Conclusions: Despite significant variation of HbA1c results between the Afinion and DCA Vantage analyzers compared to HPLC, we conclude that both analyzers should be considered in health clinics in the Peruvian Amazon for therapeutic adjustments if healthcare workers are aware of the differences relative to testing in a clinical laboratory. However, imprecision and bias were not low enough to recommend either device for screening purposes, and the local prevalence of anemia and malaria may interfere with diagnostic determinations for a substantial portion of the population.

SIRT3 is an NAD+-dependent mitochondrial protein deacetylase purported to influence cellular and systemic metabolism through modulation of the mitochondrial acetylproteome. Fuel-stimulated insulin secretion from pancreatic islets involves mitochondrial metabolism and might be susceptible to SIRT3-mediated effects. To investigate this idea, we used CRISPR/Cas9 technology to obtain complete SIRT3 knockout in the INS-1 832/13 insulinoma cell line. In the context of this SIRT3 knockout cell line, we re-expressed wild-type SIRT3, β-Galactosidase, or one of three enzymatically inactive mutant forms of SIRT3 to generate lines representing a wide range of SIRT3 expression and mitochondrial protein deacetylase activity. We performed large-scale acetylproteome profiling by mass spectrometry on the different lines, and observed wide-spread, SIRT3-dependent changes in acetylation of enzymes involved in fatty acid oxidation, the TCA cycle, and the electron transport chain. Remarkably, despite these broad changes, the cell lines had indistinguishable insulin secretion responses to glucose or pyruvate, and exhibited no differences in function or viability in response to metabolic or ER stress-inducing agents. Moreover, metabolomic profiling revealed that, when compared to SIRT3-null cell lines, expression of wild-type SIRT3 does not result in appreciable changes in a host of organic acid, amino acid or fatty acid-derived acylcarnitine metabolites during glucose stimulation.

We also studied mice with global SIRT3 knockout (KO) fed a standard chow (STD) or high-fat/high-sucrose (HFHS) diet. Importantly, we performed these studies in the C57Bl/6J background in which we replaced the mutant allele of nicotinamide nucleotide transhydrogenase (NNT) present in the “J” substrain, with the wild-type allele in order to restore endogenous NNT function. SIRT3 KO and wild-type (WT) mice fed a STD diet exhibited no differences in insulin secretion during oral or IP glucose tolerance tests, and the function of islets isolated from these mice was indistinguishable in islet perifusion studies conducted with a broad array of secretagogues. Only when chronically fed a HFHS diet did SIRT3 KO animals exhibit a modest impairment in insulin secretion, but without an effect on glycemic control. Our broad conclusion is that major changes in mitochondrial protein acetylation in response to manipulation of SIRT3 are not sufficient to cause changes in islet function or metabolism. However, under conditions of chronic nutritional stress (feeding of a HFHS diet for 12 weeks), a negative effect on function appears, suggesting that islets are more susceptible to nutrition-induced factors (oxidative stress, local cytokine production, etc.) when SIRT3 is absent. Further studies will be required to identify factors that may interact with SIRT3 deficiency and mitochondrial protein hyperacetylation to increase the risk of -cell dysfunction.

Studies in the ankyrin-B+/- mouse reveal that ankyrin-B deficiency is associated with both the benefits of enhanced cardiac contractility and the costs of arrhythmia, early senescence, reduced lifespan, and impaired glucose tolerance. This constellation of traits is known as ankyrin-B syndrome, which may have important implications for humans possessing functional ankyrin-B mutations. We found that ankyrin-B variants are surprisingly common, ranging from 2 percent of European individuals to 8 percent in individuals from West Africa. Furthermore, by studying of the metabolic phenotype associated with ankyrin-B mouse, we have uncovered a major new dimension to ankyrin-B syndrome, a link between ankyrin-B and parasympathetic control of insulin secretion. Stimulation of pancreatic beta cells by acetylcholine augments glucose-stimulated insulin secretion by inducing inositol-trisphosphate receptor (InsP3R)-mediated Ca2+ release. We report that ankyrin-B is also enriched in pancreatic beta cells. Ankyrin-B-deficient islets display impaired potentiation of insulin secretion by the muscarinic agonist carbachol, blunted carbachol-mediated intracellular Ca2+- release, and reduced InsP3R stability. Ankyrin-B(+/-) mice also display postprandial hyperglycemia, consistent with impaired parasympathetic potentiation of glucose-stimulated insulin secretion. R1788W mutation of ankyrin-B impairs its function in pancreatic islets and associates with type 2 diabetes in Caucasians and Hispanics. Finally, we have generated knockin mice corresponding to the R1788W and L1622I mutations. Functional characterization of these animals will allow us to better understand the relationship between human ankyrin-B variants and ankyrin-B syndrome.

A large proportion of the variation in traits between individuals can be attributed to variation in the nucleotide sequence of the genome. The most commonly studied traits in human genetics are related to disease and disease susceptibility. Although scientists have identified genetic causes for over 4,000 monogenic diseases, the underlying mechanisms of many highly prevalent multifactorial inheritance disorders such as diabetes, obesity, and cardiovascular disease remain largely unknown. Identifying genetic mechanisms for complex traits has been challenging because most of the variants are located outside of protein-coding regions, and determining the effects of such non-coding variants remains difficult. In this dissertation, I evaluate the hypothesis that such non-coding variants contribute to human traits and diseases by altering the regulation of genes rather than the sequence of those genes. I will specifically focus on studies to determine the functional impacts of genetic variation associated with two related complex traits: gestational hyperglycemia and fetal adiposity. At the genomic locus associated with maternal hyperglycemia, we found that genetic variation in regulatory elements altered the expression of the HKDC1 gene. Furthermore, we demonstrated that HKDC1 phosphorylates glucose in vitro and in vivo, thus demonstrating that HKDC1 is a fifth human hexokinase gene. At the fetal-adiposity associated locus, we identified variants that likely alter VEPH1 expression in preadipocytes during differentiation. To make such studies of regulatory variation high-throughput and routine, we developed POP-STARR, a novel high throughput reporter assay that can empirically measure the effects of regulatory variants directly from patient DNA. By combining targeted genome capture technologies with STARR-seq, we assayed thousands of haplotypes from 760 individuals in a single experiment. We subsequently used POP-STARR to identify three key features of regulatory variants: that regulatory variants typically have weak effects on gene expression; that the effects of regulatory variants are often coordinated with respect to disease-risk, suggesting a general mechanism by which the weak effects can together have phenotypic impact; and that nucleotide transversions have larger impacts on enhancer activity than transitions. Together, the findings presented here demonstrate successful strategies for determining the regulatory mechanisms underlying genetic associations with human traits and diseases, and value of doing so for driving novel biological discovery.

Background: According to the WHO 2020 NCD report, the COVID-19 pandemic disrupted diabetes care in 49% of countries. eHealth emerged as a solution to disease management challenges during the unprecedented outbreak. Due to the rapidly expanding nature of eHealth use during the pandemic, this study aimed to determine 1) the sociodemographic factors associated with eHealth during COVID-19 and 2) whether eHealth was associated with diabetes management and clinical outcomes. Methods: Using quantitative data from cross-sectional surveys from Kunshan and Taicang, China (n=309) and Manila, Philippines (n=150) (data sets uncombined), we performed Chi-squared and Fisher’s exact tests and univariate logistic regressions to determine the relationship of eHealth use and sociodemographic characteristics. We then performed logistic and linear regression to determine the association of eHealth use with diabetes disease outcomes. Results: In China, younger age (p=0.02), higher education level (p=0.001), married marital status (p=0.03), suburban residence type (p=0.001), and higher household monthly income during COVID-19 (p=0.004) were associated with using eHealth. In the Philippines, younger age (p= 0.009) and higher education level (p=0.01) were associated with eHealth use. eHealth use was associated with undergoing FBS testing in the last three months (OR = 2.19, 95%CI = 1.00, 4.78), undergoing HbA1c testing in the last three months (OR = 3.64, 95%CI = 1.01, 13.15), and reporting disease control per their last HbA1c test (OR = 9.98, 95%CI = 3.41, 29.18) in the Philippines, adjusting for various demographic characteristics. Conclusions: Our data indicated eHealth use could positively affect diabetes clinical and management outcomes for people with diabetes in China and the Philippines. We posit more research is needed for the impacts of eHealth on clinical outcomes as well as the methods for eHealth implementation and integration in LMIC.

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The prevalence of diabetes is on the rise, thus imposing a huge burden on our society. In addition to genetic and environmental risk factors, obesity strongly predicts the development of diabetes. Owing to the strong correlation between saturated fat and fructose intake and the incidence of metabolic diseases and obesity, dietary intervention is one of the common management strategies to control obesity and diabetes. Although there are still contentious debates on whether a low-fat or low-carbohydrate diet is beneficial to metabolic health, less is known about the interaction between lipid and carbohydrate metabolism. One of the overlooked interactions is how fat intake modulates fructose metabolism.

The effects of high-fat intake were first studied in mouse models. Coupled with fructose stable isotopic tracing, we profiled the fructose metabolism in various tissues among mice fed with either a high-fat diet or a control diet. We also surveyed the fructose-derived metabolites in the circulation to establish the systematic perturbations of fructose metabolism by the high-fat diet. We unveiled that a high-fat diet potentiates intestinal fructose metabolism and the production of glycerate. The fructose-derived glycerate then enters the circulation and reaches the pancreas.

We performed daily glycerate administration to model the high circulating glycerate level among mice. Through metabolic profiling, we found that high circulating glycerate can cause glucose intolerance but not induce insulin resistance. The observed glucose intolerance phenotype is likely related to the reduction in circulating insulin levels and that reduced insulin secretion is independent of the insulin sensitivity among the mice treated with glycerate. In the investigation of the pancreatic islets, we found glycerate treatment promotes pancreatic beta-cell damage and apoptosis.

In conclusion, we demonstrated the interaction between fat and fructose intake, in which high fat intake enhances the fructose metabolism in the small intestine. The up-regulated fructose metabolism in the small intestine enhances the production of glycerate. High circulating glycerate is a risk factor for diabetes development via depletion of insulin content in pancreatic islets. Therefore, this study discovers a novel mechanism for fructose-induced diabetes via the action of glycerate. It also supports the idea of reducing fat and fructose intake simultaneously, rather than either macronutrient alone, to achieve maximal metabolic health benefits.

Background: In recent decades there has been an escalating epidemic of diabetes in Ghana. However, there has been little research on the economic burdens associated with diabetes in Ghana, despite diabetes's costly nature. This study investigated economic burdens and financial protections of households with diabetes patient(s) in urban Ghana.

Methods: Questionnaire-based interviews were conducted with 40 diabetes patients and their household heads in two urban communities in the city of Accra, Ghana. Information was obtained regarding participants' demographic and socioeconomic characteristics, patterns of healthcare utilization, direct and indirect costs, and financial protections pertaining to diabetes treatment and management. Cost-­of-­illness analysis and catastrophic health expenditure computation were conducted to investigate the costs associated with diabetes and households' affordability. Statistical tests were also conducted to analyze the effect of the National Health Insurance Scheme (NHIS) on the costs associated with diabetes.

Results: The total cost of diabetes for 40 households was estimated to be 14,989 cedis/month, of which 66.5% was direct cost and 30.2% was indirect cost. 52.9% of the households occurred catastrophic health expenditure. The means of outpatient and inpatient expenditure were 136 and 418 Cedi/month, respectively. NHIS had a positive financial protection effect on the economic burden of diabetes, while this effect was diminished by deficiencies in NHIS. Extended family was the main resource of financial support for diabetes treatment and management.

Conclusion: The economic burden of diabetes is high in urban Ghana, with a catastrophic effect on households. Except for NHIS, patients' financial support mainly comes from personal resources rather than public resources. Social supports and improvements in NHIS are needed to protect households with diabetes patient(s) against financial risks.

Type 2 diabetes is characterized by insufficient insulin secretion to maintain euglycemia in the setting of peripheral insulin resistance. The majority of insulin-resistant diabetics are obese, yet not all insulin-resistant obese individuals develop diabetes. This obesity/diabetes dichotomy suggests that genetic factors play a pivotal role in disease pathogenesis.

Gene mapping has identified genetic quantitative trait loci (QTL) influencing disease-related phenotypes. To uncover molecular pathways leading from genotype to clinical trait, we classify phenotypes in greater depth and identify QTL that influence combinations of physiological traits, mRNA levels, and metabolite abundance. A major challenge then becomes deciphering the causal interrelationships among correlated phenotypes.

In this dissertation, we develop methods for building causal direction into an undirected network by including QTLs for each phenotype. We then apply and validate these methods in an F2 intercross between the diabetes-resistant C57BL/6 leptinob/ob (B6ob/ob) and the diabetes-susceptible BTBR leptinob/ob (BTBRob/ob) mouse strains. We show that genomic analysis can be integrated with liver transcriptional and metabolite profiling data to construct causal networks for specific metabolic processes in liver. This causal network construction led to the discovery of a pathway by which glutamine induces Phosphoenolpyruvate carboxykinase (Pck1) expression.

To investigate glutamine induction of Pck1 in the context of diabetes, we perform mRNA expression analysis and metabolic profiling in liver of the parental strains. We find glutamine is decreased with obesity in both strains; in the diabetes-resistant B6 strain, liver Pck1 expression parallels glutamine abundance, but in the diabetes-susceptible BTBR strain, Pck1 is elevated with obesity. Follow-up in vitro studies indicate that α-ketoglutarate, which is elevated nearly two fold in the livers of BTBR relative to B6 mice in vivo, may mediate the glutamine effect. We hypothesize that hepatic Pck1 is regulated by glutamine abundance in the liver of B6 animals, but in the presence of high α-ketoglutarate, Pck1 becomes uncoupled from glutamine regulation in the livers of diabetes-susceptible BTBR mice.

Our method of causal network construction led to the discovery of glutamine induction of a key hepatic gluconeogenic enzyme, a pathway potentially disrupted in the diabetes-susceptible BTBR mouse. Future studies will include identifying hepatic mediators of the glutamine effect, and applying QTL-directed networks to multiple organs to ultimately define causal relationships between tissues involved in diabetes progression.

Insulin secretion is a fundamental process of pancreatic beta-cells required for the maintenance of glucose homeostasis. Fuel-stimulated insulin secretion occurs in proportion to the rate of metabolism of fuel substrates, yet the signals generated by metabolism of these secretagogues are incompletely understood. The increased burden placed on the beta-cell in conditions of obesity and insulin resistance often leads to dysregulation of stimulous-secretion coupling. Therefore, better understanding of the metabolic events required for insulin release is likely to be helpful in development of more effective treatments for diabetes.

Previous work in our lab revealed a critical role for the pyruvate-isocitrate cycling pathway in glucose-stimulated insulin secretion. It has been our hypothesis that this series of reactions plays a unique role in the beta-cell, and may be responsible for the generation of second-messenger signals critical for insulin secretion in response to increased fuel metabolism. One of the intermediates in the pyruvate/isocitrate cycle is cytosolic 2-oxoglutarate (2OG). In an effort to better understand the components of the pyruvate/isocitrate cycle and the signals that it generates, we initially focused our studies on the transporter protein responsible for the return of 2OG to the mitochondria, the 2-oxoglutarate carrier (OGC).

OGC was overexpressed in rat insulinoma 832/13 beta-cells and suppressed in both 832/13 cells and islets, and effects on metabolism and insulin secretion were measured. While overexpression of the OGC failed to alter insulin secretion, its siRNA-mediated suppression resulted in decreased insulin secretion in response to glucose, glutamine + BCH, and dimethyl-2-oxoglutarate. Suppression of OGC did not affect core pathways of fuel metabolism such as glucose usage, glucose oxidation or ATP production during glucose-stimulated insulin secretion (GSIS) or glutamine oxidation or ATP production during amino acid-stimulated insulin secretion (AASIS). Similar to previous findings, glucose-induced NADPH production was determined to be decreased in response to OGC suppression, whereas NADPH production during AASIS in untreated cells was already much lower than for GSIS, and suppression of OGC failed to decrease NADPH further.

As an additional approach to studying the role of 2OG metabolism in insulin secretion, we also investigated the mitochondrial enzyme glutamate dehydrogenase (Glud1). Overexpression of wild-type Glud1 failed to alter insulin secretion in 832/13 cells or in islets; however, suppression of Glud1 decreased both GSIS and AASIS, but did not affect dimethyl-2OG-stimulated insulin secretion. The reduction in AASIS was most likely the result of reduced glutamine oxidation. In contrast, during GSIS, NADPH production was decrease by Glud1 suppression, similar to our observation with the OGC.

In summary, these data expand our understanding of the metabolic pathways necessary for insulin secretion, and support the idea of a common metabolic pathway required for fuel-stimulated insulin release, including flux through the OGC, Glud1, and ICDc. However, while these data support the hypothesis that NADPH production is necessary for robust GSIS, it plays a less-prominent role during AASIS, and most likely works in concert with additional coupling-factors and signals.

This study explores patients’ needs in rural Thanjavur, southern India through understanding how people with diabetes choose providers and perceive care-seeking experience. To measure perception, the study surveyed people regarding six common barriers to care-seeking behavior, selected from both literature and local expert interview. Ninety-one percent of the sampled population goes to public or private allopathic providers out of the six presented providers. The low socioeconomic group and people with more complications or comorbidities are more likely to go to private allopathic providers. What is more, there is no difference between public and private allopathic providers in patients’ perception of care except for perceived cost. Positive perceptions in both providers are very common except for perceptions in blood-sugar management, distance to facilities, and cost of care. Sixty-six percent of patients perceived their blood-sugar control to fluctuate or have no change versus improved control. Twenty-seven percent of patients perceived the distance to facilities as unreasonable, and sixty-two percent of patients perceived the cost as high for them. The results suggest that cost may affect low socioeconomic people’s choice of care significantly. However, for people in middle and higher socioeconomic groups, cost does not appear to be a major factor. For qualitative text analyses, physician’s behavior and reputation emerge as themes, which require further studies.