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Thesis
Peer Reviewed

In vivo ²H₂O administration for the measurement of adipose and hepatic triglyceride synthesis, de novo lipogenesis, adipocyte proliferation and protein synthesis reveals differences in insulin sensitive versus insulin resistant obese humans.

Allister, Candice
Advisor(s): Hellerstein, Marc K

UC Berkeley Electronic Theses and Dissertations (2014)

Obesity affects about 34% of adults in the United Sates, and 25% of these individuals will become insulin resistance. Studies have provided evidence to suggest that subcutaneous adipose tissue is protective against insulin resistance and type 2 diabetes. Anti-diabetic drugs, thiazolidinediones (TZDs), improve insulin sensitivity by redistributing fat storage into the subcutaneous fat depots and away from visceral fat depots. Unlike subcutaneous fat, visceral fat is highly lipolytic, releasing elevated concentrations of free fatty acids (FFAs) into circulation. Elevated free fatty acids are a common phenotype of insulin resistance, causing lipotoxicity in tissues and impairing insulin signaling in insulin-responsive cells. For example, hepatic lipotoxicity inhibits insulin's ability to suppress gluconeogenesis, in addition to acetyl-CoA entering into the gluconeogenic pathway. Thus, it is important to prevent the intrusion of lipids into tissues and preserve insulin action. However, not all obese individuals become insulin resistant.

It has been reported that a significant portion of obese individuals are metabolically healthy. The purpose of this dissertation was to investigate potential mechanisms that protect some obese individuals from becoming insulin resistant. Using ²H₂O in BMI-matched obese insulin sensitive (IS) and insulin resistant (IR) individuals, we quantified subcutaneous adipose lipid kinetics, cellular proliferation and the synthesis of proteins involved in metabolic pathways, all of which may play an imperative role in the etiology of insulin resistance.

We hypothesized that dysfunctional subcutaneous adipose triglyceride storage is an initiating factor in the development of systemic insulin resistance in human obesity. We tested this hypothesis using ²H₂O to quantify, in vivo, triglyceride (TG) synthesis, de novo lipogenesis (DNL), and adipocyte proliferation in subcutaneous adipose cells of insulin-sensitive (IS) and insulin-resistant (IR) subjects. Adipose cell size distribution was quantified using Beckman Multisizer III. Plasma free fatty acid (FFA) concentrations were measured under fasting and postprandial insulin concentrations during a steady state plasma glucose (SSPG) test as a measurement of insulin suppression of lipolysis. Total TG synthesis and DNL were significantly lower in adipose cells of IR compared to IS subjects, and correlated inversely with the proportion of small adipocytes. FFA concentrations were significantly greater in IR versus IS during fasting and postprandial insulin concentrations. Adipocyte proliferation did not differ significantly between groups. These results strongly support the hypothesis that decreased capacity for lipid storage in subcutaneous adipose tissue is a defining feature of obesity-associated insulin resistance and may contribute to insulin resistance in peripheral tissues due to ectopic fat deposition.

Following our findings in a predominately Caucasian cohort, we sought to determine if adipose dysfunction is predictive of insulin resistance amongst different ethnicities. In the United States, African-Americans (AA) have 51% higher rates of obesity than Caucasians (C), and the highest rates of type 2 diabetes and cardiovascular disease Obesity is associated with insulin resistance, yet the physiological mechanisms of this association and fat regional distribution as a potential contributor is not yet completely understood, particularly in minority populations most affected by this disease. Increased visceral adipose tissue (VAT) mass a significant risk factor for insulin-resistance, but recent studies have discovered that this phenotype may not be a global risk factor. VAT tends to be consistently lower in AA compared to C, yet individuals in this group are more insulin resistant for a given degree of body fat. Thus, we aimed to further investigate differences in adipose lipid synthesis between African-Americans and Caucasians. We administered ²H₂O to five African-Americans and seven Caucasians for the quantification of TG synthesis, DNL and adipocyte proliferation

Using ²H₂O, we aimed to apply dynamic proteomics to quantify protein turnover rates of proteins from isolated subcutaneous mature adipocytes. Dynamic proteomics is the large-scale measurement of protein expression and turnover in a single specimen, biological tissue or bodily fluid. Dynamic proteomics applied to humans may allow us to discover the adipoycte proteome of insulin resistance. We investigated the adipocyte proteome of 3 IS and 3 IR, equally obese subjects at steady state after 4 weeks of 2H2O consumption. Peptides were submitted through LC/MS-MS analysis to separate and identify tryspinized peptides by size and charge, followed by Spectrum Mill analysis to organize peptides based on their corresponding protein. A total of 620 proteins were identified amongst the six subjects, but after filtering for criteria (1

Cover page: In vivo <sup>2</sup>H<sub>2</sub>O administration for the measurement of adipose and hepatic triglyceride synthesis, de novo lipogenesis, adipocyte proliferation and protein synthesis reveals differences in insulin sensitive versus insulin resistant obese humans.

Article
Peer Reviewed

Longitudinal multi-omics of host–microbe dynamics in prediabetes

Type 2 diabetes mellitus (T2D) is a growing health problem, but little is known about its early disease stages, its effects on biological processes or the transition to clinical T2D. To understand the earliest stages of T2D better, we obtained samples from 106 healthy individuals and individuals with prediabetes over approximately four years and performed deep profiling of transcriptomes, metabolomes, cytokines, and proteomes, as well as changes in the microbiome. This rich longitudinal data set revealed many insights: first, healthy profiles are distinct among individuals while displaying diverse patterns of intra- and/or inter-personal variability. Second, extensive host and microbial changes occur during respiratory viral infections and immunization, and immunization triggers potentially protective responses that are distinct from responses to respiratory viral infections. Moreover, during respiratory viral infections, insulin-resistant participants respond differently than insulin-sensitive participants. Third, global co-association analyses among the thousands of profiled molecules reveal specific host-microbe interactions that differ between insulin-resistant and insulin-sensitive individuals. Last, we identified early personal molecular signatures in one individual that preceded the onset of T2D, including the inflammation markers interleukin-1 receptor agonist (IL-1RA) and high-sensitivity C-reactive protein (CRP) paired with xenobiotic-induced immune signalling. Our study reveals insights into pathways and responses that differ between glucose-dysregulated and healthy individuals during health and disease and provides an open-access data resource to enable further research into healthy, prediabetic and T2D states.

In vivo 2H2O administration for the measurement of adipose and hepatic triglyceride synthesis, de novo lipogenesis, adipocyte proliferation and protein synthesis reveals differences in insulin sensitive versus insulin resistant obese humans.

Longitudinal multi-omics of host–microbe dynamics in prediabetes

In vivo ²H₂O administration for the measurement of adipose and hepatic triglyceride synthesis, de novo lipogenesis, adipocyte proliferation and protein synthesis reveals differences in insulin sensitive versus insulin resistant obese humans.