Investigation of early predictors in insulin resistance and Type 2 Diabetes Mellitus

Obesity is an established risk factor for diabetes mellitus, and this represents a major public health concern worldwide. During the last three decades, the obesity epidemic has been paralleled by an increasing incidence of diabetes mellitus highlighting the need for safer, effective, and affordable prevention strategies.
Recent interest has turned to the importance of L-serine deficiency and its impaired metabolism in patients with diabetes. L-serine is categorised as a nutritionally non-essential (dispensable) amino acid as it can be synthesized from 3-phosphoglycerate (3-PG) and glycine or found in the range between 2 and 5% in protein sources. L-serine plays several metabolic functions in a wide variety of cellular processes involving the production of protein, sphingolipids, phospholipids, sulphur-containing amino acids as well as neurotransmission. In the blood of children with Type 1 Diabetes Mellitus (T1DM), concentrations of L-serine were decreased by 42% compared to non-diabetic controls. Similar results were also reported in patients with Type 2 Diabetes Mellitus (T2DM). Interestingly, postprandial levels of L-serine were reduced in patients with T2DM compared to non-diabetic controls. On the other hand, increased L-serine levels are correlated to enhanced insulin secretion and sensitivity in non[1]diabetic men aged between 45 and 73 years, and it is also associated with an improved glucose tolerance after a 2-hour oral glucose tolerance test.
Furthermore, mounting evidence suggests a potential link between impaired metabolism of L-serine and the production of a-typical sphingolipids, termed 1- deoxysphingolipids (1-DSL) which have been linked to the pathogenesis of insulin resistance (IR) and diabetes. Recent literature demonstrated that patients with compromised fasting glucose levels, metabolic syndrome (MS), and T2DM had considerably higher levels of 1-DSL. On the contrary, the levels of these a-typical lipids are not raised in patients with T1DM. Interestingly, these plasma levels are also significantly elevated in non-diabetic people who subsequently develop T2DM. 1-DSL have also been shown to be elevated in patients with diabetic neuropathy, and in a randomized trial of high-dose of L-serine administration, this amino acid reduced 1-DSL levels, potentially slowing the progression of the disease. Furthermore, 1-DSL have been shown to be notably raised in obese and T2DM patients compared to athletes and lean individuals, in which their 1-DSL accumulation appeared to trigger insulin resistance in vitro. Thus, high 1-DSL levels potentially are early biomarkers of T2DM, independent of the levels of glycated haemoglobin in MS and in the general population. Nevertheless, the underlying molecular pathways that contribute to the pathophysiology of T2DM in individuals with elevated atypical sphingolipids have not been completely explored.
The aims of this thesis were two-fold: i) to determine whether, given their cytotoxic properties, 1-DSL impairs skeletal myoblasts’ functionality and prevents them from differentiating into mature myotubes, which contributes to the pathophysiology of T2DM; ii) to investigate if a new, safe and affordable pharmacotherapy of L-serine supplementation could ameliorate the glucose profile of High Fat Diet (HFD) fed mice.
Study 1 aimed to investigate whether 1-DSA, a type of 1-DSL, are cytotoxic and disrupt the cellular processes of skeletal muscle precursors (myoblasts) and differentiated cells (myotubes) by performing a battery of in vitro assays including cell viability adenosine triphosphate assay, migration assay, myoblast fusion assay, glucose uptake assay, and immunocytochemistry. Our results demonstrated that 1-DSA significantly reduced the viability of myoblasts in a concentration and time-dependent manner, and induced apoptosis (97%) as well as cellular necrosis (78.5%). Importantly, myoblasts were more sensitive to the cytotoxic effects induced by 1-DSA rather than by saturated fatty acids, such as palmitate, which are critical mediators of skeletal muscle dysfunction in T2DM. Additionally, 1-DSA significantly reduced the migration ability of myoblasts and the differentiation process of myoblasts into myotubes. 1-DSA also triggered autophagy in myoblasts and significantly reduced 20 % of insulin-stimulated glucose uptake in myotubes. These findings indicate that 1-DSA directly compromises the functionality of skeletal muscle cells and suggest that increased levels of 1- DSA observed during the development of T2DM are likely to contribute to the pathophysiology of muscle dysfunction detected in this disease.
In study 2, the impact of L-serine supplementation on pre-diabetic mice was examined. Mice were fed either a standard chow diet, chow with L-serine, HFD, or HFD with L-serine. After 4 weeks of HFD, mice were then introduced 1% L-serine supplementation in drinking water, together with a standard diet or HFD for 12 weeks. L-serine treatment was shown to reduce body weight and adiposity in mice consuming a HFD by 8.51% and 23 %, respectively, with no effect on mice consuming a standard chow diet. Fasting glucose and glucose intolerance were also significantly improved by 13.9% and 15.73%, respectively. Interestingly, xxv L-serine significantly down-regulated renal mRNA expression of vasopressin biomarkers (Avp, Avpr1a, and Avpr2), and aldosterone receptor in HFD-fed mice. Additionally, we found that L-serine supplementation downregulated the expression of the kidney injury marker HAVCR1, which was also confirmed by a reduction in inflammation marker expression (Il-6, Tnf-α). Also, the mRNA expression of the purinoceptor P2ry1, a known marker of renal injury, was decreased in mice treated with L-Serine.
In summary, my in-vitro work demonstrated for the first time that 1-DSA are cytotoxic and compromise the functionality of skeletal muscle cells. 1-DSA also reduces cell viability and causes cell death in the form of apoptosis and necrosis. Further, 1-DSA was shown to reduce myoblast migration, inhibit myoblast differentiation and alter myotube structure which may lead to the reduction of insulin-stimulated glucose uptake. Overall, my animal work demonstrated that chronic L-serine supplementation improved blood glucose levels in obese mice and reduced substantial markers of kidney injury plausibly identifying the potential mechanism for its therapeutic effect.