An investigation of the anti-hyperglycaemic, biochemical and molecular effects of 4-hydroxyisoleucine and fenugreek seed extract in comparison to metformin in vitro and in vivo.

Doctoral Degree. University of KwaZulu-Natal, Durban.Type two diabetes mellitus (T2D) is a significant cause of premature death and disability, accompanied with negative socio-economic impacts. This metabolic disorder is characterized by hyperglycaemia and defective insulin signalling. Long-term exposure to hyperglycaemia gives rise to altered fat metabolism and reactive oxygen species (ROS) generation. These precursors are central to the progression of dyslipidaemia and attenuated antioxidant (AO) response and detoxification system, respectively. Diabetic dyslipidaemia and oxidative stress (OS) are risk factors for the onset and progression of cardiovascular disease (CVD) and other diabetic complications. The treatment regimen for T2D comprises self-care and anti-diabetic drugs such as metformin. However, due to the lack of compliance to self-care recommendations and some undesirable side effects of metformin, there is the necessity for alternate therapy. Natural products have been used for the treatment of many disorders, including T2D. Trigonella foenum-graecum commonly known as fenugreek is a plant that possesses anti-diabetic effects. These effects are attributed to its bioactive compound – 4-hydroxyisoleucine (4-OH-lle), which constitutes approximately 80% of the bio-composition of the fenugreek seed. Despite these effects, biochemical and molecular effects of 4-OH-lle on insulin signalling, lipid metabolism, and ROS production is not well-documented. This study investigated the effects of 4-OH-lle in comparison to metformin and fenugreek seed extract (FSE) on hyperglycaemic human hepatoma (HepG2) cells and C57BL/6 male mice. Treatments were conducted under normoglycaemic and hyperglycaemic conditions as follows; control, 4-OH-lle (in vitro: 100ng/ml; in vivo: 100mg/kg Body weight) metformin (in vitro: 20mM; in vivo: 20mg/kg Body weight) and FSE (in vitro: 100ng/ml; in vivo: 100mg/kg Body weight) treatment groups. The experiments included; blood glucose measurements, lipid profile analysis, spectrophotometric assays (in vitro), western blotting for protein expression and qPCR for mRNA expression. First, to validate the effects on insulin signalling and glucose sensing, glucose levels were measured with completion of an oral glucose tolerance test. 4-OH-lle treatment attenuated glucose levels, and elevated the mRNA levels of glycogen synthase (GS) and glucokinase (Gck). This was followed by the investigation of the protein and gene expression of insulin signalling regulators: insulin receptor β (IRβ), insulin receptor substrate 1 (IRS1), phosphorylated protein kinase B (pAkt), phosphorylated glycogen synthase kinase 3α/β (pGSK3α/β) and glucose transport 2 (GLUT2). In in vivo hyperglycaemia, 4-OH-lle increased the expression of the investigated proteins and genes. The results showed that 4-OH-lle was just as potent as MF, and FSE in stimulating the insulin signalling cascade. Second, the effect of 4-OH-lle on dyslipidaemia was investigated by measuring mRNA levels of sterol regulatory binding element 1c (SREBP1c) and fatty acid synthase (FAS) – key factors in fatty acid metabolism. Both genes were up-regulated and correlated with the changes in triglyceride and cholesterol levels. Next the protein expression of proprotein convertase subtilisin-like/kexin type (PCSK9) - a regulator of low density lipoprotein cholesterol (LDLc) and peroxisome proliferator-activated receptor gamma (PPARG) – a regulator of high density lipoprotein (HDLc) was evaluated. The data showed that 4-OH-lle down-regulated protein and mRNA expression of PCSK9 and up-regulated protein expression of PPARG. The reduction in PCSK9 levels correlated with the changes observed in low density lipoprotein receptor (LDLr) and LDLc, whereas the increase in PPARG correlated with the elevated mRNA expression of apolipoprotein A1 (Apo A1) and HDLc. Together these results provide substantial evidence for the regulatory effect of 4-OH-lle, in comparison to metformin, and FSE on PCSK9, PPARG and related lipid factors. Finally, the effect of 4-OH-lle on redox status and AO response was assessed by measuring nuclear factor E2-related factor 2 (Nrf2). In both models, there was an increase in the protein expression of phosphorylated Nrf2 accompanied by an increase in mRNA levels of superoxide dismutase 2 (SOD2) and glutathione peroxidase (GPx), and GSH levels. Mitochondria play a central role in contributing to elevated ROS levels. While nuclear responses like Nrf2 regulate ROS, mitochondria possess their own maintenance proteins. These include mitochondrial Lon protease 1 (LonP1), Sirtuin 3 (SIRT3) and peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) which play an integral role in combatting OS and mitochondrial dysfunction. The results showed that 4-OH-lle displayed a potent effect in inducing the AO response and increasing mitochondrial regulatory proteins. In conclusion, 4-OH-lle improved the compromised insulin signalling and the altered lipid profile as well as induced the AO response and mitochondrial maintenance proteins, in the presence of elevated glucose. Furthermore, the effect of 4-OH-lle was greater than the first-line drug; metformin and FSE, albeit in cultured human liver cells and a mouse model. Also, the crude seed extract displayed promising effects on all investigated parameters. Considering the active role of chronic hyperglycaemia in the onset and progression of CVD and diabetic complications, 4-OH-lle poses as a highly favourable alternate therapy in the treatment of T2D. Moreover, this has great importance in socio-economically challenged communities where T2D is a common disorder, access to healthcare facilities is limited, and plants serve as sources of easily accessible treatments

Trigonella foenum-graecum seed and 4-hydroxyisoleucine mediates glucose uptake via proximal insulin signaling activation and related downstream gene expression in liver cells.

M. Med. Sc. University of KwaZulu-Natal, Durban 2014.Fenugreek (Trigonella foenum-graecum) is one of the oldest medicinal plants used worldwide to treat a variety of ailments, including hyperglycaemia. The seed and active compound – 4-hydroxyisoleucine (4-OH-lle) is thought to aid in the treatment of insulin resistance. This study investigated the effects of fenugreek aqueous seed extract and 4-OH-lle, on human liver cells (HepG2) compared to insulin (100ng/ml) and metformin (2mM) controls. Cells were treated with fenugreek seed extract (FSE) and 4-OH-lle: 10 and 100ng/ml under normogylcaemic (5mM glucose) and hyperglycaemic (30mM) conditions for 72h. Tyrosine phosphorylation of insulin receptor-β (IR-β), protein kinase B (Akt) and glycogen synthase kinase-3α/β (GSK-3α/β) protein extracts was determined by western blotting. Gene expression of sterol regulatory element binding protein 1c (SREBP1c), glucose transporter 2 (GLUT2), glycogen synthase (GS) and glucokinase (GK) was evaluated by qPCR. Under normogylcaemic and hyperglycaemic conditions, FSE, 4-OH-lle and insulin at 100ng/ml and metformin (2mM) caused tyrosine phosphorylation of IR-β (p<0.0729; p<0.0121), Akt (p<0.0046; p<0.0005) and GSK-3α/β (p<0.0128; p<0.0048). However, FSE showed the greatest ability in positively controlling GS (*p<0.0262; *p<0.333) and GK (*p<0.333; *p<0.0213), which regulates glycogen synthesis. Also, FSE increased SREBP1c (*p<0.0157; *p<0.0012) which positively regulates GLUT2 (*p<0.0330, *p<0.0417), allowing glucose into the cell. The data suggests that FSE and 4-OH-lle causes an up-regulation of insulin signaling proteins at a proximal level and related downstream gene expression. Taken together, the study suggests that FSE has potential application in the management of chronic hyperglycaemia

Albiglutide and cardiovascular outcomes in patients with type 2 diabetes and cardiovascular disease (Harmony Outcomes): a double-blind, randomised placebo-controlled trial

Background: Glucagon-like peptide 1 receptor agonists differ in chemical structure, duration of action, and in their effects on clinical outcomes. The cardiovascular effects of once-weekly albiglutide in type 2 diabetes are unknown. We aimed to determine the safety and efficacy of albiglutide in preventing cardiovascular death, myocardial infarction, or stroke. Methods: We did a double-blind, randomised, placebo-controlled trial in 610 sites across 28 countries. We randomly assigned patients aged 40 years and older with type 2 diabetes and cardiovascular disease (at a 1:1 ratio) to groups that either received a subcutaneous injection of albiglutide (30–50 mg, based on glycaemic response and tolerability) or of a matched volume of placebo once a week, in addition to their standard care. Investigators used an interactive voice or web response system to obtain treatment assignment, and patients and all study investigators were masked to their treatment allocation. We hypothesised that albiglutide would be non-inferior to placebo for the primary outcome of the first occurrence of cardiovascular death, myocardial infarction, or stroke, which was assessed in the intention-to-treat population. If non-inferiority was confirmed by an upper limit of the 95% CI for a hazard ratio of less than 1·30, closed testing for superiority was prespecified. This study is registered with ClinicalTrials.gov, number NCT02465515. Findings: Patients were screened between July 1, 2015, and Nov 24, 2016. 10 793 patients were screened and 9463 participants were enrolled and randomly assigned to groups: 4731 patients were assigned to receive albiglutide and 4732 patients to receive placebo. On Nov 8, 2017, it was determined that 611 primary endpoints and a median follow-up of at least 1·5 years had accrued, and participants returned for a final visit and discontinuation from study treatment; the last patient visit was on March 12, 2018. These 9463 patients, the intention-to-treat population, were evaluated for a median duration of 1·6 years and were assessed for the primary outcome. The primary composite outcome occurred in 338 (7%) of 4731 patients at an incidence rate of 4·6 events per 100 person-years in the albiglutide group and in 428 (9%) of 4732 patients at an incidence rate of 5·9 events per 100 person-years in the placebo group (hazard ratio 0·78, 95% CI 0·68–0·90), which indicated that albiglutide was superior to placebo (p&lt;0·0001 for non-inferiority; p=0·0006 for superiority). The incidence of acute pancreatitis (ten patients in the albiglutide group and seven patients in the placebo group), pancreatic cancer (six patients in the albiglutide group and five patients in the placebo group), medullary thyroid carcinoma (zero patients in both groups), and other serious adverse events did not differ between the two groups. There were three (&lt;1%) deaths in the placebo group that were assessed by investigators, who were masked to study drug assignment, to be treatment-related and two (&lt;1%) deaths in the albiglutide group. Interpretation: In patients with type 2 diabetes and cardiovascular disease, albiglutide was superior to placebo with respect to major adverse cardiovascular events. Evidence-based glucagon-like peptide 1 receptor agonists should therefore be considered as part of a comprehensive strategy to reduce the risk of cardiovascular events in patients with type 2 diabetes. Funding: GlaxoSmithKline

Isoniazid resistance-conferring mutations are associated with highly variable phenotypic resistance

Background: High-dose isoniazid is recommended in the 9–12 months short-course regimen for multidrug-resistant tuberculosis with inhA mutation. However, there is insufficient evidence to support the assumption of genotypic-phenotypic concordance. This study aimed to identify the genetic mutations associated with high-level phenotypic isoniazid resistance. Methods: Clinical isolates from patients with drug-resistant tuberculosis were profiled by whole-genome sequencing and subjected to minimum inhibitory concentration (MIC) testing using MGIT based-method. MICs were performed in concentration ranges based on the mutation present: isolates with no isoniazid resistance-conferring mutations and H37Rv, 0.016–0.256 µg/ml; inhA, 0.256–4.0 µg/ml, katG 1.0–16.0 µg/ml; and inhA + katG, 4.0–64.0 µg/ml. Isolates demonstrating resistance at the upper limit of the concentration range were tested up to the maximum of 64.0 µg/ml. Bootstrap of the mean MICs was performed to increase the robustness of the estimates and an overlap index was used to compare the distributions of the MICs for each mutation profile. Results: A total of 52 clinical isolates were included in this analysis. Bootstrap MIC means for inhA, katG and inhA + katG were 33.64 (95% CI, 9.47, 56.90), 6.79 (4.45, 9.70) and 52.34 (42.750, 61.66) µg/ml, respectively. There was high overlap between inhA and inhA + katG mutations (eta = 0.45) but not with inhA and katG (eta = 0.19). Furthermore, katG showed poor overlap with inhA + katG mutations (eta = 0.09). Unexpectedly, 4/8 (50.0%) of all InhA mutants demonstrated high-level resistance, while 20/24 (83.3%) of katG mutants demonstrated moderate-level resistance. Conclusions: InhA mutations demonstrated unexpectedly high MICs and showed high overlap with inhA + katG. Contrary to the common belief that katG mutants are associated with high-level resistance, this mutation primarily showed moderate-level resistance