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In recent years, insufficiently characterised controls have been a contributing factor to irreproducibility in biomedical research including neuroscience and metabolism. There is now a growing awareness of phenotypic differences between the C57BL/6 substrains which are commonly used as control animals. We here investigated baseline metabolic characteristics such as glucose regulation, fasted serum insulin levels and hepatic insulin signalling in five different C57BL/6 substrains (N, J, JOla, JRcc) of both sexes, obtained from two commercial vendors, Charles River Laboratories (Crl) and Envigo (Env). Our results indicate systematic and tissue-specific differences between substrains, affected by both vendor and sex, in all parameters investigated, and not necessarily mediated by the presence of the NntC57BL/6J mutation. Not only were there differences between 6J and 6N as expected, all three 6J substrains exhibited different profiles, even from the same breeder. Two distinct metabolic profiles were identified, one in which low insulin levels resulted in impaired glucose clearance (6JCrl; both sexes) and the other, where sustained elevations in fasted basal insulin levels led to glucose intolerance (male 6JRccEnv). Further, 6JRccEnv displayed sex differences in both glucose clearance and hepatic insulin signalling markers. In comparison, the two 6N substrains of either sex, irrespective of vendor, did not exhibit considerable differences, with 6NCrl animals presenting a good choice as a healthy baseline 'control' for many types of experiments. Overall, our data emphasise the importance of selecting and characterising control subjects regarding background, sex, and supplier to ensure proper experimental outcomes in biomedical research., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Sil et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Dietary methionine restriction (MR) leads to loss of adiposity, improved insulin sensitivity and lifespan extension. The possibility that dietary MR can protect the kidney from age-associated deterioration has not been addressed. Aged (10-month old) male and female mice were placed on a MR (0.172% methionine) or control diet (0.86% methionine) for 8-weeks and blood glucose, renal insulin signalling, and gene expression were assessed. Methionine restriction lead to decreased blood glucose levels compared to control-fed mice, and enhanced insulin-stimulated phosphorylation of PKB/Akt and S6 in kidneys, indicative of improved glucose homeostasis. Increased expression of lipogenic genes and downregulation of PEPCK were observed, suggesting that kidneys from MR-fed animals are more insulin sensitive. Interestingly, renal gene expression of the mitochondrial uncoupling protein UCP1 was upregulated in MR-fed animals, as were the anti-ageing and renoprotective genes Sirt1, FGF21, klotho, and β-klotho. This was associated with alterations in renal histology trending towards reduced frequency of proximal tubule intersections containing vacuoles in mice that had been on dietary MR for 190days compared to control-fed mice, which exhibited a pre-diabetic status. Our results indicate that dietary MR may offer therapeutic potential in ameliorating the renal functional decline related to ageing and other disorders associated with metabolic dysfunction by enhancing renal insulin sensitivity and renoprotective gene expression., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Background: Components of the insulin receptor signaling pathway are probably some of the best studied ones. Even though methods for studying these components are well established, the in vivo effects of different fasting regimens, and the time course of insulin receptor phosphorylation and that of its downstream components in insulin-sensitive peripheral tissues have not been analyzed in detail., Rationale: When assessing insulin signaling, it may be beneficial to drive insulin levels as low as possible by performing an overnight fast before injecting a supra-physiological dose of insulin. Recent studies have shown however that 5 or 6 h fast in mice is sufficient to assess physiological responses to insulin and/or glucose in glucose tolerance tests, insulin tolerance tests and euglycemic hyperinsulinemic clamp studies. Moreover, mice are nocturnal feeders, with ∼70% of their daily caloric intake occurring during the dark cycle, and their metabolic rate is much higher than humans. Therefore, an overnight fast in mice is closer to starvation than just food withdrawal. Thus our aim was to assess insulin signaling components from the insulin receptor to downstream targets IRS1, Akt/PKB, GSK3, Erk1/2 and ribosomal protein S6 in muscle, liver and adipose tissue in 5 h versus 16 h (overnight) fasted mice, and the time course (0-30 min) of these phosphorylation events. We also assessed whether re-feeding under 5 h and 16 h fasting conditions was a more robust stimulus than insulin alone., Conclusions: Our study determines that a short food withdrawal from mice, for a period of 5 h, results in a similar insulin-stimulated response in phosphorylation events as the long overnight fast, presenting a more physiological experimental set up. We also demonstrate that in vivo, insulin-stimulated phosphorylation of its signaling components is different between different peripheral tissues, and depending on the tissue(s) and protein(s) of interest, an appropriate time course should be chosen., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Aims Methionine restriction (MR) and hepatic protein tyrosine phosphatase 1B (PTP1B) knockdown both improve hepatic insulin sensitivity by targeting different proteins within the insulin signaling pathway, as well as diminishing hepatic triglyceride content through decreasing hepatic lipogenesis. We hypothesized that a combined approach of hepatic PTP1B inhibition and methionine restriction could lead to a synergistic effect on improvements in glucose homeostasis and lipid metabolism. Methods Male and female hepatic PTP1B knockout ( Alb-Ptp1b -/- ) and control wild-type ( Ptp1b fl/fl ) mice were maintained on control diet (0.86% methionine) or MR diet (0.172% methionine) for 8 weeks. Body weight and food intake were recorded and physiological tests for whole-body glucose homeostasis were performed. Serum and tissues were analyzed biochemically. Results MR decreased body weight and increased food intake in Ptp1b fl/fl mice as expected, without changing PTP1B protein expression levels or activity. In females, MR treatment alone improved glucose tolerance in Ptp1b fl/fl mice, which was further amplified with hepatic PTP1B deficiency. However, other markers of glucose homeostasis were similar between MR-fed groups. In males, MR improved glucose homeostasis in both, Alb-Ptp1b -/- and wild-type Ptp1b fl/fl mice to a similar extent. Hepatic PTP1B inhibition in combination with MR could not further enhance insulin-stimulated hepatic protein kinase B/Akt phosphorylation compared to MR treatment alone and therefore led to no further increase in hepatic insulin signaling. The combined treatment did not further improve lipid metabolism relative to MR diet alone. Conclusions Methionine restriction improves glucose and lipid homeostasis; however, adding hepatic PTP1B inhibition to MR is unlikely to yield any additional protective effects. [ABSTRACT FROM AUTHOR]

The NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome has been implicated as a crucial component in both neurodegeneration and diabetes. However, the role of metabolic signalling pathways and the NLRP3 inflammasome in frontotemporal dementia remain largely elusive. We therefore investigated the effects of an NLRP3 inhibitor (MCC950) in a murine tau knock-in (PLB2 TAU) model vs. wild-type (PLB WT) control mice. In male PLB2 TAU mice (4 months at start of study), MCC950 treatment (20 mg/kg, for 12 weeks) improved insulin sensitivity and reduced circulating plasma insulin levels. Further molecular analysis suggested normalisation in insulin signalling pathways in both liver and muscle tissue. Treatment also resulted in improvements in inflammation and ER stress signalling, both peripherally and centrally, alongside a partial normalisation of phospho-tau levels. Overall, we provide evidence that MCC950 improved metabolic, inflammatory and frontotemporal dementia (FTD) relevant phenotypes in multiple tissues. NLRP3 inhibition may therefore offer a therapeutic approach to ameliorate FTD pathology. • The metabolic role of NLRP3 was investigated in a murine tauopathy model, PLB2 Tau. • The inhibitor MCC950 improved glucose tolerance and insulin signalling. • Inflammation and ER stress was ameliorated in brain and liver. • In the brain, a partial normalisation of phospho-tau levels was detected. • MCC950 partially recovered metabolic, inflammatory and FTD-relevant phenotypes. [ABSTRACT FROM AUTHOR]