Many people living with diabetes also have non-alcoholic fatty liver disease (NAFLD). Interleukin-6 (IL-6) is involved in both diseases, interacting with both membrane-bound (classical) and circulating soluble receptors (trans-signaling). We investigated whether secretion of IL-6 trans-signaling co-receptors are altered in NAFLD by diabetes, and whether this might associate with the severity of fatty liver disease. Secretion patterns were investigated using human hepatocyte, stellate and monocyte cell lines. Associations with liver pathology were investigated in two patient cohorts: 1) biopsy-confirmed NASH and 2) class 3 obesity. We found that exposure of stellate cells to high glucose and palmitate increased IL-6 and sgp130 secretion. In line with this, plasma sgp130 in both patient cohorts positively correlated with HbA1c, and subjects with diabetes had higher circulating levels of IL-6 and trans-signaling co-receptors. Plasma sgp130 strongly correlated with liver stiffness and was significantly increased in subjects with F4 fibrosis stage. Monocyte activation was associated with reduced sIL-6R secretion. These data suggest that the hyperglycemia and hyperlipidemia can directly impact IL-6 trans-signaling, and that this may be linked to enhanced severity of NAFLD in patients with concomitant diabetes.

The importance of glucokinase (GK) in the regulation of insulin secretion has been highlighted by the phenotypes of individuals with activating and inactivating mutations in the glucokinase gene (GCK). Here we report 10 cases of congenital hyperinsulinism (HI) caused by eight unique activating mutations of GCK. Six are novel and near previously identified activating mutations sites. The first recognized episode of hypoglycemia in these patients occurred between birth to 24 years and severity of the phenotype was also variable. Mutant enzymes were expressed and purified for enzyme kinetics in vitro. Mutant enzymes had low glucose S0.5 values and increased enzyme activity index compared to wild type GK. We performed functional evaluation of islets from the pancreata of 3 children with GCK-HI that required pancreatectomy. Basal insulin secretion in perifused GCK-HI islets was normal and the response to glyburide was preserved. However, the threshold for glucose-stimulated insulin secretion in perifused GCK-HI islets was decreased and glucagon secretion was greatly suppressed. Our evaluation of novel GCK disease-associated mutations revealed that the detrimental effects of these mutations on glucose homeostasis can be attributed not only to a lowering of the glucose threshold of insulin secretion, but also to a decreased counter-regulatory glucagon secretory response.

Type 2 diabetes is associated with cognitive impairment and a twofold increased risk of dementia compared to age-matched individuals without diabetes. Given that the eye and the brain share similar embryologic origin and anatomical features the retina offers a unique "window" to the brain. In this study we wanted to determine whether there was a difference in retinal imaging-based neuronal and vascular markers in individuals with type 2 diabetes with or without minimal cognitive impairment (MCI). We included 134 persons with type 2 diabetes. Based on neuropsychological tests the prevalence of MCI was 28%. We performed 7-field color fundus photos, optical coherence tomography (OCT), OCT-Angiography and retinal oximetry in order to analysis retinal markers. In a multivariable cluster analysis, persons with MCI had significant thinner macular retinal nerve fiber layer and macular ganglion cell layer, and less venular oxygen saturation in the nasal quadrant compared to those without MCI. There were no differences in retinal vessel density, fractal dimension, width, tortuosity or OCT-A markers. People with type 2 diabetes and MCI demonstrate alterations in retinal structure and metabolism, suggesting non-invasive retinal markers may be useful to detect those at risk of cognitive dysfunction in people with type 2 diabetes.

Insulin activates insulin receptor (IR) signaling and subsequently triggers IR endocytosis to attenuate signaling. Cell division regulators MAD2, BUBR1, and p31comet promote IR endocytosis upon insulin stimulation. Here, we show that genetic ablation of the IR-MAD2 interaction in mice delays IR endocytosis, increases IR levels, and prolongs insulin action at the cell surface. This in turn causes a defect in insulin clearance and increases circulating insulin levels, unexpectedly increasing glucagon levels, which alters glucose metabolism modestly. Disruption of the IR-MAD2 interaction increases serum fatty acid concentrations and hepatic fat accumulation in fasted male mice. Furthermore, disruption of the IR-MAD2 interaction distinctly changes metabolic and transcriptomic profiles in the liver and adipose tissues. Our findings establish the function of cell division regulators in insulin signaling and provide insights into the metabolic functions of IR endocytosis.

Maternal (m) consumption of a Western-style diet (WD) during pregnancy alters fatty-acid metabolism and reduces insulin sensitivity in fetal skeletal muscle. The long-term impact of these fetal adaptations and the pathways underlying disordered lipid metabolism are incompletely understood. Therefore, we tested whether a mWD chronically fed to lean, insulin sensitive adult Japanese macaques throughout pregnancy and lactation would impact skeletal muscle oxidative capacity and lipid metabolism in adolescent offspring fed a postweaning (pw)WD or control diet (CD). Although body weight was not different, retroperitoneal fat mass and subscapular skinfold thickness were significantly higher in pwWD offspring consistent with elevated fasting insulin and glucose. Maximal complex I-dependent respiration in muscle was lower in mWD offspring in the presence of fatty acids, suggesting that mWD impacts muscle integration of lipid with non-lipid oxidation. Abundance of all five OXPHOS complexes and VDAC, but not ETF/ETFDH, were reduced with mWD, partially explaining the lower respiratory capacity with lipids. Muscle triglycerides increased with pwWD; however, the fold increase in lipid saturation, 1,2-diacylglycerides and C18: ceramide between pwCD and pwWD was greatest in mWD offspring. Reductions in complex I abundance and VDAC correlated with reduced markers of oxidative stress suggesting these reductions may be an early life adaptation to mWD to mitigate excess reactive oxygen species. Altogether, mWD, independent of maternal obesity or insulin resistance, results in sustained metabolic reprogramming in offspring muscle despite a healthy diet intervention.

Both long- and short-term glycemic variability have been associated with incident diabetes complications. We evaluated their relative and potential additive effects on incident renal complications in the Action to Control Cardiovascular Risk in Diabetes trial. A marker of shortterm glycemic variability 1,5-anhydroglucitol (1,5-AG) was measured in 4,000 random 12-months post-randomization plasma samples (when hemoglobin A1C (HbA1c) was stable). Visit-to-visit fasting plasma glucose coefficient of variation (CV-FPG) was determined from 4 months postrandomization until the endpoints of (1) microalbuminuria or (2) macroalbuminuria. Using Cox proportional hazard models, high CV-FPG and low 1,5-AG were independently associated with microalbuminuria after adjusting for clinical risk factors. However, only the CV-FPG association remained after additional adjustments for average HbA1c. Only CV-FPG was a significant risk factor for macroalbuminuria. This post hoc analysis indicates that long-term rather than short-term glycemic variability better predicts risk for renal disease in type 2 diabetes.

There is clinical evidence that increased urinary serine proteases are associated with the disease severity in the setting of diabetic nephropathy (DN). Elevation of serine proteases may mediate [Ca2+]i dynamics in podocytes through the protease-activated receptors (PARs) pathway, including associated activation of non-specific cation channels. Cultured human podocytes and freshly isolated glomeruli were used for fluorescence and immunohistochemistry stainings, calcium imaging, Western blot analysis, scanning ionconductance microscopy, and patch-clamp analysis. Goto-Kakizaki, Wistar, type 2 diabetic nephropathy (T2DN), and a novel PAR1 knockout on T2DN rat background rats were used to test the importance of PAR1-mediated signaling in DN settings. We found that PAR1 activation increases [Ca2+]i via TRPC6 channels. Both human cultured podocytes exposed to high glucose and podocytes from freshly isolated glomeruli of T2DN rats had increased PAR1-mediated [Ca2+]i compared to controls. Imaging experiments revealed that PAR1 activation plays a role in podocyte morphological changes. T2DN rats exhibited a significantly higher response to thrombin and urokinase. Moreover, the plasma concentration of thrombin in T2DN rats was significantly elevated compared to Wistar rats. T2DNPar1-/- rats were embryonically lethal. T2DNPar1+/- rats had a significant decrease in glomerular damage associated with DN liaisons. Overall, this data provides evidence that during the development of DN, elevated levels of serine proteases promote an excessive [Ca2+]i influx in podocytes through the PAR1-TRPC6 signaling, ultimately leading to podocyte apoptosis, the development of albuminuria, and glomeruli damage.

Hemopexin (HPX) is overexpressed in the retina of diabetic patients, and induces the breakdown of the blood-retinal barrier (BRB) in vitro. The present study was aimed at evaluating whether HPX blockade by specific antibodies (aHPX) could avoid vascular leakage in vivo and microvascular angiogenesis in vitro and ex vivo. For this purpose, the effect of intravitreal injections (IVT) of aHPX on vascular leakage was evaluated in db/db mice and rats with streptozotocin-induced diabetes (D-STZ) using the Evans Blue method. Retinal neurodegeneration and inflammation were also evaluated. The antiangiogenic effect of aHPX on human retinal endothelial cells (HREC) was tested by scratch wound healing and tube formation using standardized methods, as well as choroidal sprouting assays from retinal explants obtained in rats. We found that IVT of aHPX significantly reduced vascular leakage, as well as retinal neurodegeneration and inflammation. In addition, treatment with aHPX significantly reduced the HRECs migration and tube formation induced by high glucose concentration, and suppressed choroidal sprouting even after VEGF stimulation, this effect being higher than obtained with Bevacizumab. In conclusion, the anti-permeability and antiangiogenic effects IVT of aHPX suggest the blockade or inhibition of HPX as a new strategy for the treatment of advanced stages of diabetic retinopathy.

The ability of metabolically active tissues to increase glucose uptake in response to insulin is critical to whole-body glucose homeostasis. This report describes the Dual Tracer Test, a robust method involving sequential retro-orbital injection of 14C-2-deoxyglucose (14C-2DG) alone, followed 40 min later by injection of 3H-2DG with a maximal dose of insulin to quantify both basal and insulin-stimulated 2DG uptake in the same mouse. The collection of both basal and insulin-stimulated measures from a single animal is imperative for generating high-quality data since differences in insulin action may be misinterpreted mechanistically if basal glucose uptake is not accounted for. The approach was validated in a classic diet-induced model of insulin resistance and a novel transgenic mouse with reduced GLUT4 expression that, despite ubiquitous peripheral insulin resistance, did not exhibit fasting hyperinsulinemia. This suggests that reduced insulin-stimulated glucose disposal is not a primary contributor to chronic hyperinsulinemia. The Dual Tracer Test offers a technically simple assay that enables the study of insulin action in many tissues simultaneously. By administering two tracers and accounting for both basal and insulin-stimulated glucose transport, this assay halves the required sample size for studies in inbred mice and demonstrates increased statistical power to detect insulin resistance, relative to other established approaches using a single tracer. The Dual Tracer Test is a valuable addition to the metabolic phenotyping toolbox.