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Adipocytes are key regulators of human metabolism, and their dysfunction in insulin signaling is central to metabolic diseases including type II diabetes mellitus (T2D). However, the progression of insulin resistance into T2D is still poorly understood. This limited understanding is due, in part, to the dearth of suitable models of insulin signaling in human adipocytes. Traditionally, adipocyte models fail to recapitulate in vivo insulin signaling, possibly due to exposure to supraphysiological nutrient and hormone conditions. We developed a protocol for human pluripotent stem cell–derived adipocytes that uses physiological nutrient conditions to produce a potent insulin response comparable to in vivo adipocytes. After systematic optimization, this protocol allows robust insulin-stimulated glucose uptake and transcriptional insulin response. Furthermore, exposure of sensitized adipocytes to physiological hyperinsulinemia dampens insulin-stimulated glucose uptake and dysregulates insulin-responsive transcription. Overall, our methodology provides a novel platform for the mechanistic study of insulin signaling and resistance using human pluripotent stem cell–derived adipocytes.

An estimated 70% of individuals with type 2 diabetes also suffer from nonalcoholic fatty liver disease (NAFLD) . Insulin resistance is pathologically fundamental to both NAFLD and T2D, however the mechanisms of insulin resistance linking these complex diseases remains incompletely understood. Microphysiological systems provide an avenue to simulate disease parameters in the context of human biology, however current in vitro models of hepatic insulin resistance often include nutrient and insulin concentrations orders of magnitude above in vivo levels. Here, we established an in vitro 3D model culturing primary human hepatocytes and non-parenchymal cells in physiologically relevant media conditions simulating both baseline physiological and T2D conditions. Albumin secretion and cytochrome p450 assays indicate hepatocytes remain functional over two weeks in culture for both physiological and T2D conditions. Results further show hepatocytes cultured in physiological conditions maintain insulin responsiveness after 12 days in culture, measured by glucose output following dose response of insulin stimulation. Conversely, cells cultured in T2D media conditions have significantly attenuated response to insulin stimulation compared to the physiological condition. Measuring insulin clearance from the media also showed reduced clearance over time for the T2D conditions. Together, these results demonstrate the induction of hepatic insulin resistance through culture with insulin concentrations found in the portal vein of T2D patients. Studies further applying this in vitro model to understand insulin resistance mechanisms are ongoing. Disclosure E.Tevonian: Research Support; Novo Nordisk. J.F.Jeppesen: Employee; Novo Nordisk A/S. D.Lauffenburger: None. L.G.Griffith: Research Support; Novo Nordisk A/S, Novo Nordisk A/S, Novo Nordisk A/S, Novo Nordisk A/S, Novo Nordisk A/S, Novo Nordisk A/S, Novo Nordisk A/S.

Insulin receptor (IR) signaling is central to normal metabolic control and is dysregulated in metabolic diseases such as type 2 diabetes. We have found that IR is incorporated into biomolecular condensates during the response to insulin stimulation and that the activity of IR in these condensates is altered in insulin resistance. In normal cells, insulin stimulation results in accumulation of IR in bodies that display dynamic molecular characteristics expected of liquid-like condensates. In insulin-resistant cells, both IR accumulation in condensates and the normal dynamic behavior of these condensates are reduced, suggesting a physico-mechanical link between insulin response and the dynamic molecular behavior of IR condensates. The observed defects in IR condensate behaviors are caused, at least in part, by an increase in oxidative stress in insulin-resistant cells, which is known to interfere with normal condensate dynamics. Treatment of insulin-resistant cells with metformin, a first-line drug used to treat type 2 diabetes, can rescue accumulation and dynamic behavior of IR condensates. This rescue corresponds with metformin’s effect of reducing the levels of reactive oxygen species. The observation that IR is incorporated into biomolecular condensates during the response to insulin stimulation, and evidence that changes in the physico-mechanical features of IR condensates contribute to insulin resistance, have implications for improved therapeutic approaches for patients. Disclosure A.Dall’agnese: Consultant; Dewpoint Therapeutics. J.Platt: None. T.Lee: None. J.F.Jeppesen: Employee; Novo Nordisk A/S. R.A.Young: Research Support; Novo Nordisk.

Insulin resistant individuals display elevated fasting and post-prandial glucose levels which are mainly driven by inadequate inhibition of gluconeogenesis (GNG) and are rarely normalized. The aim of this study was to investigate whether direct inhibition of GNG could be a new therapeutical approach to improve glucose homeostasis using in vitro and in vivo models of insulin resistance. Fructose 1,6-bisphosphatase (FBP1) is key in controlling GNG in liver and kidney and loss of function patients display hypoglycaemia episodes and lactate acidosis. Systemic FBP1 inhibition using small molecules has shown to improve glucose homeostasis. To assess the clinical efficacy and safety potential of silencing FBP1 selectively in the liver we dosed insulin resistant DIO rats with a hepatocyte specific GalXC-FBP1 siRNA entity. GalXC-FBP1 siRNA markedly reduced FBP1 mRNA level in the liver by over 90%. This was associated with a complete lack of glucose excursion following a pyruvate challenge indicating inhibition of GNG. Blood glucose level decreased similarly in rats treated with GalXC-FBP1 siRNA or vehicle during an insulin challenge or a prolonged fast suggesting that hepatic FBP1 silencing is not inducing hypoglycaemia. Insulin sensitivity and hyperinsulinemia were improved. Plasma lactate and liver enzymes were not elevated, but a significant 2-fold increase in liver triglycerides was observed in the GalXC-FBP1 siRNA group. Using human hepatocytes in a Liver-on-Chip in vitro model FBP1 silencing reduced glucose production by 30% supporting the relevance of this approach in humans. Collectively these data suggest that liver specific FBP1 silencing has the potential to improve insulin sensitivity in DIO rats without inducing hypoglycaemia but may be associated with a risk of liver steatosis overtime. This concept highlights the difficulty of blocking liver GNG without re-directing the metabolic intermediates fluxes toward triglycerides accumulation in liver. Disclosure C. Fledelius: Employee; Novo Nordisk A/S. D. Demozay: Employee; Novo Nordisk A/S. H. Iversen: None. R. S. Ingvorsen: None. L. B. Eriksen: None. A. Blois: Employee; Novo Nordisk. Y. Montauban: None. R. Rijnbrand: Employee; Novo Nordisk. W. Han: None. J. F. Jeppesen: Employee; Novo Nordisk A/S.

SummaryThe capacity to generate functional hepatocytes from renewable human pluripotent stem cells (hPSCs) could address limited supplies of primary human hepatocytes. However, hepatocytes differentiated from hPSCs in vitro are functionally immature. To understand mechanisms regulating maturation of in vitro derived hepatocytes, we developed a 3D spheroid differentiation system and compared gene regulatory elements in uncultured human primary hepatocytes with those in hepatocytes that were differentiated in 2D or 3D conditions from human PSCs by RNA-seq, ATAC-seq, and H3K27Ac ChIP-seq. Three-dimensional differentiation improved enhancer activity and expression of transcription factor ONECUT1, but was insufficient to upregulate human-specific mature hepatocytes marker gene CYP3A4 or super-enhancer regulated transcription factor gene NFIC. Regulome comparisons showed reduced enrichment of thyroid receptor THRB motifs in accessible chromatin and in active enhancers without reduced transcription of THRB, suggesting the regulation at the level of THRB ligands in PSC-differentiated hepatocytes. Addition of thyroid hormone T3 to the PSC-differentiated hepatocytes increased CYP3A4 expression. T3 increased binding of THRB to the CYP3A4 proximal enhancer and restored the super-enhancer status and gene expression of NFIC and reduced expression of AFP. The resultant hPSC-hepatocytes showed gene expression, epigenetic status and super-enhancer landscape closer to primary hepatocytes and activated regulatory regions including non-coding SNPs associated with liver-related diseases. Transplanting the 3D PSC-hepatocytes into immunocompromised mice resulted in engraftment of human hepatocytes in the mouse liver parenchyma without disrupting normal liver histology at 6 months after transplantation. This work provides insights into the functions of nuclear receptor THRB and highlights the importance of the environmental factors-nuclear receptors axis in regulating maturation of human PSC-differentiated cell types.

Semaglutide, a glucagon-like peptide 1 (GLP-1) analog, induces weight loss, lowers glucose levels, and reduces cardiovascular risk in patients with diabetes. Mechanistic preclinical studies suggest weight loss is mediated through GLP-1 receptors (GLP-1Rs) in the brain. The findings presented here show that semaglutide modulated food preference, reduced food intake, and caused weight loss without decreasing energy expenditure. Semaglutide directly accessed the brainstem, septal nucleus, and hypothalamus but did not cross the blood-brain barrier; it interacted with the brain through the circumventricular organs and several select sites adjacent to the ventricles. Semaglutide induced central c-Fos activation in 10 brain areas, including hindbrain areas directly targeted by semaglutide, and secondary areas without direct GLP-1R interaction, such as the lateral parabrachial nucleus. Automated analysis of semaglutide access, c-Fos activity, GLP-1R distribution, and brain connectivity revealed that activation may involve meal termination controlled by neurons in the lateral parabrachial nucleus. Transcriptomic analysis of microdissected brain areas from semaglutide-treated rats showed upregulation of prolactin-releasing hormone and tyrosine hydroxylase in the area postrema. We suggest semaglutide lowers body weight by direct interaction with diverse GLP-1R populations and by directly and indirectly affecting the activity of neural pathways involved in food intake, reward, and energy expenditure.

Acute glucagon-like peptide-1 (GLP-1) infusion reversed the high fat diet-induced microvascular insulin resistance that occurred after both 5 days and 8 weeks of a high fat diet intervention. When GLP-1 was co-infused with insulin it had overt effects on whole body insulin sensitivity as well as insulin-mediated skeletal muscle glucose uptake after 5 days of a high fat diet, but not after 8 weeks of high fat diet intervention. Acute GLP-1 infusion did not have an additive effect to that of insulin on microvascular recruitment or skeletal muscle glucose uptake in the control group. Here we demonstrate that GLP-1 potently increases the microvascular recruitment in rat skeletal muscle but does not increase glucose uptake in the fasting state. Thus, like insulin, GLP-1 increased the microvascular recruitment but unlike insulin, GLP-1 had no direct effect on skeletal muscle glucose uptake.Acute infusion of glucagon-like peptide-1 (GLP-1) has potent effects on blood flow distribution through the microcirculation in healthy humans and rats. A high fat diet induces impairments in insulin-mediated microvascular recruitment (MVR) and muscle glucose uptake, and here we examined whether this could be reversed by GLP-1. Using contrast-enhanced ultrasound, microvascular recruitment was assessed by continuous real-time imaging of gas-filled microbubbles in the microcirculation after acute (5 days) and prolonged (8 weeks) high fat diet (HF)-induced insulin resistance in rats. A euglycaemic hyperinsulinaemic clamp (3 mU min(-1) kg(-1) ), with or without a co-infusion of GLP-1 (100 pmol l(-1) ), was performed in anaesthetized rats. Consumption of HF attenuated the insulin-mediated MVR in both 5 day and 8 week HF interventions which was associated with a 50% reduction in insulin-mediated glucose uptake compared to controls. Acute administration of GLP-1 restored the normal microvascular response by increasing the MVR after both 5 days and 8 weeks of HF intervention (P 0.05). This effect of GLP-1 was associated with a restoration of both whole body insulin sensitivity and increased insulin-mediated glucose uptake in skeletal muscle by 90% (P 0.05) after 5 days of HF but not after 8 weeks of HF. The present study demonstrates that GLP-1 increases MVR in rat skeletal muscle and can reverse early stages of high fat diet-induced insulin resistance in vivo.