Laser micromachining technique offers a promising alternative method for rapid production of microfluidic devices. However, the effect of process parameters on the channel geometry and quality of channels on common microfluidic substrates has not been fully understood yet. In this research, we studied the effect of laser system parameters on the microchannel characteristics of Polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), and microscope glass substrate—three most widely used materials for microchannels. We also conducted a cell adhesion experiment using normal human dermal fibroblasts on laser-machined microchannels on these substrates. A commercial CO2 laser system consisting of a 45W laser tube, circulating water loop within the laser tube and air cooling of the substrate was used for machining microchannels in PDMS, PMMA and glass. Four laser system parameters - speed, power, focal distance, and number of passes were varied to fabricate straight microchannels. The channel characteristics such as depth, width, and shape were measured using a scanning electron microscope (SEM) and a 3D profilometer. The results show that higher speed produces lower depth while higher laser power produces deeper channels regardless of the substrate materials. Unfocused laser machining produces wider but shallower channels. For the same speed and power, PDMS channels were the widest while PMMA channels were the deepest. Results also showed that the profiles of microchannels can be controlled by increasing the number of passes. With an increased number of passes, both glass and PDMS produced uniform, wider, and more circular channels; in contrast, PMMA channels were sharper at the bottom and skewed. In rapid cell adhesion experiments, PDMS and glass microchannels performed better than PMMA microchannels. This study can serve as a quick reference in material-specific laser-based microchannel fabrications.

Hepatic insulin resistance is a hallmark feature of nonalcoholic fatty liver disease and type-2 diabetes and significantly contributes to systemic insulin resistance. Abnormal activation of nutrient and stress-sensing kinases leads to serine/threonine phosphorylation of insulin receptor substrate (IRS) and subsequent IRS proteasome degradation, which is a key underlying cause of hepatic insulin resistance. Recently, members of the cullin-RING E3 ligases (CRLs) have emerged as mediators of IRS protein turnover, but the pathophysiological roles and therapeutic implications of this cellular signaling regulation is largely unknown. CRLs are activated upon cullin neddylation, a process of covalent conjugation of a ubiquitin-like protein called Nedd8 to a cullin scaffold. Here, we report that pharmacological inhibition of cullin neddylation by MLN4924 (Pevonedistat) rapidly decreases hepatic glucose production and attenuates hyperglycemia in mice. Mechanistically, neddylation inhibition delays CRL-mediated IRS protein turnover to prolong insulin action in hepatocytes. In vitro knockdown of either cullin 1 or cullin 3, but not other cullin members, attenuates insulin-induced IRS protein degradation and enhances cellular insulin signaling activation. In contrast, in vivo knockdown of liver cullin 3, but not cullin 1, stabilizes hepatic IRS and decreases blood glucose, which recapitulates the effect of MLN4924 treatment. In summary, these findings suggest that pharmacological inhibition of cullin neddylation represents a therapeutic approach for improving hepatic insulin signaling and lowering blood glucose.

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Background and significance Hepatic insulin resistance is a hallmark feature of non‐alcoholic fatty liver disease (NAFLD) and type‐2 diabetes (T2D) and significantly contributes to systemic insulin resistance. Abnormal activation of nutrient and stress‐sensing kinases leads to serine/threonine phosphorylation of insulin receptor substrate (IRS) and subsequent IRS proteasome degradation, which is a key underlying cause of hepatic insulin resistance. Recently, members of the cullin‐RING E3 ligases (CRLs) have emerged as mediators of IRS protein turnover. CRLs are activated upon cullin neddylation, a process of covalent conjugation of a ubiquitin‐like protein called Nedd8 to a cullin scaffold. Aim To investigate the role of CRLs in regulating hepatic insulin signaling in NAFLD. Methods Neddylation inhibitor MLN4924 was used to study its effect on insulin sensitivity in liver cells. Hyperinsulinemic‐euglycemic clamp was used to assess MNL4924 effect on insulin sensitivity in vivo. siRNA‐mediated screening was used to identify the Cullin members that mediates IRS protein turnover in liver cells. Co‐immunoprecipitation was performed to study Cullin and IRS interaction. Glucose homeostasis and insulin sensitivity were studied in mice with AAV‐mediated liver specific knockdown of Cul1 or Cul3. Results Neddylation inhibition delays CRL‐mediated IRS protein turnover to prolong insulin action in cultured liver cells. Consistently, acute MLN4924 treatment rapidly decreases hepatic glucose production without causing hypoglycemia in mice, while chronic MLN4924 treatment completely normalizes hyperglycemia in obese mice independent of obesity. Hyperinsulinemic‐euglycemic clamp study revealed that MLN4924 significantly increased insulin sensitivity and improved hepatic glucose output. In vitro siRNA based screening showed that knockdown of Cul1 or Cul3, but not other cullin family members, stabilized IRS protein and increased insulin sensitivity in AML12 cells. Consistently, Co‐immunoprecipitation demonstrated IRS interaction with Cul1 and Cul3. AAV‐mediated liver specific knockdown of Cul1 in mice improved insulin sensitivity, lowered fasting glucose, and prevented Western diet‐induced obesity and NAFLD. In contrast, AAV‐mediated liver Cul3 knockdown only decreased fasting glucose in chow‐fed mice but not Western diet‐fed mice. Conclusion These findings suggest that pharmacological inhibition of cullin neddylation is a novel therapeutic strategy for hepatic insulin sensitization in NAFLD and T2D. This effect may be at least in part mediated by hepatic Cul1 and Cul3 neddylation.