Dysregulation of hepatic glucose production (HGP) serves as a major underlying mechanism for the pathogenesis of type 2 diabetes. The pancreatic hormone glucagon increases and insulin suppresses HGP, controlling blood glucose homeostasis. The forkhead transcription factor Foxo1 promotes HGP through increasing expression of genes encoding the rate-limiting enzymes responsible for gluconeogenesis. We previously established that insulin suppresses Foxo1 by Akt-mediated phosphorylation of Foxo1 at Ser in human hepatocytes. In this study, we found a novel Foxo1 regulatory mechanism by glucagon, which promotes Foxo1 nuclear translocation and stability via cAMP- and protein kinase A-dependent phosphorylation of Foxo1 at Ser Replacing Foxo1-S276 with alanine (A) or aspartate (D) to block or mimic phosphorylation, respectively, markedly regulates Foxo1 stability and nuclear localization in human hepatocytes. To establish in vivo function of Foxo1-Ser phosphorylation in glucose metabolism, we generated Foxo1-S273A and Foxo1-S273D knock-in (KI) mice. The KI mice displayed impaired blood glucose homeostasis, as well as the basal and glucagon-mediated HGP in hepatocytes. Thus, Foxo1-Ser is a new target site identified in the control of Foxo1 bioactivity and associated metabolic diseases.
Glucagon promotes hepatic glucose production maintaining glucose homeostasis in the fasting state. Glucagon maintains at high level in both diabetic animals and human, contributing to hyperglycemia. Mitochondria, a major place for glucose oxidation, are dysfunctional in diabetic condition. However, whether hepatic mitochondrial function can be affected by glucagon remains unknown. Recently, we reported that FOXO1 is an important mediator in glucagon signaling in control of glucose homeostasis. In this study, we further assessed the role of FOXO1 in the action of glucagon in the regulation of hepatic mitochondrial function. We found that glucagon decreased the heme production in a FOXO1-dependent manner, suppressed heme-dependent complex III (UQCRC1) and complex IV (MT-CO1) and inhibited hepatic mitochondrial function. However, the suppression of mitochondrial function by glucagon was largely rescued by deleting the Foxo1 gene in hepatocytes. Glucagon tends to reduce hepatic mitochondrial biogenesis by attenuating the expression of NRF1, TFAM and MFN2, which is mediated by FOXO1. In db/db mice, we found that hepatic mitochondrial function was suppressed and expression levels of UQCRC1, MT-CO1, NRF1 and TFAM were downregulated in the liver. These findings suggest that hepatic mitochondrial function can be impaired when hyperglucagonemia occurs in the patients with diabetes mellitus, resulting in organ failure.
Forkhead/winged helix transcription factor O-class member 1 (FoxO1) is a key mediator of insulin and glucagon signaling in control of glucose homeostasis. Although epigallocatechin gallate (EGCG) has attracted interest owing to its potential to combat hyperglycemic diabetes, molecular mechanisms underlying its antihyperglycemic effect, in particular the effect on FoxO1, is poorly understand. This study aims to assess the impact of EGCG on the glucagon signaling pathway in regulating glucose metabolism. Primary hepatocytes from wild-type (WT), liver-specific FoxO1 knock out (FKO), and FoxO1-S273D knock-in (KI) mice were isolated, cultured, and treated with EGCG and/or glucagon. Our data showed the treatment of 10 μM EGCG for 6 h decreased hepatic glucose production by 20 and 23% in WT and FKO primary hepatocytes, respectively. EGCG repressed both gluconeogenesis and glycogenolysis in primary hepatocytes, coupled with activating AMPK. In addition, EGCG decreased mitochondrial oxygen consumption. We further investigated the effects of EGCG on glucagon-stimulated cAMP/PKA signaling pathway. EGCG reduced p-PKA-T197/T-PKA and p-CREB-S133/T-CREB levels by 39 and 20%, blocked p-FoxO1-S273, and suppressed nuclear FoxO1 translocation, suggesting that FoxO1 and CREB were possible downstream targets. A novel mechanism of EGCG in restraining hepatic glucose production (HGP) is through antagonizing glucagon signaling and suppressing FoxO1 via Ser273. EGCG may serve as a promising compound for regulating glucose homeostasis.
The liver is a key player for maintaining glucose homeostasis. Excessive hepatic glucose production is considered to be a key for the onset of type 2 diabetes. The primary function of heme oxygenase-1 (HO1) is to catalyze the degradation of heme into biliverdin, ferrous iron, and carbon monoxide. Previous studies have demonstrated that the degradation of heme by HO1 in the liver results in mitochondrial dysfunction and drives insulin resistance. In this study, by overexpressing HO1 in hepatocytes and mice, we showed that HO1 promotes gluconeogenesis in a Foxo1-dependent manner. Importantly, HO1 overexpression increased the generation of ferrous iron in the liver, which further activates nuclear factor-κB and phosphorylates Foxo1 at Ser273 to enhance gluconeogenesis. We further assessed the role of HO1 in insulin-resistant liver-specific knockout of IRS1 and IRS2 genes (L-DKO) mice, which exhibit upregulation of HO1 in the liver and hepatic ferrous iron overload. HO1 knockdown by shRNA or treatment of iron chelator rescued the aberrant gluconeogenesis in L-DKO mice. In addition, we found that systemic iron overload promotes gluconeogenesis by activating the hepatic protein kinase A→Foxo1 axis. Thus, our results demonstrate the role of HO1 in regulating hepatic iron status and Foxo1 to control gluconeogenesis and blood glucose.
Accurate deflection readings must be obtained when using NDT devices in order to obtain correct backcalculated layer moduli. This paper provides an analysis of the sources of deflection errors and illustrates through a series of examples how random deflection errors and random pavement thickness errors affect backcalculated moduli. The consequence of these errors is most pronounced in the backcalculated modulus of the surface course. The moduli of the base and subbase layers are also affected, and the subgrade moduli are only slightly affected by these errors. To some degree this outcome may be due to the bottom-to-top approach used by the backcalculation program MODCOMP 2. The consequence of the variability of the backcalculated moduli on the required pavement overlay thicknesses was found to be minimal. This was due to the fact that, generally speaking, data errors which cause an overestimate of the base course modulus also cause an approximately equally significant, but opposite, underestimation of the surface course modulus. Thus, the needed overlay requirement remains nearly the same. The paper shows how certain FWD calibration and testing procedures can be used to minimize deflection measurement errors. The accuracy of backcalculated moduli is shown to be determinable from the RMS error of the deflection fit. The examples provide an impression of the extent to which random layer thickness errors and deflection measurement errors will influence the ability to discern point-to-point variability of layer moduli for the specific pavement structure considered in this investigation. This provides a perspective from which the practical accuracy of the backcalculation approach can be judged.
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