Pathway-selective Insulin Resistance Research Articles

SAT048 Insulin Signaling Through The Insulin Receptor Increases Bone Mineral Density And Linear Growth Through Its Effects On Bone

Abstract Disclosure: R.M. Tuska: None. M.C. Okawa: None. M. Lightbourne: None. J.C. Reynolds: None. A. Layne: None. R.J. Brown: None. Context: Overnutrition and obesity associates with increased childhood growth and higher bone mineral density (BMD), whereas undernutrition associates with impaired growth and lower BMD. Increased vs decreased insulin signaling in bone may be partly responsible for these phenotypes. Patients with lipodystrophy syndromes (LD, characterized by deficient adipose tissue) have pathway selective insulin resistance (IR) causing impairment of some insulin signaling pathways while others are overstimulated by compensatory hyperinsulinemia. These patients thus serve as models of increased insulin signaling in bone without confounding by increased body mass. By contrast, patients with homozygous pathogenic variants of the insulin receptor (INSR-/-), have impairment of all insulin signaling pathways and thus model decreased insulin signaling in bone. We hypothesized that patients with LD would have tall stature and high BMD due to hyperinsulinemia, whereas those with INSR would have short stature and low BMD due to lack of insulin signaling despite hyperinsulinemia. Methods. We compared patients with genetic forms of generalized LD (GLD, (N=30), partial LD (PLD, (N=104), and INSR-/- (N=10). Whole body and site-specific (1/3 radius, Total Hip, femoral neck, and spine) DXA scans were obtained. Height Standard Deviation Scores (SDS) were calculated based on sex and age-matched normative data for patients <20 y old and based on sex-specific 20-y-old normative data for patients ≥ 20 y. Similarly, height-adjusted BMD SDS were calculated using BMDCS reference data. Blood was collected after an 8-12 hour fast for measurement of insulin. Results. All groups had hyperinsulinemia, which was highest in INSR-/- followed by PLD and CGL (423.3 mcU/ml [161.0,1000]; 31.0 mcU/ml [17.1,62.2]; 26.1 mcU/ml [11.2, 52.4]; p<0.0001). Patients with GLD and PLD had tall stature and high BMD vs the general population, whereas INSR -/- had short stature and low BMD. GLD had the greatest whole-body and site-specific BMD SDS followed by PLD and INSR -/- (Whole Body: 1.9±0.9, 0.4±1.3, -1.9± 0.1, p <0.0001; 1/3 Radius: 1.2± 1.2, 0.1± 1.6, -1.5± 1.2, p = 0.0002; Femoral Neck: 1.4±1.0, -0.7±1.3, -1.9±0.8, p <0.0001; Total Hip: 2.0±1.0, -0.3±1.3, -2.0±1.2, p <0.0001; Spine: 1.5±1.0, 0.5±1.6, -1.7±0.7, p <0.0001). All BMD SDS in each group differed from the population mean of 0, except for 1/3 radius in PLD. Similarly, GLD had the greatest height SDS followed by PLD and INSR-/- (0.5± 1.0, 0.3± 1.2, -2.5± 1.4, p<0.0001), all of which differed from the population mean. Conclusions: Patients with non-selective insulin resistance (INSR-/-) lack insulin signaling in bone, contributing to short stature and low BMD. Pathway selective insulin resistance, as observed in patients with GLD and PLD, contributes to compensatory hyperinsulinemia thus increasing insulin signaling in bone, which contributes to tall stature and high BMD. Presentation: Saturday, June 17, 2023

THU047 Growth Hormone (GH) Signaling In Hepatocytes Controls Hepatic Insulin Sensitivity And Nutrient Disposition

Abstract Disclosure: M. del Rio-Moreno: None. M. Vazquez-Borrego: None. J. Cordoba-Chacon: None. O.P. McGuinness: None. R.D. Kineman: None. It is commonly accepted that GH reduces the actions of insulin. However, we reported plasma GH levels within a physiological range are negatively associated with insulin-mediated glucose uptake, but positively associated with insulin-mediated suppression of hepatic glucose production (HGP) [Cordoba-Chacon J. et al. 2014. AJP Endocrinol Metab]. These findings suggest that GH may promote insulin’s actions on the liver. To determine the direct contribution of GH signaling on hepatic insulin sensitivity, hyperinsulinemic-euglycemic clamps were performed with stable isotope tracers in chow-fed adult-onset, hepatocyte-specific GHR knockdown (aHepGHRkd) mice. aHepGHRkd mice show reduced IGF1 and increased GH levels [Cordoba-Chacon J. et al 2015, Diabetes] which indirectly impacts systemic insulin sensitivity and substrate flux to the liver. To normalize plasma IGF1 and GH levels, aHepGHRkd mice were co-treated with adeno-associated virus (AAV) that express IGF1 or a constitutively active form of STAT5b (STAT5bCA). These AAVs have a thyroxin binding globulin promoter, leading to hepatocyte-specific expression. Under both basal and clamped conditions, aHepGHRkd mice exhibited hyperinsulinemia, that was normalized by AAV-mediated IGF1 and STAT5bCA expression. Interestingly, glucose disappearance (Rd), tissue-specific glucose uptake, or HGP (gluconeogenesis and glycogenolysis) did not differ across all groups. However, higher insulin tone in aHepGHRkd mice was required to achieve the same level of Rd and suppression of HGP in the steady-state, which was indicative of systemic and hepatic insulin resistance. These observations are consistent with our previous findings that GH reduces tissue-specific glucose uptake but promotes insulin-mediated control of HGP. In contrast, aHepGHRkd mice exhibited an increased rate of hepatic de novo lipogenesis, and an increase in plasma levels of newly formed triglyceride and triglyceride-bound palmitate, that was normalized by IGF1 and STAT5bCA expression. Taken together, these results indicate loss of hepatocyte GH signaling leads to pathway selective hepatic insulin resistance. This pathway selective hepatic insulin resistance has also been reported in mouse models due to altered substrate delivery to the liver as consequence of excess dietary intake and/or increased adipose tissue lipolysis. However, chow-fed aHepGHRkd mice show hyperinsulinemia that was sufficient to normalize systemic glucose uptake and suppress WAT lipolysis, as noted by normal plasma NEFA levels under both basal and clamped conditions. Therefore, changes in substrate delivery to the liver cannot readily explain the changes observed, suggesting hepatocyte GHR-signaling plays a direct role in partitioning of hepatic nutrients in response to insulin. Presentation: Thursday, June 15, 2023

289-OR: Defining Pathway-Selective Insulin Resistance in Type 2 Diabetes Using iPS Cell-Derived Hepatocytes

Hepatic insulin resistance is central to type 2 diabetes (T2D), fatty liver disease, and metabolic syndrome. To identify the fundamental, cell-autonomous defects underlying hepatic insulin resistance in T2D, we have used inducible human pluripotent stem (iPS) cells derived from 16 individuals with and without T2D differentiated to hepatocytes (iHeps) and studied in vitro. Consistent with pathway selective insulin resistance, we find that insulin failed to suppress gene expression of critical gluconeogenic enzymes (PCK1, G6PC) but continued to stimulate increased expression of the lipogenic enzymes, fatty acid synthase and acetyl Co-A carboxylase 1 in T2D iHeps. iHeps from T2D also displayed reduced insulin receptor tyrosine phosphorylation and reduced phosphorylation through the IRS/Akt pathway. Global phosphoproteomics in control and T2D iHeps identified 378 unique phosphosites regulated by insulin, and many dysregulated phosphosites within the classical insulin signaling pathway (IRS, AKT1/2, GSK3, FOXO1, RPS6KB1, TSC2) and components of gene transcription regulation, membrane trafficking, Rho-GTPases, vesicle transport. Using a kinome-wide data set created with synthetic peptide libraries to profile almost all functional known human serine/threonine kinases, we found the alterations in insulin signaling mapped to AKT, P70S6K, P90S6K, RSK, PKCτ, and SGK kinases. Thus, iHeps from T2D patients show pathway-selective insulin resistance with a loss of suppression of gluconeogenesis by insulin yet persistent activation of lipogenesis, even in vitro, in the absence of any circulating factor exposure. T2D iHeps also show a dysregulated phosphorylation network that provides new clues to the kinases whose action is altered in T2D and hepatic insulin resistance. Disclosure A.Gattu: None. A.Krook: None. J.R.Zierath: None. M.Mann: None. C.Kahn: None. Funding National Institutes of Health (T32DK007260-46)

Endothelial dysfunction due to selective insulin resistance in vascular endothelium: insights from mechanistic modeling.

Previously, we have used mathematical modeling to gain mechanistic insights into insulin-stimulated glucose uptake. Phosphatidylinositol 3-kinase (PI3K)-dependent insulin signaling required for metabolic actions of insulin also regulates endothelium-dependent production of the vasodilator nitric oxide (NO). Vasodilation increases blood flow that augments direct metabolic actions of insulin in skeletal muscle. This is counterbalanced by mitogen-activated protein kinase (MAPK)-dependent insulin signaling in endothelium that promotes secretion of the vasoconstrictor endothelin-1 (ET-1). In the present study, we extended our model of metabolic insulin signaling into a dynamic model of insulin signaling in vascular endothelium that explicitly represents opposing PI3K/NO and MAPK/ET-1 pathways. Novel NO and ET-1 subsystems were developed using published and new experimental data to generate model structures/parameters. The signal-response relationships of our model with respect to insulin-stimulated NO production, ET-1 secretion, and resultant vascular tone, agree with published experimental data, independent of those used for model development. Simulations of pathological stimuli directly impairing only insulin-stimulated PI3K/Akt activity predict altered dynamics of NO and ET-1 consistent with endothelial dysfunction in insulin-resistant states. Indeed, modeling pathway-selective impairment of PI3K/Akt pathways consistent with insulin resistance caused by glucotoxicity, lipotoxicity, or inflammation predict diminished NO production and increased ET-1 secretion characteristic of diabetes and endothelial dysfunction. We conclude that our mathematical model of insulin signaling in vascular endothelium supports the hypothesis that pathway-selective insulin resistance accounts, in part, for relationships between insulin resistance and endothelial dysfunction. This may be relevant for developing novel approaches for the treatment of diabetes and its cardiovascular complications.

An oxide transport chain essential for balanced insulin action

Background and aimsPatients with overnutrition, obesity, the atherometabolic syndrome, and type 2 diabetes typically develop fatty liver, atherogenic dyslipoproteinemia, hyperglycemia, and hypertension. These features share an unexplained origin – namely, imbalanced insulin action, also called pathway-selective insulin resistance and responsiveness. To control glycemia, these patients require hyperinsulinemia that then overdrives ERK and hepatic de-novo lipogenesis. We previously reported that NADPH oxidase-4 regulates balanced insulin action, but the model appeared incomplete. MethodsWe conducted structure-function studies in liver cells to search for additional molecular mediators of balanced insulin action. ResultsWe found that NADPH oxidase-4 is part of a new limb of insulin signaling that we abbreviate “NSAPP” after its five major proteins. The NSAPP pathway is an oxide transport chain that begins when insulin stimulates NADPH oxidase-4 to generate superoxide (O2•–). NADPH oxidase-4 forms a novel, tight complex with superoxide dismutase-3, to efficiently transfer O2•– for quantitative conversion into hydrogen peroxide. The pathway ends when aquaporin-3 channels H2O2 across the plasma membrane to inactivate PTEN. Accordingly, aquaporin-3 forms a novel complex with PTEN in McArdle hepatocytes and in unpassaged human primary hepatic parenchymal cells. Molecular or chemical disruption of any component of the NSAPP chain, from NADPH oxidase-4 up to PTEN, leaves PTEN persistently active, thereby recapitulating the same deadly pattern of imbalanced insulin action seen clinically. ConclusionsThe NSAPP pathway functions as a master regulator of balanced insulin action via ERK, PI3K-AKT, and downstream targets of AKT. Unraveling its dysfunction in overnutrition might clarify the molecular cause of the atherometabolic syndrome and type 2 diabetes.

283-LB: Dissociating Insulin Signaling and SREBP1c Action from the Lipogenic Drive Seen in Human and Murine Hepatic Insulin Resistance

The ongoing debate regarding pathway selective hepatic insulin resistance (IR) focuses on potential insulin signaling branchpoints beyond which regulation of glucose metabolism is attenuated and regulation of de novo lipogenesis (DNL) remains intact. These models suggest that impaired insulin receptor kinase activity, proximal to any potential branchpoint, only plays a minor role in clinical hepatic IR. We compared insulin signaling in liver biopsies from human subjects with nonalcoholic fatty liver disease (NAFLD) and hepatic IR vs. weight matched controls, using glucose ingestion to drive insulin secretion and fructose ingestion as a low insulin comparator. Insulin signaling through phosphorylation of insulin receptor β, Akt, and GSK3β was suppressed in NAFLD (all P < 0.05). Insulin regulation of mTORC1 via PRAS40 phosphorylation was absent in NAFLD subjects; no differences were seen between controls and NAFLD subjects in AMPK dependent RAPTOR phosphorylation. Pathway selective IR implies SREBP1c is primarily responsible for DNL in the insulin resistant liver; we probed the transcriptional regulation of DNL by assessing the in vivo occupancy of lipogenic target gene promoters by SREBP1c and ChREBP using ChIP experiments in mice with diet-induced IR. Fasted-refed mice were refed with high fat diet (HFD) and 1% dextrose drinking water. Mice with hepatic IR (9 day HFD fed) or hepatic and peripheral IR (4 week HFD fed) were compared with more insulin sensitive 2 day HFD fed mice. SREBP1c was not bound to the Fasn promoter in insulin resistant livers, while ChREBP was bound to the Fasn promoter equally in all three groups of mice. Conclusions: There is a proximal block in insulin signaling in humans with hepatic IR and NAFLD, dissociating previously measured changes in DNL rates from insulin signaling. SREBP does not mediate DNL in insulin resistant mice while ChREBP action can explain the maintenance of the transcriptional program of lipogenesis. Disclosure D.F. Vatner: None. K.W. ter Horst: None. L. Goedeke: None. D. Zhang: None. V. Samuel: None. M.J. Serlie: Advisory Panel; Self; Fresenius Medical Care. G.I. Shulman: Advisory Panel; Self; AstraZeneca, Janssen Research & Development, Merck & Co., Inc. Advisory Panel; Spouse/Partner; Merck & Co., Inc. Board Member; Self; Novo Nordisk A/S. Consultant; Self; Aegerion Pharmaceuticals, IMetabolic BioPharma Corporation, Longitude Capital, Nimbus Discovery, Inc., Staten Biotechnology B.V. Funding National Institutes of Health

Evidence against Pathway-Selective Hepatic Insulin Resistance in Mice

Insulin suppresses hepatic glucose production and increases hepatic de novo lipogenesis (DNL). Paradoxically, hepatic DNL remains elevated in insulin-resistant subjects, leading to the hypothesis that hepatic insulin resistance is pathway-selective. Prior studies of DNL in hepatic insulin resistance are complicated by confounders, such as use of markedly unphysiologic animal models or comparison of animals on different diets and thus different availability of DNL precursors. We measured DNL in InsrT1150A knockin (KI) mice, a strain protected from diacylglycerol-mediated hepatic insulin resistance, and in littermate controls. Diets were matched: 60% high fat diet (HFD) with 1% dextrose in drinking water. After two days of fat-feeding, before the development of significant insulin resistance, KI and WT mice displayed similar rates of DNL (KI 24.3% ± 3.6; WT 22.2% ± 3.5). After 9 days of HFD, when WT but not KI mice have developed hepatic insulin resistance, rates of DNL were reduced in WT mice but preserved in KI mice (KI 16.4% ± 2.4; WT 7.4% ± 1.3; P < 0.01). After 4 weeks of HFD, with the development of skeletal muscle resistance, rates of DNL increased in WT mice to reach those observed in KI mice (KI 19.3% ± 2.6; WT 15.9% ± 2.3). Consistent with the insulin-resistant phenotype, Srebp-1c cleavage was reduced in the setting of hepatic insulin resistance in WT mice; this effect was attenuated in KI mice. The Srebp-regulated lipogenic proteins Fasn and Scd1 were both reduced in 9d HFD fed livers regardless of genotype; at 4 weeks, protein abundance remained suppressed in WT mice but was restored in KI mice. Pklr, a protein associated with DNL but not regulated by Srebp, was unaffected by HFD-induced hepatic insulin resistance. Conclusion: Regulation of DNL is subject to lipid-induced hepatic insulin resistance, challenging the selective hepatic insulin resistance hypothesis. Increased DNL seen in insulin resistant subjects may be due to hyperinsulinemia and diversion of substrate from the insulin resistant periphery. Disclosure D.F. Vatner: None. M.C. Petersen: None. X. Li: None. J.C. Rogers: None. G. Cline: None. V. Samuel: None. G.I. Shulman: Advisory Panel; Self; AstraZeneca, Janssen Research & Development, Merck & Co., Inc., Novo Nordisk Inc.. Research Support; Self; Gilead Sciences, Inc..

Aquaporins-1 and -3 Play Novel, Distinct Roles in Normal Balanced Insulin Signaling

Patients with overnutrition, obesity, the atherometabolic syndrome, and type 2 diabetes exhibit imbalanced insulin action, also called pathway-selective insulin resistance. To control glycemia, they require hyperinsulinemia that then overdrives ERK and hepatic de-novo lipogenesis (DNL). We discovered an oxide transport chain that is required for balanced insulin action (Atherosclerosis 2016;247:225). The chain, abbreviated “NSAPP”, begins when insulin stimulates NOX4 to generate O2•-. NOX4 hands O2•- to SOD3 for conversion into H2O2. Aquaporins (AQPs) channel this H2O2 to inactivate PTEN. Disruption of any component, from NOX4 up to PTEN, leaves PTEN persistently active, thereby producing the same deadly pattern of imbalanced insulin action seen clinically. We now sought to identify which aquaporins mediate balanced insulin signaling in hepatocytes. Four AQPs had been reported to act as pores for H2O2: AQP1, 3, 8, and 9. We found that global inhibition of all APQs with low-dose AgNO3 recapitulated the clinical pattern of imbalanced insulin signaling, i.e., full two-site phosphorylation of ERK (pT202/Y204-ERK); monophosphorylation of AKT (pT308-AKT) with only weak phosphorylation at Ser473; robust phosphorylation of AKT targets leading to DNL; but impaired AKT-mediated phosphorylation of FOXO1 (needed to suppress hepatic gluconeogenesis). Our knockdown of AQP3 recapitulated this pattern of imbalanced insulin signaling. Knockdown of AQP1 produced the mirror-image: strongly inhibited pT202/Y204-ERK and pT308-AKT, but intact pS473-AKT. Knockdowns of AQP8 or AQP9 did not affect insulin signaling to either site on ERK or AKT. Importantly, immunoprecipitation of AQP3 brought down PTEN, a previously unknown partner of any aquaporin. Thus, AQP3 participates in the NSAPP oxide transport chain. AQP1 plays an unexpected role, perhaps as a scaffold. Our findings will facilitate unraveling the molecular basis for NSAPP dysfunction in overnutrition and hence imbalanced insulin action. Disclosure X. Wu: None. K. Williams: Stock/Shareholder; Self; Hygieia, Gemphire, Inc.. Advisory Panel; Self; Gemphire, Inc..

Estrogen signaling prevents diet-induced hepatic insulin resistance in male mice with obesity

The development of insulin resistance in the liver is a key event that drives dyslipidemia and predicts diabetes and cardiovascular risk with obesity. Clinical data show that estrogen signaling in males helps prevent adiposity and insulin resistance, which may be mediated through estrogen receptor-α (ERα). The tissues and pathways that mediate the benefits of estrogen signaling in males with obesity are not well defined. In female mice, ERα signaling in the liver helps to correct pathway-selective insulin resistance with estrogen treatment after ovariectomy. We assessed the importance of liver estrogen signaling in males using liver ERα-knockout (LKO) mice fed a high-fat diet (HFD). We found that the LKO male mice had decreased insulin sensitivity compared with their wild-type floxed (fl/fl) littermates during hyperinsulinemic euglycemic clamps. Insulin failed to suppress endogenous glucose production in LKO mice, indicating liver insulin resistance. Insulin promoted glucose disappearance in LKO and fl/fl mice similarly. In the liver, insulin failed to induce phosphorylation of Akt-Ser(473) and exclude FOXO1 from the nucleus in LKO mice, a pathway important for liver glucose and lipid metabolism. Liver triglycerides and diacylglycerides were also increased in LKO mice, which corresponded with dysregulation of insulin-stimulated ACC phosphorylation and DGAT1/2 protein levels. Our studies demonstrate that estrogen signaling through ERα in the liver helps prevent whole body and hepatic insulin resistance associated with HFD feeding in males. Augmenting hepatic estrogen signaling through ERα may lessen the impact of obesity on diabetes and cardiovascular risk in males.

The vascular endothelium in diabetes and its potential as a therapeutic target

The vascular endothelium in diabetes and its potential as a therapeutic target

Estrogen Treatment After Ovariectomy Protects Against Fatty Liver and May Improve Pathway-Selective Insulin Resistance

Pathway-selective insulin resistance where insulin fails to suppress hepatic glucose production but promotes liver fat storage may underlie glucose and lipid abnormalities after menopause. We tested the mechanisms by which estrogen treatment may alter the impact of a high-fat diet (HFD) when given at the time of ovariectomy (OVX) in mice. Female C57BL/6J mice underwent sham operation, OVX, or OVX with estradiol (E2) treatment and were fed an HFD. Hyperinsulinemic-euglycemic clamps were used to assess insulin sensitivity, tracer incorporation into hepatic lipids, and liver triglyceride export. OVX mice had increased adiposity that was prevented with E2 at the time of OVX. E2 treatment increased insulin sensitivity with OVX and HFD. In sham and OVX mice, HFD feeding induced fatty liver, and insulin reduced hepatic apoB100 and liver triglyceride export. E2 treatment reduced liver lipid deposition and prevented the decrease in liver triglyceride export during hyperinsulinemia. In mice lacking the liver estrogen receptor α, E2 after OVX limited adiposity but failed to improve insulin sensitivity, to limit liver lipid deposition, and to prevent insulin suppression of liver triglyceride export. In conclusion, estrogen treatment may reverse aspects of pathway-selective insulin resistance by promoting insulin action on glucose metabolism but limiting hepatic lipid deposition.

The role of pathway-selective insulin resistance and responsiveness in diabetic dyslipoproteinemia

Type 2 diabetes mellitus (T2DM) and related syndromes exhibit a deadly triad of dyslipoproteinemia, which leads to atherosclerosis, hyperglycemia, which causes microvascular disease, and hypertension. These features share a common, but unexplained, origin--namely, pathway-selective insulin resistance and responsiveness (SEIRR). Here, we review recent work on hepatic SEIRR indicating that deranged insulin signaling may have a remarkably simple molecular basis. Comprehensive examination of a set of 18 insulin targets revealed that T2DM liver in vivo exhibits a specific defect in the ability of the NAD(P)H oxidase 4 (NOX4) to inactivate protein tyrosine phosphatase gene family members after stimulation with insulin, and that impairment of this single molecule, NOX4, in cultured hepatocytes recapitulates all features of hepatic SEIRR in vivo. These features include insulin-stimulated generation of an unusual monophosphorylated form of AKT at Thr308 (pT308-AKT) with only weak phosphorylation at Ser473, impaired insulin-stimulated pathways for lowering plasma levels of lipids and glucose, but continued lipogenic pathways and robust extracellular signal-regulated kinase activation. This new study, in combination with important prior work, provides clues to several long-standing mysteries, such as how AKT might regulate lipid-lowering and glucose-lowering pathways that become insulin-resistant but also lipogenic pathways that remain insulin-responsive, as well as a potential role for NOX4 in insulin-stimulated generation of oxysterol ligands for LXR, a key lipogenic factor. These findings suggest a unified molecular explanation for fatty liver, atherogenic dyslipoproteinemia, hyperglycemia, and hence accelerated atherosclerosis and microvascular disease in T2DM, obesity, and related syndromes of positive caloric imbalance.

NOX4 Pathway as a Source of Selective Insulin Resistance and Responsiveness

Type 2 diabetes mellitus and related syndromes exhibit a deadly triad of dyslipoproteinemia, which leads to atherosclerosis; hyperglycemia, which causes microvascular disease; and hypertension. These features share a common, but unexplained, origin-namely, pathway-selective insulin resistance and responsiveness. Here, we undertook a comprehensive characterization of pathway-selective insulin resistance and responsiveness in liver and hepatocytes by examining 18 downstream targets of the insulin receptor, surveying the AKT, ERK, and NAD(P)H oxidase 4 pathways. Injection of insulin into hyperphagic, obese type 2 diabetic db/db mice failed to inactivate hepatic protein tyrosine phosphatase gene family members, a crucial action of NAD(P)H oxidase 4 previously thought to be required for all signaling through AKT and ERK. Insulin-stimulated type 2 diabetic livers unexpectedly produced an unusual form of AKT that was phosphorylated at Thr308 (pT308), with only weak insulin-stimulated phosphorylation at Ser473. Remarkably, knockdown or inhibition of NAD(P)H oxidase 4 in cultured hepatocytes recapitulated the entire complicated pattern of pathway-selective insulin resistance and responsiveness seen in vivo-namely, monophosphorylated pT308-AKT, impaired insulin-stimulated pathways for lowering plasma lipids and glucose, but continued lipogenic pathways and robust ERK activation. Functional disturbance of a single molecule, NAD(P)H oxidase 4, is sufficient to induce the key harmful features of deranged insulin signaling in type 2 diabetes mellitus, obesity, and other conditions associated with hyperinsulinemia and pathway-selective insulin resistance and responsiveness.

Oxidative Stress and Diabetic Complications

Oxidative stress plays a pivotal role in the development of diabetes complications, both microvascular and cardiovascular. The metabolic abnormalities of diabetes cause mitochondrial superoxide overproduction in endothelial cells of both large and small vessels, as well as in the myocardium. This increased superoxide production causes the activation of 5 major pathways involved in the pathogenesis of complications: polyol pathway flux, increased formation of AGEs (advanced glycation end products), increased expression of the receptor for AGEs and its activating ligands, activation of protein kinase C isoforms, and overactivity of the hexosamine pathway. It also directly inactivates 2 critical antiatherosclerotic enzymes, endothelial nitric oxide synthase and prostacyclin synthase. Through these pathways, increased intracellular reactive oxygen species (ROS) cause defective angiogenesis in response to ischemia, activate a number of proinflammatory pathways, and cause long-lasting epigenetic changes that drive persistent expression of proinflammatory genes after glycemia is normalized ("hyperglycemic memory"). Atherosclerosis and cardiomyopathy in type 2 diabetes are caused in part by pathway-selective insulin resistance, which increases mitochondrial ROS production from free fatty acids and by inactivation of antiatherosclerosis enzymes by ROS. Overexpression of superoxide dismutase in transgenic diabetic mice prevents diabetic retinopathy, nephropathy, and cardiomyopathy. The aim of this review is to highlight advances in understanding the role of metabolite-generated ROS in the development of diabetic complications.

Mechanisms of Disease: pathway-selective insulin resistance and microvascular complications of diabetes

Resistance to the actions of insulin is strongly associated with the microvascular complications of diabetes. To the extent that insulin resistance leads to hyperglycemia, dyslipidemia and hypertension, this association is not surprising. It is now clear that insulin also has direct actions in the microvasculature that influence the development and progression of microvascular disease. In the healthy state, insulin appears to have only minor effects on vascular function, because of the activation of opposing mediators such as nitric oxide and endothelin-1. Diabetes and obesity, however, are associated with selective insulin resistance in the phosphatidylinositol-3-kinase signaling pathway, which leads to reduced synthesis of nitric oxide, impaired metabolic control and compensatory hyperinsulinemia. By contrast, insulin signaling via extracellular signal-regulated kinase dependent pathways is relatively unaffected in diabetes, tipping the balance of insulin's actions so that they favor abnormal vasoreactivity, angiogenesis, and other pathways implicated in microvascular complications and hypertension. In addition, preferential impairment of nonoxidative glucose metabolism leads to increased intracellular formation of advanced glycation end products, oxidative stress and activation of other pathogenic mediators. Despite a strong temporal association, a causal link between pathway-selective insulin resistance and microvascular damage remains to be established. It is possible that this association reflects a common genotype or phenotype. Nonetheless, insulin resistance remains an important marker of risk and a key target for intervention, because those patients who achieve a greater improvement of insulin sensitivity achieve better microvascular outcomes.