Control-IQ Technology Use in Individuals With High Insulin Requirements: Results From the Multicenter Higher-IQ Trial.

BACKGROUND: Control-IQ technology version 1.5 allows for a wider range of weight and total daily insulin (TDI) entry, in addition to other changes to enhance performance for users with high basal rates. This study evaluated the safety and performance of the updated Control-IQ system for users with basal rates >3 units/h and high TDI in a multicenter, single arm, prospective study. METHODS: Adults with type 1 diabetes (T1D) using continuous subcutaneous insulin infusion (CSII) and at least one basal rate over 3 units/h (N = 34, mean age = 39.9 years, 41.2% female, diabetes duration = 21.8 years) used the t:slim X2 insulin pump with Control-IQ technology version 1.5 for 13 weeks. Primary outcome was safety events (severe hypoglycemia and diabetic ketoacidosis (DKA)). Central laboratory hemoglobin A1c (HbA1c) was measured at system initiation and 13 weeks. Participants continued using glucagon-like peptide-1 (GLP-1) receptor agonists, sodium-glucose transport protein 2 (SGLT-2) inhibitors, or other medications for glycemic control and/or weight loss if on a stable dose. RESULTS: All 34 participants completed the study. Fifteen participants used a basal rate >3 units/h for all 24 hours of the day. Nine participants used >300 units TDI on at least one day during the study. There were no severe hypoglycemia or DKA events. Time in range 70-180 mg/dL was 64.8% over the 13 weeks, with 1.0% time <70 mg/dL. Hemoglobin A1c decreased from 7.69% at baseline to 6.87% at 13 weeks (-0.82%, P < .001). CONCLUSIONS: Control-IQ technology version 1.5, with wider range of weight and TDI input and enhancements for users with high insulin requirements, was safe in individuals with T1D in this study.

A Novel INS Mutation in the C-Peptide Region Causing Hyperproinsulinemic Maturity Onset Diabetes of Youth Type 10.

Monogenetic diabetes mellitus (DM) describes a collection of single-gene diseases marked by hyperglycemia presenting in childhood or adulthood and the absence of immunological markers of type 1 DM. Mutations in the human insulin gene INS give rise to two separate clinical syndromes: permanent neonatal DM, type 4 (PNDM4), and maturity-onset diabetes of youth, type 10 (MODY10); the former presents shortly after birth and the latter presents in childhood and adulthood. We describe a 40-year-old man in a kindred with high prevalence of DM who presented with severe hyperglycemia but not ketoacidosis or hypertriglyceridemia. Twelve years after initial presentation, the patient had elevated proinsulin and normal plasma C-peptide when nearly euglycemic on treatment with insulin glargine. A novel INS mutation, Gln65Arg, within the C-peptide region was identified. The INS (p.Gln65Arg) mutation may cause MODY10 by disrupting proinsulin maturation.

Autophagy Ablation in Adipocytes Induces Insulin Resistance and Reveals Roles for Lipid Peroxide and Nrf2 Signaling in Adipose-Liver Crosstalk.

Autophagy is a homeostatic cellular process involved in the degradation of long-lived or damaged cellular components. The role of autophagy in adipogenesis is well recognized, but its role in mature adipocyte function is largely unknown. We show that the autophagy proteins Atg3 and Atg16L1 are required for proper mitochondrial function in mature adipocytes. In contrast to previous studies, we found that post-developmental ablation of autophagy causes peripheral insulin resistance independently of diet or adiposity. Finally, lack of adipocyte autophagy reveals cross talk between fat and liver, mediated by lipid peroxide-induced Nrf2 signaling. Our data reveal a role for autophagy in preventing lipid peroxide formation and its transfer in insulin-sensitive peripheral tissues.

Endothelial Cell Autophagy Maintains Shear Stress-Induced Nitric Oxide Generation via Glycolysis-Dependent Purinergic Signaling to Endothelial Nitric Oxide Synthase.

OBJECTIVE: Impaired endothelial cell (EC) autophagy compromises shear stress-induced nitric oxide (NO) generation. We determined the responsible mechanism. APPROACH AND RESULTS: On autophagy compromise in bovine aortic ECs exposed to shear stress, a decrease in glucose uptake and EC glycolysis attenuated ATP production. We hypothesized that decreased glycolysis-dependent purinergic signaling via P2Y1 (P2Y purinoceptor 1) receptors, secondary to impaired autophagy in ECs, prevents shear-induced phosphorylation of eNOS (endothelial nitric oxide synthase) at its positive regulatory site S1117 (p-eNOSS1177) and NO generation. Maneuvers that restore glucose transport and glycolysis (eg, overexpression of GLUT1 [glucose transporter 1]) or purinergic signaling (eg, addition of exogenous ADP) rescue shear-induced p-eNOSS1177 and NO production in ECs with impaired autophagy. Conversely, inhibiting glucose transport via GLUT1 small interfering RNA, blocking purinergic signaling via ectonucleotidase-mediated ATP/ADP degradation (eg, apyrase), or inhibiting P2Y1 receptors using pharmacological (eg, MRS2179 [2'-deoxy-N6-methyladenosine 3',5'-bisphosphate tetrasodium salt]) or genetic (eg, P2Y1-receptor small interfering RNA) procedures inhibit shear-induced p-eNOSS1177 and NO generation in ECs with intact autophagy. Supporting a central role for PKCδT505 (protein kinase C delta T505) in relaying the autophagy-dependent purinergic-mediated signal to eNOS, we find that (1) shear stress-induced activating phosphorylation of PKCδT505 is negated by inhibiting autophagy, (2) shear-induced p-eNOSS1177 and NO generation are restored in autophagy-impaired ECs via pharmacological (eg, bryostatin) or genetic (eg, constitutively active PKCδ) activation of PKCδT505, and (3) pharmacological (eg, rottlerin) and genetic (eg, PKCδ small interfering RNA) PKCδ inhibition prevents shear-induced p-eNOSS1177 and NO generation in ECs with intact autophagy. Key nodes of dysregulation in this pathway on autophagy compromise were revealed in human arterial ECs. CONCLUSIONS: Targeted reactivation of purinergic signaling and PKCδ has strategic potential to restore compromised NO generation in pathologies associated with suppressed EC autophagy.

The Inhibitory G Protein α-Subunit, Gαz, Promotes Type 1 Diabetes-Like Pathophysiology in NOD Mice.

The α-subunit of the heterotrimeric Gz protein, Gαz, promotes ß-cell death and inhibits ß-cell replication when pancreatic islets are challenged by stressors. Thus, we hypothesized that loss of Gαz protein would preserve functional ß-cell mass in the nonobese diabetic (NOD) model, protecting from overt diabetes. We saw that protection from diabetes was robust and durable up to 35 weeks of age in Gαz knockout mice. By 17 weeks of age, Gαz-null NOD mice had significantly higher diabetes-free survival than wild-type littermates. Islets from these mice had reduced markers of proinflammatory immune cell infiltration on both the histological and transcript levels and secreted more insulin in response to glucose. Further analyses of pancreas sections revealed significantly fewer terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL)-positive ß-cells in Gαz-null islets despite similar immune infiltration in control mice. Islets from Gαz-null mice also exhibited a higher percentage of Ki-67-positive ß-cells, a measure of proliferation, even in the presence of immune infiltration. Finally, ß-cell-specific Gαz-null mice phenocopy whole-body Gαz-null mice in their protection from developing hyperglycemia after streptozotocin administration, supporting a ß-cell-centric role for Gαz in diabetes pathophysiology. We propose that Gαz plays a key role in ß-cell signaling that becomes dysfunctional in the type 1 diabetes setting, accelerating the death of ß-cells, which promotes further accumulation of immune cells in the pancreatic islets, and inhibiting a restorative proliferative response.

Hepatocytes Are the Principal Source of Circulating RBP4 in Mice.

RBP4 is produced mainly by hepatocytes. In type 2 diabetes and obesity, circulating RBP4 is increased and may act systemically to cause insulin resistance and glucose intolerance. Observations that adipocyte RBP4 mRNA increases in parallel with circulating RBP4 in these conditions, whereas liver RBP4 mRNA does not, led to a widely held hypothesis that elevated circulating RBP4 is a direct result of increased production by adipocytes. To test this, we generated mice with hepatocyte-specific deletion of RBP4 (liver RBP4 knockout or LRKO mice). Adipose tissue RBP4 expression and secretion remained intact in LRKO mice and increased as expected in the setting of diet-induced insulin resistance. However, circulating RBP4 was undetectable in LRKO mice. We conclude that adipocyte RBP4 is not a significant source of circulating RBP4, even in the setting of insulin resistance. Adipocyte RBP4, therefore, may have a more important autocrine or paracrine function that is confined within the adipose tissue compartment.

Serum α-hydroxybutyrate (α-HB) predicts elevated 1†h glucose levels and early-phase ß-cell dysfunction during OGTT.

OBJECTIVE: Serum α-hydroxybutyrate (α-HB) is elevated in insulin resistance and diabetes. We tested the hypothesis that the α-HB level predicts abnormal 1 h glucose levels and ß-cell dysfunction inferred from plasma insulin kinetics during a 75 g oral glucose tolerance test (OGTT). RESEARCH DESIGN AND METHODS: This cross-sectional study included 217 patients at increased risk for diabetes. 75 g OGTTs were performed with multiple postload glucose and insulin measurements over a 30-120 min period. OGTT responses were analyzed by repeated measures analysis of variance (ANOVA). Multivariable logistic regression was used to predict 1 h glucose ≥155 mg/dL with α-HB added to traditional risk factors. RESULTS: Mean±SD age was 51±15 years (44% male, 25% with impaired glucose tolerance). Fasting glucose and insulin levels, but not age or body mass index (BMI), were significantly higher in the second/third α-HB tertiles (>3.9 µg/mL) than in the first tertile. Patients in the second/third α-HB tertiles exhibited a higher glucose area under the receiver operating characteristics curve (AUC) and reduced initial slope of insulin response during OGTT. The AUC for predicting 1 h glucose ≥155 mg/dL was 0.82 for a base model that included age, gender, BMI, fasting glucose, glycated hemoglobin (HbA1c), and insulin, and increased to 0.86 with α-HB added (p=0.015), with a net reclassification index of 52% (p<0.0001). CONCLUSIONS: Fasting serum α-HB levels predicted elevated 1 h glucose during OGTT, potentially due to impaired insulin secretion kinetics. This association persisted even in patients with an otherwise normal insulin-glucose homeostasis. Measuring serum α-HB could thus provide a rapid, inexpensive screening tool for detecting early subclinical hyperglycemia, ß-cell dysfunction, and increased risk for diabetes.

Exercise increases cutaneous nerve density in diabetic patients without neuropathy.

Early diabetic neuropathy is characterized by loss of unmyelinated axons, resulting in pain, numbness, and progressive decline in intraepidermal nerve fiber density. Patients with type 2 diabetes, without neuropathy, were assigned to quarterly lifestyle counseling (N = 40) or structured, supervised weekly exercise (N = 60) for 1 year. Distal leg IENFD significantly increased in the exercise cohort and remained unchanged in the counseling cohort (1.5 ± 3.6 vs. -0.1 ± 3.2 fibers/mm, P = 0.03). These results suggest preclinical injury to unmyelinated axons is potentially reversible, and that IENFD may be a responsive biomarker useful in future neuropathy prevention clinical trials.

Mitochondrial function/dysfunction in white adipose tissue.

The role of mitochondria in white adipocytes has long been neglected due in part to their lower abundance in these cells. However, accumulating evidence suggests that mitochondria are vital for maintaining metabolic homeostasis in white adipocytes because of their involvement in adipogenesis, fatty acid synthesis and esterification, branched-chain amino acid catabolism and lipolysis. It is therefore not surprising that white adipose tissue function can be perturbed by altering mitochondrial components or oxidative capacity. Moreover, studies in humans and animals with significantly altered fat mass, such as in obesity or lipoatrophy, indicate that impaired mitochondrial function in adipocytes may be linked directly to the development of metabolic diseases such as diabetes and insulin resistance. However, recent studies that specifically targeted mitochondrial function in adipocytes indicated dissociation between impaired mitochondrial oxidative capacity and systemic insulin sensitivity.

Impairment of autophagy in endothelial cells prevents shear-stress-induced increases in nitric oxide bioavailability.

Autophagy is a lysosomal catabolic process by which cells degrade or recycle their contents to maintain cellular homeostasis, adapt to stress, and respond to disease. Impairment of autophagy in endothelial cells studied under static conditions results in oxidant stress and impaired nitric oxide (NO) bioavailability. We tested the hypothesis that vascular autophagy is also important for induction of NO production caused by exposure of endothelial cells to shear stress (i.e., 3 h × ≈20 dyn/cm(2)). Atg3 is a requisite autophagy pathway mediator. Control cells treated with non-targeting control siRNA showed increased autophagy, reactive oxygen species (ROS) production, endothelial NO synthase (eNOS) phosphorylation, and NO production upon exposure to shear stress (p < 0.05 for all). In contrast, cells with >85% knockdown of Atg3 protein expression (via Atg3 siRNA) exhibited a profound impairment of eNOS phosphorylation, and were incapable of increasing NO in response to shear stress. Moreover, ROS accumulation and inflammatory cytokine production (MCP-1 and IL-8) were exaggerated (all p < 0.05) in response to shear stress. These findings reveal that autophagy not only plays a critical role in maintaining NO bioavailability, but may also be a key regulator of oxidant-antioxidant balance and inflammatory-anti-inflammatory balance that ultimately regulate endothelial cell responses to shear stress.

Liver retinol transporter and receptor for serum retinol-binding protein (RBP4).

Vitamin A (retinol) is absorbed in the small intestine, stored in liver, and secreted into circulation bound to serum retinol-binding protein (RBP4). Circulating retinol may be taken up by extrahepatic tissues or recycled back to liver multiple times before it is finally metabolized or degraded. Liver exhibits high affinity binding sites for RBP4, but specific receptors have not been identified. The only known high affinity receptor for RBP4, Stra6, is not expressed in the liver. Here we report discovery of RBP4 receptor-2 (RBPR2), a novel retinol transporter expressed primarily in liver and intestine and induced in adipose tissue of obese mice. RBPR2 is structurally related to Stra6 and highly conserved in vertebrates, including humans. Expression of RBPR2 in cultured cells confers high affinity RBP4 binding and retinol transport, and RBPR2 knockdown reduces RBP4 binding/retinol transport. RBPR2 expression is suppressed by retinol and retinoic acid and correlates inversely with liver retinol stores in vivo. We conclude that RBPR2 is a novel retinol transporter that potentially regulates retinol homeostasis in liver and other tissues. In addition, expression of RBPR2 in liver and fat suggests a possible role in mediating established metabolic actions of RBP4 in those tissues.

Adipose tissue dysregulation and reduced insulin sensitivity in non-obese individuals with enlarged abdominal adipose cells.

BACKGROUND: Obesity contributes to Type 2 diabetes by promoting systemic insulin resistance. Obesity causes features of metabolic dysfunction in the adipose tissue that may contribute to later impairments of insulin action in skeletal muscle and liver; these include reduced insulin-stimulated glucose transport, reduced expression of GLUT4, altered expression of adipokines, and adipocyte hypertrophy. Animal studies have shown that expansion of adipose tissue alone is not sufficient to cause systemic insulin resistance in the absence of adipose tissue metabolic dysfunction. To determine if this holds true for humans, we studied the relationship between insulin resistance and markers of adipose tissue dysfunction in non-obese individuals. METHOD: 32 non-obese first-degree relatives of Type 2 diabetic patients were recruited. Glucose tolerance was determined by an oral glucose tolerance test and insulin sensitivity was measured with the hyperinsulinaemic-euglycaemic clamp. Blood samples were collected and subcutaneous abdominal adipose tissue biopsies obtained for gene/protein expression and adipocyte cell size measurements. RESULTS: Our findings show that also in non-obese individuals low insulin sensitivity is associated with signs of adipose tissue metabolic dysfunction characterized by low expression of GLUT4, altered adipokine profile and enlarged adipocyte cell size. In this group, insulin sensitivity is positively correlated to GLUT4 mRNA (R = 0.49, p = 0.011) and protein (R = 0.51, p = 0.004) expression, as well as with circulating adiponectin levels (R = 0.46, 0 = 0.009). In addition, insulin sensitivity is inversely correlated to circulating RBP4 (R = -0.61, 0 = 0.003) and adipocyte cell size (R = -0.40, p = 0.022). Furthermore, these features are inter-correlated and also associated with other clinical features of the metabolic syndrome in the absence of obesity. No association could be found between the hypertrophy-associated adipocyte dysregulation and HIF-1alpha in this group of non-obese individuals. CONCLUSIONS: In conclusion, these findings support the concept that it is not obesity per se, but rather metabolic dysfunction of adipose tissue that is associated with systemic insulin resistance and the metabolic syndrome.

Quantitative measurement of full-length and C-terminal proteolyzed RBP4 in serum of normal and insulin-resistant humans using a novel mass spectrometry immunoassay.

Serum retinol-binding protein 4 (RBP4) levels are increased in insulin-resistant humans and correlate with severity of insulin resistance in metabolic syndrome. Quantitative Western blotting (qWestern) has been the most accurate method for serum RBP4 measurements, but qWestern is technically complex and labor intensive. The lack of a reliable, high-throughput method for RBP4 measurements has resulted in variability in findings in insulin-resistant humans. Many commonly used ELISAs have limited dynamic range. Neither the current ELISAs nor qWestern distinguish among full-length and carboxyl terminus proteolyzed forms of circulating RBP4 that are altered in different medical conditions. Here, we report the development of a novel quantitative mass spectrometry immunoaffinity assay (qMSIA) to measure full-length and proteolyzed forms of RBP4. qMSIA and qWestern of RBP4 were performed in identical serum aliquots from insulin-sensitive/normoglycemic or insulin-resistant humans with impaired glucose tolerance or type 2 diabetes. Total RBP4 qMSIA measurements were highly similar to qWestern and correlated equally well with clinical severity of insulin resistance (assessed by clamp glucose disposal rate, r = -0.74), hemoglobin A1c (r = 0.63), triglyceride/high-density lipoprotein (r = 0.55), waist/hip (r = 0.61), and systolic blood pressure (r = 0.53, all P < 0.001). Proteolyzed forms of RBP4 accounted for up to 50% of total RBP4 in insulin-resistant subjects, and des(Leu)-RBP4 (cleavage of last leucine) correlated highly with insulin resistance (assessed by glucose disposal rate, r = -0.69). In multiple regression analysis, insulin resistance but not glomerular filtration rate was the strongest, independent predictor of serum RBP4 levels. Thus, qMSIA provides a novel tool for accurately measuring serum RBP4 levels as a biomarker for severity of insulin resistance and risk for type 2 diabetes and metabolic syndrome.

Long-term Fenretinide treatment prevents high-fat diet-induced obesity, insulin resistance, and hepatic steatosis.

The synthetic retinoid Fenretinide (FEN) increases insulin sensitivity in obese rodents and is in early clinical trials for treatment of insulin resistance in obese humans with hepatic steatosis (46). We aimed to determine the physiological mechanisms for the insulin-sensitizing effects of FEN. Wild-type mice were fed a high-fat diet (HFD) with or without FEN from 4-5 wk to 36-37 wk of age (preventive study) or following 22 wk of HF diet-induced obesity (12 wk intervention study). Retinol-binding protein-4 (RBP4) knockout mice were also fed the HFD with or without FEN in a preventive study. FEN had minimal effects on HFD-induced body weight gain but markedly reduced HFD-induced adiposity and hyperleptinemia in both studies. FEN-HFD mice gained epididymal fat but not subcutaneous or visceral fat mass in contrast to HFD mice without FEN. FEN did not have a measurable effect on energy expenditure, food intake, physical activity, or stool lipid content. Glucose infusion rate during hyperinsulinemic-euglycemic clamp was reduced 86% in HFD mice compared with controls and was improved 3.6-fold in FEN-HFD compared with HFD mice. FEN improved insulin action on glucose uptake and glycogen levels in muscle, insulin-stimulated suppression of hepatic glucose production, and suppression of serum FFA levels in HFD mice. Remarkably, FEN also reduced hepatic steatosis. In RBP4 knockout mice, FEN reduced the HFD-induced increase in adiposity and hyperleptinemia. In conclusion, long-term therapy with FEN partially prevents or reverses obesity, insulin resistance, and hepatic steatosis in mice on HFD. The anti-adiposity effects are independent of the RBP4 lowering effect.

The relationship of retinol binding protein 4 to changes in insulin resistance and cardiometabolic risk in overweight black adolescents.

OBJECTIVE: To assess, among overweight non-hispanic black adolescents the relationship of changes in plasma retinol binding protein 4 (RBP4) over 3 years to changes in insulin resistance (IR) and 4 associated cardiometabolic risks. STUDY DESIGN: Nested, retrospective study of 51 overweight, post-pubertal non-Hispanic black participants in the Princeton School District Study. Participants were in the top (worsening IR) or bottom (improved IR) quartile for 3-year change in IR. RBP4 was measured by quantitative Western blot with frozen plasma. Regression analyses adjusted for age, sex, and adiposity (baseline and change). Three measures of adiposity were assessed (waist circumference, body mass index, and weight) in separate regression models. RESULTS: RBP4 increased in one third (n = 17). In logistic regression analyses, increased RBP4 was associated with significantly higher odds of worsening as opposed to improved IR independent of age, sex, or adiposity. Odds ratios were 5.6 (weight, P = .024), 6.0 (BMI, P = .025) and 7.4 (waist circumference, P = .015). Initial RBP4 (beta = 0.81, P = .005) and change in RBP4 (beta = 0.56, P = .046) also predicted change in triglycerides, but not change in high-density lipoprotein-cholesterol, low-density lipoprotein-cholesterol, or fibrinogen. CONCLUSION: This retrospective cohort study provides evidence that RBP4 may be a mechanism through which obesity influences insulin resistance and hypertriglyceridemia in overweight postpubertal black youth and suggests utility of RBP4 as a biomarker of risk.

Decreased clearance of serum retinol-binding protein and elevated levels of transthyretin in insulin-resistant ob/ob mice.

Serum retinol-binding protein (RBP4) is secreted by liver and adipocytes and is implicated in systemic insulin resistance in rodents and humans. RBP4 normally binds to the larger transthyretin (TTR) homotetramer, forming a protein complex that reduces renal clearance of RBP4. To determine whether alterations in RBP4-TTR binding contribute to elevated plasma RBP4 levels in insulin-resistant states, we investigated RBP4-TTR interactions in leptin-deficient ob/ob mice and high-fat-fed obese mice (HFD). Gel filtration chromatography of plasma showed that 88-94% of RBP4 is contained within the RBP4-TTR complex in ob/ob and lean mice. Coimmunoprecipitation with an RBP4 antibody brought down stoichiometrically equal amounts of TTR and RBP4, indicating that TTR was not more saturated with RBP4 in ob/ob mice than in controls. However, plasma TTR levels were elevated approximately fourfold in ob/ob mice vs. controls. RBP4 injected intravenously in lean mice cleared rapidly, whereas the t(1/2) for disappearance was approximately twofold longer in ob/ob plasma. Urinary fractional excretion of RBP4 was reduced in ob/ob mice, consistent with increased retention. In HFD mice, plasma TTR levels and clearance of injected RBP4 were similar to chow-fed controls. Hepatic TTR mRNA levels were elevated approximately twofold in ob/ob but not in HFD mice. Since elevated circulating RBP4 causes insulin resistance and glucose intolerance in mice, these findings suggest that increased TTR or alterations in RBP4-TTR binding may contribute to insulin resistance by stabilizing RBP4 at higher steady-state concentrations in circulation. Lowering TTR levels or interfering with RBP4-TTR binding may enhance insulin sensitivity in obesity and type 2 diabetes.

Effects of genetic variation in the human retinol binding protein-4 gene (RBP4) on insulin resistance and fat depot-specific mRNA expression.

OBJECTIVE: Serum retinol binding protein 4 (RBP4) is a new liver- and adipocyte-derived signal that may contribute to insulin resistance. Therefore, the RBP4 gene represents a plausible candidate gene involved in susceptibility to type 2 diabetes. RESEARCH DESIGN AND METHODS: In this study, the RBP4 gene was sequenced in DNA samples from 48 nonrelated Caucasian subjects. Five novel and three known single nucleotide polymorphisms (SNPs) were identified. Furthermore, five recently reported SNPs were genotyped in 90 subjects. Six SNPs, representative of their linkage disequilibrium groups, were then genotyped in 934 diabetic and 716 nondiabetic subjects. RESULTS: A haplotype of six common SNPs (A-G-G-T-G-C) was significantly increased in 934 case subjects with type 2 diabetes compared with 537 healthy control subjects with normal glucose tolerance (P = 0.02; odds ratio 1.37 [95% CI 1.05-1.79]). Furthermore, in the cohort of 716 nondiabetic Caucasian subjects, carriers of the A-G-G-T-G-C haplotype had significantly higher mean fasting plasma insulin and 2-h plasma glucose than subjects without the haplotype. Two single SNPs (rs10882283 and rs10882273) were also associated with BMI, waist-to-hip ratio, and fasting plasma insulin, and several SNPs were associated with circulating free fatty acids (all adjusted P < 0.05). In addition, subjects carrying a previously reported diabetes-associated haplotype had significantly higher mRNA levels in visceral adipose tissue (adjusted P < 0.05) in a subgroup of nondiabetic subjects (n = 170) with measurements of RBP4 mRNA expression in visceral and subcutaneous fat depots. CONCLUSIONS: Our data indicate a role of RBP4 genetic variation in susceptibility to type 2 diabetes and insulin resistance, possibly through an effect on RBP4 expression.

Serum retinol-binding protein is more highly expressed in visceral than in subcutaneous adipose tissue and is a marker of intra-abdominal fat mass.

Intra-abdominal fat is associated with insulin resistance and cardiovascular risk. Levels of serum retinol-binding protein (RBP4), secreted by fat and liver cells, are increased in obesity and type 2 diabetes (T2D). Here we report that, in 196 subjects, RBP4 is preferentially expressed in visceral (Vis) versus subcutaneous (SC) fat. Vis fat RBP4 mRNA was increased approximately 60-fold and 12-fold in Vis and SC obese subjects respectively versus lean subjects, and approximately 2-fold with impaired glucose tolerance/T2D subjects versus normoglycemic subjects. In obese subjects, serum RBP4 was increased 2- to 3-fold, and serum transthyretin, which stabilizes RBP4 in the circulation, was increased 35%. Serum RBP4 correlated positively with adipose RBP4 mRNA and intra-abdominal fat mass and inversely with insulin sensitivity, independently of age, gender, and body mass index. RBP4 mRNA correlated inversely with GLUT4 mRNA in Vis fat and positively with adipocyte size in both depots. RBP4 levels are therefore linked to Vis adiposity, and Vis fat may be a major source of RBP4 in insulin-resistant states.

The adipokine lipocalin 2 is regulated by obesity and promotes insulin resistance.

OBJECTIVE: We identified lipocalin 2 (Lcn2) as a gene induced by dexamethasone and tumor necrosis factor-alpha in cultured adipocytes. The purpose of this study was to determine how expression of Lcn2 is regulated in fat cells and to ascertain whether Lcn2 could be involved in metabolic dysregulation associated with obesity. RESEARCH DESIGN AND METHODS: We examined Lcn2 expression in murine tissues and in 3T3-L1 adipocytes in the presence and absence of various stimuli. We used quantitative Western blotting to observe Lcn2 serum levels in lean and obese mouse models. To assess effects on insulin action, we used retroviral delivery of short hairpin RNA to reduce Lcn2 levels in 3T3-L1 adipocytes. RESULTS: Lcn2 is highly expressed by fat cells in vivo and in vitro. Expression of Lcn2 is elevated by agents that promote insulin resistance and is reduced by thiazolidinediones. The expression of Lcn2 is induced during 3T3-L1 adipogenesis in a CCAAT/enhancer-binding protein-dependent manner. Lcn2 serum levels are elevated in multiple rodent models of obesity, and forced reduction of Lcn2 in 3T3-L1 adipocytes improves insulin action. Exogenous Lcn2 promotes insulin resistance in cultured hepatocytes. CONCLUSIONS: Lcn2 is an adipokine with potential importance in insulin resistance associated with obesity.

Reduction of elevated serum retinol binding protein in obese children by lifestyle intervention: association with subclinical inflammation.

CONTEXT: Retinol binding protein (RBP4), secreted primarily from the liver and adipose tissues, was recently proposed as a link between obesity and insulin resistance. The role of RBP4 in pediatric obesity, its relationship with subclinical inflammation, and its response to lifestyle changes are not elucidated. OBJECTIVE: The objective of the study was to determine in children: 1) the status of RBP4 levels in lean vs. obese; 2) the relationship between RBP4 levels and subclinical inflammation; and 3) the effect of lifestyle-only intervention on RBP4 levels. DESIGN, SETTING, AND PATIENTS: Lean and obese children (n = 21) matched for age (>14 yr to < 18 yr) and maturity stage (Tanner IV) were studied at baseline and with lifestyle intervention in obese subjects only (n = 15). INTERVENTION: Patients received 3 months of randomized and controlled physical activity-based lifestyle intervention. MAIN OUTCOME MEASURE: RBP4 levels in children before and after intervention and the relationship between RBP4 and subclinical inflammation were measured. RESULTS: Higher RBP4 levels were found in the obese group vs. lean group (P = 0.005). RBP4 correlated with not only indices of obesity and insulin resistance but also inflammatory factors (r = 0.63 and 0.64 for C-reactive protein and IL-6, respectively, P < 0.01). Intervention reduced RBP4 levels by approximately 30% (P = 0.001), and RBP4 reduction was correlated with the magnitude of decrease in inflammatory factors (P = 0.01). CONCLUSION: Alterations in serum RBP4 occur at an early age in the clinical course of obesity and appear to correlate with subclinical inflammation. Lifestyle intervention almost entirely reversed the raised RBP4 levels in obese children. Future studies should determine whether elevation of RBP4 is a direct trigger for the insulin resistance and subclinical inflammation implicated in the premature development of cardiovascular disease and diabetes.