Cutaneous Collagenous Vasculopathy

Cutaneous collagenous vasculopathy is a rare microangiopathy of dermal blood vessels. Clinically indistinguishable from generalized essential telangiectasia, this condition is diagnosed by its unique histological appearance. In contrast to other primary telangiectatic processes, cutaneous collagenous vasculopathy has dilated vascular structures that contain deposits of eosinophilic hyaline material within the vessel walls. To date, cutaneous collagenous vasculopathy has been described in a total of 19 cases in the medical literature. The first several cases were described exclusively in middle-aged to elderly men. Though it has now been described in both men and women, cutaneous collagenous vasculopathy is still most often described in middle-aged to older adults. No particular disease or medication has been linked to the development of cutaneous collagenous vasculopathy, and the etiology remains unknown. In this case series, the authors present three additional patients diagnosed with cutaneous collagenous vasculopathy and discuss their clinical and histopathologic features.Cutaneous collagenous vasculopathy (CCV) is a rare, idiopathic microangiopathy first reported in 2000 by Salama and Rosenthal.1 CCV has characteristic microscopic findings, including dilated capillaries and post-capillary venules with marked collagen deposition,2 which are features essential to diagnosis. Clinically, CCV presents as blanchable, non-urticating macules that typically begin on the lower extremities and then spread to the trunk and upper extremities. 1,3-6 Due to its clinical similarity to generalized essential telangiectasia (GET), dermatologists may not biopsy these patients, potentially causing CCV to be underdiagnosed and under-reported. 4,5,7,8 To date, CCV has been described in approximately equal numbers in men and women of Caucasian race with patients ranging from 16 to 83 years of age.4,9 The majority of cases have been diagnosed in patients with other concomitant diseases, most commonly hypertension and cardiovascular disease,3,5,6,9,10 but also in patients with autoimmune conditions2,5,8,9 and diabetes mellitus.5,10 Additionally, in most described cases, patients were taking at least one medication on an intermittent or ongoing basis. 1,2,4-7,9,1° Despite this observation, specific medical conditions or medications are yet to be linked to the development of CCV. In this article, the authors present three female patients who have been diagnosed with CCV along with their clinical and histopathological features.     

Vascular Endothelial Growth Factor Inhibition by dRK6 Causes Endothelial Apoptosis, Fibrosis, and Inflammation in the Heart via the Akt/eNOS Axis in db/db Mice

OBJECTIVE

Vascular endothelial growth factor (VEGF), which is associated with the stimulation of angiogenesis and collateral vessel synthase, is one of the crucial factors involved in cardiac remodeling in type 2 diabetes.

RESEARCH DESIGN AND METHODS

We investigated VEGF inhibition by dRK6 on the heart in an animal model of type 2 diabetes. Male db/db and db/m mice either were treated with dRK6 starting at 7 weeks of age for 12 weeks (db/db-dRK6 and db/m-dRK6) or were untreated.

RESULTS

Cardiac dysfunction and hypertrophy were noted by echocardiogram and molecular markers in the db/db-dRK6 mice. The presence of diabetes significantly suppressed the expression of VEGF receptor (VEGFR)-1 and VEGFR-2, phospho-Akt, and phospho-endothelial nitric oxide synthase (eNOS) in the heart. In db/db-dRK6 mice, dRK6 completely inhibited VEGFR-2, phospho-Akt, and phospho-eNOS expression, whereas no effect on VEGFR-1 was observed. Cardiac fibrosis, microvascular scarcity associated with an increase in apoptotic endothelial cells, and inflammation were prominent, as well as increase in antiangiogenic growth factors. Cardiac 8-hydroxy-deoxyguanine and hypoxia-inducible factor-1α expression were significantly increased. No such changes were found in the other groups, including the db/m-dRK6 mice. The number of apoptotic human umbilical vein endothelial cells was increased by dRK6 in a dose-dependent manner only at high glucose concentrations, and this was associated with a decrease in phospho-Akt and phospho-eNOS related to oxidative stress.

CONCLUSIONS

Our results demonstrated that systemic blockade of VEGF by dRK6 had deleterious effects on the heart in an animal model of type 2 diabetes; dRK6 induced downregulation of the VEGFR-2 and Akt-eNOS axis and enhancement of oxidative stress.Vascular endothelial growth factor (VEGF), especially VEGF-A, is an important mediator of neovascularization, as well as a key factor for maintaining the integrity of endothelial cells in physiologic and pathologic conditions. Two important VEGF receptor tyrosine kinases have been identified and their functions described (VEGF receptor [VEGFR]-1 or Flt-1 and VEGFR-2 or KDR/Flk-1) (1). The deregulation of VEGF results in reduced activation of endothelial nitric oxide synthase (eNOS) and increased production of reactive oxygen species (ROS), which upregulates the expression of procoagulant, prothrombotic, and proinflammatory mediators (2). Several VEGF inhibitors are currently under evaluation or have already been approved for the treatment of age-related macular degeneration and macular edema (3), various cancers (4,5), and diabetic microvascular complications (3). However, there is a growing realization that VEGF inhibitors have significant side effects on the kidney and blood pressure and have been associated with thrombotic microangiopathy, which are typically downstream consequences of suppression of the endothelial Akt-eNOS signaling pathway (4). Impaired cardiac function has been observed in some patients on anti-VEGF therapy (4).Diabetic cardiomyopathy (DCM) is a clinical condition associated with ventricular dysfunction; DCM develops in patients with diabetes in the absence of atherosclerosis and hypertension (6–9). Left ventricular diastolic and systolic dysfunction and alterations in the coronary microcirculation, as well as interstitial fibrosis, are characteristics of DCM because of a reduced microvasculature (10–12). There continues to be debate about the role of VEGF with regard to the heart in diabetic patients. Expression of VEGF and VEGFR-2 in the human heart, and in streptozotocin-induced diabetic animals, has been shown to be significantly decreased because of loss of insulin-induced VEGFR-eNOS axis activation (13–15), which is followed by an increase of apoptosis in endothelial cells, a decrease in the number of circulating endothelial progenitor cells, decreased capillary density, and impaired myocardial perfusion (14–16). Downregulation of VEGF in the heart is associated with an increased risk for cardiovascular morbidity and mortality in diabetic patients (16). Both increased and unaltered serum or plasma VEGF levels were observed in type 1 and 2 diabetic patients versus control subjects (17,18). On the other hand, the serum VEGF level is increased in patients with diabetic symptomatic polyneuropathy, overt diabetic nephropathy, and coronary artery disease, particularly related to the extent of myocardial damage (19–21).dRK6 is an arginine-rich anti-VEGF hexapeptide and a d-amino acid derivative of RK6 (Arg-Arg-Lys-Arg-Arg-Arg). dRK6 binds with VEGF-A and can thereby block the interaction between VEGF-A (mainly VEGF₁₆₅ and VEGF₁₂₁) and the VEGFRs. In a previous study, dRK6 showed significant inhibition of VEGF-induced angiogenesis and retarded the growth and metastasis of colon carcinoma cells without direct cytotoxicity (22). dRK6 is stable, and its half-life at 37°C in the serum is 13.5 h (22). Previous results demonstrated that a subcutaneous injection of dRK6 (25–50 μg) every other day for 3 weeks reduced the incidence and severity of collagen-induced arthritis in a dose-dependent manner without any toxicity (22,23).Therefore, we determined whether systemic VEGF blockade, using dRK6, could lead to structural and functional deterioration of the heart in db/db mice in a murine model of type 2 diabetes.     

Association of 18 Confirmed Susceptibility Loci for Type 2 Diabetes With Indices of Insulin Release, Proinsulin Conversion, and Insulin Sensitivity in 5,327 Nondiabetic Finnish Men

OBJECTIVE

We investigated the effects of 18 confirmed type 2 diabetes risk single nucleotide polymorphisms (SNPs) on insulin sensitivity, insulin secretion, and conversion of proinsulin to insulin.

RESEARCH DESIGN AND METHODS

A total of 5,327 nondiabetic men (age 58 ± 7 years, BMI 27.0 ± 3.8 kg/m²) from a large population-based cohort were included. Oral glucose tolerance tests and genotyping of SNPs in or near PPARG, KCNJ11, TCF7L2, SLC30A8, HHEX, LOC387761, CDKN2B, IGF2BP2, CDKAL1, HNF1B, WFS1, JAZF1, CDC123, TSPAN8, THADA, ADAMTS9, NOTCH2, KCNQ1, and MTNR1B were performed. HNF1B rs757210 was excluded because of failure to achieve Hardy-Weinberg equilibrium.

RESULTS

Six SNPs (TCF7L2, SLC30A8, HHEX, CDKN2B, CDKAL1, and MTNR1B) were significantly (P < 6.9 × 10⁻⁴) and two SNPs (KCNJ11 and IGF2BP2) were nominally (P < 0.05) associated with early-phase insulin release (InsAUC_0–30/GluAUC_0–30), adjusted for age, BMI, and insulin sensitivity (Matsuda ISI). Combined effects of these eight SNPs reached −32% reduction in InsAUC_0–30/GluAUC_0–30 in carriers of ≥11 vs. ≤3 weighted risk alleles. Four SNPs (SLC30A8, HHEX, CDKAL1, and TCF7L2) were significantly or nominally associated with indexes of proinsulin conversion. Three SNPs (KCNJ11, HHEX, and TSPAN8) were nominally associated with Matsuda ISI (adjusted for age and BMI). The effect of HHEX on Matsuda ISI became significant after additional adjustment for InsAUC_0–30/GluAUC_0–30. Nine SNPs did not show any associations with examined traits.

CONCLUSIONS

Eight type 2 diabetes–related loci were significantly or nominally associated with impaired early-phase insulin release. Effects of SLC30A8, HHEX, CDKAL1, and TCF7L2 on insulin release could be partially explained by impaired proinsulin conversion. HHEX might influence both insulin release and insulin sensitivity.Impaired insulin secretion and insulin resistance, two main pathophysiological mechanisms leading to type 2 diabetes, have a significant genetic component (1). Recent studies have confirmed 20 genetic loci reproducibly associated with type 2 diabetes (2–13). Three were previously known (PPARG, KCNJ11, and TCF7L2), whereas 17 loci were recently discovered either by genome-wide association studies (SLC30A8, HHEX-IDE, LOC387761, CDKN2A/2B, IGF2BP2, CDKAL1, FTO, JAZF1, CDC123/CAMK1D, TSPAN8/LGR5, THADA, ADAMTS9, NOTCH2, KCNQ1, and MTNR1B), or candidate gene approach (WFS1 and HNF1B). The mechanisms by which these genes contribute to the development of type 2 diabetes are not fully understood.PPARG is the only gene from the 20 confirmed loci previously associated with insulin sensitivity (14,15). Association with impaired β-cell function has been reported for 14 loci (KCNJ11, SLC30A8, HHEX-IDE, CDKN2A/2B, IGF2BP2, CDKAL1, TCF7L2, WFS1, HNF1B, JAZF1, CDC123/CAMK1D, TSPAN8/LGR5, KCNQ1, and MTNR1B) (6,12,13,16–38). Although associations of variants in HHEX (16–22), CDKAL1 (6,21–26), TCF7L2 (22,27–30), and MTNR1B (13,31,32) with impaired insulin secretion seem to be consistent across different studies, information concerning other genes is limited (12,18–25,27,33–38). The mechanisms by which variants in these genes affect insulin secretion are unknown. However, a few recent studies suggested that variants in TCF7L2 (22,39–42), SLC30A8 (22), CDKAL1 (22), and MTNR1B (31) might influence insulin secretion by affecting the conversion of proinsulin to insulin. Variants of FTO have been shown to confer risk for type 2 diabetes through their association with obesity (7,16) and therefore were not included in this study.Large population-based studies can help to elucidate the underlying mechanisms by which single nucleotide polymorphisms (SNPs) of different risk genes predispose to type 2 diabetes. Therefore, we investigated confirmed type 2 diabetes–related loci for their associations with insulin sensitivity, insulin secretion, and conversion of proinsulin to insulin in a population-based sample of 5,327 nondiabetic Finnish men.     

SH2B1 Enhances Insulin Sensitivity by Both Stimulating the Insulin Receptor and Inhibiting Tyrosine Dephosphorylation of Insulin Receptor Substrate Proteins

OBJECTIVE

SH2B1 is a SH2 domain-containing adaptor protein expressed in both the central nervous system and peripheral tissues. Neuronal SH2B1 controls body weight; however, the functions of peripheral SH2B1 remain unknown. Here, we studied peripheral SH2B1 regulation of insulin sensitivity and glucose metabolism.

RESEARCH DESIGN AND METHODS

We generated TgKO mice expressing SH2B1 in the brain but not peripheral tissues. Various metabolic parameters and insulin signaling were examined in TgKO mice fed a high-fat diet (HFD). The effect of SH2B1 on the insulin receptor catalytic activity and insulin receptor substrate (IRS)-1/IRS-2 dephosphorylation was examined using in vitro kinase assays and in vitro dephosphorylation assays, respectively. SH2B1 was coexpressed with PTP1B, and insulin receptor–mediated phosphorylation of IRS-1 was examined.

RESULTS

Deletion of peripheral SH2B1 markedly exacerbated HFD-induced hyperglycemia, hyperinsulinemia, and glucose intolerance in TgKO mice. Insulin signaling was dramatically impaired in muscle, liver, and adipose tissue in TgKO mice. Deletion of SH2B1 impaired insulin signaling in primary hepatocytes, whereas SH2B1 overexpression stimulated insulin receptor autophosphorylation and tyrosine phosphorylation of IRSs. Purified SH2B1 stimulated insulin receptor catalytic activity in vitro. The SH2 domain of SH2B1 was both required and sufficient to promote insulin receptor activation. Insulin stimulated the binding of SH2B1 to IRS-1 or IRS-2. This physical interaction inhibited tyrosine dephosphorylation of IRS-1 or IRS-2 and increased the ability of IRS proteins to activate the phosphatidylinositol 3-kinase pathway.

CONCLUSIONS

SH2B1 is an endogenous insulin sensitizer. It directly binds to insulin receptors, IRS-1 and IRS-2, and enhances insulin sensitivity by promoting insulin receptor catalytic activity and by inhibiting tyrosine dephosphorylation of IRS proteins.Insulin decreases blood glucose both by promoting glucose uptake into skeletal muscle and adipose tissue and by suppressing hepatic glucose production. In type 2 diabetes, the ability of insulin to reduce blood glucose is impaired (insulin resistance) because of a combination of genetic and environmental factors, resulting in hyperglycemia. Insulin resistance is not only the hallmark but also a determinant of type 2 diabetes.Insulin binds to and activates the insulin receptor. Insulin receptor tyrosyl phosphorylates insulin receptor substrates (IRS-1, -2, -3, and -4). IRS proteins, particularly IRS-1 and IRS-2, initiate and coordinate multiple downstream pathways, including the phosphatidylinositol 3-kinase/Akt pathway (1). Genetic deletion of IRS-1, IRS-2, or Akt2 causes insulin resistance in mice, indicating that the IRS protein/phosphatidylinositol 3-kinase/Akt2 pathway is required for regulation of glucose homeostasis by insulin (2–5). Insulin receptor and IRS proteins are negatively regulated by various intracellular molecules, including PTP1B, Grb10, Grb14, SOCS1, SOCS3, JNK, PKCθ, S6K, and IKKβ (6–23). The relative contribution of these negative regulators to the progression of insulin resistance has been extensively studied (6–24). However, insulin signaling is likely to also be modulated by positive regulators. In this study, we demonstrate that SH2B1 is a novel endogenous insulin sensitizer.SH2B1 is a member of the SH2B family of adapter proteins that also includes SH2B2 (APS) and SH2B3 (Lnk). SH2B1 and SH2B2 are expressed in multiple tissues, including insulin target tissues (e.g., skeletal muscle, adipose tissue, liver, and the brain); by contrast, SH2B3 expression is restricted to hematopoietic tissue (25,26). Structurally, SH2B family members have an NH₂-terminal dimerization domain, a central pleckstrin homology domain, and a COOH-terminal Src homology 2 (SH2) domain. The dimerization domain mediates homodimerization or heterodimerization between different SH2B proteins (27). SH2B1 and SH2B2 bind via their SH2 domains to a variety of tyrosine phosphorylated proteins, including JAK2 and insulin receptor, in cultured cells (28). Genetic deletion of SH2B1 results in marked leptin resistance, obesity, insulin resistance, and type 2 diabetes in mice, demonstrating that SH2B1 is required for the maintenance of normal body weight, insulin sensitivity, and glucose metabolism (29–32). Surprisingly, SH2B2-null mice have normal body weight and slightly improved insulin sensitivity (32,33), suggesting that SH2B1 and SH2B2 have distinct functions in vivo. However, it remains unclear whether SH2B1 cell autonomously regulates insulin sensitivity in peripheral insulin target tissues because systemic deletion of SH2B1 causes obesity, which may cause insulin resistance in SH2B1-null mice.We generated a mouse model in which recombinant SH2B1 is specifically expressed in the brain of SH2B1-null mice (TgKO) using transgenic approaches (31). Neuron-specific restoration of SH2B1 corrects both leptin resistance and obesity, suggesting that neuronal SH2B1 regulates energy balance and body weight by enhancing leptin sensitivity (31). Consistent with these conclusions, polymorphisms in the SH2B1 loci are linked to leptin resistance and obesity in humans (34–36). In this work, we demonstrate that deletion of SH2B1 in peripheral tissues impairs insulin sensitivity independent of obesity in TgKO mice. Moreover, we demonstrate that SH2B1 directly promotes insulin responses by stimulating insulin receptor catalytic activity and by protecting IRS proteins from tyrosine dephosphorylation.     

Glucagon-Like Peptide 1/Glucagon Receptor Dual Agonism Reverses Obesity in Mice

OBJECTIVE

Oxyntomodulin (OXM) is a glucagon-like peptide 1 (GLP-1) receptor (GLP1R)/glucagon receptor (GCGR) dual agonist peptide that reduces body weight in obese subjects through increased energy expenditure and decreased energy intake. The metabolic effects of OXM have been attributed primarily to GLP1R agonism. We examined whether a long acting GLP1R/GCGR dual agonist peptide exerts metabolic effects in diet-induced obese mice that are distinct from those obtained with a GLP1R-selective agonist.

RESEARCH DESIGN AND METHODS

We developed a protease-resistant dual GLP1R/GCGR agonist, DualAG, and a corresponding GLP1R-selective agonist, GLPAG, matched for GLP1R agonist potency and pharmacokinetics. The metabolic effects of these two peptides with respect to weight loss, caloric reduction, glucose control, and lipid lowering, were compared upon chronic dosing in diet-induced obese (DIO) mice. Acute studies in DIO mice revealed metabolic pathways that were modulated independent of weight loss. Studies in Glp1r^−/− and Gcgr^−/− mice enabled delineation of the contribution of GLP1R versus GCGR activation to the pharmacology of DualAG.

RESULTS

Peptide DualAG exhibits superior weight loss, lipid-lowering activity, and antihyperglycemic efficacy comparable to GLPAG. Improvements in plasma metabolic parameters including insulin, leptin, and adiponectin were more pronounced upon chronic treatment with DualAG than with GLPAG. Dual receptor agonism also increased fatty acid oxidation and reduced hepatic steatosis in DIO mice. The antiobesity effects of DualAG require activation of both GLP1R and GCGR.

CONCLUSIONS

Sustained GLP1R/GCGR dual agonism reverses obesity in DIO mice and is a novel therapeutic approach to the treatment of obesity.Obesity is an important risk factor for type 2 diabetes, and ∼90% of patients with type 2 diabetes are overweight or obese (1). Among new therapies for type 2 diabetes, peptidyl mimetics of the gut-derived incretin hormone glucagon-like peptide 1 (GLP-1) stimulate insulin biosynthesis and secretion in a glucose-dependent manner (2,3) and cause modest weight loss in type 2 diabetic patients. The glucose-lowering and antiobesity effects of incretin-based therapies for type 2 diabetes have prompted evaluation of the therapeutic potential of other glucagon-family peptides, in particular oxyntomodulin (OXM). The OXM peptide is generated by post-translational processing of preproglucagon in the gut and is secreted postprandially from l-cells of the jejuno-ileum together with other preproglucagon-derived peptides including GLP-1 (4,5). In rodents, OXM reduces food intake and body weight, increases energy expenditure, and improves glucose metabolism (6–8). A 4-week clinical study in obese subjects demonstrated that repeated subcutaneous administration of OXM was well tolerated and caused significant weight loss with a concomitant reduction in food intake (9). An increase in activity-related energy expenditure was also noted in a separate study involving short-term treatment with the peptide (10).OXM activates both, the GLP-1 receptor (GLP1R) and glucagon receptor (GCGR) in vitro, albeit with 10- to 100-fold reduced potency compared with the cognate ligands GLP-1 and glucagon, respectively (11–13). It has been proposed that OXM modulates glucose and energy homeostasis solely by GLP1R agonism, because its acute metabolic effects in rodents are abolished by coadministration of the GLP1R antagonist exendin(9–39) and are not observed in Glp1r^−/− mice (7,8,14,15). Other aspects of OXM pharmacology, however, such as protective effects on murine islets and inhibition of gastric acid secretion appear to be independent of GLP1R signaling (14). In addition, pharmacological activation of GCGR by glucagon, a master regulator of fasting metabolism (16), decreases food intake in rodents and humans (17–19), suggesting a potential role for GCGR signaling in the pharmacology of OXM. Because both OXM and GLP-1 are labile in vivo (T_1/2 ∼12 min and 2–3 min, respectively) (20,21) and are substrates for the cell surface protease dipeptidyl peptidase 4 (DPP-4) (22), we developed two long-acting DPP-4–resistant OXM analogs as pharmacological agents to better investigate the differential pharmacology and therapeutic potential of dual GLP1R/GCGR agonism versus GLP1R-selective agonism. Peptide DualAG exhibits in vitro GLP1R and GCGR agonist potency comparable to that of native OXM and is conjugated to cholesterol via a Cys sidechain at the C-terminus for improved pharmacokinetics. Peptide GLPAG differs from DualAG by only one residue (Gln³→Glu) and is an equipotent GLP1R agonist, but has no significant GCGR agonist or antagonist activity in vitro. The objective of this study was to leverage the matched GLP1R agonist potencies and pharmacokinetics of peptides DualAG and GLPAG in comparing the metabolic effects and therapeutic potential of a dual GLP1R/GCGR agonist with a GLP1R-selective agonist in a mouse model of obesity.     

Kallikrein 5-Mediated Inflammation in Rosacea: Clinically Relevant Correlations with Acute and Chronic Manifestations in Rosacea and How Individual Treatments May Provide Therapeutic Benefit

Rosacea is a chronic inflammatory condition of facial skin estimated to affect more than 16 million Americans. Although the pathogenesis of rosacea is not fully understood, recent evidence in vitro as well as in vivo has supported the role of increased levels of the trypsin-like serine protease, kallikrein 5, in initiating an augmented inflammatory response in rosacea. The increase in the quantity and magnitude of biological activity of kallikrein 5 leads to production of greater quantities of cathelicidin (LL-37), an antimicrobial peptide associated with increases in innate cutaneous inflammation, vasodilation, and vascular proliferation, all of which are characteristic features of rosacea. In this article, the authors review the literature supporting the role of kallikrein 5 in the pathophysiology of rosacea, including how therapeutic interventions modulate the effects of kallikrein 5, thus providing further support for this pathophysiological model that at least partially explains many of the clinical features of cutaneous rosacea.Cutaneous rosacea (rosacea) is a chronic inflammatory facial skin disorder noted most commonly in individuals of northern European descent, although people of any ethnicity or skin color may be affected.1-4 The visible manifestations with central facial predominance are characteristic of rosacea, including erythema, papules, pustules, telangiectasias, and phymatous changes.1-4 However, persistent (nontransient) erythema involving the central face that intensifies during flares and the presence of telangiectasias, which are also accentuated mostly on the central face, are the core clinical features that support a diagnosis of rosacea.1-9 Papules and pustules are not consistently present in rosacea, characterizing only those individuals with rosacea who exhibit the papulopustular subtype of the disease.3-6 In fact, papulopustular lesions never emerge in many individuals affected by rosacesa, and phymatous changes affect only a relatively small number of the rosacea-affected population; however, central facial erythema is present to some extent in essentially all people with rosacea.1-8Why do some people get rosacea and others do not? Although the entire explanation that would fully answer this question remains elusive, current evidence suggests that individuals affected by rosacea exhibit rosacea-prone skin, which inherently displays dysregulation of two main systems present within skin—the neurovascular/neuroimmune system and the immune detection/response system (innate immunity).3,5-8,1–9 Both of these systems normally serve physiological functions related to how skin responds to exogenous changes or insults (i.e., changes in temperature, exposure to microbial pathogens). However, in rosacea, both the cutaneous neurovascular/neuroimmune system and the immune detection/response system are dysregulated, with both demonstrating augmented responses that correlate with clinical manifestations commonly seen in patients with cutaneous rosacea.Neurovascular/neuroimmune dysregulation, which includes both anatomic and physiochemical differences present in rosacea-prone skin as compared to healthy facial skin, appears to be a major contributor that exacerbates the vasodilation of facial skin vasculature with increased facial blood flow that occurs during a rosacea flare.3,5-7,17,18,20 This increased vasodilation in rosacea-affected skin, which can be acute or subacute in onset, is commonly referred to as flushing.1,3,4,6,7,17,20 Neurosensory symptoms (i.e., stinging, burning) are often associated with or exacerbated during a rosacea flare.1,3-8 Exogenous factors that are commonly recognized by patients as triggers, which seem to induce a flare, include increased ambient heat/warmth and certain spices (i.e., capsaicin), all of which can induce signaling of neurogenic inflammation via specific receptor channels (transient receptor potential vanilloid [TRPV] subfamily) shown to be increased in rosacea-prone skin.3,6,17,18 The immune detection/response dysregulation of rosacea is evidenced by the upregulation of the pattern recognition receptor, toll-like receptor 2 (TLR2) and the cathelicidin innate immunity pathway.3,5-17,19,21,22 Ultraviolet light (UV) exposure, another recognized trigger factor associated with flares of rosacea, produces changes that induce ligand-binding of TLR2, which signals innate inflammation.3,5-16,19,21 Lastly, upregulated production of several matrix metalloproteases (MMPs) has been demonstrated in rosacea, further contributing to cascades of inflammation and degradation of the dermal matrix.1,3,5-7,19 Accentuated immune detection/response as a major component of the pathophysiology of rosacea has been discussed extensively in the literature and is addressed in more detail as a major subject of this article.5-7,10-16,19,21,22Although the pathophysiology of rosacea is not completely understood, dysregulation of the innate immune detection/response system plays a significant role in the inflammatory and vascular responses seen in this condition.5-7,10-16,19,21,22 As a known inducer of innate and cellular inflammation, increased vascularity, and angiogenesis, cathelicidin (LL-37), an antimicrobial peptide that physiologically provides near-immediate innate defense against several microbial organisms, has been investigated to determine its potential role in the pathophysiology of rosacea.10,11,23,24 Results have shown that patients with rosacea express elevated levels of LL-37 in facial skin, with this increased expression attributed to abnormally high levels of the trypsin-like serine protease enzyme, kallikrein 5 (KLK5), which selectively cleaves an inactive precursor protein (hCAP18) to form the biologically active antimicrobial peptide (LL-37).10,22 Investigations of the mechanism of action of two agents proven to be effective in reducing papulopustular lesions and perilesional erythema in rosacea, topical azelaic acid (AzA) and oral doxycycline, demonstrated direct and indirect inhibition of KLK5, respectively.25-29 In one study with AzA 15% gel, the reduction in KLK5 activity correlated with clinical improvement of rosacea.29 In this review, the authors further describe the role of KLK5 in the pathophysiology of rosacea, including the inflammatory cascades that result from increased KLK5 expression, as well as a more detailed discussion of different therapies shown to inhibit the progression of this cascade.     

The C3a Anaphylatoxin Receptor Is a Key Mediator of Insulin Resistance and Functions by Modulating Adipose Tissue Macrophage Infiltration and Activation

OBJECTIVE

Significant new data suggest that metabolic disorders such as diabetes, obesity, and atherosclerosis all posses an important inflammatory component. Infiltrating macrophages contribute to both tissue-specific and systemic inflammation, which promotes insulin resistance. The complement cascade is involved in the inflammatory cascade initiated by the innate and adaptive immune response. A mouse genomic F2 cross biology was performed and identified several causal genes linked to type 2 diabetes, including the complement pathway.

RESEARCH DESIGN AND METHODS

We therefore sought to investigate the effect of a C3a receptor (C3aR) deletion on insulin resistance, obesity, and macrophage function utilizing both the normal-diet (ND) and a diet-induced obesity mouse model.

RESULTS

We demonstrate that high C3aR expression is found in white adipose tissue and increases upon high-fat diet (HFD) feeding. Both adipocytes and macrophages within the white adipose tissue express significant amounts of C3aR. C3aR^−/− mice on HFD are transiently resistant to diet-induced obesity during an 8-week period. Metabolic profiling suggests that they are also protected from HFD-induced insulin resistance and liver steatosis. C3aR^−/− mice had improved insulin sensitivity on both ND and HFD as seen by an insulin tolerance test and an oral glucose tolerance test. Adipose tissue analysis revealed a striking decrease in macrophage infiltration with a concomitant reduction in both tissue and plasma proinflammatory cytokine production. Furthermore, C3aR^−/− macrophages polarized to the M1 phenotype showed a considerable decrease in proinflammatory mediators.

CONCLUSIONS

Overall, our results suggest that the C3aR in macrophages, and potentially adipocytes, plays an important role in adipose tissue homeostasis and insulin resistance.The complement system is an integral part of both the innate and adaptive immune response involved in the defense against invading pathogens (1). Complement activation culminates in a massive amplification of the immune response leading to increased cell lysis, phagocytosis, and inflammation (1). C3 is the most abundant component of the complement cascade and the convergent point of all three major complement activation pathways. C3 is cleaved into C3a and C3b by the classical and lectin pathways, and iC3b is generated by the alternative pathway (2,3). C3a has potent anaphylatoxin activity, directly triggering degranulation of mast cells, inflammation, chemotaxis, activation of leukocytes, as well as increasing vascular permeability and smooth muscle contraction (3). C3a mediates its downstream signaling effects by binding to the C3a receptor (C3aR), a Gi-coupled G protein–coupled receptor. Several studies have demonstrated a role for C3a and C3aR in asthma, sepsis, liver regeneration, and autoimmune encephalomyelitis (1,3). Therefore, targeting C3aR may be an attractive therapeutic option for the treatment of several inflammatory diseases.Increasing literature suggests that metabolic disorders such as diabetes, obesity, and atherosclerosis also possess an important inflammatory component (4–7). Several seminal reports have demonstrated that resident macrophages can constitute as much as 40% of the cell population of adipose tissue (7–9) and can significantly affect insulin resistance (10–18). Several proinflammatory cytokines, growth factors, acute-phase proteins, and hormones are produced by the adipose tissue and implicated in insulin resistance and vascular homeostasis (4–7,19). An integrated genomics approach was performed with several mouse strains to infer causal relationships between gene expression and complex genetic diseases such as obesity/diabetes. This approach identified the C3aR gene as being causal for omental fat pad mass (20). The C3aR^−/− mice were shown to have decreased adiposity as compared with wild-type mice on a regular diet (20). Monocytes and macrophages express the C3aR (21–28). Increased C3a levels also correlate with obesity, diabetes, cholesterol, and lipid levels (29–34). We therefore sought to investigate the specific role of the C3aR in insulin resistance, obesity, and macrophage function utilizing both normal diet and the diet-induced obesity model.     

C-Peptide Activates AMPKα and Prevents ROS-Mediated Mitochondrial Fission and Endothelial Apoptosis in Diabetes

Vasculopathy is a major complication of diabetes; however, molecular mechanisms mediating the development of vasculopathy and potential strategies for prevention have not been identified. We have previously reported that C-peptide prevents diabetic vasculopathy by inhibiting reactive oxygen species (ROS)-mediated endothelial apoptosis. To gain further insight into ROS-dependent mechanism of diabetic vasculopathy and its prevention, we studied high glucose–induced cytosolic and mitochondrial ROS production and its effect on altered mitochondrial dynamics and apoptosis. For the therapeutic strategy, we investigated the vasoprotective mechanism of C-peptide against hyperglycemia-induced endothelial damage through the AMP-activated protein kinase α (AMPKα) pathway using human umbilical vein endothelial cells and aorta of diabetic mice. High glucose (33 mmol/L) increased intracellular ROS through a mechanism involving interregulation between cytosolic and mitochondrial ROS generation. C-peptide (1 nmol/L) activation of AMPKα inhibited high glucose–induced ROS generation, mitochondrial fission, mitochondrial membrane potential collapse, and endothelial cell apoptosis. Additionally, the AMPK activator 5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside and the antihyperglycemic drug metformin mimicked protective effects of C-peptide. C-peptide replacement therapy normalized hyperglycemia-induced AMPKα dephosphorylation, ROS generation, and mitochondrial disorganization in aorta of diabetic mice. These findings highlight a novel mechanism by which C-peptide activates AMPKα and protects against hyperglycemia-induced vasculopathy.C-peptide and insulin are cosecreted in equimolar amounts into the circulation from the pancreatic β-cells of Langerhans (1). C-peptide deficiency is a prominent attribute of type 1 diabetes (1). Deficiencies of C-peptide and insulin may also occur in the late stages of type 2 diabetes as a result of progressive loss of β-cells (2–4). Recent evidence demonstrates a beneficial role for C-peptide in diabetic neuropathy (1,5,6), nephropathy (1,6,7), and vascular dysfunction (1,5) and inflammation (1). C-peptide protects against diabetic vascular damage by promoting nitric oxide (NO) release (8) and suppressing nuclear factor-κB (9), which suppresses leukocyte-endothelium interactions (8,9). C-peptide may prevent atherosclerosis by inhibiting vascular smooth muscle proliferation and migration (10) and reducing venous neointima formation (11). However, the molecular mechanism by which C-peptide prevents diabetes complications is not understood well enough to permit its clinical implementation.Generation of reactive oxygen species (ROS) in response to high glucose is the leading cause of endothelial damage and diabetic vasculopathy (12). Protein kinase C (PKC)-dependent NADPH oxidase is considered a major cytosolic mediator of ROS generation in endothelial cells (13,14) that play a central role in hyperglycemia-induced endothelial cell apoptosis and vascular complications (15–17). Overproduction of intracellular ROS by mitochondria also occurs during the development of hyperglycemia-induced vascular complications (12,18,19). Altered mitochondrial dynamics due to mitochondrial fission were recently linked with endothelial dysfunction in diabetes (20,21). However, the mechanisms regulating production of cytosolic and mitochondrial ROS and their individual functions in regulating mitochondrial dynamics and apoptosis remain to be elucidated.AMP-activated protein kinase (AMPK) is an intracellular energy and stress sensor (22) and is an emerging target for preventing diabetes complications (23), as exhibited by the most common antihyperglycemic drugs, rosiglitazone (24) and metformin (25). AMPK prevents apoptosis of endothelial cells (26–28) by inhibiting ROS generation by NADPH oxidase (24,29) and mitochondria (30). Additionally, AMPK dephosphorylation is associated with diabetes (22,31,32). It has been reported that C-peptide inhibits high glucose–induced mitochondrial superoxide generation in renal microvascular endothelial cells (7). We recently demonstrated a key role for C-peptide in preventing high glucose–induced ROS generation and apoptosis of endothelial cells through inhibition of transglutaminase (17). However, the mechanism underlying C-peptide–mediated inhibition of intracellular ROS production and subsequent apoptosis remains unclear. Thus, we hypothesized that the potential protective role of C-peptide could be attributed to activation of AMPK, which results in reduced hyperglycemia-induced production of intracellular ROS and altered mitochondrial dynamics that suppress apoptosis of endothelial cells.In this study, we sought to elucidate the mechanism by which C-peptide protects against hyperglycemia-induced ROS production and subsequent endothelial cell damage. We examined the beneficial effect of C-peptide through AMPKα activation and subsequent protection against hyperglycemia-induced production of intracellular ROS, dysregulation of mitochondrial dynamics, mitochondrial membrane potential (∆Ψ_m) collapse, and apoptosis of endothelial cells. These studies were confirmed in vivo in mice with streptozotocin-induced diabetes using C-peptide supplement therapy delivered through osmotic pumps. Thus, our study implicates C-peptide replacement therapy as a potentially significant approach for preventing diabetes complications.     

Autoimmune Polyendocrinopathy-Candidiasis-Ectodermal Dystrophy

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy is a rare autoimmune disorder. The clinical spectrum of symptoms is diverse; the diagnosis relying on the presence of at least two out of the three main conditions defining the syndrome: chronic mucocutaneous candidiasis, hypoparathyroidism, and Addison''s disease.Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), also called autoimmune polyendocrine syndrome type I (APS-1), is a rare organ-specific autosomal recessive disease (OMIM 240300).1–3 Although APECED may occur worldwide, it remains most common in Iranian Jews (1:9,000),4 Sardinians (1:14,500),5–12 Finns (1:25,000),13 Slovenians (1:43,000),14 Norwegians (1:80,000),15 Polish (1:1,29,000),16 and Japanese (1:10,000,000).17 Multiple organ failures in APECED are caused by a progressive loss of tolerance against self-antigens resulting in an immunological attack and secondary destruction of the adrenals, parathyroid glands, β cells of islet of Langerhans,18 stomach, and small intestine.13 Many of the auto-antigens have been identified and characterized.9,12,19     

Glomerular Structure and Function Require Paracrine,Not Autocrine,VEGF–VEGFR-2 Signaling

VEGF is a potent vascular growth factor produced by podocytes in the developing and mature glomerulus. Specific deletion of VEGF from podocytes causes glomerular abnormalities including profound endothelial cell injury, suggesting that paracrine signaling is critical for maintaining the glomerular filtration barrier (GFB). However, it is not clear whether normal GFB function also requires autocrine VEGF signaling in podocytes. In this study, we sought to determine whether an autocrine VEGF–VEGFR-2 loop in podocytes contributes to the maintenance of the GFB in vivo. We found that induced, whole-body deletion of VEGFR-2 caused marked abnormalities in the kidney and also other tissues, including the heart and liver. By contrast, podocyte-specific deletion of the VEGFR-2 receptor had no effect on glomerular development or function even up to 6 months old. Unlike cell culture models, enhanced expression of VEGF by podocytes in vivo caused foot process fusion and alterations in slit diaphragm-associated proteins; however, inhibition of VEGFR-2 could not rescue this defect. Although VEGFR-2 was dispensable in the podocyte, glomerular endothelial cells depended on VEGFR-2 expression: postnatal deletion of the receptor resulted in global defects in the glomerular microvasculature. Taken together, our results provide strong evidence for dominant actions of a paracrine VEGF–VEGFR-2 signaling loop both in the developing and in the filtering glomerulus. VEGF produced by the podocyte regulates the structure and function of the adjacent endothelial cell.Vascular endothelial growth factor (VEGF) was initially discovered as a tumor permeability factor in 1989.^1,2 Subsequently, it has been shown that VEGF has major angiogenic stimulatory properties in development and pathologic angiogenesis of tumors, wound healing, diabetes, and arthritis. In the kidney, altered VEGF expression is associated with a variety of renal diseases that include thrombotic microangiopathy, diabetes, and primary glomerular disease.^3–7 VEGF is a secreted dimeric glycoprotein that exists in a number of splice variants, each displaying different properties with regard to diffusibility and heparin-binding affinities.^8–11 The most abundant isoform expressed by the kidney is VEGF-165 (VEGF-164 in mouse).^12,13 Two receptor tyrosine kinases have been identified for VEGF: VEGFR-1 (Flt1, fms-like tyrosyl kinase-1 where fms refers to feline McDonough sarcoma virus) and VEGFR-2 (human KDR, kinase domain receptor; mouse Flk1, fetal liver kinase-1).^14,15 The majority of VEGF signaling described to date is mediated through VEGFR-2 whereas the signaling capacities of VEGFR-1 are still unclear. VEGF also binds to neuropilin-1 and neuropilin-2, both of which may function to enhance VEGFR-2 signaling.^16,17In the glomerulus, podocytes function as vasculature support cells and produce VEGF in vast amounts. Podocyte-specific deletion or overexpression of VEGF during development leads to dramatic and distinct glomerular phenotypes.^18,19 Here, the major defects occur within the glomerular endothelial cell compartment, indicating that VEGF paracrine signaling is critical for the formation of a functional glomerular filtration, barrier (GFB). More recently, Eremina et al. demonstrated that VEGF production by the podocyte is also necessary to maintain the integrity of the GFB once fully formed as pharmacologic and genetic inhibtion of VEGF in mature podocytes also results in glomerular endothelial cell damage and thrombotic microangiopathy.⁵ Although the role of VEGF production by podocytes is well established, the expression and/or function(s) of VEGF receptors within the podocyte is less clear and is an area of much discussion. Treating cultured mouse podocytes with VEGF-164 resulted in an eightfold increase in VEGFR-2 expression, enhanced cell survival, and upregulation of podocin and mediated the interaction between CD2-associated protein and podocin.²⁰ Another group reported the expression of VEGFR-2 in podocyte foot processes in vivo using immunogold electron microscopic staining.²¹ In contrast, other groups have found VEGFR-1 but not VEGFR-2 in cultured mouse and human podocytes and have shown that VEGFR-1 mediates podocyte cell survival and production of extracellular matrix proteins.^22,23Although the existence of a paracrine interaction between podocytes and endothelial cells is well established, it remains unclear whether VEGF signals in an autocrine fashion in podocytes in vivo. In this study, we utilized an inducible gene-targeting system to eliminate VEGFR-2 from all cells postdevelopmentally. Interestingly, whole body deletion of VEGFR-2 resulted in multiorgan vascular defects that affected only specific vascular beds, such as the glomerulus, while sparing others completely. Importantly, deletion of VEGFR-2 recapitulated the glomerular phenotype observed in mice with a podocyte-specific deletion of the VEGF ligand. To determine whether autocrine and/or paracrine VEGF–VEGFR-2 signaling is important in the glomerulus, we examined the expression of VEGFR-2 in vivo in podocytes using available mouse reporter lines and in FACS-sorted primary podocytes and glomerular endothelial cells. To test for biologic function, we generated podocyte-specific VEGFR-2 knockout mice. We also sought to determine whether the genetic deletion of VEGFR-2 from podocytes could rescue the glomerular disease that develops in mice where VEGF levels are increased. In contrast to the whole body deletion studies, deletion of VEGFR-2 specifically from podocytes has no effect on glomerular function. Taken together, our data provide genetic evidence that VEGFR-2 autocrine signaling is not required for normal podocyte function in vivo. Instead, we propose that VEGF paracrine signaling through VEGFR-2 plays a critical and specific role in the maintenance of the integrity of certain highly specialized endothelial cells, including those in the glomerular microvasculature.     

Distinct Effects of Leptin and a Melanocortin Receptor Agonist Injected Into Medial Hypothalamic Nuclei on Glucose Uptake in Peripheral Tissues

OBJECTIVE

The medial hypothalamus mediates leptin-induced glucose uptake in peripheral tissues, and brain melanocortin receptors (MCRs) mediate certain central effects of leptin. However, the contributions of the leptin receptor and MCRs in individual medial hypothalamic nuclei to regulation of peripheral glucose uptake have remained unclear. We examined the effects of an injection of leptin and the MCR agonist MT-II into medial hypothalamic nuclei on glucose uptake in peripheral tissues.

RESEARCH DESIGN AND METHODS

Leptin or MT-II was injected into the ventromedial (VMH), dorsomedial (DMH), arcuate nucleus (ARC), or paraventricular (PVH) hypothalamus or the lateral ventricle (intracerebroventricularly) in freely moving mice. The MCR antagonist SHU9119 was injected intracerebroventricularly. Glucose uptake was measured by the 2-[³H]deoxy-d-glucose method.

RESULTS

Leptin injection into the VMH increased glucose uptake in skeletal muscle, brown adipose tissue (BAT), and heart, whereas that into the ARC increased glucose uptake in BAT, and that into the DMH or PVH had no effect. SHU9119 abolished these effects of leptin injected into the VMH. Injection of MT-II either into the VMH or intracerebroventricularly increased glucose uptake in skeletal muscle, BAT, and heart, whereas that into the PVH increased glucose uptake in BAT, and that into the DMH or ARC had no effect.

CONCLUSIONS

The VMH mediates leptin- and MT-II–induced glucose uptake in skeletal muscle, BAT, and heart. These effects of leptin are dependent on MCR activation. The leptin receptor in the ARC and MCR in the PVH regulate glucose uptake in BAT. Medial hypothalamic nuclei thus play distinct roles in leptin- and MT-II–induced glucose uptake in peripheral tissues.Leptin is an adipocyte hormone that inhibits food intake and increases energy expenditure (1). The hypothalamus is a principal target of leptin in its regulation of energy metabolism (2–5). The arcuate nucleus (ARC) is the most well characterized of hypothalamic nuclei in terms of its role in the central effects of leptin (2–5). The ARC contains two populations of leptin-responsive neurons: pro-opiomelanocortin (POMC)-expressing neurons, which release the potent anorexic peptide α-melanocyte–stimulating hormone, and neurons that release two potent orexigenic peptides, agouti-related peptide (AgRP) and neuropeptide Y (NPY) (2–5). α-Melanocyte–stimulating hormone activates the melanocortin receptor (MCR), whereas AgRP competitively inhibits this receptor and NPY functionally antagonizes MCR signaling (6). Both sets of neurons project to second-order MCR-expressing neurons within the hypothalamus, including the paraventricular (PVH), ventromedial (VMH), dorsomedial (DMH), and lateral hypothalamus, as well as to other brain regions such as the brain stem (2,4,7,8). Leptin inhibits food intake through reciprocal regulation of POMC and AgRP/NPY neurons in the ARC and consequent activation of MCR in hypothalamic nuclei, including the PVH (5,6,7,9). Mice lacking the melanocortin 3 (MC3R) or 4 (MC4R) receptor show increased adiposity and feeding efficiency (4). Restoration of MC4R expression in certain sets of PVH neurons prevented hyperphagia and reduced body weight in MC4R-null mice (9). In addition to that in the ARC, the leptin receptor Ob-Rb in other hypothalamic nuclei has also been shown to regulate energy intake and adiposity. Neurons positive for steroidogenic factor 1 (SF1; also known as Ad4BP) (10,11) are largely restricted to the VMH in the adult brain. Leptin depolarizes these neurons, and specific ablation of the leptin receptor in SF1-positive cells induced obesity and increased susceptibility to a high-fat diet in mice (12).The leptin receptor in the brain also regulates glucose metabolism in certain peripheral tissues (13–17). Treatment with leptin ameliorates diabetes in lipodystrophic mice and humans (18,19). Intravenous or intracerebroventricular administration of leptin markedly increased whole-body glucose turnover and glucose uptake by certain tissues in mice without any substantial change in plasma insulin or glucose levels (13). We have also previously shown that microinjection of leptin into the medial hypothalamus, such as into the VMH, but not into the lateral hypothalamus, preferentially increased glucose uptake in skeletal muscle, heart, and brown adipose tissue (BAT) (14–16). Restoration of Ob-Rb expression in the ARC and the VMH of the Ob-Rb–mutated Koletsky rat by adenovirus- or adeno-associated virus–mediated gene transfer improved peripheral insulin sensitivity and reduced plasma glucose concentration (17,20). Ablation of suppressor of cytokine signaling 3 (SOCS3) in SF1-positive cells (10,11) improved glucose homeostasis in mice fed a high-fat diet (21). Furthermore, intracerebroventricular injection of the MCR agonist (MT-II) increased whole-body glucose turnover and expression of GLUT4 in skeletal muscle (22). Ob-Rb in the ARC and the VMH as well as the brain melanocortin pathway are thus implicated in the regulation of glucose uptake in peripheral tissues as well as in energy metabolism. However, little is known about the contributions of the leptin receptor and MCR in individual medial hypothalamic nuclei to regulation of glucose uptake in peripheral tissues, as opposed to their roles in the regulation of food intake and leanness.We have now examined the acute effects of microinjection of leptin and MT-II into the VMH, ARC, DMH, and PVH, all of which express Ob-Rb, MC3R, and MC4R at a high level (3–7,23–25), on glucose uptake in peripheral tissues of mice in vivo. Our results suggest that the VMH mediates stimulatory actions of leptin and MT-II on glucose uptake in skeletal muscle, heart, and BAT, whereas the leptin receptor in the ARC as well as MCRs in PVH regulate glucose uptake in BAT. The medial hypothalamic nuclei thus appear to play distinct roles in the regulation of glucose uptake in peripheral tissues by leptin and MT-II.     

Mesenchymal Stem Cells Enhance Allogeneic Islet Engraftment in Nonhuman Primates

OBJECTIVE

To test the graft-promoting effects of mesenchymal stem cells (MSCs) in a cynomolgus monkey model of islet/bone marrow transplantation.

RESEARCH DESIGN AND METHODS

Cynomolgus MSCs were obtained from iliac crest aspirate and characterized through passage 11 for phenotype, gene expression, differentiation potential, and karyotype. Allogeneic donor MSCs were cotransplanted intraportally with islets on postoperative day (POD) 0 and intravenously with donor marrow on PODs 5 and 11. Recipients were followed for stabilization of blood glucose levels, reduction of exogenous insulin requirement (EIR), C-peptide levels, changes in peripheral blood T regulatory cells, and chimerism. Destabilization of glycemia and increases in EIR were used as signs of rejection; additional intravenous MSCs were administered to test the effect on reversal of rejection.

RESULTS

MSC phenotype and a normal karyotype were observed through passage 11. IL-6, IL-10, vascular endothelial growth factor, TGF-β, hepatocyte growth factor, and galectin-1 gene expression levels varied among donors. MSC treatment significantly enhanced islet engraftment and function at 1 month posttransplant (n = 8), as compared with animals that received islets without MSCs (n = 3). Additional infusions of donor or third-party MSCs resulted in reversal of rejection episodes and prolongation of islet function in two animals. Stable islet allograft function was associated with increased numbers of regulatory T-cells in peripheral blood.

CONCLUSIONS

MSCs may provide an important approach for enhancement of islet engraftment, thereby decreasing the numbers of islets needed to achieve insulin independence. Furthermore, MSCs may serve as a new, safe, and effective antirejection therapy.Multipotent mesenchymal stem cells (MSCs) (1,2) can deliver immunomodulatory signals (3–7) that inhibit allogeneic T-cell responses through downregulation of the proinflammatory cytokines TNF-α and IFN-γ and production of the regulatory cytokines/molecules IL-10, hepatocyte growth factor (HGF), TGF-β, vascular endothelial growth factor (VEGF), indoleamine 2,3-dioxygenase, galectin-1, prostaglandin E2, nitric oxide, and matrix metalloproteinase-2 and -9 (3,8–12). Inflammatory signals, such as IFN-γ, can activate and upregulate MSC suppressive activities (9,13). These cells are able to migrate to sites of injury after intravenous injection (14,15). Their use in clinical trials and experimental models is based on their immunomodulatory and regenerative properties (1,7,16). Clinically, MSCs have been observed to enhance donor bone marrow cell (DBMC) engraftment and chimerism (17,18). Therefore, cotransplantation of MSCs that secrete immunomodulatory cytokines and growth factors might enhance islet survival and function. In experimental mouse models, intravenously infused MSCs are capable of migrating to pancreatic islets (19,20). Systemic infusion of MSCs in murine models of diabetes was accompanied by delayed onset of diabetes, improved glycemic levels, reduced pancreatic insulitis, and pancreatic tissue regeneration (19,21–25), as well as prevention of autoimmune destruction of β-cells via induction of regulatory T-cells (Tregs) (26). Cotransplantation of syngeneic MSCs with a marginal mass of allogeneic islets under the kidney capsule of streptozotocin (STZ)-induced diabetic mice resulted in prolonged normoglycemia (11). Cotransplantation of syngeneic MSC with a marginal mass of allogeneic islets has been performed in the omentum (27) and kidney capsule (28) of STZ-induced diabetic rats, with enhanced islet graft survival as compared with animals receiving islets alone. In this study, cynomolgus monkey MSCs were characterized and donor MSCs were examined for the ability to promote intraportal islet engraftment as well as chimerism in recipients of islet/DBMC transplants. In addition, we tested the use of donor or third-party MSCs to reverse episodes of islet allograft rejection.     

Confirmation of Multiple Risk Loci and Genetic Impacts by a Genome-Wide Association Study of Type 2 Diabetes in the Japanese Population

OBJECTIVE

To identify novel type 2 diabetes gene variants and confirm previously identified ones, a three-staged genome-wide association study was performed in the Japanese population.

RESEARCH DESIGN AND METHODS

In the stage 1 scan, we genotyped 519 case and 503 control subjects with 482,625 single nucleotide polymorphism (SNP) markers; in the stage 2 panel comprising 1,110 case subjects and 1,014 control subjects, we assessed 1,456 SNPs (P < 0.0025, stage 1); additionally to direct genotyping, 964 healthy control subjects formed the in silico control panel. Along with genome-wide exploration, we aimed to replicate the disease association of 17 SNPs from 16 candidate loci previously identified in Europeans. The associated and/or replicated loci (23 SNPs; P < 7 × 10^–5 for genome-wide exploration and P < 0.05 for replication) were examined in the stage 3 panel comprising 4,000 case subjects and 12,569 population-based samples, from which 4,889 nondiabetic control subjects were preselected. The 12,569 subjects were used for overall risk assessment in the general population.

RESULTS

Four loci—1 novel with suggestive evidence (PEPD on 19q13, P = 1.4 × 10^–5) and three previously reported—were identified; the association of CDKAL1, CDKN2A/CDKN2B, and KCNQ1 were confirmed (P < 10^–19). Moreover, significant associations were replicated in five other candidate loci: TCF7L2, IGF2BP2, SLC30A8, HHEX, and KCNJ11. There was substantial overlap of type 2 diabetes susceptibility genes between the two populations, whereas effect size and explained variance tended to be higher in the Japanese population.

CONCLUSIONS

The strength of association was more prominent in the Japanese population than in Europeans for more than half of the confirmed type 2 diabetes loci.The predisposition to and the course of type 2 diabetes vary according to ethnic group (1–3). In Japan, the incidence of type 2 diabetes has increased recently and is now comparable to that of other countries; this is supposedly attributable to the gradual spread of Western habits, such as consuming a high-fat diet, and the lower insulin secretory capacity of Japanese subjects (4,5). Recent technological developments have allowed the successful identification of gene regions involved in the development of type 2 diabetes in genome-wide association (GWA) studies (6–17). Several susceptibility gene loci identified by GWA studies to date have been used to obtain reproducible evidence of disease association in different populations of European descent and Asians, but not necessarily in African Americans (18–24). A number of GWA studies on type 2 diabetes have been conducted on populations of European descent (6–12). Two GWA scans in the Japanese population simultaneously reported the discovery of type 2 diabetes susceptibility gene (KCNQ1) variants in non-European populations; this result was also obtained in Scandinavian samples (25,26). Thus far, the replicated associations for a limited number of candidate genes have broadly indicated the tendency of interethnic similarity. Even though the common (or cosmopolitan) effect of type 2 diabetes risk variants is known, the extent to which the causation of this disease differs or overlaps between populations remains unknown. Here, besides comparing the genetic associations between European-descent and Japanese populations, we aimed to identify new genetic variants using a three-staged GWA study design.     

Additive Effects of Genetic Variation in GCK and G6PC2 on Insulin Secretion and Fasting Glucose

OBJECTIVE

Glucokinase (GCK) and glucose-6-phosphatase catalytic subunit 2 (G6PC2) regulate the glucose-cycling step in pancreatic β-cells and may regulate insulin secretion. GCK rs1799884 and G6PC2 rs560887 have been independently associated with fasting glucose, but their interaction on glucose-insulin relationships is not well characterized.

RESEARCH DESIGN AND METHODS

We tested whether these variants are associated with diabetes-related quantitative traits in Mexican Americans from the BetaGene Study and attempted to replicate our findings in Finnish men from the METabolic Syndrome in Men (METSIM) Study.

RESULTS

rs1799884 was not associated with any quantitative trait (corrected P > 0.1), whereas rs560887 was significantly associated with the oral glucose tolerance test 30-min incremental insulin response (30′ Δinsulin, corrected P = 0.021). We found no association between quantitative traits and the multiplicative interaction between rs1799884 and rs560887 (P > 0.26). However, the additive effect of these single nucleotide polymorphisms was associated with fasting glucose (corrected P = 0.03) and 30′ Δinsulin (corrected P = 0.027). This additive association was replicated in METSIM (fasting glucose, P = 3.5 × 10⁻¹⁰ 30′ Δinsulin, P = 0.028). When we examined the relationship between fasting glucose and 30′ Δinsulin stratified by GCK and G6PC2, we noted divergent changes in these quantitative traits for GCK but parallel changes for G6PC2. We observed a similar pattern in METSIM.

CONCLUSIONS

Our data suggest that variation in GCK and G6PC2 have additive effects on both fasting glucose and insulin secretion.Genome-wide association (GWA) studies have identified several loci for type 2 diabetes (1–5) and type 2 diabetes–related quantitative traits (6–20). Two of these loci, glucokinase (GCK) (21–23) and glucose-6-phosphatase catalytic subunit 2 (G6PC2) (9,10), regulate the critical glucose-sensing mechanism within pancreatic β-cells. Mutations in GCK confer susceptibility to maturity-onset diabetes of the young (MODY)-2 (24–26), and a −30 GCK promoter variant (rs1799884) has been shown to be associated with β-cell function (21), fasting glucose, and birth weight (23). Chen et al. (9) demonstrated an association between the G6PC2 region and fasting glucose, an observation replicated by Bouatia-Naji et al. (10). Although fasting glucose levels are associated with both GCK and G6PC2, there has been no evidence that genetic variation at these two loci contribute a risk for type 2 diabetes, suggesting contribution to mild elevations in glycemia.GCK phosphorylates glucose to glucose-6-phosphate, whereas G6PC2 dephosphorylates glucose-6-phosphate back to glucose, forming a glucose cycle previously demonstrated to exist within pancreatic β-cells (27,28). The important role of GCK in glucose sensing by pancreatic islets has been demonstrated by numerous studies, and other studies suggest a role for glucose cycling in insulin secretion and diabetes (28–30), implying that the balance between GCK and G6PC2 activity is important for determining glycolytic flux, ATP production, and subsequent insulin secretion. This was validated by a demonstration that direct manipulation of glucose cycling alters insulin secretion (31,32).BetaGene is a study in which we are performing detailed phenotyping of Mexican American probands with recent gestational diabetes mellitus (GDM) and their family members to obtain quantitative estimates of body composition, insulin sensitivity (S_I), acute insulin response (AIR), and β-cell compensation (disposition index) with the goal of identifying genes influencing variations in type 2 diabetes–related quantitative traits (33–35). Based on the evidence that variation in GCK (rs1799884) and G6PC2 (rs560887) are independently associated with fasting glucose concentrations and both are crucial to glucose cycling in β-cells, we hypothesized that interaction between these loci may be associated not just with fasting glucose but also with measures of insulin secretion or β-cell function. We tested this hypothesis in the BetaGene Study and, for replication, in a separate sample of Finnish men participating in the METabolic Syndrome in Men (METSIM) Study (36).     

Why Do SGLT2 Inhibitors Inhibit Only 30–50% of Renal Glucose Reabsorption in Humans?

Sodium glucose cotransporter 2 (SGLT2) inhibition is a novel and promising treatment for diabetes under late-stage clinical development. It generally is accepted that SGLT2 mediates 90% of renal glucose reabsorption. However, SGLT2 inhibitors in clinical development inhibit only 30–50% of the filtered glucose load. Why are they unable to inhibit 90% of glucose reabsorption in humans? We will try to provide an explanation to this puzzle in this perspective analysis of the unique pharmacokinetic and pharmacodynamic profiles of SGLT2 inhibitors in clinical trials and examine possible mechanisms and molecular properties that may be responsible.Type 2 diabetes is a serious global health issue that has reached epidemic proportions in both developed and developing countries over the last two decades (1). With currently available medicines, many diabetic patients fail to achieve optimal glycemic control (HbA_1c <6.5–7.0%). With the exception of the glucagon-like peptide 1 analogs and the thiazolidinediones (2), other antidiabetic medications lose their effectiveness to control hyperglycemia over time, partially due to the progressive decline of β-cell function (2–4). As a consequence, many patients receive multiple antidiabetic medicines and eventually require insulin therapy, which often fails to achieve the desired glycemic goal and is associated with weight gain and hypoglycemia (5,6). Failure to achieve glycemic targets is the primary factor responsible for the microvascular complications (retinopathy, neuropathy, nephropathy) and, to a lesser extent, macrovascular complications (2,7). In addition, the majority of diabetic patients are overweight or obese, and many of the current therapies are associated with weight gain, which causes insulin resistance and deterioration in glycemic control (2).Given the difficulty in achieving optimal glycemic control (8,9) for many diabetic patients using current therapies, there is an unmet medical need for new antidiabetic agents. Although it has been known for 50 years (10,11) that renal glucose reabsorption is increased in type 2 diabetic patients, only recently have the clinical therapeutic implications of this observation been recognized (2,12). Inhibition of renal tubular glucose reabsorption, leading to a reduction in blood glucose concentration through enhanced urinary glucose excretion, provides a novel insulin-independent therapy (2,12) that in animal models of diabetes has been shown to reverse glucotoxicity and improve insulin sensitivity and β-cell function (13,14). The majority (∼80–90%) of filtered plasma glucose is reabsorbed in the early proximal tubule by the high-capacity, low-affinity sodium glucose cotransporter (SGLT) 2 (15,16). The remaining 10–20% of filtered glucose is reabsorbed by the high-affinity, low-capacity SGLT1 transporter in the more distal portion of the proximal tubule. After glucose is actively reabsorbed by SGLT2 and SGLT1 into the proximal tubular cells, it is diffused out of the cells from the basolateral side into blood through facilitative GLUT 2 and 1 (15). Because the majority of glucose reabsorption occurs via the SGLT2 transporter, pharmaceutical companies have focused on the development of SGLT2 inhibitors, and multiple SGLT2 inhibitors currently are in human phase II and III clinical trials (17). This class of antidiabetic medication effectively lowers blood glucose levels and offers additional benefits, including weight loss, low propensity for causing hypoglycemia, and reduction in blood pressure. The SGLT2 inhibitors are effective as monotherapy and in combination with existing therapies (2,12,14,15,17), including insulin (18). Because of their unique mechanism of action (12,15), which is independent of the severity of insulin resistance and β-cell failure, type 2 diabetic individuals with recent-onset diabetes (<1 year) respond equally well as type 2 diabetic patients with long-standing diabetes (>10 years) (19).Dapagliflozin is the most advanced SGLT2 inhibitor in clinical trials (12,17,20). In addition, multiple other SGLT2 inhibitors are in phase II to III trials (Fig. 1) (17,21). However, none of these SGLT2 inhibitors are able to inhibit >30–50% of the filtered glucose load, despite in vitro studies indicate that 100% inhibition of the SGLT2 transporter should be achieved at the drug concentrations in humans (22,23). In this perspective, we shall examine potential explanations for this apparent paradox. Resolution of the paradox has important clinical implications with regard to the efficacy of this class of drugs and the development of more efficacious SGLT2 inhibitors.Open in a separate windowFIG. 1.SGLT2 inhibitors in late-stage clinical trials.     

Induction of Human ��-Cell Proliferation and Engraftment Using a Single G1/S Regulatory Molecule, cdk6

OBJECTIVE

Most knowledge on human β-cell cycle control derives from immunoblots of whole human islets, mixtures of β-cells and non-β-cells. We explored the presence, subcellular localization, and function of five early G1/S phase molecules—cyclins D1–3 and cdk 4 and 6—in the adult human β-cell.

RESEARCH DESIGN AND METHODS

Immunocytochemistry for the five molecules and their relative abilities to drive human β-cell replication were examined. Human β-cell replication, cell death, and islet function in vivo were studied in the diabetic NOD-SCID mouse.

RESULTS

Human β-cells contain easily detectable cdks 4 and 6 and cyclin D3 but variable cyclin D1. Cyclin D2 was only marginally detectable. All five were principally cytoplasmic, not nuclear. Overexpression of the five, alone or in combination, led to variable increases in human β-cell replication, with the cdk6/cyclin D3 combination being the most robust (15% versus 0.3% in control β-cells). A single molecule, cdk6, proved to be capable of driving human β-cell replication in vitro and enhancing human islet engraftment/proliferation in vivo, superior to normal islets and as effectively as the combination of cdk6 plus a D-cyclin.

CONCLUSIONS

Human β-cells contain abundant cdk4, cdk6, and cyclin D3, but variable amounts of cyclin D1. In contrast to rodent β-cells, they contain little or no detectable cyclin D2. They are primarily cytoplasmic and likely ineffective in basal β-cell replication. Unexpectedly, cyclin D3 and cdk6 overexpression drives human β-cell replication most effectively. Most importantly, a single molecule, cdk6, supports robust human β-cell proliferation and function in vivo.While broadly similar, human pancreatic β- cells differ from their rodent counterparts in many ways. For example, rodent β-cells can be induced to replicate using many strategies, including partial pancreatectomy, induction of obesity and insulin resistance, infusion of glucose, administration of growth factors, and activation of signaling pathways downstream of these growth factors and nutrients (1–8). In contrast, while evidence suggests that human fetal and early neonatal β-cells are able to replicate (9–11), no investigator has been able to demonstrate or induce robust rates of proliferation in adult human β-cells using the same growth factors, nutrients, signaling pathways, and maneuvers that have been effective in rodents (12–19). For example, unlike events in rodents, obesity or type 2 diabetes in human adults is not associated with accelerated β-cell proliferation (19), and even partial pancreatectomy does not induce β-cell replication or regeneration in humans (17). This inability to induce substantial human β-cell replication is problematic, for it is now clear that β-cell replacement therapy for diabetes will require large numbers of human or xenogeneic β-cells (20).While much has been learned from mouse genetic models regarding the molecular control of β-cell replication (21–35), little is known regarding the molecules that regulate the G1/S transition in the human adult β-cell. We have begun to explore these molecules in adult human islets, mapping the members of the G1/S proteome that are present in human cadaveric islets (12,36). Human islets are believed to contain most of the key molecules that govern the G1/S transition in other cell types (12,36,37). These include the three members of the pocket protein family (pRb, p107, and p130); cyclins D1 and D3; cdks 1, 2, 4, and 6; the four members of the INK4 family (p15, p16, p18, and p19); the three members of the KIP/CIP family (p21, p27, and p57); several E2F family members; and p53 and its E3 ligase, HDM2 (supplementary Fig 1, available in an online appendix at http://diabetes.diabetesjournals.org/cgi/content/full/db09-1776/DC1). Upstream of these G1/S molecules are other regulatory molecules such as skp2, menin, FoxM1, bmi1, and ezh2 (26–29,34). We and others have also shown that human islets differ from their rodent counterparts in that they express cdk6, a cdk that is not detectable in mouse islets (12,35). We have also shown that overexpression of cdk6 in association with a D-cyclin, cyclin D1, markedly increases the replication of adult human β-cells (12), and it does so in a way that does not lead to dedifferentiation or accelerated β-cell death but instead leads to enhanced engraftment and function of adult human cadaveric islets in vivo (12). We also have shown that cdk4 (36) and cdk6 (12) in conjunction with cyclin D1 can enhance pRb phosphorylation and replication in human β-cells. Despite these advances, it is important to underscore that, of the 30+ molecules that regulate the human β-cell G1/S transition (supplementary Fig. 1), the therapeutic potential has been explored for only three: cdk4, cdk6, and cyclin D1. For example, nothing is known about the efficacy of the other two D-cyclins, cyclins D2 and D3, in driving proliferation of human β-cells.Another difference between human and rodent islets may involve cyclin D2. In mice, excellent evidence indicates that cyclin D2 is indispensible for β-cell growth and islet development and function: animals that lack cyclin D2 develop islet hypoplasia, hypoinsulinemia, and diabetes (32,33). In contrast to mouse studies, in humans, cyclin D2 has been reported to be difficult to detect or undetectable, despite the use of multiple antisera from multiple vendors and appropriate positive controls (12,37).Here, we asked four questions. 1) Which D-cyclins are definitively present in human islets? 2) Which of the three D-cyclins might be the optimal partner for cdk4 or cdk6 for enhancing adult human β-cell replication? 3) Are cdk4, cdk6, and the three D-cyclins actually present in β-cells [in contrast to immunoblot studies of whole islet extracts described previously (12,37)], and if so, in which subcellular compartment? 4) From a therapeutic standpoint, is it essential to deliver both a D-cyclin and a cdk4/6 family member to enhance human islet engraftment, or can this be simplified by using only a single member of the cdk4/6–D-cyclin complex?We report here that 1) cyclin D1 is variably present, D2 is only marginally detectable, and D3 is readily detectable in human islets; 2) cyclin D3 appears to be surprisingly effective in inducing human β-cell proliferation and, in combination with cdk6, may be the most effective in driving human β-cell replication; 3) cdks 4 and 6, and cyclins D1 and D3, are located principally in the cytoplasm and not the nucleus; and 4) a single cdk, cdk6, is surprisingly as effective in driving human islet engraftment as the previously reported combination of cdk6 plus cyclin D1.     

Identification of Body Fat Mass as a Major Determinant of Metabolic Rate in Mice

OBJECTIVE

Analysis of energy expenditure (EE) in mice is essential to obesity research. Since EE varies with body mass, comparisons between lean and obese mice are confounded unless EE is normalized to account for body mass differences. We 1) assessed the validity of ratio-based EE normalization involving division of EE by either total body mass (TBM) or lean body mass (LBM), 2) compared the independent contributions of LBM and fat mass (FM) to EE, and 3) investigated whether leptin contributes to the link between FM and EE.

RESEARCH DESIGN AND METHODS

We used regression modeling of calorimetry and body composition data in 137 mice to estimate the independent contributions of LBM and FM to EE. Subcutaneous administration of leptin or vehicle to 28 obese ob/ob mice and 32 fasting wild-type mice was used to determine if FM affects EE via a leptin-dependent mechanism.

RESULTS

Division of EE by either TBM or LBM is confounded by body mass variation. The contribution of FM to EE is comparable to that of LBM in normal mice (expressed per gram of tissue) but is absent in leptin-deficient ob/ob mice. When leptin is administered at physiological doses, the plasma leptin concentration supplants FM as an independent determinant of EE in both ob/ob mice and normal mice rendered leptin-deficient by fasting.

CONCLUSIONS

The contribution of FM to EE is substantially greater than predicted from the metabolic cost of adipose tissue per se, and the mechanism underlying this effect is leptin dependent. Regression-based approaches that account for variation in both FM and LBM are recommended for normalization of EE in mice.The maintenance of stable body weight is achieved through a process termed “energy homeostasis” that matches energy intake to energy expenditure (EE) over long time intervals (1). Accordingly, when animals experience a sustained increase of energy intake (e.g., during consumption of an energy-rich highly palatable diet), an adaptive increase of metabolic rate can help to limit the associated weight gain (2). However, the ability to quantify adaptive changes of EE is confounded in that larger animals tend to have a higher metabolic rate than smaller ones. Therefore, to reliably detect changes in EE that are not due simply to differences in body size per se, EE must be normalized to body mass using a method that eliminates this confounding effect. To date, most rodent studies of obesity use ratio-based normalization methods whereby EE is divided by either total body mass (TBM) or lean body mass (LBM) (3–6). However, these two methods can give widely divergent results when applied to the same data (3,4,6,7).A recent Diabetes Perspectives article (7) cogently reviewed the confounding effect of normalizing EE via division by TBM in mice, particularly when groups being compared differ in fat mass (FM). Accompanying this caution was the recommendation that EE be normalized via division by LBM instead (7) on grounds that FM consumes much less energy than LBM. Despite its intuitive appeal, dividing EE by LBM is theoretically problematic as a means to remove the influence of body size variation from group comparisons. The linear relationship between EE and either TBM or LBM is typically characterized by a positive y (EE) intercept term (8–13) due to heterogeneity inherent in the EE of various tissues comprising LBM (14). Consequently, dividing resting or average EE by either TBM or LBM mathematically forces heavier individuals to have a lower normalized EE than smaller ones (8–13), a concept first articulated >60 years ago (8). One approach that has been forwarded to obviate this mathematical bias is to use allometric scaling (15) wherein a TBM scaling exponent b and scaling coefficient a must be identified based on the data (15,16) such that EE divided by TBM^b assumes the constant expected value a. This approach, however, is limited by interpretational and other difficulties (17), and the notion that a predetermined fixed TBM scaling exponent can be applied universally has been challenged (16,18).Normalizing EE in human studies is now accomplished using multiple regression methods that adjust group comparisons of EE for differences in body mass so as to eliminate the influence of body size variation per se from evaluations of key independent variables such as ethnicity, sex, genotype, or nutritional status (9,19–28). Although multiple regression has been used in animal studies (11,29–35), the relative importance of FM and LBM as determinants of metabolic rate in mice remains an open question. Indeed, both human and animal investigations suggest that the energy cost of FM in vivo is greater than expected on the basis of its intrinsic metabolic rate (20,28,31,36). This possibility is consistent with evidence that changes in FM can influence metabolic rate at least in part through homeostatic adjustments of EE that promote body weight stability (2,37–42). Testing this hypothesis, however, requires the application of valid strategies for normalizing EE to body size.In the current work, we demonstrate in a large sample of mice that ratio-based normalization of EE is problematic, even when LBM is used in lieu of TBM. Moreover, multiple regression analysis indicates that variation in FM makes a surprisingly large contribution to EE. These findings lead us to support recommendations for the broad use of regression-based approaches to normalizing EE in mice that take both FM and LBM into account (9–12,19,43,44). Based on the hypothesis that the effect of FM on EE reflects adaptive responses involving the adipocyte hormone leptin, we asked 1) whether the effect of FM on EE is absent in ob/ob mice that lack a leptin signal, 2) if the plasma leptin level supplants FM as an independent determinant of EE when leptin is administered to ob/ob mice at physiological doses, and 3) whether in wild-type (WT) mice rendered leptin-deficient by fasting, the plasma leptin level emerges as a determinant of EE when physiological replacement is achieved by exogenous leptin administration. Our results confirm each of these predictions and therefore implicate circulating leptin in the mechanism whereby FM variation affects EE.     

Insulin resistance, defective insulin-mediated fatty acid suppression, and coronary artery calcification in subjects with and without type 1 diabetes: The CACTI study

OBJECTIVE

To assess insulin action on peripheral glucose utilization and nonesterified fatty acid (NEFA) suppression as a predictor of coronary artery calcification (CAC) in patients with type 1 diabetes and nondiabetic controls.

RESEARCH DESIGN AND METHODS

Insulin action was measured by a three-stage hyperinsulinemic-euglycemic clamp (4, 8, and 40 mU/m²/min) in 87 subjects from the Coronary Artery Calcification in Type 1 Diabetes cohort (40 diabetic, 47 nondiabetic; mean age 45 ± 8 years; 55% female).

RESULTS

Peripheral glucose utilization was lower in subjects with type 1 diabetes compared with nondiabetic controls: glucose infusion rate (mg/kg FFM/min) = 6.19 ± 0.72 vs. 12.71 ± 0.66, mean ± SE, P < 0.0001, after adjustment for age, sex, BMI, fasting glucose, and final clamp glucose and insulin. Insulin-induced NEFA suppression was also lower in type 1 diabetic compared with nondiabetic subjects: NEFA levels (μM) during 8 mU/m²/min insulin infusion = 370 ± 27 vs. 185 ± 25, P < 0.0001, after adjustment for age, sex, BMI, fasting glucose, and time point insulin. Lower glucose utilization and higher NEFA levels, correlated with CAC volume (r = −0.42, P < 0.0001 and r = 0.41, P < 0.0001, respectively) and predicted the presence of CAC (odds ratio [OR] = 0.45, 95% CI = 0.22–0.93, P = 0.03; OR = 2.4, 95% CI = 1.08–5.32, P = 0.032, respectively). Insulin resistance did not correlate with GHb or continuous glucose monitoring parameters.

CONCLUSIONS

Type 1 diabetic patients are insulin resistant compared with nondiabetic subjects, and the degree of resistance is not related to current glycemic control. Insulin resistance predicts the extent of coronary artery calcification and may contribute to the increased risk of cardiovascular disease in patients with type 1 diabetes as well as subjects without diabetes.Cardiovascular disease (CVD) remains the leading cause of death in individuals with type 1 diabetes (1–4). Although hyperglycemia appears to be the primary mediator of microvascular disease (5,6), its role in macrovascular disease is less clear (4). Tight glycemic control improves, but does not normalize CVD risk, and correlation of GHb to CVD risk remains controversial (7–15). In addition, standard prediction rules for CVD risk do not accurately predict CVD in type 1 diabetic populations (16). Thus, the mechanism of accelerated atherosclerosis in type 1 diabetes is unclear and identification of those patients at highest risk and most in need of aggressive risk factor modification is inaccurate.In the general population, insulin resistance has been implicated as an important contributor to accelerated atherosclerosis (17–25). Although type 1 diabetes is primarily a disease of insulin deficiency, previous studies have demonstrated insulin resistance and suggested that CVD may also be linked to insulin resistance in type 1 diabetes (10,26–32). As early as 1968, Martin et al. (30) demonstrated an “impaired glucose assimilation index” and an inverse association between this index and prevalent macrovascular disease in type 1 diabetic subjects. More recently, the Pittsburgh Epidemiology of Diabetes Complications Study (10) found no correlation between GHb and coronary artery disease outcomes. However, in addition to other known CVD risk factors, estimated glucose disposal rate correlated inversely with these outcomes. Similar correlations of estimated insulin resistance or a surrogate of insulin resistance (waist-to-hip ratio) to coronary artery disease were also found in the Diabetes Control and Complications Trial (DCCT) and the EURODIAB study (33). These data suggest that an estimate of insulin resistance may add to CVD risk prediction in type 1 diabetes. In addition, elevated nonesterified fatty acid (NEFA) levels have been proposed to mediate the increased atherosclerotic risk associated with insulin resistance in the general population (18,34–37). It is not known whether the defects in insulin action in type 1 diabetes extend beyond glucose utilization to NEFA suppression.The Coronary Artery Calcification in Type 1 Diabetes (CACTI) study has followed a cohort of type 1 diabetic subjects and similar nondiabetic controls with electron beam computed tomography for measurement of coronary artery calcification (CAC) and CVD outcomes for 6 years (15,38). We hypothesized that type 1 diabetic subjects would be more insulin resistant than nondiabetic controls in terms of both glucose utilization and NEFA suppression, and that both measures of insulin resistance would correlate with CAC, a marker of the extent of coronary atherosclerosis.