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ContextCanagliflozin has been reported to increase the risk of bone fracture – possibly mediated by decreasing 1,25-dihydroxyvitamin D [1,25(OH)2D] and increasing PTH.ObjectiveTo investigate whether baseline vitamin D (VitD) deficiency renders individuals vulnerable to this adverse effect and whether VitD3 supplementation is protective.DesignThis study had a paired design comparing individual participants before and after VitD3 supplementation.SettingCommunity-based outpatient.Patients11 VitD deficient (25-hydroxyvitamin D [25(OH)D] ≤ 20 ng/mL) individuals recruited from the Amish population in Lancaster PA.InterventionsParticipants underwent two canagliflozin challenge protocols (300 mg daily for five days): the first before and the second after VitD3 supplementation. In the VitD3 supplementation protocol, participants received VitD3 supplementation (50,000 IU once or twice a week depending on BMI for 4-6 weeks) to achieve 25(OH)D ≥ 30 ng/mL.Main Outcome MeasuresTwo co-primary endpoints were identified: effects of VitD3 supplementation on canagliflozin-induced changes in 1,25(OH)2D and PTH. Secondary endpoints included effects of VitD3 supplementation on baseline levels of VitD metabolites and PTH.ResultsVitD3 supplementation increased mean 25(OH)D from 16.5±1.6 to 44.3±5.5 ng/mL (p=0.0006) and 24,25-dihydroxyvitamin D [24,25(OH)2D] from 1.0±0.1 to 4.3±0.6 ng/mL (p=0.0002). Mean 1,25(OH)2D and PTH were unchanged. VitD3 supplementation decreased the magnitude of canagliflozin-induced changes in 1,25(OH)2D (from -31.3%±4.7% to -9.3%±8.3%; p=0.04) and PTH (from +36.2%±6.2% to +9.7%±3.7%; p=0.005).ConclusionsVitD deficiency rendered individuals more vulnerable to adverse effects of canagliflozin on biomarkers associated with bone health. VitD3 supplementation was protective against canagliflozin’s short-term adverse effects on 1,25(OH)2D and PTH.

BackgroundSGLT2 inhibitors provide multiple benefits to patients with type 2 diabetes – including improved glycemic control and decreased risks of cardiorenal disease. Because drug responses vary among individuals, we initiated investigations to identify genetic variants associated with the magnitude of drug responses.MethodsCanagliflozin (300 mg) was administered to 30 healthy volunteers. Several endpoints were measured to assess clinically relevant responses – including drug-induced increases in urinary excretion of glucose, sodium, and uric acid.ResultsThis pilot study confirmed that canagliflozin (300 mg) triggered acute changes in mean levels of several biomarkers: fasting plasma glucose (-4.1 mg/dL; p=6×10-5), serum creatinine (+0.05 mg/dL; p=8×10-4), and serum uric acid (-0.90 mg/dL; p=5×10-10). The effects of sex on glucosuria depended upon how data were normalized. Whereas males’ responses were ∼60% greater when data were normalized to body surface area, males and females exhibited similar responses when glucosuria was expressed as grams of urinary glucose per gram-creatinine. The magnitude of glucosuria was not significantly correlated with fasting plasma glucose, estimated GFR, or age in these healthy non-diabetic individuals with estimated GFR>60 mL/min/1.73m2.ConclusionsNormalizing data relative to creatinine excretion will facilitate including data from males and females in a single analysis. Furthermore, because our ongoing pharmacogenomic study (NCT02891954) is conducted in healthy individuals, this will facilitate detection of genetic associations with limited confounding by other factors such as age and renal function.RegistrationNCT02462421 (clinicaltrials.gov)FundingResearch grants from the National Institute of Diabetes and Digestive and Kidney Diseases: R21DK105401, R01DK108942, T32DK098107, and P30DK072488.

Identifying genes that result in monogenic diabetes can provide insights that can build a scientific foundation for precision medicine. At present, nearly 20% of neonatal diabetes cases have unknown causes. In this issue of the JCI, De Franco and Lytrivi et al. sequenced the genome of two probands with a rare neonatal diabetes subtype that also associated with microcephaly and epilepsy. The authors revealed mutations in the YIPF5 gene. YIPF5 resides in the Golgi apparatus and is thought to play a critical role in vesicular trafficking. Notably, disrupting YIPF5 in β cell-based models induced ER stress signaling and resulted in the accumulation of intracellular proinsulin. We believe that utilizing registries and biobanks to reveal other monogenic atypical forms of diabetes is an important approach to gaining insight and suggest that an insulin sensitizer may alleviate ER stress associated with YIPF5 disruption by decreasing the demand for insulin secretion.

Insulin-resistant diabetes in Rabson–Mendenhall syndrome (RMS) is relatively unresponsive to first-line antidiabetic treatments, including metformin and insulin. We report 2 patients with RMS treated with 2 different sodium-glucose cotransporter inhibitors 2: empagliflozin in an 11-year-old boy and dapagliflozin in a 12-year-old girl. In the first patient, we began empagliflozin at 2.5 mg/day and increased the dose to 10 mg/day over 3 months. During treatment with empagliflozin, the amount of time during which the patient maintained serum glucose in the 70 to 180 mg/dL target range increased by 2 hours per day. Hemoglobin A1C dropped from >14% to 11.9%, urinary calcium increased almost twofold, and β-hydroxybutyrate remained 50%. In 2 patients with RMS, empagliflozin and dapagliflozin were well tolerated and improved glycemic control without significantly increasing ketonemia. Renal calcium excretion should be carefully monitored.

Increased blood levels of low-density lipoprotein cholesterol (LDL-C) and fibrinogen are independent risk factors for cardiovascular disease. We identified associations between an Amish-enriched missense variant (p.Asn352Ser) in a functional domain of beta-1,4-galactosyltransferase 1 (

> The arc of the moral universe is long, but it bends toward justice. > > —Martin Luther King, Jr. Diabetes exacts a high cost in human suffering, including increased burdens of cardiovascular disease, blindness, end-stage kidney disease, and amputations. Landmark clinical trials have demonstrated the value of intensive pharmacotherapy to delay or prevent chronic complications of diabetes (1–6). Over the past decade, clinical trials demonstrated that several sodium–glucose cotransporter 2 (SGLT2) inhibitors and glucagon-like peptide 1 (GLP-1) receptor agonists decrease the risks of major adverse cardiovascular events, hospitalization for heart failure, and progression of diabetic kidney disease (7–12). In this issue of Diabetes Care , Zhou et al. (13) estimate the economic cost of glucose-lowering drugs at $57.6 billion per year in the U.S. in 2015–2017 (∼15–20% of the estimated annual cost for all prescription drugs in the U.S.). At a human level, the financial burden has a devastating impact on people without health insurance and people whose insurance imposes high deductibles—the people least able to afford the high cost of diabetes drugs. Thus, the high cost of diabetes drugs has important implications for both public policy and social justice. Zhou et al. (13) obtained data from the Medical Expenditure Panel Survey, a nationally representative survey for the civilian noninstitutionalized population in the U.S. National spending on glucose-lowering drugs was estimated by extrapolating to the entire U.S. population. The authors estimated that total national spending on glucose-lowering medications increased by 240% (from $16.9 to $57.6 billion per year expressed in 2017 dollars) in 2015–2017 compared with 2005–2007. Over the same time period, the authors estimated a 38% increase in the number of people using glucose-lowering drugs (from 15.3 to 21.1 million) and a 147% increase in the average annual cost per user (from $1,106 to $2,727). Further analysis …

Summary Sodium-glucose co-transporter-2 (SGLT2) inhibitors have several beneficial effects in patients with type 2 diabetes, including glucose lowering, weight loss, blood pressure lowering, and a reduced risk of major adverse cardiovascular events. To address high unmet medical need via improved glycaemic control, several clinical trials have been done to assess the efficacy and safety of SGLT2 inhibitors in combination with insulin therapy in patients with type 1 diabetes. In this Personal View, we summarise data from eight clinical trials of canagliflozin, dapagliflozin, empagliflozin, and sotagliflozin in patients with type 1 diabetes. HbA1c-lowering efficacy was greatest at 8–12 weeks of therapy, but the magnitude of HbA1c lowering waned with longer duration of treatment (up to 52 weeks). Data are not yet available to establish for how long glycaemic efficacy could be sustained during long-term therapy in patients with type 1 diabetes. Moreover, SGLT2 inhibitor therapy induces serious adverse events, including a roughly six-times increased risk of diabetic ketoacidosis. The US Food and Drug Administration estimated that one additional case of ketoacidosis will occur for every 26 patient-years of exposure of patients with type 1 diabetes to sotagliflozin therapy. Assuming a case mortality of 0·4%, this estimate translates into 16 additional deaths per year per 100 000 patients with type 1 diabetes undergoing treatment. These considerations raise important questions about the risk-to-benefit profile of SGLT2 inhibitors when used as adjunctive therapy in patients with type 1 diabetes.

Sodium–glucose cotransporter 2 (SGLT2) inhibitors are the most recently approved class of diabetes drugs. Unlike other agents, SGLT2 inhibitors act on the kidney to promote urinary glucose excretion. SGLT2 inhibitors provide multiple benefits, including decreased HbA1c, body weight, and blood pressure. These drugs have received special attention because they decrease the risk of major adverse cardiovascular events and slow progression of diabetic kidney disease (1–3). Balanced against these impressive benefits, the U.S. Food and Drug Administration–approved prescribing information describes a long list of side effects: genitourinary infections, ketoacidosis, bone fractures, amputations, acute kidney injury, perineal necrotizing fasciitis, and hyperkalemia. This review provides a physiological perspective to understanding the multiple actions of these drugs complemented by a clinical perspective toward balancing benefits and risks.

Diabetes mellitus is a major public health problem, affecting about 10% of the population. Pharmacotherapy aims to protect against microvascular complications, including blindness, end-stage kidney disease, and amputations. Landmark clinical trials have demonstrated that intensive glycemic control slows progression of microvascular complications (retinopathy, nephropathy, and neuropathy). Long-term follow-up has demonstrated that intensive glycemic control also decreases risk of macrovascular disease, albeit rigorous evidence of macrovascular benefit did not emerge for over a decade. The US FDA's recent requirement for dedicated cardiovascular outcome trials ushered in a golden age for understanding the clinical profiles of new type 2 diabetes drugs. Some clinical trials with sodium-glucose cotransporter-2 (SGLT2) inhibitors and glucagon-like peptide 1 (GLP1) receptor agonists reported data demonstrating cardiovascular benefit (decreased risk of major adverse cardiovascular events and hospitalization for heart failure) and slower progression of diabetic kidney disease. This Review discusses current guidelines for use of the 12 classes of drugs approved to promote glycemic control in patients with type 2 diabetes. The Review also anticipates future developments with potential to improve the standard of care: availability of generic dipeptidylpeptidase-4 (DPP4) inhibitors and SGLT2 inhibitors; precision medicine to identify the best drugs for individual patients; and new therapies to protect against chronic complications of diabetes.

> I dream things that never were, and I say “Why not?” > > —George Bernard Shaw Pancreatic β-cells secrete insulin to maintain physiological health. While the glucose level is the principle driver of insulin secretion, β-cells also respond to other regulatory inputs including circulating metabolites (most notably amino acids), regulatory hormones, gut peptides, and neurotransmitters. Insulin secretion increases when glucose levels are high, thereby increasing total body glucose utilization and suppressing endogenous glucose production. Of critical importance, insulin secretion is suppressed when glucose levels are low, which protects against life-threatening hypoglycemia. There have been remarkable advances to enhance efficacy and safety of insulin therapy since the discovery of insulin 100 years ago. Insulin pharmacokinetics has been optimized through both formulation and modification of the peptide itself. Sophisticated mechanical devices can provide continuous insulin delivery and continuous glucose monitoring. Pancreatic islets can be transplanted, and bihormonal pumps more closely mimic normal physiology. Despite technological advances, most insulin-dependent patients do not achieve physiological glucose control. The risk of severe hypoglycemia is particularly troublesome because it limits the ability to safely achieve optimal glycemic control, which in turn makes it harder to protect against the long-term risk of microvascular complications (1,2). To underscore this point, half of the Diabetes Control and Complications Trial (DCCT)/Epidemiology of Diabetes Interventions and Complications (EDIC) cohort experienced severe hypoglycemia (∼40 episodes per 100 patient-years) (3). Furthermore, self-reported severe hypoglycemia is associated with 3.4-fold increased risk of death during a 5-year follow-up period (4). The pursuit of a glucose-responsive insulin (GRI) therapy is an ambitious technical objective. In type 2 diabetes, incretin therapy enhances insulin secretion when glucose levels are high but does not cause hypoglycemia because it does not promote insulin secretion when glucose levels are low. Similarly, GRI technology aims to mimic the ability of β-cells …

Summary A patient with atypical partial lipodystrophy who had a transient initial response to metreleptin experienced acute worsening of her metabolic state when neutralizing antibodies against metreleptin appeared. Because her metabolic status continued to deteriorate, a therapeutic trial with melanocortin-4 receptor agonist setmelanotide, that is believed to function downstream from leptin receptor in the leptin signaling system, was undertaken in an effort to improve her metabolic status for the first time in a patient with lipodystrophy. To achieve this, a compassionate use (investigational new drug application; IND) was initiated (NCT03262610). Glucose control, body fat by dual-energy X-ray absorptiometry and MRI, and liver fat by proton density fat fraction were monitored. Daily hunger scores were assessed by patient filled questionnaires. Although there was a slight decrease in hunger scales and visceral fat, stimulating melanocortin-4 receptor by setmelanotide did not result in any other metabolic benefit such as improvement of hypertriglyceridemia or diabetes control as desired. Targeting melanocortin-4 receptor to regulate energy metabolism in this setting was not sufficient to obtain a significant metabolic benefit. However, complex features of our case make it difficult to generalize these observations to all cases of lipodystrophy. It is still possible that melanocortin-4 receptor agonistic action may offer some therapeutic benefits in leptin-deficient patients. Learning points: A patient with atypical lipodystrophy with an initial benefit with metreleptin therapy developed neutralizing antibodies to metreleptin (Nab-leptin), which led to substantial worsening in metabolic control. The neutralizing activity in her serum persisted for longer than 3 years. Whether the worsening in her metabolic state was truly caused by the development of Nab-leptin cannot be fully ascertained, but there was a temporal relationship. The experience noted in our patient at least raises the possibility for concern for substantial metabolic worsening upon emergence and persistence of Nab-leptin. Further studies of cases where Nab-leptin is detected and better assay systems to detect and characterize Nab-leptin are needed. The use of setmelanotide, a selective MC4R agonist targeting specific neurons downstream from the leptin receptor activation, was not effective in restoring metabolic control in this complex patient with presumed diminished leptin action due to Nab-leptin. Although stimulating the MC4R pathway was not sufficient to obtain a significant metabolic benefit in lowering triglycerides and helping with her insulin resistance as was noted with metreleptin earlier, there was a mild reduction in reported food intake and appetite. Complex features of our case make it difficult to generalize our observation to all leptin-deficient patients. It is possible that some leptin-deficient patients (especially those who need primarily control of food intake) may still theoretically benefit from MC4R agonistic action, and further studies in carefully selected patients may help to tease out the differential pathways of metabolic regulation by the complex network of leptin signaling system.

Lipoprotein(a), Lp(a), is a modified low-density lipoprotein particle that contains apolipoprotein(a), encoded by LPA, and is a highly heritable, causal risk factor for cardiovascular diseases that varies in concentrations across ancestries. Here, we use deep-coverage whole genome sequencing in 8392 individuals of European and African ancestry to discover and interpret both single-nucleotide variants and copy number (CN) variation associated with Lp(a). We observe that genetic determinants between Europeans and Africans have several unique determinants. The common variant rs12740374 associated with Lp(a) cholesterol is an eQTL for SORT1 and independent of LDL cholesterol. Observed associations of aggregates of rare non-coding variants are largely explained by LPA structural variation, namely the LPA kringle IV 2 (KIV2)-CN. Finally, we find that LPA risk genotypes confer greater relative risk for incident atherosclerotic cardiovascular diseases compared to directly measured Lp(a), and are significantly associated with measures of subclinical atherosclerosis in African Americans., Circulating lipoprotein(a) is an important risk factor for cardiovascular disease and shows variability between different ethnic groups. Here, Zekavat et al. perform whole-genome sequencing in individuals of European and African ancestries and find ancestry-specific genetic determinants for lipoprotein(a) levels.

There are more than 30 million Americans with diabetes, a disease that costs the U.S. more than $327 billion per year (1,2). Achieving glycemic control and controlling cardiovascular risk factors have been conclusively shown to reduce diabetes complications, comorbidities, and mortality. To achieve these desired outcomes, the medical community now has available many classes of medications and many formulations of insulin to effectively manage the metabolic abnormalities for people with diabetes. However, the affordability of medications in general, and for insulin specifically, is currently of great concern to people with diabetes, their families, health care providers, insurers, and employers. For millions of people living with diabetes, including all individuals with type 1 diabetes, access to insulin is literally a matter of life and death. The average list price of insulin has skyrocketed in recent years, nearly tripling between 2002 and 2013 (3). The reasons for this increase are not entirely clear but are due in part to the complexity of drug pricing in general and of insulin pricing in particular. As the price of insulin continues to rise, individuals with diabetes are often forced to choose between purchasing their medications or paying for other necessities, exposing them to serious short- and long-term health consequences (4–9). To find solutions to the issue of insulin affordability, there must be a better understanding of the transactions throughout the insulin supply chain, the impact each stakeholder has on what people with diabetes pay for insulin, and the relative efficacy of therapeutic options. Thus, as the nation’s leading voluntary health organization whose mission is “to prevent and cure diabetes and to improve the lives of all people affected by diabetes,” the American Diabetes Association (ADA) is committed to finding ways to provide relief for individuals and families who lack affordable access …

BACKGROUND: In the Joslin Medalist Study (Medalists), we determined whether significant associations exist between β cell function and pathology and clinical characteristics. METHODS: Individuals with type 1 diabetes (T1D) for 50 or more years underwent evaluation including HLA analysis, basal and longitudinal autoantibody (AAb) status, and β cell function by a mixed-meal tolerance test (MMTT) and a hyperglycemia/arginine clamp procedure. Postmortem analysis of pancreases from 68 Medalists was performed. Monogenic diabetes genes were screened for the entire cohort. RESULTS: Of the 1019 Medalists, 32.4% retained detectable C-peptide levels (>0.05 ng/mL, median: 0.21 ng/mL). In those who underwent a MMTT (n = 516), 5.8% responded with a doubling of baseline C-peptide levels. Longitudinally (n = 181, median: 4 years), C-peptide levels increased in 12.2% (n = 22) and decreased in 37% (n = 67) of the Medalists. Among those with repeated MMTTs, 5.4% (3 of 56) and 16.1% (9 of 56) had waxing and waning responses, respectively. Thirty Medalists with baseline C-peptide levels of 0.1 ng/mL or higher underwent the clamp procedure, with HLA(–)/AAb(–) and HLA(+)/AAb(–) Medalists being most responsive. Postmortem examination of pancreases from 68 Medalists showed that all had scattered insulin-positive cells; 59 additionally had few insulin-positive cells within a few islets; and 14 additionally had lobes with multiple islets with numerous insulin-positive cells. Genetic analysis revealed that 280 Medalists (27.5%) had monogenic diabetes variants; in 80 (7.9%) of these Medalists, the variants were classified as “likely pathogenic” (rare exome variant ensemble learner [REVEL] >0.75). CONCLUSION: All Medalists retained insulin-positive β cells, with many responding to metabolic stimuli even after 50 years of T1D. The Medalists were heterogeneous with respect to β cell function, and many with HLA(+) diabetes risk alleles also had monogenic diabetes variants, indicating the importance of genetic testing for clinically diagnosed T1D. FUNDING: Funding for this work was provided by the Dianne Nunnally Hoppes Fund; the Beatson Pledge Fund; the NIH, National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK); and the American Diabetes Association (ADA).

Alleles associated with lower levels of LDL cholesterol (LDL-C) have recently been associated with an increased risk of type 2 diabetes (T2D), highlighting the complex relationship between LDL-C and diabetes. This observation begs the question of whether LDL-C–raising alleles are associated with a decreased risk of T2D. This issue was recently addressed in a large familial hypercholesterolemia (FH) screening study, which reported a lower prevalence of self-reported diabetes in FH subjects than in age-matched relatives without FH. To extend this observation, we tested the association of FH with diabetes status and glycemia in a large Amish population enriched for the FH-associated APOB R3527Q variant that included 640 APOB R3527Q carriers and 4,683 noncarriers. Each copy of the R3527Q T allele was associated with a 74.9 mg/dL increase in LDL-C. There was little difference in T2D prevalence between subjects with (5.2%) and without (4.5%) the R3527Q allele (P = 0.23), and there was no association between R3527Q variant and impaired fasting glucose, fasting glucose or insulin, or oral glucose tolerance test–derived measures. Our data provide no evidence supporting an association between the APOB R3527Q variant and T2D or glycemia and highlight the asymmetry of the LDL-C–T2D relationship and/or the gene/variant-dependent specificity of the LDL-C–T2D association.

Increased LDL-cholesterol (LDL-C) and fibrinogen are independent risk factors for cardiovascular disease (CVD). We identified novel associations between an Amish-enriched missense variant (p.Asn352Ser) in a functional domain of beta-1,4-galactosyltransferase 1 (B4GALT1) and 13.5 mg/dl lower LDL-C (p=1.6E-15), and 26 mg/dl lower plasma fibrinogen (p= 9.8E-05). N-linked glycan profiling found p.Asn352Ser to be associated (p-values from 1.4E-06 to 1.0E-17) with decreased glycosylation of glycoproteins including: fibrinogen, ApoB100, immunoglobulin G (IgG), and transferrin.In vitroassays found that the mutant (352Ser) protein had 50% lower galactosyltransferase activity compared to wild type (352Asn) protein. Knockdown ofb4galt1in zebrafish embryos resulted in significantly lower LDL-C compared to control, which was fully rescued by co-expression of 352Asn humanB4GALT1mRNA but only partially rescued by co-expression of 352Ser humanB4GALT1mRNA. Our findings establishB4GALT1as a novel gene associated with lower LDL-C and fibrinogen and suggest that targeted modulation of protein glycosylation may represent a therapeutic approach to decrease CVD risk.

Apolipoprotein CIII (apoCIII), an inhibitor of lipoprotein lipase, plays an important role in triglyceride metabolism. However, the role of apoCIII in hypertriglyceridemia in lipodystrophy and the effects of leptin replacement on apoCIII levels are unknown.The objective of the study was to test the hypotheses that apoCIII is elevated in hypertriglyceridemic patients with lipodystrophy and that leptin replacement in these patients lowers circulating apoCIII.Using a post hoc cross-sectional case-control design, we compared serum apoCIII levels from patients with lipodystrophy not associated with HIV (n = 60) and age-, gender-, race-, and ethnicity-matched controls (n = 54) participating in ongoing studies at the National Institutes of Health. In a prospective, open-label, ongoing study, we studied the effects of 6–12 months of leptin replacement on apoCIII in lipodystrophy patients as an exploratory outcome.ApoCIII was higher in lipodystrophy patients (geometric mean [25th and 75th percentiles]) (23.9 mg/dL [14.6, 40.3]) compared with controls (14.9 mg/dL [12.3, 17.7]) (P.0001). ApoCIII and triglyceride levels were positively correlated in patients with lipodystrophy (R = 0.72, P.0001) and healthy controls (R = 0.6, P.0001). Leptin replacement (6–12 mo) did not significantly alter apoCIII (before leptin: 23.4 mg/dL [14.5, 40.1]; after leptin: 21.4 mg/dL [16.7, 28.3]; P = .34).Leptin replacement in lipodystrophy did not alter serum apoCIII levels. Elevated apoCIII may play a role in the hypertriglyceridemia of lipodystrophy independent of leptin deficiency and replacement.

Studies including ours indicate that systemic inhibition of dipeptidyl peptidase 4 (DPP4), an ubiquitously expressed peptidase, by pharmaceutical inhibitors improved atherosclerosis (1,2). However, the role of immune cell–derived DPP4 is not well defined in atherosclerosis. Our previous work demonstrated that DPP4 expression on monocytes/macrophages was increased in obesity and associated with the degree of insulin resistance (3). To test if the obesity-related increase of monocyte DPP4 plays a role in vascular disease, we investigated the relationship of monocyte DPP4 expression with human aortic atherosclerosis, obesity, and lipid metabolism. A total of 14 control volunteers and 27 atherosclerotic patients without diagnosed heart, lung, or liver diseases and without prescription drugs (except ACE inhibitors and angiotensin receptor blockers) were selected from Aliskiren Effect on Aortic Plaque Progression (ALPINE), a phase 4 clinical trial (clinical trial reg. no. NCT01417104, clinicaltrials.gov). Presence of aortic atherosclerotic plaque (Fig. 1 A ) was confirmed by high-resolution three-dimensional MRI. Figure 1 A : High-resolution three-dimensional dark-blood MRI was used to confirm the aortic atherosclerotic plaque in patients with atherosclerotic disease. Arrows indicate the thickening of the aortic wall. Scale bars, 10 mm. B and C : DPP4 expression on circulating immune cells. Peripheral blood mononuclear cells were isolated from healthy volunteers, and CD11b+ monocyte and CD3+ T cells were gated for …

BACKGROUND: Post-prandial lipemia (PPL), defined as a prolonged or elevated rise in triglycerides that accompanies fat feeding, is a significant risk factor for coronary heart disease (CHD) and associated comorbidities. The impact of PPL on CHD risk is underscored by the preponderance of each day spent in the post-prandial state. OBJECTIVE: In this study, we evaluated cross-sectionally the association between usual (i.e., non-interventional) physical activity and the 6-hour triglyceride response to a standardized high-fat meal. METHODS: The high-fat meal intervention was carried out in 671 apparently healthy individuals as part of the Hereditary and Phenotype Intervention (HAPI) Heart Study. Triglyceride levels were measured in the fasting state and during six hours following administration of a standardized fat challenge. We defined PPL response as the triglyceride area under the fat load curve (AUC) and measured physical activity using accelerometers that were worn continuously over a 7-day period. RESULTS: Physical activity levels decreased with increasing age and were higher in men than women (both p < 0.001). The triglyceride AUC increased with increasing age in both men and women (both p

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Chronic inflammation may contribute to insulin resistance via molecular cross-talk between pathways for pro-inflammatory and insulin signaling. Interleukin 1 receptor-associated kinase 1 (IRAK-1) mediates pro-inflammatory signaling via IL-1 receptor/Toll-like receptors, which may contribute to insulin resistance, but this hypothesis is untested. Here, we used male Irak1 null (k/o) mice to investigate the metabolic role of IRAK-1. C57BL/6 wild-type (WT) and k/o mice had comparable body weights on low-fat and high-fat diets (LFD and HFD, respectively). After 12 weeks on LFD (but not HFD), k/o mice (versus WT) had substantially improved glucose tolerance (assessed by the intraperitoneal glucose tolerance test (IPGTT)). As assessed with the hyperinsulinemic euglycemic glucose clamp technique, insulin sensitivity was 30% higher in the Irak1 k/o mice on chow diet, but the Irak1 deletion did not affect IPGTT outcomes in mice on HFD, suggesting that the deletion did not overcome the impact of obesity on glucose tolerance. Moreover, insulin-stimulated glucose-disposal rates were higher in the k/o mice, but we detected no significant difference in hepatic glucose production rates (± insulin infusion). Positron emission/computed tomography scans indicated higher insulin-stimulated glucose uptake in muscle, but not liver, in Irak1 k/o mice in vivo. Moreover, insulin-stimulated phosphorylation of Akt was higher in muscle, but not in liver, from Irak1 k/o mice ex vivo. In conclusion, Irak1 deletion improved muscle insulin sensitivity, with the effect being most apparent in LFD mice.

MFN2 encodes mitofusin 2, a membrane-bound mediator of mitochondrial membrane fusion and inter-organelle communication. MFN2 mutations cause axonal neuropathy, with associated lipodystrophy only occasionally noted, however homozygosity for the p.Arg707Trp mutation was recently associated with upper body adipose overgrowth. We describe similar massive adipose overgrowth with suppressed leptin expression in four further patients with biallelic MFN2 mutations and at least one p.Arg707Trp allele. Overgrown tissue was composed of normal-sized, UCP1-negative unilocular adipocytes, with mitochondrial network fragmentation, disorganised cristae, and increased autophagosomes. There was strong transcriptional evidence of mitochondrial stress signalling, increased protein synthesis, and suppression of signatures of cell death in affected tissue, whereas mitochondrial morphology and gene expression were normal in skin fibroblasts. These findings suggest that specific MFN2 mutations cause tissue-selective mitochondrial dysfunction with increased adipocyte proliferation and survival, confirm a novel form of excess adiposity with paradoxical suppression of leptin expression, and suggest potential targeted therapies. DOI: http://dx.doi.org/10.7554/eLife.23813.001, eLife digest Obesity and the diseases associated with it are among the biggest healthcare problems in developed countries. The word obesity means, simply, the accumulation of too much fat tissue in the body, but this ignores growing evidence that fat tissue is highly complex. Fat tissue is important for “mopping up” and storing excess calories safely, but also sends messages to the brain and other organs to report how full the body’s energy stores are. Understanding how fat tissues perform these roles will aid the development of strategies to treat or prevent obesity. A hormone called leptin acts as a signal of the status of the body’s fat stores. High levels of leptin in the blood tell the brain that the body has plenty of fat stored. On the other hand, if the levels of leptin in the blood become very low it tells the brain to prioritize finding food and shut down any nonessential processes. This helps to prevent the body from starving. It is not clear how the production of leptin is controlled, in part because fat tissues in different parts of the body behave very differently. Individuals who have a particular rare genetic mutation accumulate large amounts of fat tissue in their upper bodies and gradually lose fat tissue in their arms and legs. Despite accumulating a lot of fat tissue in the upper body, these individuals have extremely low levels of leptin in their blood. To investigate this genetic condition, Rocha et al. studied two children with the mutation and their healthy parents. The experiments show that this mutation alters a protein called mitofusin 2, which is found in cell compartments called mitochondria. Mitofusin 2 helps the mitochondria to bind to each other and to other parts of the cell, which is important for the mitochondria to generate the energy needed for vital cell processes. The mitochondria in the fat cells of the children are less closely linked to each other and have an unusual appearance compared to the mitochondria in the parents’ fat cells. Further experiments showed that some genes, including the one that produces leptin, are less active in the children compared to their parents – while other genes that are involved in starvation or stress responses are more active. This work suggests that mitochondria play an important role in regulating the production of leptin. Furthermore, it suggests that leptin or drugs that switch off stress-related genes may have the potential to be used to treat individuals with this particular mutation. DOI: http://dx.doi.org/10.7554/eLife.23813.002

Sodium–glucose cotransporter 2 (SGLT2) inhibitors offer attractive metabolic and cardiorenal benefits for patients with type 2 diabetes, but are associated with a number of safety issues. A recent study documented that SGTL2 inhibition indirectly triggers the FGF23/1,25-dihydroxyvitamin D/parathyroid hormone axis, which may contribute to adverse effects on bone health.

On 15 May 2015, the U.S. Food and Drug Administration (FDA) warned that administration of sodium-glucose cotransporter-2 (SGLT2) inhibitors could lead to ketoacidosis in patients with diabetes mellitus. This announcement came more than 2 years after the FDA's first approval of an SGLT2 inhibitor, although the phenomenon had been known for more than 125 years. Luminaries of diabetes research (including Josef von Mering, Frederick Allen, I. Arthur Mirsky, and George Cahill) had described ketosis and ketoacidosis induced by administration of the phytochemical phlorizin, the prototypical SGLT inhibitor, as well as in patients with familial renal glucosuria, a condition that is considered a natural model of SGLT2 inhibition. Neither government regulators nor manufacturers of SGLT2 inhibitors evinced an awareness of this extensive historical record. The absence of historical inquiry delayed notice of ketoacidosis as an adverse reaction, which could have reduced the burden of illness from these drugs.

SGLT2 inhibitors decrease plasma glucose concentrations by inhibiting proximal tubular reabsorption of glucose in the kidney.1 The attractive efficacy profile of glucose-lowering plus weight loss must be balanced against possible side effects, including an increase in the incidence of treatment-emergent bone fractures observed in clinical studies. In a study of moderate renal impairment, 9.4% of patients treated with dapagliflozin (10 mg) experienced bone fractures while no fractures were observed in placebo-treated patients.2 Furthermore, a ~30% increase in bone fractures was observed in canagliflozin-treated patients in eight pooled clinical trials with longer mean duration (68 weeks).3 Although these data suggest the possibility that SGLT2 inhibitors might increase the risk of bone fractures, additional data will be required before drawing a firm conclusion. SGLT2 inhibitors increase tubular reabsorption of phosphate, thereby increasing serum phosphate levels3,4 (Fig. 1A). The body has evolved complex homeostatic mechanisms to regulate phosphate, and an increase in serum phosphate has the potential to exert an adverse impact upon bone (Fig. 1B). For example, phosphate administration increases PTH secretion.6 Furthermore, SGLT2 inhibitors increase levels of both phosphate and PTH.3,4 While canagliflozin caused a small increase in mean PTH (+7.9%), the standard deviation (SD) was relatively large (39.3%).3 Thus, a substantial number of canagliflozin-treated patients might experience a >50% increase in PTH levels – a change that could be clinically significant. In addition, phosphate administration has been reported to increase FGF23 levels in healthy volunteers,7 which would decrease 1,25-dihydroxyvitamin D levels. However, the mechanisms whereby phosphate regulates FGF23 remain controversial.8 In in vitro studies, phosphate exerts a direct effect to increase FGF23 mRNA levels in primary human fetal bone cells.9 In contrast, some evidence suggests phosphate decreases FGF23 expression by an indirect mechanism – possibly mediated by PTH.8 In light of the controversy, it is critical to conduct clinical studies to assess the effect of SGLT2 inhibitors upon FGF23 levels. Figure 1 Proposed mechanisms whereby SGTL2 inhibitors exert adverse effects on bone Phosphate administration increases both PTH and FGF23, two hormones that exert opposing effects upon vitamin D metabolism8 with PTH increasing and FGF23 decreasing 1,25-dihydroxyvitamin D levels. Accordingly, if SGLT2 inhibitors increase levels of both PTH and FGF23, one could not predict a priori the net impact upon 1,25-dihydroxyvitamin D. Nevertheless, available data suggest that SGLT2 inhibitors decrease mean 1,25-dihydroxyvitamin D levels.3 Canagliflozin caused a small decrease in mean 1,25-dihydroxyvitamin D levels (−12%), but the SD was relatively large (42.4%).3 Thus, a significant percentage of canagliflozin-treated patients could experience a clinically significant ~50% decrease in 1,25-dihydroxyvitamin D levels. In contrast to clinical data suggesting an increase in PTH, suprapharmacological doses of dapagliflozin decreased PTH in rats.10 An investigative toxicology study elucidated the mechanism.10 Dapagliflozin is relatively selective for SGLT2, but high doses administered in toxicology experiments were sufficient to inhibit intestinal SGLT1, which led to glucose malabsorption. Colonic bacteria fermented the unabsorbed glucose, which acidified the intestinal contents. Acid pH increased solubility of calcium, promoted calcium absorption, suppressed PTH, and promoted ectopic calcification. These suprapharmacologic doses of SGLT2 inhibitor are unlikely to be relevant to human pharmacology with approved doses of selective SGLT2 inhbitors.10 Sustained increases in PTH enhance bone resorption, and increase the risk of bone fractures. Similarly, increased levels of FGF23 have been associated with bone disease.8 Finally, decreased levels of 1,25-dihydroxyvitamin D may decrease absorption of Ca+2 from the GI tract, and impair bone calcification. Consistent with these mechanisms, canagliflozin was observed to increase bone turnover as reflected by increases in levels of both collagen type 1 beta-carboxy-telopeptide levels and osteocalcin (biomarkers for bone resorption and bone formation, respectively). In addition, both DXA and quantitative CT detected a decrease in bone mineral density in the lumbar spine and total hip after 52 weeks of therapy with canagliflozin (300 mg).3 In contrast, there was no statistically significant change in bone mineral density at the distal forearm and femoral neck. Additional data could place these observations into context – including a correlation of clinical outcomes (i.e., fractures) with changes in bone biomarkers and bone mineral density in individual patients. The existence of numerous homeostatic mechanisms creates challenges in interpretation of studies of mechanisms whereby SGLT2 inhibitors could affect bone health. For example, an increase in serum phosphate is predicted to increase FGF23 and PTH – both of which promote phosphaturia. Because of this negative feedback, the maximum increase in mean serum phosphate may be transient and/or small in magnitude. Nevertheless, even small changes may significantly affect bone health over years of drug exposure. Finally, it is important to recognize that most drug-treated patients do not experience bone fractures. Bone fractures may be most likely to occur in the subpopulation of “outlier” patients with above average changes in bone-related parameters. In short, mean data may not provide a full picture of the impact upon bone. Patients with type 2 diabetes are especially susceptible to adverse effects of drugs upon bone because of coexisting bone diseases (including post-menopausal osteoporosis, renal osteodystrophy, and diabetes-associated bone fragility). As reported in the ADOPT trial, thiazolidinediones increase the risk of bone fractures.11 After a one year lag, rosiglitazone-treated patients experienced an increase in bone fractures (hazard ratio, 1.6). Similarly, the fracture rate was not increased during the first year of treatment with canagliflozin, but patients experienced more fractures during the second year of therapy.3 The median follow-up for rosiglitazone-treated patients was four years, suggesting that the average duration of the pooled canagliflozin studies (68 weeks) was likely too short to provide conclusive data on fracture risk. Fortunately, ongoing FDA-mandated cardiovascular outcome studies with SGLT2 inhibitors are of sufficient size and duration to assess the risk of bone fractures. Several arguments have been advanced suggesting that the observed increase in fracture rate might be a “chance phenomenon”.2 For example, some fractures occur in the feet, hands, and patella, which were stated not to be associated with bone health.2,3 At the time of the FDA’s Dapagliflozin Advisory Committee, eight fractures had been observed among patients treated with dapagliflozin (10 mg) in the special study of patients with moderate renal impairment. These included one patient with a fractured patella and one with a foot fracture. It is impossible to draw firm conclusions based upon these two patients – either about the distribution of fractures among anatomical sites or whether the increased number of fractures will ultimately be confirmed as a toxicity associated with SGLT2 inhibitors. In contrast to biomarker data suggesting canagliflozin increases bone turnover,3 it has been reported that dapagliflozin does not affect mean bone mineral density or biomarkers of bone turnover in patients with normal to mildly impaired renal function.3,4 These obervations raise the question of whether the bone effects might be a compound-specific rather than mechanism-based. However, the literature suggests that any differences among compounds may be the consequence of dose selection rather than an intrinsic difference between the two compounds. Whereas the 300 mg dose of canagliflozin delivers maximal inhibition of SGLT2,3 the 10 mg dose of dapagliflozin delivers only sub-maximal inhibition.12 Specifically, the 10 mg dose was reported to cause ~35 g/day of urinary glucose excretion – approximately 35% less than the urinary glucose excretion caused by maximally effective doses of dapagliflozin (50–100 mg).4 Thus, canagliflozin (300 mg) might be more efficacious than dapagliflozin (10 mg) with respect to all mechanism-based pharmacology (both mechanism-based toxicity and glucose-lowering). It would require head-to-head trials to draw firm conclusions about comparative benefit:risk profiles of individual SGLT2 inhibitors. In conclusion, SGLT2 inhibitors have been observed to increase the incidence of treatment-emergent bone fractures, and the risk of fractures appears to increase over time. We have hypothesized plausible pathophysiologic mechanisms with potential to mediate adverse effects upon bone. Future mechanistic research may identify patients who are most vulnerable to develop drug-induced bone fractures (e.g., post-menopausal women), and may suggest therapeutic approaches to minimize the risk.

Diabetic ketoacidosis is a serious metabolic condition that may occur in patients with either Type 1 or Type 2 diabetes. The accumulation of ketoacids in the serum is a consequence of insulin deficiency and glucagon excess. Sodium Glucose Transporter 2 (SGLT2) inhibitors are novel therapeutic treatments for improving glucose homeostasis in patients with diabetes. Through reductions in glucose reabsorption by the kidney, they lower serum glucose in patients with Type 2 diabetes and they improve glucose control whether used alone or in combination with other therapies. Mechanistically, these drugs increase serum ketoacids and increase glucagon production, which in some individuals, can lead to formation of diabetic ketoacidosis. This review will first focus in how the kidney normally handles ketoacids, and second will discuss how the SGLT2 inhibitors affect the kidney in such a way so as to enhance the risk for development of ketoacidosis in susceptible individuals.

Sodium glucose cotransporter 2 (SGLT2) inhibitors are antidiabetic drugs that increase urinary excretion of glucose, thereby improving glycemic control and promoting weight loss. Since approval of the first-in-class drug in 2013, data have emerged suggesting that these drugs increase the risk of diabetic ketoacidosis. In May 2015, the Food and Drug Administration issued a warning that SGLT2 inhibitors may lead to ketoacidosis.Using PubMed and Google, we conducted Boolean searches including terms related to ketone bodies or ketoacidosis with terms for SGLT2 inhibitors or phlorizin. Priority was assigned to publications that shed light on molecular mechanisms whereby SGLT2 inhibitors could affect ketone body metabolism.SGLT2 inhibitors trigger multiple mechanisms that could predispose to diabetic ketoacidosis. When SGLT2 inhibitors are combined with insulin, it is often necessary to decrease the insulin dose to avoid hypoglycemia. The lower dose of insulin may be insufficient to suppress lipolysis and ketogenesis. Furthermore, SGLT2 is expressed in pancreatic α-cells, and SGLT2 inhibitors promote glucagon secretion. Finally, phlorizin, a nonselective inhibitor of SGLT family transporters decreases urinary excretion of ketone bodies. A decrease in the renal clearance of ketone bodies could also increase the plasma ketone body levels.Based on the physiology of SGLT2 and the pharmacology of SGLT2 inhibitors, there are several biologically plausible mechanisms whereby this class of drugs has the potential to increase the risk of developing diabetic ketoacidosis. Future research should be directed toward identifying which patients are at greatest risk for this side effect and also to optimizing pharmacotherapy to minimize the risk to patients.

Context: Sodium glucose cotransporter 2 (SGLT2) inhibitors are antidiabetic drugs that increase urinary excretion of glucose, thereby improving glycemic control and promoting weight loss. Since approval of the first-in-class drug in 2013, data have emerged suggesting that these drugs increase the risk of diabetic ketoacidosis. In May 2015, the Food and Drug Administration issued a warning that SGLT2 inhibitors may lead to ketoacidosis. Evidence Acquisition: Using PubMed and Google, we conducted Boolean searches including terms related to ketone bodies or ketoacidosis with terms for SGLT2 inhibitors or phlorizin. Priority was assigned to publications that shed light on molecular mechanisms whereby SGLT2 inhibitors could affect ketone body metabolism. Evidence Synthesis: SGLT2 inhibitors trigger multiple mechanisms that could predispose to diabetic ketoacidosis. When SGLT2 inhibitors are combined with insulin, it is often necessary to decrease the insulin dose to avoid hypoglycemia. The lower dose of insuli...

The interaction of insulin with its cell surface receptor is the first step in insulin action and the first identified target of insulin resistance. The insulin resistance in several syndromic forms of extreme insulin resistance has been shown to be caused by mutations in the receptor gene. We studied 8 female patients with the type A form of extreme insulin resistance and 3 patients (2 male and 1 female) with the Rabson-Mendenhall syndrome and followed the natural history of these patients for up to 30 years. The 11 patients ranged in age from 7 to 32 years at presentation. All 11 patients had extreme insulin resistance, acanthosis nigricans, and hyperandrogenism in the female patients, and all but 1 were of normal body weight. This phenotype strongly predicts mutations in the insulin receptor: of the 8 patients studied, 7 were found to have mutations. Similar results from the literature are found in other patients with type A and Rabson-Mendenhall syndromes and leprechaunism. The hyperandrogenic state resulting from hyperinsulinemia and insulin resistance in these patients was extreme: 6 of 8 patients had ovarian surgery to correct the polycystic ovarian syndrome and elevation of serum testosterone. By contrast, a larger group of insulin-resistant patients who were obese with hyperandrogenism, insulin resistance, and acanthosis nigricans (HAIR-AN syndrome) did not have a high probability of mutations in the insulin receptor. The morbidity and mortality of these patients were high: 3 of 11 died, 9 of 11 were diabetic and 1 had impaired glucose tolerance, and 7 of 9 patients had 1 or more severe complication of diabetes. Our literature review revealed that the mortality of leprechaunism is so high that the term leprechaunism should be restricted to infants or young children under 2 years of age. Analogous to patients with the common forms of type 2 diabetes, these patients had a heterogeneous course. In 2 patients who were able to maintain extremely high endogenous insulin production, the fasting blood glucose remained normal even though post-glucose-challenge levels were elevated. Most patients, however, required large doses of exogenous insulin to ameliorate the severe hyperglycemia. Preliminary results of a recent study suggest that recombinant leptin administration may benefit these patients with severe insulin resistance.

311 ipoatrophic–lipodystrophic syndromes comprise a heterogeneous group of disorders associated with insulin-resistant diabetes and hypertriglyceridemia [1]. Unlike the more common form of diabetes that is associated with obesity, the lipoatrophic–lipodystrophic syndromes all cause either a lack of or an abnormal distribution of adipose tissue. The similar metabolic abnormalities in diabetes and lipoatrophic– lipodystrophic syndromes (i.e., obesity and lipoatrophy) underscore the importance of adipose tissue in energy homeostasis. In lipoatrophic–lipodystrophic syndromes, hypertension, polycystic ovary syndrome, and acanLipoatrophic–Lipodystrophic Syndromes: The Spectrum of Findings on MR Imaging

Background: The lipodystrophies are characterized by loss of body fat and metabolic disturbances, but the CNS is seldom affected. Methods: An investigation of a family with partial lipodystrophy and neurologic abnormalities included lipid analysis, dual-energy x-ray absorbtiometry (DEXA) for adiposity, insulin resistance, karyotype and other genetic analyses, peroxisomal function, glycosylation pattern of transferrin and thyroglobulin, and muscle biopsy. Results: The propositus, a 28-year-old woman with congenital partial lipodystrophy and cataracts, presented with a spastic–ataxic gait and lower extremity paresthesiae at age 18. Laboratory investigation revealed a type V hyperlipidemia pattern, insulin resistance, and high α-tocopherol levels. A similar syndrome in other family members suggested an autosomal dominant pattern of inheritance. Discussion: The progressive neurologic degenerative condition associated with this autosomal dominant, partial lipodystrophy may be misdiagnosed as MS or spinocerebellar degeneration. Search for a few relevant candidate genes was unrevealing. A genome-wide search to determine the molecular etiology can be undertaken if other similar cases are identified.

Background Regulatory agencies have concluded that sodium glucose cotransporter 2 (SGLT2) inhibitors lead to ketoacidosis, but published literature on this point remains controversial. Methods We searched the FDA Adverse Event Reporting System (FAERS) for reports of acidosis in patients treated with canagliflozin, dapagliflozin, or empagliflozin (from the date of each drug's FDA approval until May 15, 2015). We compared the number of SGLT2 inhibitor-related reports to reports of acidosis in patients treated with the 2 most commonly used DPP4 inhibitors: sitagliptin and saxagliptin. We estimated relative risks of acidosis by relating the number of reports to cumulative drug sales (a surrogate for patient exposure). Results FAERS contained 259 reports of acidosis (including 192 reports of ketoacidosis) for SGLT2 inhibitors compared with 477 reports of acidosis for DPP4 inhibitors (including 71 reports of ketoacidosis). Based on estimated patient exposure, the overall risk of developing acidosis was ~14-fold higher for SGLT2 inhibitors. Among 51 SGLT2 inhibitor-related reports with quantifiable metabolic information, 20 cases occurred in patients with type 1 diabetes (T1D), 25 in type 2 diabetes (T2D), and 6 in patients with unspecified type of diabetes. After excluding patients with T1D and focusing on patients identified as having T2D, we estimate that SGLT2 inhibitors were associated with ~7-fold increase in developing acidosis. Seventy-one percent had euglycemic ketoacidosis. Conclusions Our results support the FDA's warning that SGLT2 inhibitors lead to ketoacidosis, as evidenced by an increased reporting rate for acidosis above that in a comparator population treated with DPP4 inhibitors.

Sorting nexins (SNX) comprise a family of proteins with homology to several yeast proteins, including Vps5p and Mvp1p, that are required for the sorting of proteins to the yeast vacuole. Human SNX1, -2, and -4 have been proposed to play a role in receptor trafficking and have been shown to bind to several receptor tyrosine kinases, including receptors for epidermal growth factor, platelet-derived growth factor, and insulin as well as the long form of the leptin receptor, a glycoprotein 130-associated receptor. We now describe a novel member of this family, SNX6, which interacts with members of the transforming growth factor-beta family of receptor serine-threonine kinases. These receptors belong to two classes: type II receptors that bind ligand, and type I receptors that are subsequently recruited to transduce the signal. Of the type II receptors, SNX6 was found to interact strongly with ActRIIB and more moderately with wild type and kinase-defective mutants of TbetaRII. Of the type I receptors, SNX6 was found to interact only with inactivated TbetaRI. SNXs 1-4 also interacted with the transforming growth factor-beta receptor family, showing different receptor preferences. Conversely, SNX6 behaved similarly to the other SNX proteins in its interactions with receptor tyrosine kinases. Strong heteromeric interactions were also seen among SNX1, -2, -4, and -6, suggesting the formation in vivo of oligomeric complexes. These findings are the first evidence for the association of the SNX family of molecules with receptor serine-threonine kinases.

Sorting nexins are a family of phox homology domain containing proteins that are homologous to yeast proteins involved in protein trafficking. We have identified a novel 342-amino acid residue sorting nexin, SNX15, and a 252-amino acid splice variant, SNX15A. Unlike many sorting nexins, a SNX15 ortholog has not been identified in yeast or Caenorhabditis elegans. By Northern blot analysis, SNX15 mRNA is widely expressed. Although predicted to be a soluble protein, both endogenous and overexpressed SNX15 are found on membranes and in the cytosol. The phox homology domain of SNX15 is required for its membrane association and for association with the platelet-derived growth factor receptor. We did not detect association of SNX15 with receptors for epidermal growth factor or insulin. However, overexpression of SNX15 led to a decrease in the processing of insulin and hepatocyte growth factor receptors to their mature subunits. Immunofluorescence studies showed that SNX15 overexpression resulted in mislocalization of furin, the endoprotease responsible for cleavage of insulin and hepatocyte growth factor receptors. Based on our data and the existing findings with yeast orthologs of other sorting nexins, we propose that overexpression of SNX15 disrupts the normal trafficking of proteins from the plasma membrane to recycling endosomes or the trans-Golgi network.

Sorting nexin (SNX) 15 is a novel member of the SNX family of proteins. Although the functions of most SNXs have not yet been determined, several family members (e.g., SNX1, SNX2, SNX3, and SNX8) are orthologs of yeast proteins involved in protein trafficking. Overexpression of myc-tagged SNX15 in COS-7 cells altered the morphology of several endosomal compartments. In transient transfection experiments, myc-SNX15 was first seen in small punctate spots and small ring structures. Later, myc-SNX15 was found in larger rings. Finally, myc-SNX15 was observed in large, amorphous membrane-limited structures. These structures contained proteins from lysosomes, late endosomes, early endosomes, and the trans-Golgi network. However, the morphology of the endoplasmic reticulum and Golgi was not affected by overexpression of myc-SNX15. In myc-SNX15-overexpressing cells, the endocytosis of transferrin was severely inhibited and endocytosis of tac-trans-Golgi network (TGN) 38 and tac-furin was slowed. In addition, the recycling of internalized tac-TGN38 and tac-furin was also inhibited. Both the morphological and biochemical data indicate that SNX15 plays a crucial role in trafficking through the endocytic pathway. This is the first demonstration that a mammalian SNX protein is involved in protein trafficking.

Familial partial lipodystrophy (FPLD), Dunnigan variety, is an autosomal dominant disorder characterized by marked loss of subcutaneous adipose tissue from the extremities and trunk but by excess fat deposition in the head and neck. The disease is frequently associated with profound insulin resistance, dyslipidemia, and diabetes. We have localized a gene for FPLD to chromosome 1q21-q23, and it has recently been proposed that nuclear lamin A/C is altered in FPLD, on the basis of a novel missense mutation (R482Q) in five Canadian probands. This gene had previously been shown to be altered in autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD-AD) and in dilated cardiomyopathy and conduction-system disease. We examined 15 families with FPLD for mutations in lamin A/C. Five families harbored the R482Q alteration that segregated with the disease phenotype. Seven families harbored an R482W alteration, and one family harbored a G465D alteration. All these mutations lie within exon 8 of the lamin A/C gene-an exon that has also been shown to harbor different missense mutations that are responsible for EDMD-AD. Mutations could not be detected in lamin A/C in one FPLD family in which there was linkage to chromosome 1q21-q23. One family with atypical FPLD harbored an R582H alteration in exon 11 of lamin A. This exon does not comprise part of the lamin C coding region. All mutations in FPLD affect the globular C-terminal domain of the lamin A/C protein. In contrast, mutations responsible for dilated cardiomyopathy and conduction-system disease are observed in the rod domain of the protein. The FPLD mutations R482Q and R482W occurred on different haplotypes, indicating that they are likely to have arisen more than once.

A type 1 diabetes has its highest incidence in the pediatric age range, there are also other forms of diabetes that affect children. From a clinical point of view, it is important to recognize these other forms of diabetes because of the important implications with respect to both prognosis and therapy. In addition, these relatively uncommon forms of diabetes provide an instructive model for the pathophysiological mechanisms that cause diabetes. Some syndromes are caused by insulin deficiency [e.g. maturityonset type diabetes of the young (MODY)]. Unlike patients with type 1 diabetes who exhibit near total absence of insulin due to autoimmune destruction of the b cell, MODY is characterized by a milder form of insulin deficiency caused by mutations in genes required for normal b cell function (1–6). However, not all cases of diabetes are caused by insulin deficiency. Indeed, some patients develop diabetes even though their b cells secrete sufficient insulin to achieve significant hyperinsulinemia (e.g. lipoatrophic diabetes and syndromes due to mutations in the insulin receptor gene) (7–10).

Throughout human history, people have struggled to understand the causes of human suffering and human illness. For example, in the Book of Job, Satan afflicted Job with painful skin sores. Job, his family, and his friends all inquired why Job became ill despite the fact that he is described as a man who was upright, feared God, and avoided evil. In recent years, the search for explanations has been extended from the realm of religion to include scientific inquiry. In the case of genetic diseases, scientists have inquired why Evolution did not eliminate disease-causing mutations through natural selection. For example, 60 years ago, Allison suggested that heterozygosity for HbS (“sickle trait”) provides partial protection from malaria 1. This protection from endemic malaria provided a positive selection to maintain the HbS mutation in the gene pool – despite the fact that homyzygosity for the same mutation causes a severe illness (i.e., sickle cell disease). More recently, a genetic variant form of apolipoprotein L1 has been demonstrated to lyse Trypanosoma brucei rhodesiense 2,3, and the same variant has been reported to increase the risk of developing chronic renal disease – including focal segmental glomerulosclerosis 2,4 . In a parallel vein, over 50 years ago, Neel 5,6 proposed a “thrifty gene” hypothesis – that evolution selected for the ability to store energy efficiently to prepare for famine which was a constant threat during most of human history. In recent years, such a thrifty gene may have become maladaptive by promoting obesity in modern times when food has become quite abundant for many (albeit not all) people. In the present issue of Diabetes and Metabolism Reviews, Brima et al. 7 have provided another example whereby a predisposition to develop a chronic disease may be associated with resistance to an infectious disease. Specifically, they report that high fat diet induced metabolic syndrome but also protected CD-1 mice from the lethality associated with Trypanosoma cruzi infection. As emphasized by the authors, there is a complex relationship between nutrition and the state of host defenses to fight infection. At one extreme, starvation and under-nutrition compromise host defenses, and render the individual highly susceptible to infectious disease. At the other extreme, a surfeit of calories produces metabolic syndrome, which is associated with multiple abnormalities including obesity, dyslipidemia, insulin resistance, and a pro-inflammatory state. In the study by Brima et al. 7, a high fat diet dramatically decreased mortality due to T. cruzi infection by ~65% (i.e., from 55% to 20% mortality). Based upon this seminal observation, the investigators initiated an inquiry into the mechanisms whereby high fat feeding protected CD-1 mice from lethality due to T. cruzi infection. Unexpectedly, metformin therapy provided added protection from lethality even though the drug partially mitigated the metabolic abnormalities induced by high fat feeding. It is likely that future studies with additional anti-diabetic drugs (e.g., PPAR-gamma agonists) may shed additional light on the mechanisms whereby high fat feeding protects T. cruzi infected mice from lethality. In the Discussion section of the paper, the authors refer to one specific molecular mechanism that could potentially contribute to the protective effect of a high fat diet. Trypanomastigotes hijack the LDL receptor, which mediates entry into adipocytes 8. Thus, alterations in lipoprotein metabolism have potential to alter expression or function of LDL receptors, and indirectly inhibit the entry of the infectious agent into cells. A growing literature has suggested complex interactions between hepatitis C virus (HCV) and lipoprotein metabolism 9. Thus, this type of mechanism may be broadly relevant to multiple infectious diseases. In conclusion, the paper of Brima et al. 7 is a thought-provoking study that highlights the complex interactions between nutrition and host defenses against infectious disease. According to a modern understanding of systems biology, there are complex regulatory networks that mediate multiple physiological changes in response to simple perturbation. In the case of high fat feeding, it is most common to emphasize the adverse effects such as obesity, dyslipidemia, insulin resistance, diabetes, and cardiovascular disease. Brima et al. 7 remind us that high fat feeding represents an ill wind that may blow some good – in this case, protection from lethality due to T. cruzi infection.

Dileucine-containing motifs are involved in trans-Golgi sorting, lysosomal targeting, and internalization. Previously, we have shown that the dileucine motif (EKITLL, residues 982-987) in the juxtamembrane region of the insulin receptor is involved in receptor internalization. Substitution of alanine residues for Leu986 and Leu987 led to a 3- to 5-fold decrease in the ability of the receptors to mediate insulin uptake. In the current study, we show that mutation of the same motif to Met986Ser987, the sequence found in the homologous position in the type I insulin-like growth factor receptor, did not affect insulin uptake. Therefore, we inquired whether the sequence EKITMS as an isolated motif could mediate the targeting of a reporter molecule to endosomes and then lysosomes, as was shown previously with the EKITLL motif of the normal receptor. Chimeric molecules containing Tac antigen fused to different hexapeptide sequences showed distinct patterns of subcellular localization by immunofluorescence microscopy. Tac-EKITLL and Tac-EKITAA were found predominantly in lysosomes and the plasma membrane, respectively. In contrast, Tac-EKITMS was found at the plasma membrane, in the trans-Golgi network, and in endosomes, but only small amounts were found in lysosomes. Thus, the dileucine motif (EKITLL) plays an important role in directing endocytosis of the intact insulin receptor and in mediating efficient endocytosis and lysosomal targeting as an isolated motif. Substitution of AA for LL inhibits endocytosis and lysosomal targeting in both systems. In contrast, substitution of MS for LL permits rapid endocytosis in the intact receptor, but mediates modest endocytosis and very little targeting to lysosomes as an isolated motif. Our observations support the idea that sorting signals are recognized at multiple steps in the cell, and that specific amino acid substitutions may differentially affect each of these sorting steps.

Several polymorphisms have been identified in the amino acid sequence of human insulin receptor substrate-1 (IRS-1). Some of the variant sequences have been reported to be increased in prevalence among patients with noninsulin-dependent diabetes mellitus (NIDDM). This observation led to the hypothesis that these amino acid substitutions may impair the function of IRS-1, thereby causing the insulin resistance seen in patients with NIDDM. To address this question, we have designed studies to evaluate the effects of three variant sequences identified in our laboratory: Gly819→Arg, Gly972→Arg, and Arg1221→Cys. We constructed four IRS-1 expression vectors for transfection in COS-7 cells: wild-type, single mutant (Gly819→Arg), double mutant (Gly819→Arg; Gly972→Arg), and triple mutant (Gly819→Arg; Gly972→Arg; Arg1221→Cys) IRS-1. The mutations did not alter the level of expression or the extent of insulin receptor-mediated tyrosine phosphorylation of recombinant IRS-1. Moreover, the mutations did not lead to a de...