Maternal and Neonatal Circulating Markers of Metabolic and Cardiovascular Risk in the Metformin in Gestational Diabetes (MiG) Trial: Responses to maternal metformin versus insulin treatment

OBJECTIVE

This study was designed to compare glucose, lipids, and C-reactive protein (CRP) in women with gestational diabetes mellitus treated with metformin or insulin and in cord plasma of their offspring and to examine how these markers relate to infant size at birth.

RESEARCH DESIGN AND METHODS

Women with gestational diabetes mellitus were randomly assigned to metformin or insulin in the Metformin in Gestational Diabetes trial. Fasting maternal plasma glucose, lipids, and CRP were measured at randomization, 36 weeks’ gestation, and 6–8 weeks postpartum as well as in cord plasma. Women with available cord blood samples (metformin n = 236, insulin n = 242) were included.

RESULTS

Maternal plasma triglycerides increased more from randomization to 36 weeks’ gestation in women treated with metformin (21.93%) versus insulin (9.69%, P < 0.001). Maternal and cord plasma lipids, CRP, and neonatal anthropometry did not differ between treatments. In logistic regression analyses adjusted for confounders, the strongest associations with birth weight >90th centile were maternal triglycerides and measures of glucose control at 36 weeks.

CONCLUSIONS

There were few differences in circulating maternal and neonatal markers of metabolic status and no differences in measures of anthropometry between the offspring of women treated with metformin and the offspring of women treated with insulin. There may be subtle effects of metformin on maternal lipid function, but the findings suggest that treating gestational diabetes mellitus with metformin does not adversely affect lipids or CRP in cord plasma or neonatal anthropometric measures.Gestational diabetes mellitus is carbohydrate intolerance first diagnosed during pregnancy (1) and affects up to 18% of pregnancies. The prevalence varies depending on maternal demographics and diagnostic criteria (2). The prevalence of gestational diabetes mellitus is increasing, which is likely driven by the rising population prevalence of overweight and obesity and increasing maternal age at pregnancy (3). Gestational diabetes mellitus increases maternal and infant morbidity and mortality during pregnancy (4). Women with a history of gestational diabetes mellitus are at risk for metabolic syndrome, type 2 diabetes (5), and cardiovascular disease in later life (6). Children born to women with gestational diabetes mellitus have higher rates of type 2 diabetes and obesity (7).Treating gestational diabetes mellitus improves pregnancy outcomes for both mother and infant (8). Current therapies include modification of diet, increased physical activity, and drug therapy with insulin and oral hypoglycemic agents, including metformin. In addition to improving insulin sensitivity and hyperglycemia, metformin therapy in the setting of type 2 diabetes reduces triglycerides (9), total cholesterol, LDL cholesterol (10), and VLDL cholesterol; increases HDL cholesterol (9); and reduces markers of inflammation and thrombosis (11). Metformin therapy in gestational diabetes mellitus achieves maternal glucose control and pregnancy outcomes similar to insulin therapy (12,13).In contrast to insulin, metformin crosses the placenta (14) and, therefore, could directly influence fetal metabolism. Our recent follow-up studies in 2-year-old offspring of women enrolled in the Metformin in Gestational Diabetes (MiG) trial showed increased subcutaneous fat measurements with no increase in abdominal adiposity or total fat (15). Further assessments are required to determine whether metformin actually reduces visceral/ectopic fat. Therefore, we hypothesized that metformin would be more effective than insulin in improving markers of insulin sensitivity and cardiovascular risk during pregnancy and postpartum in women with gestational diabetes mellitus and in their newborns.     

Lower risk of cancer in patients on metformin in comparison with those on sulfonylurea derivatives: results from a large population-based follow-up study

OBJECTIVE

Numerous studies have suggested a decreased risk of cancer in patients with diabetes on metformin. Because different comparison groups were used, the effect magnitude is difficult to estimate. Therefore, the objective of this study was to further analyze whether, and to what extent, use of metformin is associated with a decreased risk of cancer in a cohort of incident users of metformin compared with users of sulfonylurea derivatives.

RESEARCH DESIGN AND METHODS

Data for this study were obtained from dispensing records from community pharmacies individually linked to hospital discharge records from 2.5 million individuals in the Netherlands. The association between the risk of cancer in those using metformin compared with those using sulfonylurea derivatives was analyzed using Cox proportional hazard models with cumulative duration of drug use as a time-varying determinant.

RESULTS

Use of metformin was associated with a lower risk of cancer in general (hazard ratio 0.90 [95% CI 0.88–0.91]) compared with use of sulfonylurea derivatives. When specific cancers were used as end points, similar estimates were found. Dosage-response relations were identified for users of metformin but not for users of sulfonylurea derivatives.

CONCLUSIONS

In our study, cumulative exposure to metformin was associated with a lower risk of specific cancers and cancer in general, compared with cumulative exposure to sulfonylurea derivatives. However, whether this should indeed be seen as a decreased risk of cancer for the use of metformin or as an increased risk of cancer for the use sulfonylurea derivatives remains to be elucidated.As the drug of first choice in type 2 diabetes, metformin is the most widely prescribed oral glucose-lowering drug (OGLD) (1,2). However, the decision to prescribe metformin also depends on patient characteristics: metformin use is contraindicated in those with renal failure, cardiac, or hepatic failure (2).A statistically nonsignificant relationship between use of metformin and the risk of colon cancer was described in 2004 (3). However, 1 year later, metformin was found to be associated with a decreased risk of cancer in general in a case-control study in a diabetic population (4). Numerous studies followed; among which studies confirming the association between use of metformin and a decreased risk of cancer in general (5–8) or in specific cancers (5,6,9–14). However, for breast cancer (5,6) and prostate cancer (5,14), the decreased risk was not consistently demonstrated; for other cancers, no association with use of metformin was found (6,12). Hence, there is heterogeneity among published studies on cancer in patients with diabetes on metformin (15), partly because different comparison groups were used, such as nonmetformin users, users of other OGLDs, or users of insulin. Higher endogenous insulin levels have been linked to an increased risk of certain cancers (16). Moreover, specifically for insulin glargine, the debate whether this specific insulin increases the risk of cancer is ongoing (17–21).Owing to factors such as different drugs used to attain metabolic control, the duration of diabetes, and the presence of other diseases, the assessment of cancer risk in diabetic patients remains difficult. Therefore, the objective of this study was to analyze whether, and to what extent, use of metformin is associated with a decreased risk of cancer in a cohort of incident users of metformin compared with use of sulfonylurea derivatives.     

Long-Term Safety and Efficacy of Empagliflozin,Sitagliptin, and Metformin: An active-controlled,parallel-group,randomized, 78-week open-label extension study in patients with type 2 diabetes

OBJECTIVE

To investigate the long-term safety and efficacy of empagliflozin, a sodium glucose cotransporter 2 inhibitor; sitagliptin; and metformin in patients with type 2 diabetes.

RESEARCH DESIGN AND METHODS

In this randomized, open-label, 78-week extension study of two 12-week, blinded, dose-finding studies of empagliflozin (monotherapy and add-on to metformin) with open-label comparators, 272 patients received 10 mg empagliflozin (166 as add-on to metformin), 275 received 25 mg empagliflozin (166 as add-on to metformin), 56 patients received metformin, and 56 patients received sitagliptin as add-on to metformin.

RESULTS

Changes from baseline in HbA_1c at week 90 were −0.34 to −0.63% (−3.7 to −6.9 mmol/mol) with empagliflozin, −0.56% (−6.1 mmol/mol) with metformin, and −0.40% (−4.4 mmol/mol) with sitagliptin. Changes from baseline in weight at week 90 were −2.2 to −4.0 kg with empagliflozin, −1.3 kg with metformin, and −0.4 kg with sitagliptin. Adverse events (AEs) were reported in 63.2–74.1% of patients on empagliflozin and 69.6% on metformin or sitagliptin; most AEs were mild or moderate in intensity. Hypoglycemic events were rare in all treatment groups, and none required assistance. AEs consistent with genital infections were reported in 3.0–5.5% of patients on empagliflozin, 1.8% on metformin, and none on sitagliptin. AEs consistent with urinary tract infections were reported in 3.8–12.7% of patients on empagliflozin, 3.6% on metformin, and 12.5% on sitagliptin.

CONCLUSIONS

Long-term empagliflozin treatment provided sustained glycemic and weight control and was well tolerated with a low risk of hypoglycemia in patients with type 2 diabetes.Type 2 diabetes is characterized by insulin resistance and progressive deterioration of β-cell function (1). Metformin is the recommended first-line antidiabetes agent for patients with type 2 diabetes (2). However, in order to achieve and maintain glycemic control as the disease progresses, patients often require therapies in addition to metformin (2,3).Despite the availability of a number of antihyperglycemic agents, the side effects associated with existing treatments and their gradual loss of efficacy over time (2,3) mean that many patients with type 2 diabetes do not reach therapeutic goals (3,4). In addition, treatment is often complicated by common comorbidities of type 2 diabetes such as obesity and hypertension, which are not addressed by existing oral antidiabetes agents (5–7).Inhibition of sodium glucose cotransporter 2 (SGLT2), located in the proximal tubule of the kidney, represents an approach for the treatment of type 2 diabetes that is independent of β-cell function and insulin resistance (8,9). SGLT2 mediates most of renal glucose reabsorption, and inhibition of this transporter leads to reduced reabsorption of filtered glucose and increased urinary glucose excretion (8,10), resulting in reduced plasma glucose levels in patients with type 2 diabetes (8–10). In addition, this mechanism leads to weight loss owing to the loss of calories via urinary glucose excretion (8,11).Empagliflozin is a potent and selective inhibitor of SGLT2 (12), which in patients with type 2 diabetes causes urinary glucose excretion of up to 90 g/day (13). In two placebo- and active-controlled, dose-finding trials, treatment with empagliflozin for 12 weeks in patients with type 2 diabetes was generally well tolerated and resulted in placebo-corrected reductions in HbA_1c of up to 0.72% (7.9 mmol/mol) and placebo-corrected reductions in weight of up to 1.7 kg (14,15). In these studies, reductions in HbA_1c were comparable to those of the active comparators metformin and sitagliptin (14,15). The objective of this study was to assess the long-term safety and efficacy of empagliflozin, sitagliptin, and metformin in a 78-week, open-label extension study of two dose-finding trials.     

Independent and combined effects of exercise training and metformin on insulin sensitivity in individuals with prediabetes

OBJECTIVE

Physical activity or metformin enhances insulin sensitivity and opposes the progression from prediabetes to type 2 diabetes. The combination may be more effective because each treatment stimulates AMP-activated protein kinase activity in skeletal muscle. We evaluated the effects of exercise training plus metformin on insulin sensitivity in men and women with prediabetes, compared with each treatment alone.

RESEARCH DESIGN AND METHODS

For 12 weeks, men and women with prediabetes were assigned to the following groups: placebo (P), 2,000 mg/day metformin (M), exercise training with placebo (EP), or exercise training with metformin (EM) (n = 8 per group). Before and after the intervention, insulin sensitivity was measured by euglycemic hyperinsulinemic (80 mU/m²/min) clamp enriched with [6,6-²H]glucose. Changes due to intervention were compared across groups by repeated-measures ANOVA.

RESULTS

All three interventions increased insulin sensitivity (P < 0.05) relative to the control group. The mean rise was 25–30% higher after EP than after either EM or M, but this difference was not significant.

CONCLUSIONS

Insulin sensitivity was considerably higher after 12 weeks of exercise training and/or metformin in men and women with prediabetes. Subtle differences among condition means suggest that adding metformin blunted the full effect of exercise training.Before developing overt diabetes, most individuals spend years in an intermediate condition called prediabetes. Prediabetes is defined by impaired glucose tolerance (IGT), impaired fasting glucose (IFG), or the combination of IGT plus IFG (1). Approximately 79 million individuals in the U.S. have prediabetes and are at risk to develop type 2 diabetes (2). The progression is not inevitable, however. The U.S. Diabetes Prevention Program (DPP) demonstrated that either lifestyle modification (i.e., low-fat diet and increased physical activity) or the antihyperglycemic medication metformin reduced the transition from prediabetes to type 2 diabetes (3).Habitual exercise and metformin each increase peripheral (mainly skeletal muscle) insulin sensitivity in part by stimulating AMP-activated protein kinase (AMPK) (4–8). Combining exercise plus metformin, compared with either treatment alone, may more effectively activate the key regulatory enzyme AMPK and oppose the transition from prediabetes to type 2 diabetes.The American Diabetes Association strongly recommends exercise as a cornerstone therapy for diabetes prevention and, recently, suggested that some individuals with prediabetes be considered for metformin treatment (9,10). The efficacy of combining lifestyle modification with metformin has been tested only a few times (11–15). Results suggest 2–5 kg more weight loss with the addition of metformin compared with lifestyle modification alone (11,12), but little (11,14) or no further (15,16) improvement to insulin sensitivity. However, the use of self-reports to estimate physical activity and surrogates (via fasting glucose and insulin concentrations or responses to oral carbohydrate) (11,13–15) rather than direct measurement of insulin sensitivity using the glucose clamp limits our understanding of the interaction between exercise and metformin. There is considerable need to better understand the potential for additive effects when physical activity and metformin are used concurrently because the scope of the public health problem is so pressing. Therefore, the purpose of this study was to determine the effect of combining exercise training with metformin (EM) on insulin sensitivity in individuals with prediabetes, compared with either treatment alone.     

Efficacy and Safety of the Human Glucagon-Like Peptide-1 Analog Liraglutide in Combination With Metformin and Thiazolidinedione in Patients With Type 2 Diabetes (LEAD-4 Met+TZD)

OBJECTIVE

To determine the efficacy and safety of liraglutide (a glucagon-like peptide-1 receptor agonist) when added to metformin and rosiglitazone in type 2 diabetes.

RESEARCH DESIGN AND METHODS

This 26-week, double-blind, placebo-controlled, parallel-group trial randomized 533 subjects (1:1:1) to once-daily liraglutide (1.2 or 1.8 mg) or liraglutide placebo in combination with metformin (1 g twice daily) and rosiglitazone (4 mg twice daily). Subjects had type 2 diabetes, A1C 7–11% (previous oral antidiabetes drug [OAD] monotherapy ≥3 months) or 7–10% (previous OAD combination therapy ≥3 months), and BMI ≤45 kg/m².

RESULTS

Mean A1C values decreased significantly more in the liraglutide groups versus placebo (mean ± SE −1.5 ± 0.1% for both 1.2 and 1.8 mg liraglutide and −0.5 ± 0.1% for placebo). Fasting plasma glucose decreased by 40, 44, and 8 mg/dl for 1.2 and 1.8 mg and placebo, respectively, and 90-min postprandial glucose decreased by 47, 49, and 14 mg/dl, respectively (P < 0.001 for all liraglutide groups vs. placebo). Dose-dependent weight loss occurred with 1.2 and 1.8 mg liraglutide (1.0 ± 0.3 and 2.0 ± 0.3 kg, respectively) (P < 0.0001) compared with weight gain with placebo (0.6 ± 0.3 kg). Systolic blood pressure decreased by 6.7, 5.6, and 1.1 mmHg with 1.2 and 1.8 mg liraglutide and placebo, respectively. Significant increases in C-peptide and homeostasis model assessment of β-cell function and significant decreases in the proinsulin-to-insulin ratio occurred with liraglutide versus placebo. Minor hypoglycemia occurred more frequently with liraglutide, but there was no major hypoglycemia. Gastrointestinal adverse events were more common with liraglutide, but most occurred early and were transient.

CONCLUSIONS

Liraglutide combined with metformin and a thiazolidinedione is a well-tolerated combination therapy for type 2 diabetes, providing significant improvements in glycemic control.Type 2 diabetes is characterized by insulin resistance and progressive β-cell failure. Treatment often must be intensified over time, usually by a combination of agents that address both insulin resistance and β-cell dysfunction (1,2). However, several available therapies increase the risk for hypoglycemia and weight gain, which may reduce patient adherence and lead to poor glycemic control (3).Glucagon-like peptide-1 (GLP-1) stimulates insulin secretion and suppression of glucagon secretion in a glucose-dependent manner, delays gastric emptying, and decreases appetite (4). GLP-1 is rapidly degraded by dipeptidyl peptidase-4 (4). Liraglutide is a human GLP-1 analog with 97% homology to native GLP-1 (5). Liraglutide has a half-life in humans of 13 h compared with 1–2 min for native GLP-1, making liraglutide suitable as a once-daily treatment for patients with type 2 diabetes (6).In previously published phase 3 trials (the Liraglutide Effect and Action in Diabetes [LEAD] Program), treatment with liraglutide produced substantial and clinically significant reductions in A1C and fasting and postprandial glucose (PPG) levels, with a low risk of hypoglycemia, and moderate weight loss (7–10). Liraglutide treatment alone or in combination with oral antidiabetes drugs (OADs) demonstrated significantly larger A1C reductions compared with glimepiride (monotherapy) (7), rosiglitazone (in combination with a sulfonylurea) (8), and insulin glargine (in combination with metformin and sulfonylurea) (10). When initiated as monotherapy in a subgroup of previously treatment-naïve patients with type 2 diabetes, a mean A1C reduction of 1.6% was observed, with mean A1C values sustained below 7.0% over 52 weeks (7). In combination with metformin, liraglutide reduced body weight by 2–3 kg, with the majority of the weight loss being fat (11). In addition, a decrease in systolic blood pressure (SBP) has been previously demonstrated (7–10). No major hypoglycemic events occurred during the randomized treatment period when liraglutide was used as monotherapy or with metformin (7,9). The current study investigated liraglutide treatment in combination with metformin and a thiazolidinedione (TZD) (rosiglitazone) as part of the LEAD program. These three glucose-lowering agents are of particular interest, as they have complementary modes of action and are not generally associated with increased risk of hypoglycemia.     

Reduced risk of colorectal cancer with metformin therapy in patients with type 2 diabetes: a meta-analysis

OBJECTIVE

Both in vitro and in vivo studies indicate that metformin inhibits cancer cell growth and reduces cancer risk. Recent epidemiological studies suggest that metformin therapy may reduce the risks of cancer and overall cancer mortality among patients with type 2 diabetes. However, data on its effect on colorectal cancer are limited and inconsistent. We therefore pooled data currently available to examine the association between metformin therapy and colorectal cancer among patients with type 2 diabetes.

RESEARCH DESIGN AND METHODS

The PubMed and SciVerse Scopus databases were searched to identify studies that examined the effect of metformin therapy on colorectal cancer among patients with type 2 diabetes. Summary effect estimates were derived using a random-effects meta-analysis model.

RESULTS

The analysis included five studies comprising 108,161 patients with type 2 diabetes. Metformin treatment was associated with a significantly lower risk of colorectal neoplasm (relative risk [RR] 0.63 [95% CI 0.50–0.79]; P < 0.001). After exclusion of one study that investigated colorectal adenoma, the remaining four studies comprised 107,961 diabetic patients and 589 incident colorectal cancer cases during follow-up. Metformin treatment was associated with a significantly lower risk of colorectal cancer (0.63 [0.47–0.84]; P = 0.002). There was no evidence for the presence of significant heterogeneity between the five studies (Q = 4.86, P = 0.30; I² = 18%).

CONCLUSIONS

From observational studies, metformin therapy appears to be associated with a significantly lower risk of colorectal cancer in patients with type 2 diabetes. Further investigation is warranted.Colorectal cancer is one of the most frequent malignant tumors and a leading cause of cancer-related death worldwide (1). The incidence of colorectal cancer continues to increase in economically transitioning countries such as Asia, Eastern Europe, and selected countries in South America (2,3), whereas a declining trend has been noted in several developed countries in recent years (1).Type 2 diabetes is also a common disease, and it is well established that type 2 diabetes is associated with a higher risk of colorectal cancer (4–8). Metformin is a relative of isoamylene guanidine and has been recommended as the initial glucose-lowering therapy for diabetes. Emerging evidence from both in vitro and in vivo studies indicates that metformin may inhibit cancer cell growth and reduce cancer risks. Previous research suggests that metformin may be involved in the tumor suppressor pathway by indirectly activating AMP-activated protein kinase (9)—a key sensor of cellular ATP and AMP balance—and plays a role on activating tumor suppressor genes, e.g., LKB1. Subsequent in vitro studies have shown that metformin inhibits cancer cell proliferation (10,11) and selectively kills cancer stem cells (12). Animal experiments concur with these findings. Rodent models have shown that metformin suppresses colonic epithelial proliferation and colorectal aberrant crypt foci formation (13,14). Similarly, animal models of colon cancer have shown that metformin inhibits colon carcinoma growth (11,15). Given these encouraging findings, interest has arisen that metformin could potentially serve as a new antineoplasm drug to prevent colorectal cancer.Results from preliminary studies conducted in humans are encouraging. In a short-term randomized clinical trial among nondiabetic patients with rectal aberrant crypt foci, a significant decrease in the mean number of aberrant crypt foci was observed after metformin treatment for 1 month as compared with no significant changes in the control group (16). Findings from several epidemiological studies also support an antineoplastic role of metformin on cancer risks (17,18). If metformin therapy ultimately proves effective on reducing the risk of colorectal cancer, it would likely be recommended for the overwhelming majority of diabetes patients for both blood glucose control and cancer prevention. Nonetheless, despite accumulating evidence from population studies that indicate a lower risk of cancer at large with metformin therapy (17,19,20), data on its effect on colorectal cancer are limited and inconsistent. Accordingly, we performed a meta-analysis to pool studies currently available to examine the effect of metformin treatment on colorectal cancer risk among patients with type 2 diabetes.     

A New Mechanism of NK Cell Cytotoxicity Activation: The CD40–CD40 Ligand Interaction

NK recognition is regulated by a delicate balance between positive signals initiating their effector functions, and inhibitory signals preventing them from proceeding to cytolysis. Knowledge of the molecules responsible for positive signaling in NK cells is currently limited. We demonstrate that IL-2–activated human NK cells can express CD40 ligand (CD40L) and that recognition of CD40 on target cells can provide an activation pathway for such human NK cells. CD40-transfected P815 cells were killed by NK cell lines expressing CD40L, clones and PBLderived NK cells cultured for 18 h in the presence of IL-2, but not by CD40L-negative fresh NK cells. Cross-linking of CD40L on IL-2–activated NK cells induced redirected cytolysis of CD40-negative but Fc receptor-expressing P815 cells. The sensitivity of human TAP-deficient T2 cells could be blocked by anti-CD40 antibodies as well as by reconstitution of TAP/MHC class I expression, indicating that the CD40-dependent pathway for NK activation can be downregulated, at least in part, by MHC class I molecules on the target cells. NK cell recognition of CD40 may be important in immunoregulation as well as in immune responses against B cell malignancies.NK cells represent a distinct lineage of lymphocytes that are able to kill a variety of tumor (1), virus-infected (2), bone marrow transplanted (3), and allogeneic target cells (4). NK cells do not express T cell receptors or immunoglobulins and are apparently normal in mice with defects in the recombinase machinery (5, 6).Our knowledge about NK cell specificity has increased considerably in the last years. NK cells can probably interact with target cells by a variety of different cell surface molecules, some involved in cell adhesion, some activating the NK cytolytic program (7, 8), and other ones able to inhibit this activation by negative signaling (as reviewed in reference 9).A common feature of several inhibitory NK receptors is the capability to bind MHC class I molecules (10, 11), as predicted by the effector inhibition model within the missing self hypothesis of recognition by NK cells (12–14). Interestingly, the MHC class I receptors identified so far belong to different gene families in mouse and man; these are the p58/p70/NKAT or killer cell inhibitory receptors (KIR)¹ of the immunoglobulin superfamily in man and the Ly49 receptors of the C-type lectin family in the mouse. There is also evidence that MHC class I molecules can be recognized as triggering signals in NK cells of humans, rats as well as mice (13). The inhibitory receptors allow NK cells to kill tumor or normal cell targets with deficient MHC class I expression (12, 14). This does not exclude that other activating pathways can override inhibition by MHC class I molecules (15) and, even in their absence, there must be some activating target molecules that initiate the cytolytic program. Several surface molecules are able to mediate positive signals in NK cells. Some of these structures, like NKRP1 (16), CD69 (17), and NKG2 (18) map to the NK complex region (NKC) of chromosome 6 in mice and of chromosome 12 in humans (13). CD2 (19) and CD16 (20) molecules can also play a role in the activation pathway.NK cells resemble T cells in many respects, both may arise from an immediate common progenitor (21, 22), and share the expression of several surface molecules (23). NK cells produce cytokines resembling those secreted by some helper T cell subsets (24) and contain CD3 components in the cytoplasm (21). The expression of some surface structures, involved in TCR-dependent T cell costimulation, like CD28 in human (25), has been described on NK cells, but the functional relevance of these molecules for NK activation processes has not been fully established.Another T cell molecule of interest is CD40L, which interacts with CD40, a 50-kD membrane glycoprotein expressed on B cells (26), dendritic cells (27), and monocytes (28). CD40 is a member of the tumor necrosis factor/nerve growth factor receptor family (29) which includes CD27 (30), CD30 (31), and FAS antigen (32). Murine and human forms of CD40L had been cloned and found to be membrane glycoproteins with a molecular mass of ∼39 kD induced on T cells after activation (33). Also mast cells (34), eosinophils (35), and B cells (36) can be induced to express a functional CD40L. The CD40L–CD40 interaction has been demonstrated to be necessary for T cell–dependent B cell activation (33, 37). Mutations in the CD40L molecule cause a hyper-IgM immunodeficiency condition in man (38, 39, 40). On the other hand, CD40–CD40L interactions also orchestrate the response of regulatory T cells during both their development (41, 42) and their encounter with antigen (43, 44).NK cells have also been suggested to play a role in B cell differentiation and immunoglobulin production (45). Therefore, it was of interest to investigate whether NK cells could use a CD40-dependent pathway in their interactions with other cells. Therefore, we have investigated the ability of target cells expressing CD40 to induce activation of NK cytotoxicity.     

TRAF-interacting Protein (TRIP): A Novel Component of the Tumor Necrosis Factor Receptor (TNFR)- and CD30-TRAF Signaling Complexes That Inhibits TRAF2-mediated NF-κB Activation

Through their interaction with the TNF receptor–associated factor (TRAF) family, members of the tumor necrosis factor receptor (TNFR) superfamily elicit a wide range of biological effects including differentiation, proliferation, activation, or cell death. We have identified and characterized a novel component of the receptor–TRAF signaling complex, designated TRIP (TRAF-interacting protein), which contains a RING finger motif and an extended coiled-coil domain. TRIP associates with the TNFR2 or CD30 signaling complex through its interaction with TRAF proteins. When associated, TRIP inhibits the TRAF2-mediated NF-κB activation that is required for cell activation and also for protection against apoptosis. Thus, TRIP acts as a receptor–proximal regulator that may influence signals responsible for cell activation/proliferation and cell death induced by members of the TNFR superfamily.Members of the TNF receptor (TNFR)¹ superfamily play important roles in the induction of diverse signals leading to cell growth, activation, and apoptosis (1). Whether the signals induced by a given receptor leads to cell activation or death is, however, highly cell-type specific and tightly regulated during differentiation of cells. For example, the TNFRs can exert costimulatory signals for proliferation of naive lymphocytes but also induce death signals required for deletion of activated T lymphocytes (1). The cytoplasmic domains of these receptors lack intrinsic catalytic activity and also exhibit no significant homology to each other or to other known proteins. Exceptions to this include Fas(CD95) and TNFR1 that share a significant homology within an 80–amino acid region of their cytoplasmic tails (called the “death domain”; 2, 3). Therefore, it is suggested that the TNFR family members can initiate different signal transduction pathways by recruiting different types of intracellular signal transducers to the receptor complex (1).Indeed, several types of intracellular signal transducers have been identified that initiate distinct signal transduction pathways when recruited to the members of TNFR superfamily (4–19). Recent biochemical and molecular studies showed that a class of signal-transducing molecules are recruited to Fas(CD95) or TNFR1 via interaction of the death domains (2, 3, 6, 12, 17, 20). For example, Fas(CD95) and TNFR1 recruit FADD(MORT1)/RIP or TRADD/FADD (MORT1)/RIP through the interactions of their respective death domains (2, 3, 6, 12, 17, 20, 21). Clustering of these signal transducers leads to the recruitment of FLICE/ MACH, and subsequently, to cell death (13, 14).The TNFR family members can also recruit a second class of signal transducers called TRAFs (TNFR-associated factor), some of which are responsible for the activation of NF-κB or JNK (9, 20, 22). TRAF proteins were identified by their biochemical ability to interact with TNFR2, CD40, CD30, or LT-βR (4, 5, 10, 11, 15, 23–27). These receptors interact directly with TRAFs via a short stretch of amino acids within their cytoplasmic tails, but do not interact with the death domain containing proteins (4, 5, 15, 24–27). To date, five members of the TRAF family have been identified as signaling components of the TNFR family members. All TRAF members contain a conserved TRAF domain, ∼230 amino acids in length, that is used for either homo- or heterooligomerization among the TRAF family, for interactions with the cytoplasmic regions of the TNFR superfamily, or for interactions with downstream signal transducers (4, 5, 8, 10, 11, 19, 23–25, 28). In addition to the TRAF domain, most of the TRAF family members contain an NH₂-terminal RING finger and several zinc finger structures, which appear to be important for their effector functions (4, 5, 10, 11, 23–25).Several effector functions of TRAFs were revealed by recent experiments based on a transfection system. TRAF2, first identified by its interaction with TNFR2 (4), was subsequently shown to mediate NF-κB activation induced by two TNF receptors, CD40 and CD30 (9, 28–30). TRAF5 was also implicated in NF-κB activation mediated by LTβR (10), whereas TRAF3 (also known as CRAF1, CD40bp, or LAP1; references 5, 11, 24, and 25) was shown to be involved in the regulation of CD40-mediated CD23 upregulation in B cells (5). The role of other TRAF members in the TNFR family–mediated signal transduction is not clear. They may possess some effector functions as yet to be revealed, or work as adapter proteins to recruit different downstream signal transducers to the receptor complex. For example, TRAF1 is required for the recruitment of members of the cellular inhibitor of apoptosis protein (c-IAP) family to the TNFR2-signaling complex (7). In addition to the signal transduction by the TNFR family members, TRAFs may regulate other receptor-mediated signaling pathways. For example, TRAF6 is a component of IL-1 receptor (IL1R)–signaling complex, in which it mediates the activation of NF-κB by IL-1R (31). Since TRAFs form homo- or heterooligomers, it is suggested that the repertoire of TRAF members in a given cell type may differentially affect the intracellular signals triggered by these receptors. This may be accomplished by the selective interaction of TRAFs with a specific set of downstream signal transducers. Although many aspects of TRAF-mediated effector functions leading to cellular activation have been defined, it needs to be determined whether TRAF proteins will also mediate the apoptotic signals induced by the “death-domain-less” members of the TNFR superfamily (1, 27, 32–36).Here we report the isolation and characterization of a novel component of the TNFR superfamily/TRAFs signaling complex, named TRIP (TRAF-interacting protein). TRIP associates with the receptor/TRAF signaling complex, and inhibits the TRAF2-mediated NF-κB activation. Biochemical studies indicate that TRIP associates with the TNFR2 or CD30 receptor complex via its interaction with TRAF proteins, suggesting a model which can explain why the ligation of these receptors can promote different cell fates: proliferation or death.     

The Efficacy and Safety of Saxagliptin When Added to Metformin Therapy in Patients With Inadequately Controlled Type 2 Diabetes With Metformin Alone

OBJECTIVE

This 24-week trial assessed the efficacy and safety of saxagliptin as add-on therapy in patients with type 2 diabetes with inadequate glycemic control with metformin alone.

RESEARCH DESIGN AND METHODS

This was a randomized, double-blind, placebo-controlled study of saxagliptin (2.5, 5, or 10 mg once daily) or placebo plus a stable dose of metformin (1,500–2,500 mg) in 743 patients (A1C ≥7.0 and ≤10.0%). Efficacy analyses were performed using an ANCOVA model using last observation carried forward methodology on primary (A1C) and secondary (fasting plasma glucose [FPG] and postprandial glucose [PPG] area under the curve [AUC]) end points.

RESULTS

Saxagliptin (2.5, 5, and 10 mg) plus metformin demonstrated statistically significant adjusted mean decreases from baseline to week 24 versus placebo in A1C (−0.59, −0.69, and −0.58 vs. +0.13%; all P < 0.0001), FPG (−14.31, −22.03, and −20.50 vs. +1.24 mg/dl; all P < 0.0001), and PPG AUC (−8,891, −9,586, and −8,137 vs. −3,291 mg · min/dl; all P < 0.0001). More than twice as many patients achieved A1C <7.0% with 2.5, 5, and 10 mg saxagliptin versus placebo (37, 44, and 44 vs. 17%; all P < 0.0001). β-Cell function and postprandial C-peptide, insulin, and glucagon AUCs improved in all saxagliptin treatment groups at week 24. Incidence of hypoglycemic adverse events and weight reductions were similar to those with placebo.

CONCLUSIONS

Saxagliptin once daily added to metformin therapy was generally well tolerated and led to statistically significant improvements in glycemic indexes versus placebo added to metformin in patients with type 2 diabetes inadequately controlled with metformin alone.Saxagliptin is a potent, selective dipeptidyl peptidase-4 (DPP-4) inhibitor, specifically designed for extended inhibition of the DPP-4 enzyme (1,2). DPP-4 rapidly cleaves and inactivates the incretin hormones glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) (1). GLP-1 and GIP regulate blood glucose homeostasis by stimulation of glucose-dependent insulin secretion (3). GLP-1 also delays gastric emptying and inhibits glucagon secretion (3,4). In rodents, GLP-1 has been shown to stimulate β-cell growth and differentiation and inhibit β-cell apoptosis (5). Such an approach is needed because the majority of patients with type 2 diabetes fail to achieve recommended glycemic targets with existing therapies, owing to safety and tolerability issues and loss of efficacy over time (6).Metformin is the most widely prescribed first-line agent for the management of type 2 diabetes and is standard first-line pharmacotherapy, along with diet and exercise (7). Mechanistically, metformin reduces hepatic glucose production and improves insulin sensitivity (8); however, metformin alone is frequently insufficient to maintain glycemic goals in the face of progressive β-cell failure and increasing insulin resistance (9). Consequently, many patients require multiple oral antihyperglycemic agents (9,10). Metformin works through pathways complementary to saxagliptin, and the combination of saxagliptin with metformin may improve glycemic control (11,12). Studies of other DPP-4 inhibitors in combination with metformin over 24 weeks have demonstrated increased efficacy versus placebo (13–15). The safety and efficacy of saxagliptin monotherapy in treatment-naive patients were established previously in a 12-week study across a dose range of 2.5 to 40 mg/day. Significant A1C reductions were demonstrated in all active treatment groups with maximal A1C efficacy observed with 5 mg saxagliptin. A test for log-linear trend across the treatment groups did not demonstrate a statistically significant dose response after 12 weeks of treatment. The overall frequency of adverse events was comparable across all treatment groups and placebo and did not appear to be dose related (16). The current trial (CV181-014) examined the efficacy and safety of saxagliptin in combination with metformin administered for up to 24 weeks in patients with type 2 diabetes inadequately controlled with metformin alone.     

Pleiotropic Action of Short-Term Metformin and Fenofibrate Treatment, Combined With Lifestyle Intervention, in Type 2 Diabetic Patients With Mixed Dyslipidemia

OBJECTIVE

To compare the effect of short-term metformin and fenofibrate treatment, administered alone or in sequence, on glucose and lipid metabolism, cardiovascular risk factors, and monocyte cytokine release in type 2 diabetic patients with mixed dyslipidemia.

RESEARCH DESIGN AND METHODS

We studied 128 type 2 diabetic patients with mixed dyslipidemia complying throughout the study with lifestyle intervention who were randomized twice, initially to either metformin or placebo, and then to micronized fenofibrate or placebo.

RESULTS

Fenofibrate alleviated diabetic dyslipidemia–induced changes in plasma high-sensitivity C-reactive protein, fibrinogen, and plasminogen activator inhibitor (PAI)-1 and in monocyte cytokine release, whereas metformin or lifestyle intervention improved mainly glucose and lipid metabolism. The strongest pleiotropic effect was observed when fenofibrate was added to metformin.

CONCLUSIONS

Fenofibrate, particularly administered together with metformin, is superior to metformin and lifestyle intervention in exhibiting beneficial effects on systemic inflammation, hemostasis, and monocyte secretory function in type 2 diabetic patients with mixed dyslipidemia.Peroxisome proliferator–activated receptor (PPAR)-α activators (fibrates) administered to patients with dyslipidemia (1–4) or early glucose metabolism abnormalities (5) produce many non–lipid-related effects, including anti-inflammatory, antioxidant, and antithrombotic actions and improvement in endothelial function. Apart from normalizing glucose metabolism, metformin, the only oral antidiabetic medication shown to decrease cardiovascular events independent of glycemic control (6), improved dyslipidemia, hemostasis, and systemic inflammation (7). To the best of our knowledge, no previous clinical study has ever compared clinical benefits of metformin and fibrates when it comes to their pleiotropic effects and assessed whether metformin-fibrate combination is superior to treatment with only one of these drugs.     

Further Improvement in Postprandial Glucose Control With Addition of Exenatide or Sitagliptin to Combination Therapy With Insulin Glargine and Metformin: A proof-of-concept study

OBJECTIVE

To assess the effect of a 4-week adjunctive therapy of exenatide (EXE) (5–10 μg b.i.d.) or sitagliptin (SITA) (100 mg once daily) in response to a standardized breakfast meal challenge in 48 men or women with type 2 diabetes receiving insulin glargine (GLAR) + metformin (MET).

RESEARCH DESIGN AND METHODS

This was a single-center, randomized, open-label, active comparator–controlled study with a three-arm parallel group design, consisting of: screening, 4- to 8-week run-in period, 4-week treatment period, and follow-up. In all three groups, the GLAR dose was titrated according to an algorithm (fasting blood glucose ≤100 mg/dl).

RESULTS

The unadjusted 6-h postprandial blood glucose excursion of both GLAR + MET + EXE and GLAR + MET + SITA was statistically significantly smaller than that of GLAR + MET (606 ± 104 vs. 612 ± 133 vs. 728 ± 132 mg/dl/h; P = 0.0036 and 0.0008). A1C significantly decreased in all three groups (P < 0.0001), with the greatest reduction of −1.9 ± 0.7 under GLAR + MET + EXE (GLAR + MET + SITA −1.5 ± 0.7; GLAR + MET −1.2 ± 0.5%-points; GLAR + MET + EXE vs. GLAR + MET P = 0.0154). The American Diabetes Association A1C target of <7.0% was reached by 80.0, 87.5, and 62.5% of subjects, respectively. GLAR + MET + EXE had the highest number (47) of adverse events, mostly gastrointestinal (56%) with one dropout. GLAR + MET or GLAR + MET + SITA only had 10 and 12 adverse events, respectively, and no dropouts. Hypoglycemia (blood glucose <50 mg/dl) rates were low and comparable among groups. Weight decreased with GLAR + MET + EXE (−0.9 ± 1.7 kg; P = 0.0396) and increased slightly with GLAR + MET (0.4 ± 1.5 kg; NS; GLAR + MET + EXE vs. GLAR + MET P = 0.0377).

CONCLUSIONS

EXE or SITA added to GLAR + MET further substantially reduced postprandial blood glucose excursions. Longer-term studies in a larger population are warranted to confirm these findings.The UK Prospective Diabetes Study (UKPDS) demonstrated that good glycemic control in type 2 diabetes is associated with a reduced risk of diabetes complications (1). After lifestyle modifications (diet and exercise) and oral hypoglycemic agents (OHAs) the addition of basal insulin to OHAs is common practice (2), because this kind of regimen requires only a single injection in most cases and can improve glycemic control. Its use, however, may not adequately control postprandial hyperglycemia or may be associated with hypoglycemia and/or weight gain (3,4). Because obesity is frequently present in subjects with type 2 diabetes (5) and represents a factor contributing to insulin resistance (5) and cardiovascular risk (5), weight gain may be particularly undesirable.A significant advance in basal insulin therapy was the introduction of insulin glargine, a long-acting insulin analog with an extended duration of action of ∼24 h without exhibiting a pronounced peak (6,7). In subjects with type 2 diabetes, insulin glargine was shown to confer glycemic control at least equivalent to that of NHP insulin with a lower incidence of hypoglycemia (3,8,9). However, insulin glargine still has the drawbacks of insulin treatment such as weight gain (3,8,9) and a lower effect on postprandial glucose excursions (8) than on fasting glucose values.Exenatide is the first-in-class glucagon-like peptide 1 (GLP-1) receptor agonist (or incretin mimetic) approved in the U.S. and Europe (10). Compared with placebo, exenatide statistically reduced A1C, whereas there was no difference in A1C improvement between exenatide and insulin glargine or biphasic insulin aspart (11–14). However, postprandial glycemia as well as weight was further reduced with exenatide compared with insulin glargine or biphasic insulin, with a similar risk of hypoglycemia (12,13).Sitagliptin is an approved once-daily, potent, and highly selective dipeptidyl peptidase-4 (DPP-4) inhibitor (15). When added to metformin, sitagliptin, given at a dose of 100 mg once daily over 24 weeks, led to significant reductions in A1C, fasting, and 2-h postprandial plasma glucose and was weight-neutral (16).With this background, a therapy controlling both fasting blood glucose (FBG) and postprandial glucose excursions seems to be a promising approach for subjects with type 2 diabetes (17–21). Therefore, in the present study we investigated the influence of a 4-week adjunctive therapy of either a GLP-1 receptor agonist (exenatide) or a DPP-4 inhibitor (sitagliptin) to titrated basal insulin (insulin glargine) plus metformin versus the continuation with titrated insulin glargine plus metformin alone as active comparator in subjects with type 2 diabetes.     

��-Cell�CMediated Signaling Predominates Over Direct ��-Cell Signaling in the Regulation of Glucagon Secretion in Humans

OBJECTIVE

Given evidence of both indirect and direct signaling, we tested the hypothesis that increased β-cell–mediated signaling of α-cells negates direct α-cell signaling in the regulation of glucagon secretion in humans.

RESEARCH DESIGN AND METHODS

We measured plasma glucagon concentrations before and after ingestion of a formula mixed meal and, on a separate occasion, ingestion of the sulfonylurea glimepiride in 24 basal insulin-infused, demonstrably β-cell–deficient patients with type 1 diabetes and 20 nondiabetic, demonstrably β-cell–sufficient individuals; the latter were infused with glucose to prevent hypoglycemia after glimepiride.

RESULTS

After the mixed meal, plasma glucagon concentrations increased from 22 ± 1 pmol/l (78 ± 4 pg/ml) to 30 ± 2 pmol/l (103 ± 7 pg/ml) in the patients with type 1 diabetes but were unchanged from 27 ± 1 pmol/l (93 ± 3 pg/ml) to 26 ± 1 pmol/l (89 ± 3 pg/ml) in the nondiabetic individuals (P < 0.0001). After glimepiride, plasma glucagon concentrations increased from 24 ± 1 pmol/l (83 ± 4 pg/ml) to 26 ± 1 pmol/l (91 ± 4 pg/ml) in the patients with type 1 diabetes and decreased from 28 ± 1 pmol/l (97 ± 5 pg/ml) to 24 ± 1 pmol/l (82 ± 4 pg/ml) in the nondiabetic individuals (P < 0.0001). Thus, in the presence of both β-cell and α-cell secretory stimuli (increased amino acid and glucose levels, a sulfonylurea) glucagon secretion was prevented when β-cell secretion was sufficient but not when β-cell secretion was deficient.

CONCLUSIONS

These data indicate that, among the array of signals, indirect reciprocal β-cell–mediated signaling predominates over direct α-cell signaling in the regulation of glucagon secretion in humans.The regulation of pancreatic islet α-cell glucagon secretion is complex (1–10). It involves direct signaling of α-cells (1) and indirect signaling of α-cells by β-cell (2–6) and δ-cell (7) secretory products, the autonomic nervous system (8,9), and gut incretins (10).Appropriate glucagon secretory responses occur from the perfused pancreas (3,5) and perifused islets (2). Low plasma glucose concentrations stimulate glucagon secretion from the transplanted (i.e., denervated) human pancreas (11) and the denervated dog pancreas (12). Therefore, we have focused on the intraislet regulation of glucagon secretion. Furthermore, because selective destruction of β-cells results in loss of the glucagon response to hypoglycemia in type 1 diabetes (13), and partial reduction of the β-cell mass in minipigs results in impaired postprandial suppression of glucagon secretion (14), we have focused on the role of β-cell–mediated signaling in the regulation of glucagon secretion.Findings from studies of the perfused rat (3,4) and human (5) pancreas, rats in vivo (6), rat islets (2), isolated rat α-cells (2), and humans (15–18) have been interpreted to indicate that a β-cell secretory product or products tonically restrains basal α-cell glucagon secretion during euglycemia and that a decrease in β-cell secretion, coupled with low glucose concentrations at the α-cells, signals an increase in glucagon secretion in response to hypoglycemia. Parenthetically, the relative roles of the candidate β-cell secretory products (insulin, zinc, γ-aminobutyric acid, and amylin, among others) (2) that normally restrain α-cell glucagon secretion remain to be determined. However, that interpretation rests, in part, on results of studies in isolated rat α-cells (2), which are debated (1), and on the evidence that the islet microcirculation flows from β-cells to α-cells to δ-cells (4), which is also debated (19). Furthermore, it does not address the plausible possibility that a decrease in intraislet δ-cell somatostatin secretion might also signal an increase in α-cell glucagon secretion during hypoglycemia (7).Given that interpretation, it follows that an increase in β-cell secretion would signal a decrease in glucagon secretion in the postprandial state (14). The concept is an interplay of indirect reciprocal β-cell–mediated signaling of α-cells and of direct α-cell signaling in the regulation of glucagon secretion.There is, in our view, compelling evidence that, among other mechanisms, both indirect reciprocal β-cell–mediated signaling of α-cells (2–6) and direct α-cell signaling (1) are involved in the regulation of glucagon secretion by nutrients, hormones, neurotransmitters, and drugs. Given that premise, we posed the question: Which of these predominates in humans? Accordingly, we tested the hypothesis that increased β-cell–mediated signaling of α-cells negates direct α-cell signaling in the regulation of glucagon secretion in humans. To do so, we measured plasma glucagon responses to ingestion of a mixed meal and, on a separate occasion, to ingestion of the sulfonylurea glimepiride in patients with type 1 diabetes and in nondiabetic individuals. We conceptualized patients with type 1 diabetes as a model of α-cells isolated from β-cells because their β-cells had been destroyed but they have functioning α-cells. (Their α-cells are not, of course, isolated from other islet cells, including δ-cells.) Increased plasma amino acid and glucose levels after a mixed meal and sulfonylureas normally stimulate β-cell secretion; increased plasma amino acid and perhaps glucose (2) levels after a mixed meal and sulfonylureas (1) stimulate α-cell secretion. Our hypothesis predicts that such factors that normally stimulate both β-cells and α-cells would stimulate glucagon secretion in patients with type 1 diabetes but not in nondiabetic individuals, i.e., in the virtual absence and the presence of β-cell function, respectively. Indeed, a mixed meal (20,21) and the secretagogues tolbutamide (22), glyburide (23), and repaglinide (23) have been reported to raise plasma glucagon concentrations in patients with type 1 diabetes, but all of those studies lacked nondiabetic control subjects.     

Association of Metformin, Elevated Homocysteine, and Methylmalonic Acid Levels and Clinically Worsened Diabetic Peripheral Neuropathy

OBJECTIVE

The severity of peripheral neuropathy in diabetic patients varies for unclear reasons. Long-term use of metformin is associated with malabsorption of vitamin B¹² (cobalamin [Cbl]) and elevated homocysteine (Hcy) and methylmalonic acid (MMA) levels, which may have deleterious effects on peripheral nerves. The intent of this study was to clarify the relationship among metformin exposure, levels of Cbl, Hcy, and MMA, and severity of peripheral neuropathy in diabetic patients. We hypothesized that metformin exposure would be associated with lower Cbl levels, elevated Hcy and MMA levels, and more severe peripheral neuropathy.

RESEARCH DESIGN AND METHODS

This was a prospective case-control study of patients with type 2 diabetes and concurrent symptomatic peripheral neuropathy, comparing those who had received >6 months of metformin therapy (n = 59) with those without metformin exposure (n = 63). Comparisons were made using clinical (Toronto Clinical Scoring System and Neuropathy Impairment Score), laboratory (serum Cbl, fasting Hcy, and fasting MMA), and electrophysiological measures (nerve conduction studies).

RESULTS

Metformin-treated patients had depressed Cbl levels and elevated fasting MMA and Hcy levels. Clinical and electrophysiological measures identified more severe peripheral neuropathy in these patients; the cumulative metformin dose correlated strongly with these clinical and paraclinical group differences.

CONCLUSIONS

Metformin exposure may be an iatrogenic cause for exacerbation of peripheral neuropathy in patients with type 2 diabetes. Interval screening for Cbl deficiency and systemic Cbl therapy should be considered upon initiation of, as well as during, metformin therapy to detect potential secondary causes of worsening peripheral neuropathy.Diabetes is an increasingly prevalent disorder with a range of systemic complications including diabetic peripheral neuropathy (DPN), which occurs in up to 50% of diabetic patients and causes sensory, motor, and/or autonomic dysfunction (1). Several pathogenic mechanisms contribute to DPN severity, including microangiopathy, oxidative stress, polyol flux, mitochondrial dysfunction, insulin deficiency, and advanced glycation end products and ligand activation of their receptor (2–5). The course and severity of DPN are further affected by a wide range of comorbid conditions.Vitamin B₁₂ (cobalamin [Cbl]) deficiency may co-occur with diabetes. Although it is most classically associated with subacute combined degeneration, an exclusive peripheral neuropathy presentation can occur, typically manifesting as axonal neuropathy based on electrophysiology and pathology (6–8). Accumulating evidence suggests that Cbl-associated metabolites methylmalonic acid (MMA) and homocysteine (Hcy) are more sensitive (MMA and Hcy) and specific (MMA) indicators of early symptomatic Cbl deficiency than serum Cbl itself (9,10).Metformin, a biguanide, is perennially reported as a pharmacological cause of Cbl deficiency (11–13). The responsible mechanism has been controversial; proposed contributors have included competitive inhibition or inactivation of Cbl absorption, alterations in intrinsic factor levels, bacterial flora, gastrointestinal motility, or ileal morphological structure, and interaction with the cubulin endocytic receptor (11,14,15). Biguanides have recently been shown to impair calcium-dependent membrane activity in the ileum, including uptake of the Cbl-intrinsic factor complex (16).Metformin is recommended by the American Diabetes Association and the European Association for the Study of Diabetes as initial medical therapy for type 2 diabetes at diagnosis (17). Despite its wide use and its known effects on Cbl, metformin has not been systematically studied as a potential iatrogenic cause of or contributor to DPN. The potentially reversible effect of cobalamin deficiency may increase the clinical burden for a population of patients with DPN whose sensory function, gait, and balance frequently are already compromised.We designed a prospective case-control study to assess the effects of prolonged metformin intake in patients with type 2 diabetes matched for disease duration and disease control. We specifically examined the relationship among metformin use, levels of Cbl and its metabolites, and clinical and electrophysiological markers of peripheral neuropathy severity. We hypothesized first that metformin use would be associated with biochemical evidence of Cbl deficiency (lower serum Cbl levels and elevated MMA and Hcy) and second that metformin use would be associated with more severe peripheral neuropathy. Decreases in Cbl have been shown to depend on the dose and duration of metformin therapy in a previous case-control study (18); this finding led us to further hypothesize that biochemical abnormalities and severity of neuropathy would correlate with cumulative lifetime metformin dose.     

Changes in Insulin Secretion and Insulin Sensitivity in Relation to the Glycemic Outcomes in Subjects With Impaired Glucose Tolerance in the Indian Diabetes Prevention Programme-1 (IDPP-1)

OBJECTIVE

The Indian Diabetes Prevention Programme-1 (IDPP-1) showed that lifestyle modification (LSM) and metformin were effective for primary prevention of diabetes in subjects with impaired glucose tolerance (IGT). Among subjects followed up for 3 years (n = 502), risk reductions versus those for the control group were 28.5, 26.4, and 28.2% in LSM, metformin (MET), and LSM plus MET groups, respectively. In this analysis, the roles of changes in secretion and action of insulin in improving the outcome were studied.

RESEARCH DESIGN AND METHODS

For this analysis, 437 subjects (93 subjects with normoglycemia [NGT], 150 subjects with IGT, and 194 subjects with diabetes) were included. Measurements of anthropometry, plasma glucose, and plasma insulin at baseline and at follow-up were available for all of them. Indexes of insulin resistance (homeostasis model assessment of insulin resistance) and β-cell function (insulinogenic index [ΔI/G]: 30-min fasting insulin divided by 30-min glucose) were also analyzed in relation to the outcome.

RESULTS

Subjects with IGT showed a deterioration in β-cell function with time. Individuals with higher insulin resistance and/or low β-cell function at baseline had poor outcome on follow-up. In relation to no abnormalities, the highest incidence of diabetes occurred when both abnormalities coexisted (54.9 vs. 33.7%, χ² = 7.53, P = 0.006). Individuals having abnormal insulin resistance (41.1%) or abnormal ΔI/G (51.2%, χ² = 4.87, P = 0.027 vs. no abnormalities) had lower incidence. Normal β-cell function with improved insulin sensitivity facilitated reversal to NGT, whereas deterioration in both resulted in diabetes. The beneficial changes were better with intervention than in the control group. Intervention groups had higher rates of NGT and lower rates of diabetes.

CONCLUSIONS

In the IDPP-1 subjects, beneficial outcomes occurred because of improved insulin action and sensitivity caused by the intervention strategies.Primary prevention studies in diabetes have been done in subjects with a high risk for diabetes, such as those with impaired glucose tolerance (IGT) (1–6) or with a history of gestational diabetes mellitus (7). Lifestyle modification (LSM) (1–5) and/or pharmacological agents such as metformin (MET) (1,5) and glitazones (6) have been shown to be effective in reducing the rate of conversion of IGT to diabetes in different ethnic groups. The benefits are seen in association with weight reduction in the obese population (1,2) or without significant weight changes in relatively nonobese population (3,5). The mechanisms that result in the beneficial changes are associated with two important pathophysiological components, namely impaired secretion and impaired action of insulin.The Indian Diabetes Prevention Programme-1 (IDPP-1) had shown that moderate, but consistent, LSM or use of MET reduced the risk of deterioration of IGT to diabetes by 28% in relation to that in a control group who had no intervention in a 3-year follow-up period (5). Combining LSM with MET showed no added benefit.IGT, an intermediate state in the natural history of type 2 diabetes, is characterized by a worsening in insulin resistance and insulin secretion (8). Asian Indians have higher rates of insulin resistance than Europeans and other white populations despite being relatively nonobese (9,10).The chief pathophysiological components of type 2 diabetes, namely impaired secretion and action of insulin are detectable many years before the diagnosis of clinical diabetes (11). A combined occurrence of both defects due to gradual deterioration, eventually results in diabetes. This analysis was done to identify the changes in insulin secretion and insulin action that produced the improved outcome with the primary prevention strategies in the IDPP-1 cohort.     

Meat consumption and its association with C-reactive protein and incident type 2 diabetes: the Rotterdam Study

OBJECTIVE

To investigate whether intake of different types of meat is associated with circulating C-reactive protein (CRP) and risk of type 2 diabetes in a prospective cohort study.

RESEARCH DESIGN AND METHODS

Our analysis included 4,366 Dutch participants who did not have diabetes at baseline. During a median follow-up period of 12.4 years, 456 diabetes cases were confirmed. Intake of red meat, processed meat, and poultry was derived from a food frequency questionnaire, and their association with serum high-sensitivity CRP was examined cross-sectionally using linear regression models. Their association with risk of type 2 diabetes was examined using multivariate Cox proportional hazards model, including age, sex, family history of diabetes, and lifestyle and dietary factors.

RESULTS

An increment of 50 g of processed meat was associated with increased CRP concentration (β_{processed meat} = 0.12; P = 0.01), whereas intake of red meat and poultry was not. When comparing the highest to the lowest category of meat intake with respect to diabetes incidence, the adjusted relative risks were as follows: for red meat (1.42 [95% CI 1.06–1.91]), for processed meat (1.87 [1.26–2.78]), and for poultry (0.95 [0.74–1.22]). Additional analysis showed that the associations were not affected appreciably after inclusion of CRP into the model. After adjustment for BMI, however, the association for red meat attenuated to 1.18 (0.88–1.59).

CONCLUSIONS

Intake of processed meat is associated with higher risk of type 2 diabetes. It appears unlikely that CRP mediates this association.Since the prevalence of type 2 diabetes has increased rapidly over the last decades, investigations into the effect of dietary and other lifestyle factors on type 2 diabetes have become important (1). One of the dietary factors of interest is meat. Three meta-analyses of prospective cohort studies showed that intake of processed meat is associated with a higher risk of type 2 diabetes (2–4). For red meat, two of these meta-analyses observed an adverse association (2,4), whereas one did not (3). For poultry, no data from meta-analyses were available. Results from six prospective studies on poultry, however, showed that it is not likely that poultry is associated with a higher risk of type 2 diabetes; three studies observed an inverse association (5–7), whereas three did not observe an association (8–10).Intake of red meat and processed meat may increase risk of type 2 diabetes by mechanisms that increase circulating proinflammatory markers. Positive associations have been observed between red meat or processed meat and the proinflammatory blood marker C-reactive protein (CRP), which in turn has been associated with higher risk of type 2 diabetes (11,12,13). The positive association between intake of meat and CRP might be explained by several biological pathways. The binding capacity of iron in the body could be exceeded by the intake of meat, which contains high amounts of heme iron. Free iron can increase oxidative stress, thereby acting as proinflammatory agent (14). Advanced glycation end products (AGEs), which occur naturally in meat and are formed through heat processing (15), may also have proinflammatory actions (16). Thus, the observed positive associations between intake of red meat and processed meat and CRP, and CRP and risk of type 2 diabetes may indicate that CRP mediates the association between intake of meat, especially red and processed meat, and risk of type 2 diabetes. Therefore, we investigated whether intake of red meat, processed meat, and poultry was associated with CRP and risk of type 2 diabetes in a Dutch population.     

Association of biochemical B₁₂ deficiency with metformin therapy and vitamin B₁₂ supplements: the National Health and Nutrition Examination Survey, 1999-2006

OBJECTIVE

To describe the prevalence of biochemical B₁₂ deficiency in adults with type 2 diabetes taking metformin compared with those not taking metformin and those without diabetes, and explore whether this relationship is modified by vitamin B₁₂ supplements.

RESEARCH DESIGN AND METHODS

Analysis of data on U.S. adults ≥50 years of age with (n = 1,621) or without type 2 diabetes (n = 6,867) from the National Health and Nutrition Examination Survey (NHANES), 1999–2006. Type 2 diabetes was defined as clinical diagnosis after age 30 without initiation of insulin therapy within 1 year. Those with diabetes were classified according to their current metformin use. Biochemical B₁₂ deficiency was defined as serum B₁₂ concentrations ≤148 pmol/L and borderline deficiency was defined as >148 to ≤221 pmol/L.

RESULTS

Biochemical B₁₂ deficiency was present in 5.8% of those with diabetes using metformin compared with 2.4% of those not using metformin (P = 0.0026) and 3.3% of those without diabetes (P = 0.0002). Among those with diabetes, metformin use was associated with biochemical B₁₂ deficiency (adjusted odds ratio 2.92; 95% CI 1.26–6.78). Consumption of any supplement containing B₁₂ was not associated with a reduction in the prevalence of biochemical B₁₂ deficiency among those with diabetes, whereas consumption of any supplement containing B₁₂ was associated with a two-thirds reduction among those without diabetes.

CONCLUSIONS

Metformin therapy is associated with a higher prevalence of biochemical B₁₂ deficiency. The amount of B₁₂ recommended by the Institute of Medicine (IOM) (2.4 μg/day) and the amount available in general multivitamins (6 μg) may not be enough to correct this deficiency among those with diabetes.It is well known that the risks of both type 2 diabetes and B₁₂ deficiency increase with age (1,2). Recent national data estimate a 21.2% prevalence of diagnosed diabetes among adults ≥65 years of age and a 6 and 20% prevalence of biochemical B₁₂ deficiency (serum B₁₂ <148 pmol/L) and borderline deficiency (serum B₁₂ ≥148–221 pmol/L) among adults ≥60 years of age (3,4).The diabetes drug metformin has been reported to cause a decrease in serum B₁₂ concentrations. In the first efficacy trial, DeFronzo and Goodman (5) demonstrated that although metformin offers superior control of glycosylated hemoglobin levels and fasting plasma glucose levels compared with glyburide, serum B₁₂ concentrations were lowered by 22% compared with placebo, and 29% compared with glyburide therapy after 29 weeks of treatment. A recent, randomized control trial designed to examine the temporal relationship between metformin and serum B₁₂ found a 19% reduction in serum B₁₂ levels compared with placebo after 4 years (6). Several other randomized control trials and cross-sectional surveys reported reductions in B₁₂ ranging from 9 to 52% (7–16). Although classical B₁₂ deficiency presents with clinical symptoms such as anemia, peripheral neuropathy, depression, and cognitive impairment, these symptoms are usually absent in those with biochemical B₁₂ deficiency (17).Several researchers have made recommendations to screen those with type 2 diabetes on metformin for serum B₁₂ levels (6,7,14–16,18–21). However, no formal recommendations have been provided by the medical community or the U.S. Prevention Services Task Force. High-dose B₁₂ injection therapy has been successfully used to correct the metformin-induced decline in serum B₁₂ (15,21,22). The use of B₁₂ supplements among those with type 2 diabetes on metformin in a nationally representative sample and their potentially protective effect against biochemical B₁₂ deficiency has not been reported. It is therefore the aim of the current study to use the nationally representative National Health and Nutrition Examination Survey (NHANES) population to determine the prevalence of biochemical B₁₂ deficiency among those with type 2 diabetes ≥50 years of age taking metformin compared with those with type 2 diabetes not taking metformin and those without diabetes, and to explore how these relationships are modified by B₁₂ supplement consumption.     

A Single Nucleotide Polymorphism in STK11 Influences Insulin Sensitivity and Metformin Efficacy in Hyperinsulinemic Girls With Androgen Excess

OBJECTIVE

Serine-threonine kinase STK11 catalyzes the AMP-activated protein kinase complex. We tested the hypothesis that a gene variant in STK11 contributes to variation in insulin sensitivity and metformin efficacy.

RESEARCH DESIGN AND METHODS

We studied the effects of a single nucleotide polymorphism (SNP) (rs8111699) in STK11 on endocrine-metabolic and body composition indexes before and after 1 year of metformin in 85 hyperinsulinemic girls with androgen excess, representing a continuum from prepuberal girls with a combined history of low birth weight and precocious pubarche over to postmenarchial girls with hyperinsulinemic ovarian hyperandrogenism. Metformin was dosed at 425 mg/day in younger girls and 850 mg/day in older girls. STK11 rs8111699 was genotyped. Endocrine-metabolic features were assessed in the fasting state; body composition was estimated by absorptiometry.

RESULTS

Genotype effects were similar in younger and older girls. At baseline, the mutated G allele in STK11 rs8111699 was associated with higher insulin and IGF-I levels (both P < 0.005). The response to metformin differed by STK11 genotype: GG homozygotes (n = 24) had robust metabolic improvements, GC heterozygotes (n = 38) had intermediate responses, and CC homozygotes (n = 23) had almost no response. Such differences were found for 1-year changes in body composition, circulating insulin, IGF-I, free androgen index, and lipids (all P < 0.005).

CONCLUSIONS

In hyperinsulinemic girls with androgen excess, the STK11 rs8111699 SNP influences insulin sensitivity and metformin efficacy, so that the girls with the least favorable endocrine-metabolic profile improve most with metformin therapy.Genetic variation in enzymes and transporters mediating the actions and metabolism of medications contribute to interindividual variation in therapeutic response, on the efficacy as well as on the safety side (1).Polycystic ovary syndrome (PCOS) is a common endocrinopathy that affects ∼5–10% of young women; PCOS is characterized by androgen excess plus either anovulation or polycystic ovaries (2,3). A majority of patients with PCOS are insulin resistant, and, accordingly, metformin is often prescribed for this condition, also in adolescents (4,5). In selected girls at high risk for developing hyperinsulinemic ovarian androgen excess, metformin is even under exploration as a potentially preventive treatment; among these high-risk girls are those with a combined history of low birth weight (LBW) and precocious pubarche (6–9).The actions of metformin seem to be largely exerted through activation of AMP-activated protein kinase (AMPK), a conserved regulator of the cellular response to low energy, in many organs, including liver and skeletal muscle (10,11). The activation of AMPK in the liver is catalyzed by serine-threonine kinase (STK11, formerly known as LKB1), a tumor suppressor gene defective in Peutz-Jeghers syndrome (12); deletion of hepatic STK11 in mice results in a nearly complete loss of AMPK activity, leading to adipogenesis and lipogenic gene expression (13). STK11 serves as a mediator of metformin effects, rather than as a direct target of metformin (14).Recently, the C allele of a single nucleotide polymorphism (SNP) (rs8111699) in STK11 has been associated with a reduced ovulatory response to metformin in women with PCOS (15). In a pilot study, we have tested the hypothesis that the same SNP in STK11 also influences the endocrine-metabolic and body composition changes after metformin therapy in girls with hyperinsulinemic androgen excess.     

Inflammation and Cognitive Dysfunction in Type 2 Diabetic Carotid Endarterectomy Patients

OBJECTIVE

Type 2 diabetic patients have a high incidence of cerebrovascular disease, elevated inflammation, and high risk of developing cognitive dysfunction following carotid endarterectomy (CEA). To elucidate the relationship between inflammation and the risk of cognitive dysfunction in type 2 diabetic patients, we aim to determine whether elevated levels of systemic inflammatory markers are associated with cognitive dysfunction 1 day after CEA.

RESEARCH DESIGN AND METHODS

One hundred fifteen type 2 diabetic CEA patients and 156 reference surgical patients were recruited with written informed consent in this single-center cohort study. All patients were evaluated with an extensive battery of neuropsychometric tests. Preoperative monocyte counts, HbA_1c, C-reactive protein (CRP), intercellular adhesion molecule 1, and matrix metalloproteinase 9 activity levels were obtained.

RESULTS

In a multivariate logistic regression model constructed to identify predictors of cognitive dysfunction in type 2 diabetic CEA patients, each unit of monocyte counts (odds ratio [OR] 1.76 [95% CI 1.17–2.93]; P = 0.005) and CRP (OR 1.17 [1.10–1.29]; P < 0.001) was significantly associated with higher odds of developing cognitive dysfunction 1 day after CEA in type 2 diabetic patients.

CONCLUSIONS

Type 2 diabetic patients with elevated levels of preoperative systemic inflammatory markers exhibit more cognitive dysfunction 1 day after CEA. These observations have implications for the preoperative medical management of this high-risk group of surgical patients undergoing carotid revascularization with CEA.The incidence of ischemic stroke is significantly higher in type 2 diabetic patients (1,2), as type 2 diabetes is an independent risk factor for stroke and its recurrence (3,4). Carotid artery stenosis is a major cause of ischemic stroke and can be surgically treated with carotid endarterectomy (CEA). In previous work, we have demonstrated that ∼25% of CEA patients exhibit cognitive dysfunction, a subtle form of neurologic injury, within 1 day of CEA (5,6). Glial markers of neuronal injury (S100B) are elevated in patients who exhibit cognitive dysfunction within 1 day of CEA (7) and reflect opening of the blood–brain barrier (8). Additionally, we have data that demonstrate cognitive dysfunction exhibited within 1 day of CEA is associated with earlier mortality after CEA (9); patients who exhibit cognitive dysfunction within 1 day of CEA experience mortality 4 years earlier than those who do not exhibit cognitive dysfunction within 1 day of CEA. We have also demonstrated that type 2 diabetes is an independent risk factor for cognitive dysfunction (10). In this study, we will investigate factors that might contribute to the increased risk of type 2 diabetic patients undergoing CEA to exhibit the subtle, but significant, cognitive dysfunction.Type 2 diabetes has been associated with accelerated atherosclerosis (11) and elevated systemic inflammation (12–14). Inflammation may play a significant role in accounting for the increased risk of cognitive dysfunction in type 2 diabetic patients. Studies have shown that monocyte activation and infiltration are specifically implicated in the initiation of chronic inflammation and atherosclerosis (12). C-reactive protein (CRP) is a nonspecific marker of systemic inflammation that has been strongly associated with adverse cardiovascular outcomes in both healthy patients and those with coronary artery disease (15–18). Intercellular adhesion molecule-1 (ICAM-1) is a glycoprotein expressed on endothelial cells and cells of the immune system (19). Matrix metalloproteinase-9 (MMP-9) is secreted from macrophages (20), is involved in the breakdown of vasculature extracellular matrices, and has also been investigated for its various roles in inflammation (21–24). Many previous studies have demonstrated that CRP, ICAM-1, monocytes, and MMP-9 activity are all elevated in type 2 diabetic patients compared with nondiabetic patients. In our previous work, we have demonstrated that type 2 diabetes is a risk factor for cognitive dysfunction (10). We have also previously shown that elevated levels of ICAM-1 (25), monocyte counts (26), and MMP-9 activity (27) are associated with higher incidences of cognitive dysfunction in nondiabetic patients following CEA. We have yet to demonstrate a relationship between CRP and cognitive dysfunction. However, given the previous work done on CRP, we are inspired to do so in this study.Considering previous findings that 1) type 2 diabetes is a risk factor for cognitive dysfunction following CEA; 2) elevated ICAM-1, MMP-9, and monocyte counts are associated with more cognitive dysfunction in CEA patients; and 3) type 2 diabetic patients have elevated levels of CRP, ICAM-1, MMP-9 activity, and monocytes, we hypothesize that type 2 diabetic patients with elevated preoperative systemic inflammation are more likely to exhibit cognitive dysfunction following CEA than those with lower preoperative systemic inflammation. To date, there are no studies that investigate this relationship.We will evaluate preoperative systemic inflammation by measuring CRP, ICAM-1, MMP-9 activity, and monocytes and compare these levels between type 2 diabetic patients with and without cognitive dysfunction 1 day after CEA.