Hyperglycemia causes micro- and macrovascular complications in diabetic patients. Elevated glucose concentrations lead to increased formation of the highly reactive dicarbonyl methylglyoxal (MG), yet the early consequences of MG for development of vascular complications in vivo are poorly understood. In this study, zebrafish were used as a model organism to analyze early vascular effects and mechanisms of MG in vivo. High tissue glucose increased MG concentrations in tg(fli:EGFP) zebrafish embryos and rapidly induced several additional malformed and uncoordinated blood vessel structures that originated out of existing intersomitic blood vessels (ISVs). However, larger blood vessels, including the dorsal aorta and common cardinal vein, were not affected. Expression silencing of MG-degrading enzyme glyoxalase (glo) 1 elevated MG concentrations and induced a similar vascular hyperbranching phenotype in zebrafish. MG enhanced phosphorylation of vascular endothelial growth factor (VEGF) receptor 2 and its downstream target Akt/protein kinase B (PKB). Pharmacological inhibitors for VEGF receptor 2 and Akt/PKB as well as MG scavenger aminoguanidine and glo1 activation prevented MG-induced hyperbranching of ISVs. Taken together, MG acts on smaller blood vessels in zebrafish via the VEGF receptor signaling cascade, thereby describing a new mechanism that can explain vascular complications under hyperglycemia and elevated MG concentrations.

Previous research has shown that type 2 diabetes mellitus (T2DM) is associated with white matter microstructural changes, cognitive impairment, and decreased resting-state functional connectivity and spontaneous brain activity. This study used magnetization transfer imaging to examine, for the first time, the integrity of macromolecular protein pools in fronto-striato-thalamic circuits and its clinical and cognitive correlates in patients with T2DM. T2DM patients without mood disorders (n = 20, aged 65.05 ± 11.95 years) and healthy control subjects (HCs; n = 26, aged 62.92 ± 12.71 years) were recruited. Nodes of fronto-striato-thalamic circuits-head of the caudate nucleus (hCaud), putamen, globus pallidus, thalamus-and four cortical regions-rostral and dorsal anterior cingulate cortex, dorsolateral prefrontal cortex, and lateral orbitofrontal cortex-were examined. Compared with HCs, patients with T2DM had significantly lower magnetization transfer ratio (MTR) in bilateral anterior cingulate and hCaud. Reduced MTRs in the above regions showed correlations with T2DM-related clinical measures, including hemoglobin A1c level and vascular risk factors, and neuropsychological task performance in the domains of learning and memory, executive function, and attention and information processing. The impaired biophysical integrity of brain macromolecular protein pools and their local microenvironments in T2DM patients may provide insights into the neurological pathophysiology underlying diabetes-associated clinical and cognitive deficits.

Combined use of metformin and a sodium glucose cotransporter 2 inhibitor (SGLT2I) is a promising treatment strategy for type 2 diabetes. The mechanism by which combination treatment provides better glycemic control than metformin or SGLT2I monotherapy remains elusive. Therefore, we investigated the physiological mechanism by which both compounds lower blood glucose concentrations in diabetic mice. We compared the potential of metformin and the SGLT2I AVE2268 alone or in combination to mitigate hyperglycemia and modulate glucose fluxes in db/db and diabetic Tallyho/JngJ mice. SGLT2I treatment alone elicited a rapid decline in circulating blood glucose levels, which appeared to induce endogenous glucose production. Supplementation of metformin dampened this counterresponse, and therefore, combination therapy more efficiently maintained glycemic control. Finally, combination treatment blunted postprandial glucose excursions and improved HbA1c levels within 2 weeks. We conclude that coapplication of metformin enhances the glucose-lowering actions of SGLT2I by restraining endogenous glucose production, which may provide long-term improvement of glycemic control in type 2 diabetic patients.

Numerous studies have characterized the antidiabetic effects of adiponectin, yet the precise cellular mechanisms in skeletal muscle, in particular, changes in autophagy, require further clarification. In the current study, we used a high-fat diet (HFD) to induce obesity and insulin resistance in wild-type (WT) or adiponectin knockout (Ad-KO) mice with and without adiponectin replenishment. Temporal analysis of glucose tolerance and insulin sensitivity using hyperinsulinemic-euglycemic clamp and muscle insulin receptor substrate and Akt phosphorylation demonstrated exaggerated and more rapid HFD-induced insulin resistance in skeletal muscle of Ad-KO mice. Superoxide dismutase activity, the reduced glutathione-to-glutathione disulfide ratio, and lipid peroxidation indicated that HFD-induced oxidative stress was corrected by adiponectin. Gene array analysis implicated several antioxidant enzymes, including Gpxs, Prdx, Sod, and Nox4, in mediating this effect. Adiponectin also attenuated palmitate-induced reactive oxygen species production in cultured myotubes and improved insulin-stimulated glucose uptake in primary muscle cells. Increased LC3-II and decreased p62 expression suggested that HFD induced autophagy in muscle of WT mice; however, these changes were not observed in Ad-KO mice. Replenishing adiponectin in Ad-KO mice increased LC3-II and Beclin1 and decreased p62 protein levels, induced fibroblast growth factor-21 expression, and corrected HFD-induced decreases in LC3, Beclin1, and ULK1 gene expression. In vitro studies examining changes in phospho-ULK1 (Ser555), LC3-II, and lysosomal enzyme activity confirmed that adiponectin directly induced autophagic flux in cultured muscle cells in an AMPK-dependent manner. We overexpressed an inactive mutant of Atg5 to create an autophagy-deficient cell model, and together with pharmacological inhibition of autophagy, demonstrated reduced insulin sensitivity under these conditions. In summary, adiponectin stimulated skeletal muscle autophagy and antioxidant potential to reduce insulin resistance caused by HFD.

The impaired capacity of skeletal muscle to switch between the oxidation of fatty acid (FA) and glucose is linked to disordered metabolic homeostasis. To understand how muscle FA oxidation affects systemic glucose, we studied mice with a skeletal muscle-specific deficiency of long-chain acyl-CoA synthetase (ACSL)1. ACSL1 deficiency caused a 91% loss of ACSL-specific activity and a 60-85% decrease in muscle FA oxidation. Acsl1(M-/-) mice were more insulin sensitive, and, during an overnight fast, their respiratory exchange ratio was higher, indicating greater glucose use. During endurance exercise, Acsl1(M-/-) mice ran only 48% as far as controls. At the time that Acsl1(M-/-) mice were exhausted but control mice continued to run, liver and muscle glycogen and triacylglycerol stores were similar in both genotypes; however, plasma glucose concentrations in Acsl1(M-/-) mice were ∼40 mg/dL, whereas glucose concentrations in controls were ∼90 mg/dL. Excess use of glucose and the likely use of amino acids for fuel within muscle depleted glucose reserves and diminished substrate availability for hepatic gluconeogenesis. Surprisingly, the content of muscle acyl-CoA at exhaustion was markedly elevated, indicating that acyl-CoAs synthesized by other ACSL isoforms were not available for β-oxidation. This compartmentalization of acyl-CoAs resulted in both an excessive glucose requirement and severely compromised systemic glucose homeostasis.

The branched-chain amino acids (BCAA) accumulated in type 2 diabetes are independent contributors to insulin resistance. The activity of branched-chain α-keto acid dehydrogenase (BCKD) complex, rate-limiting enzyme in BCAA catabolism, is reduced in diabetic states, which contributes to elevated BCAA concentrations. However, the mechanisms underlying decreased BCKD activity remain poorly understood. Here, we demonstrate that mitochondrial phosphatase 2C (PP2Cm), a newly identified BCKD phosphatase that increases BCKD activity, was significantly downregulated in ob/ob and type 2 diabetic mice. Interestingly, in adiponectin (APN) knockout (APN(-/-)) mice fed with a high-fat diet (HD), PP2Cm expression and BCKD activity were significantly decreased, whereas BCKD kinase (BDK), which inhibits BCKD activity, was markedly increased. Concurrently, plasma BCAA and branched-chain α-keto acids (BCKA) were significantly elevated. APN treatment markedly reverted PP2Cm, BDK, BCKD activity, and BCAA and BCKA levels in HD-fed APN(-/-) and diabetic animals. Additionally, increased BCKD activity caused by APN administration was partially but significantly inhibited in PP2Cm knockout mice. Finally, APN-mediated upregulation of PP2Cm expression and BCKD activity were abolished when AMPK was inhibited. Collectively, we have provided the first direct evidence that APN is a novel regulator of PP2Cm and systematic BCAA levels, suggesting that targeting APN may be a pharmacological approach to ameliorating BCAA catabolism in the diabetic state.

Type 2 diabetes mellitus (T2DM) is associated with brain atrophy, but the mechanisms underlying this link are unknown. Advanced glycation end products (AGEs) accumulate in T2DM, resulting in inflammation, oxidative stress, and protein cross-linking, which are known contributors to neurodegeneration. We aimed to study whether tissue AGE accumulation is associated with T2DM-related brain atrophy. We performed brain magnetic resonance imaging, cognitive tests, and noninvasive skin autofluorescence (SAF; a measure of tissue AGE levels) on people aged >55 years with and without T2DM. Multivariable linear regression was used to study the relationships among T2DM, SAF, and gray matter volume (GMV). There were 486 people included in the study. T2DM was associated with greater SAF. Greater SAF, T2DM, and cognitive impairment were each associated with lower GMV independently of age, sex, and total intracranial volume. SAF partially mediated the association between T2DM and GMV. Longitudinal studies may help confirm whether tissue AGE accumulation is associated with brain atrophy in T2DM.

Glucose-dependent insulinotropic polypeptide (GIP) is glucagonotropic, and glucagon-like peptide-1 (GLP-1) is glucagonostatic. We studied the effects of GIP and GLP-1 on glucagon responses to insulin-induced hypoglycemia in patients with type 1 diabetes mellitus (T1DM). Ten male subjects with T1DM (C-peptide negative, age [mean ± SEM] 26 ± 1 years, BMI 24 ± 0.5 kg/m(2), HbA1c 7.3 ± 0.2%) were studied in a randomized, double-blinded, crossover study, with 2-h intravenous administration of saline, GIP, or GLP-1. The first hour, plasma glucose was lowered by insulin infusion, and the second hour constituted a "recovery phase." During the recovery phase, GIP infusions elicited larger glucagon responses (164 ± 50 [GIP] vs. 23 ± 25 [GLP-1] vs. 17 ± 46 [saline] min ⋅ pmol/L, P < 0.03) and endogenous glucose production was higher with GIP and lower with GLP-1 compared with saline (P < 0.02). On the GIP days, significantly less exogenous glucose was needed to keep plasma glucose above 2 mmol/L (155 ± 36 [GIP] vs. 232 ± 40 [GLP-1] vs. 212 ± 56 [saline] mg ⋅ kg(-1), P < 0.05). Levels of insulin, cortisol, growth hormone, and noradrenaline, as well as hypoglycemic symptoms and cognitive function, were similar on all days. Our results suggest that during hypoglycemia in patients with T1DM, exogenous GIP increases glucagon responses during the recovery phase after hypoglycemia and reduces the need for glucose administration.

Obesity is often regarded as the primary cause of metabolic syndrome. However, many lines of evidence suggest that obesity may develop as a protective mechanism against tissue damage during caloric surplus and that it is only when the maximum fat accumulation capacity is reached and fatty acid spillover occurs into to peripheral tissues that metabolic diseases develop. In this regard, identifying the molecular mechanisms that modulate adipocyte fat accumulation and fatty acid spillover is imperative. Here we identify the deleted in breast cancer 1 (DBC1) protein as a key regulator of fat storage capacity of adipocytes. We found that knockout (KO) of DBC1 facilitated fat cell differentiation and lipid accumulation and increased fat storage capacity of adipocytes in vitro and in vivo. This effect resulted in a "healthy obesity" phenotype. DBC1 KO mice fed a high-fat diet, although obese, remained insulin sensitive, had lower free fatty acid in plasma, were protected against atherosclerosis and liver steatosis, and lived longer. We propose that DBC1 is part of the molecular machinery that regulates fat storage capacity in adipocytes and participates in the "turn-off" switch that limits adipocyte fat accumulation and leads to fat spillover into peripheral tissues, leading to the deleterious effects of caloric surplus.

Chromogranin A knockout (Chga-KO) mice exhibit enhanced insulin sensitivity despite obesity. Here, we probed the role of the chromogranin A-derived peptide pancreastatin (PST: CHGA(273-301)) by investigating the effect of diet-induced obesity (DIO) on insulin sensitivity of these mice. We found that on a high-fat diet (HFD), Chga-KO mice (KO-DIO) remain more insulin sensitive than wild-type DIO (WT-DIO) mice. Concomitant with this phenotype is enhanced Akt and AMPK signaling in muscle and white adipose tissue (WAT) as well as increased FoxO1 phosphorylation and expression of mature Srebp-1c in liver and downregulation of the hepatic gluconeogenic genes, Pepck and G6pase. KO-DIO mice also exhibited downregulation of cytokines and proinflammatory genes and upregulation of anti-inflammatory genes in WAT, and peritoneal macrophages from KO mice displayed similarly reduced proinflammatory gene expression. The insulin-sensitive, anti-inflammatory phenotype of KO-DIO mice is masked by supplementing PST. Conversely, a PST variant peptide PSTv1 (PST-NΔ3: CHGA(276-301)), lacking PST activity, simulated the KO phenotype by sensitizing WT-DIO mice to insulin. In summary, the reduced inflammation due to PST deficiency prevented the development of insulin resistance in KO-DIO mice. Thus, obesity manifests insulin resistance only in the presence of PST, and in its absence obesity is dissociated from insulin resistance.

Obesity-related insulin resistance is associated with an influx of pathogenic T cells into visceral adipose tissue (VAT), but the mechanisms regulating lymphocyte balance in such tissues are unknown. Here we describe an important role for the immune cytotoxic effector molecule perforin in regulating this process. Perforin-deficient mice (Prf1(null)) show early increased body weight and adiposity, glucose intolerance, and insulin resistance when placed on high-fat diet (HFD). Regulatory effects of perforin on glucose tolerance are mechanistically linked to the control of T-cell proliferation and cytokine production in inflamed VAT. HFD-fed Prf1(null) mice have increased accumulation of proinflammatory IFN-γ-producing CD4(+) and CD8(+) T cells and M1-polarized macrophages in VAT. CD8(+) T cells from the VAT of Prf1(null) mice have increased proliferation and impaired early apoptosis, suggesting a role for perforin in the regulation of T-cell turnover during HFD feeding. Transfer of CD8(+) T cells from Prf1(null) mice into CD8-deficient mice (CD8(null)) resulted in worsening of metabolic parameters compared with wild-type donors. Improved metabolic parameters in HFD natural killer (NK) cell-deficient mice (NK(null)) ruled out a role for NK cells as a single source of perforin in regulating glucose homeostasis. The findings support the importance of T-cell function in insulin resistance and suggest that modulation of lymphocyte homeostasis in inflamed VAT is one possible avenue for therapeutic intervention.

Experimental data suggest a role for advanced glycation end products (AGEs) in cardiovascular disease (CVD), particularly in type 2 diabetes (T2DM). However, epidemiological evidence of an association between high plasma AGEs and increased cardiovascular risk remains inconclusive. Therefore, in a case-cohort study comprising 134 cardiovascular case subjects and a random subcohort of 218 individuals (including 65 cardiovascular case subjects), all with T2DM and nested in the European Prospective Investigation into Cancer and Nutrition in the Netherlands (EPIC-NL) study, plasma levels of protein-bound Nε-(carboxymethyl)lysine, Nε-(carboxyethyl)lysine, and pentosidine were measured with liquid chromatography. AGEs were loge-transformed, combined in a z-score, and the association with incident cardiovascular events was analyzed with Cox proportional hazard regression, adapted for case-cohort design (Prentice method). After multivariable adjustment (sex, age, cohort status, diabetes duration, total cholesterol to HDL-cholesterol ratio, smoking, systolic blood pressure, BMI, blood pressure-, cholesterol- and glucose-lowering treatment, prior cardiovascular events, and triglycerides), higher plasma AGE z-scores were associated with higher risk of incident cardiovascular events in individuals without prior cardiovascular events (hazard ratio 1.31 [95% CI: 1.06-1.61]). A similar trend was observed in individuals with prior cardiovascular events (1.37 [0.63-2.98]). In conclusion, high plasma AGEs were associated with incident cardiovascular events in individuals with T2DM. These results underline the potential importance of AGEs in development of CVD.