Aims/hypothesis The aim of the study was to compare the effect of six (A6 regimen) vs two meals a day, breakfast and lunch (B2 regimen), on body weight, hepatic fat content (HFC), insulin resistance and beta cell function. Methods In a randomised, open, crossover, single-centre study (conducted in Prague, Czech Republic), we assigned 54 patients with type 2 diabetes treated with oral hypoglycaemic agents, both men and women, age 30–70 years, BMI 27–50 kg/m² and HbA_1c 6–11.8% (42–105 mmol/mol), to follow two regimens of a hypoenergetic diet, A6 and B2, each for 12 weeks. Randomisation and allocation to trial groups (n = 27 and n = 27) were carried out by a central computer system. Individual calculations of energy requirements for both regimens were based on the formula: (resting energy expenditure × 1.5) − 2,092 kJ. The diet in both regimens had the same macronutrient and energy content. HFC was measured by proton magnetic resonance spectroscopy. Insulin sensitivity was measured by isoglycaemic–hyperinsulinaemic clamp and calculated by mathematical modelling as oral glucose insulin sensitivity (OGIS). Beta cell function was assessed during standard meal tests by C-peptide deconvolution and was quantified with a mathematical model. For statistical analysis, 2 × 2 crossover ANOVA was used. Results The intention-to-treat analysis included all participants (n = 54). Body weight decreased in both regimens (p < 0.001), more for B2 (−2.3 kg; 95% CI −2.7, −2.0 kg for A6 vs −3.7 kg; 95% CI −4.1, −3.4 kg for B2; p < 0.001). HFC decreased in response to both regimens (p < 0.001), more for B2 (−0.03%; 95% CI −0.033%, −0.027% for A6 vs −0.04%; 95% CI −0.041%, −0.035% for B2; p = 0.009). Fasting plasma glucose and C-peptide levels decreased in both regimens (p < 0.001), more for B2 (p = 0.004 and p = 0.04, respectively). Fasting plasma glucagon decreased with the B2 regimen (p < 0.001), whereas it increased (p = 0.04) for the A6 regimen (p < 0.001). OGIS increased in both regimens (p < 0.01), more for B2 (p = 0.01). No adverse events were observed for either regimen. Conclusions/interpretation Eating only breakfast and lunch reduced body weight, HFC, fasting plasma glucose, C-peptide and glucagon, and increased OGIS, more than the same caloric restriction split into six meals. These results suggest that, for type 2 diabetic patients on a hypoenergetic diet, eating larger breakfasts and lunches may be more beneficial than six smaller meals during the day. Trial registration ClinicalTrials.gov number, NCT01277471, completed. Funding Grant NT/11238-4 from Ministry of Health, Prague, Czech Republic and the Agency of Charles University – GAUK No 702312.

We have cloned a novel pancreatic beta cell and neuroendocrine cell-specific calcium-binding protein termed secretagogin. The cDNA obtained by immunoscreening a human pancreatic cDNA library using the recently described murine monoclonal antibody D24 contains an open reading frame of 828 base pairs. This codes for a cytoplasmic protein with six putative EF finger hand calcium-binding motifs. The gene could be localized to chromosome 6 by alignment with GenBank genomic sequence data. Northern blot analysis demonstrated abundant expression of this protein in the pancreas and to a lesser extent in the thyroid, adrenal medulla, and cortex. In addition it was expressed in scant quantity in the gastrointestinal tract (stomach, small intestine, and colon). Thyroid tissue expression of secretagogin was restricted to C-cells. Using a sandwich capture enzyme-linked immunosorbent assay with a detection limit of 6.5 pg/ml, considerable amounts of constitutively secreted protein could be measured in tissue culture supernatants of stably transfected RIN-5F and dog insulinoma (INS-H1) cell clones; however, in stably transfected Jurkat cells, the protein was only secreted upon CD3 stimulation. Functional analysis of transfected cell lines expressing secretagogin revealed an influence on calcium flux and cell proliferation. In RIN-5F cells, the antiproliferative effect is possibly due to secretagogin-triggered down-regulation of substance P transcription.

Nonalcoholic fatty liver disease is associated with chronic oxidative stress. In our study, we explored the antioxidant effect of antidiabetic metformin on chronic [high-fat diet (HFD)-induced] and acute oxidative stress induced by short-term warm partial ischemia-reperfusion (I/R) or on a combination of both in the liver. Wistar rats were fed a standard diet (SD) or HFD for 10 wk, half of them being administered metformin (150 mg·kg body wt⁻¹·day⁻¹). Metformin treatment prevented acute stress-induced necroinflammatory reaction, reduced alanine aminotransferase and aspartate aminotransferase serum activity, and diminished lipoperoxidation. The effect was more pronounced in the HFD than in the SD group. The metformin-treated groups exhibited less severe mitochondrial damage (markers: cytochrome c release, citrate synthase activity, mtDNA copy number, mitochondrial respiration) and apoptosis (caspase 9 and caspase 3 activation). Metformin-treated HFD-fed rats subjected to I/R exhibited increased antioxidant enzyme activity as well as attenuated mitochondrial respiratory capacity and ATP resynthesis. The exposure to I/R significantly increased NADH- and succinate-related reactive oxygen species (ROS) mitochondrial production in vitro. The effect of I/R was significantly alleviated by previous metformin treatment. Metformin downregulated the I/R-induced expression of proinflammatory (TNF-α, TLR4, IL-1β, Ccr2) and infiltrating monocyte (Ly6c) and macrophage (CD11b) markers. Our data indicate that metformin reduces mitochondrial performance but concomitantly protects the liver from I/R-induced injury. We propose that the beneficial effect of metformin action is based on a combination of three contributory mechanisms: increased antioxidant enzyme activity, lower mitochondrial ROS production, and reduction of postischemic inflammation.

The role of folate deficiency and associated hyperhomocysteinemia in the pathogenesis of metabolic syndrome is not fully established. In the current study, we analyzed the role of folate deficiency in pathogenesis of the metabolic syndrome in the spontaneously hypertensive rat (SHR). Metabolic and hemodynamic traits were assessed in SHR/Ola rats fed either folate-deficient or control diet for 4 weeks starting at the age of 3 months. Compared to SHRs fed a folate-replete diet, SHRs fed a folate-deficient diet showed significantly reduced serum folate (104±5 vs. 11±1 nmol/L, P < 0.0005) and urinary folate excretion (4.3±0.6 vs. 1.2±0.1 nmol/16h, P < 0.0005) together with a near 3-fold increase in plasma total homocysteine concentration (4.5±0.1 vs 13.1±0.7 μmol/L, P < 0.0005), ectopic fat accumulation in liver, and impaired glucose tolerance. Folate deficiency also increased systolic blood pressure by approximately 15mm Hg (P < 0.01). In addition, the low-folate diet was accompanied by significantly reduced activity of antioxidant enzymes and increased concentrations of lipoperoxidation products in liver, renal cortex, and heart. These findings demonstrate that the SHR model is susceptible to the adverse metabolic and hemodynamic effects of low dietary intake of folate. The results are consistent with the hypothesis that folate deficiency can promote oxidative stress and multiple features of the metabolic syndrome that are associated with increased risk for diabetes and cardiovascular disease.

Major controversy exists as to whether increased C-reactive protein (CRP) contributes to individual components of the metabolic syndrome or is just a secondary response to inflammatory disease processes. We measured blood pressure and metabolic phenotypes in spontaneously hypertensive rats (SHRs) in which we transgenically expressed human CRP in the liver under control of the apolipoprotein E promoter. In transgenic SHRs, serum levels of human CRP approximated the endogenous levels of CRP normally found in the rat. Systolic and diastolic blood pressures measured by telemetry were 10 to 15 mm Hg greater in transgenic SHRs expressing human CRP than in SHR controls ( P <0.01). During oral glucose tolerance testing, transgenic SHRs exhibited hyperinsulinemia compared with controls (insulin area under the curve: 36±7 versus 8±2 nmol/L per 2 hours, respectively; P <0.05). Transgenic SHRs also exhibited resistance to insulin stimulated glycogenesis in skeletal muscle (174±18 versus 278±32 nmol of glucose per gram per 2 hours; P <0.05), hypertriglyceridemia (0.84±0.05 versus 0.64±0.03 mmol/L; P <0.05), reduced serum adiponectin (2.4±0.3 versus 4.3±0.6 mmol/L; P <0.05), and microalbuminuria (200±35 versus 26±5 mg of albumin per gram of creatinine, respectively; P <0.001). Transgenic SHRs had evidence of inflammation and oxidative tissue damage with increased serum levels of interleukin 6 (36.4±5.2 versus 18±1.7 pg/mL; P <0.005) and increased hepatic and renal thiobarbituric acid reactive substances (1.2±0.09 versus 0.8±0.07 and 1.5±0.1 versus 1.1±0.05 nmol/L per milligram of protein, respectively; P <0.01), suggesting that oxidative stress may be mediating adverse effects of increased human CRP. These findings are consistent with the hypothesis that increased CRP is more than just a marker of inflammation and can directly promote multiple features of the metabolic syndrome.

Recently, the relationship of mitochondrial DNA (mtDNA) variants to metabolic risk factors for diabetes and other common diseases has begun to attract increasing attention. However, progress in this area has been limited because (1) the phenotypic effects of variation in the mitochondrial genome are difficult to isolate owing to confounding variation in the nuclear genome, imprinting phenomena, and environmental factors; and (2) few animal models have been available for directly investigating the effects of mtDNA variants on complex metabolic phenotypes in vivo. Substitution of different mitochondrial genomes on the same nuclear genetic background in conplastic strains provides a way to unambiguously isolate effects of the mitochondrial genome on complex traits. Here we show that conplastic strains of rats with identical nuclear genomes but divergent mitochondrial genomes that encode amino acid differences in proteins of oxidative phosphorylation exhibit differences in major metabolic risk factors for type 2 diabetes. These results (1) provide the first direct evidence linking naturally occurring variation in the mitochondrial genome, independent of variation in the nuclear genome and other confounding factors, to inherited variation in known risk factors for type 2 diabetes; and (2) establish that spontaneous variation in the mitochondrial genome per se can promote systemic metabolic disturbances relevant to the pathogenesis of common diseases.