KEY POINTS: The heat of activation of cardiac muscle reflects the metabolic cost of restoring ionic homeostasis following a contraction. The accuracy of its measurement depends critically on the abolition of crossbridge cycling. We abolished crossbridge activity in isolated rat ventricular trabeculae by use of blebbistatin, an agent that selectively inhibits myosin II ATPase. We found cardiac activation heat to be muscle length independent and to account for 15-20% of total heat production at body temperature. We conclude that it can be accurately estimated at minimal muscle length. ABSTRACT: Activation heat arises from two sources during the contraction of striated muscle. It reflects the metabolic expenditure associated with Ca2+ pumping by the sarcoplasmic reticular Ca2+ -ATPase and Ca2+ translocation by the Na+ /Ca2+ exchanger coupled to the Na+ ,K+ -ATPase. In cardiac preparations, investigators are constrained in estimating its magnitude by reducing muscle length to the point where macroscopic twitch force vanishes. But this experimental protocol has been criticised since, at zero force, the observed heat may be contaminated by residual crossbridge cycling activity. To eliminate this concern, the putative thermal contribution from crossbridge cycling activity must be abolished, at least at minimal muscle length. We achieved this using blebbistatin, a selective inhibitor of myosin II ATPase. Using a microcalorimeter, we measured the force production and heat output, as functions of muscle length, of isolated rat trabeculae from both ventricles contracting isometrically at 5 Hz and at 37°C. In the presence of blebbistatin (15 μmol l-1 ), active force was zero but heat output remained constant, at all muscle lengths. Activation heat measured in the presence of blebbistatin was not different from that estimated from the intercept of the heat-stress relation in its absence. We thus reached two conclusions. First, activation heat is independent of muscle length. Second, residual crossbridge heat is negligible at zero active force; hence, the intercept of the cardiac heat-force relation provides an estimate of activation heat uncontaminated by crossbridge cycling. Both results resolve long-standing disputes in the literature.

KEY POINTS: Uteroplacental insufficiency compromises maternal mammary development, milk production and pup organ development; this is ameliorated by cross-fostering, which improves pup growth and organ development and prevents adult diseases in growth-restricted (Restricted) offspring by enhancing postnatal nutrition. Leptin is transported to the fetus from the mother by the placenta; we report reduced plasma leptin concentrations in Restricted fetuses associated with sex-specific alterations in placental leptin transporter expression. Pup plasma leptin concentrations were also reduced during suckling, which may suggest reduced milk leptin transport or leptin reabsorption. Mothers suckled by Restricted pups had impaired mammary development and changes in milk fatty acid composition with no alterations in milk leptin; cross-fostering restored pup plasma leptin concentrations, which may be correlated to improved milk composition and intake. Increased plasma leptin and altered milk fatty acid composition in Restricted pups suckling mothers with normal lactation may improve postnatal growth and prevent adult diseases. ABSTRACT: Uteroplacental insufficiency reduces birth weight and adversely affects fetal organ development, increasing adult disease risk. Cross-fostering improves postnatal nutrition and restores these deficits. Mothers with growth-restricted pups have compromised milk production and composition; however, the impact cross-fostering has on milk production and composition is unknown. Plasma leptin concentrations peak during the completion of organogenesis, which occurs postnatally in rats. Leptin is transferred to the fetus via the placenta and to the pup via the lactating mammary gland. This study investigated the effect of uteroplacental insufficiency on pup plasma leptin concentrations and placental leptin transporters. We additionally examined whether cross-fostering improves mammary development, milk composition and pup plasma leptin concentrations. Fetal growth restriction was induced by bilateral uterine vessel ligation surgery on gestation day 18 in Wistar Kyoto rats (termed uteroplacental insufficiency surgery mothers). Growth-restricted (Restricted) fetuses had reduced plasma leptin concentrations, persisting throughout lactation, and sex-specific alterations in placental leptin transporters. Mothers suckled by Restricted pups had impaired mammary development, altered milk fatty acid composition and increased plasma leptin concentrations, despite no changes in milk leptin. Milk intake was reduced in Restricted pups suckling uteroplacental insufficiency surgery mothers compared to Restricted pups suckling sham-operated mothers. Cross-fostering Restricted pups onto a sham-operated mother improved postnatal growth and restored plasma leptin concentrations compared to Restricted pups suckling uteroplacental insufficiency surgery mothers. Uteroplacental insufficiency alters leptin homeostasis. This is ameliorated with cross-fostering and enhanced milk fatty acid composition and consumption, which may protect the pups from developing adverse health conditions in adulthood.

Babies born small are at an increased risk of developing myriad adult diseases. While growth restriction increases disease risk in all individuals, often a second hit is required to unmask 'programmed' impairments in physiology. Programmed disease outcomes are demonstrated more commonly in male offspring compared with females, with these sex-specific outcomes partly attributed to different placenta-regulated growth strategies of the male and female fetus. Pregnancy is known to be a major risk factor for unmasking a number of conditions and can be considered a 'second hit' for women who were born small. As such, female offspring often develop impairments of physiology for the first time during pregnancy that present as pregnancy complications. Numerous maternal stressors can further increase the risk of developing a maternal complication during pregnancy. Importantly, these maternal complications can have long-term consequences for both the mother after pregnancy and the developing fetus. Conditions such as preeclampsia, gestational diabetes and hypertension as well as thyroid, liver and kidney diseases are all conditions that can complicate pregnancy and have long-term consequences for maternal and offspring health. Babies born to mothers who develop these conditions are often at a greater risk of developing disease in adulthood. This has implications as a mechanism for transmission of disease across generations. In this review, we discuss the evidence surrounding long-term intergenerational implications of being born small and/or experiencing stress during pregnancy on programming outcomes.

This study seeks to determine whether knockdown of basal forebrain p75 neurotrophin receptor (p75(NTR) ) expression elicits increased hippocampal choline acetyltransferase (ChAT) activity in mature animals. Antisense (AS) oligonucleotides (oligos) targeting p75(NTR) were infused into the medial septal area of mature rats continuously for 4 weeks. In all rats, the cannula outlet was placed equidistant between the left and the right sides of the vertical diagonal band of Broca. We tested phosphorothioate (PS), morpholino (Mo), and gapmer (mixed PS/RNA) oligos. Gapmer AS infusions of 7.5 and 22 μg/day decreased septal p75(NTR) mRNA by 34% and 48%, respectively. The same infusions increased hippocampal ChAT activity by 41% and 55%. Increased hippocampal ChAT activity correlated strongly with septal p75(NTR) downregulation in individual rats. Infusions of PS and Mo AS oligos did not downregulate p75(NTR) mRNA or stimulate ChAT activity. These results demonstrate that p75(NTR) can dynamically regulate hippocampal ChAT activity in the mature CNS. They also reveal the different efficacies of three diverse AS oligo chemistries when infused intracerebrally. Among the three types, gapmer oligos worked best.

OBJECTIVE: To develop a frailty index (FI) and explore the relationship of frailty to subsequent adverse outcomes on the effectiveness and safety of more intensive control of both blood glucose and blood pressure (BP), among participants with type 2 diabetes in the Action in Diabetes and Vascular Disease: Preterax and Diamicron Modified Release Controlled Evaluation (ADVANCE) trial. RESEARCH DESIGN AND METHODS: Cox proportional hazard models were used to estimate the effectiveness and safety of intensive glucose control and BP intervention according to frailty (defined as FI >0.21) status. The primary outcomes were macro- and microvascular events. The secondary outcomes were all-cause mortality, cardiovascular mortality, severe hypoglycemia, and discontinuation of BP treatment due to hypotension/dizziness. RESULTS: There were 11,140 participants (mean age, 65.8 years; 42.5% women, 25.7% frail). Frailty was an independent predictor of all primary outcomes and secondary outcomes. The effect of intensive glucose treatment on primary outcomes showed some evidence of attenuation in the frail: hazard ratios for combined major macro- and microvascular events 1.03 (95% CI 0.90-1.19) in the frail versus 0.84 (95% CI 0.74-0.94) in the nonfrail (P = 0.02). A similar trend was observed with BP intervention. Severe hypoglycemia rates (per 1,000 person-years) were higher in the frail: 8.39 (6.15-10.63) vs. 4.80 (3.84-5.76) in nonfrail (P < 0.001). There was no significant difference in discontinuation of BP treatment between frailty groups. CONCLUSIONS: It was possible to retrospectively estimate frailty in a trial population, and this FI identified those at higher risk of poor outcomes. Participants with frailty had some attenuation of benefit from intensive glucose-lowering and BP-lowering treatments.

AIMS/HYPOTHESIS: Type 2 diabetes increases the risk of cardiovascular and renal complications, but early risk prediction could lead to timely intervention and better outcomes. Genetic information can be used to enable early detection of risk. METHODS: We developed a multi-polygenic risk score (multiPRS) that combines ten weighted PRSs (10 wPRS) composed of 598 SNPs associated with main risk factors and outcomes of type 2 diabetes, derived from summary statistics data of genome-wide association studies. The 10 wPRS, first principal component of ethnicity, sex, age at onset and diabetes duration were included into one logistic regression model to predict micro- and macrovascular outcomes in 4098 participants in the ADVANCE study and 17,604 individuals with type 2 diabetes in the UK Biobank study. RESULTS: The model showed a similar predictive performance for cardiovascular and renal complications in different cohorts. It identified the top 30% of ADVANCE participants with a mean of 3.1-fold increased risk of major micro- and macrovascular events (p = 6.3 × 10-21 and p = 9.6 × 10-31, respectively) and a 4.4-fold (p = 6.8 × 10-33) higher risk of cardiovascular death. While in ADVANCE overall, combined intensive blood pressure and glucose control decreased cardiovascular death by 24%, the model identified a high-risk group in whom it decreased the mortality rate by 47%, and a low-risk group in whom it had no discernible effect. High-risk individuals had the greatest absolute risk reduction with a number needed to treat of 12 to prevent one cardiovascular death over 5 years. CONCLUSIONS/INTERPRETATION: This novel multiPRS model stratified individuals with type 2 diabetes according to risk of complications and helped to target earlier those who would receive greater benefit from intensive therapy.

Exercise stimulates cellular and physiological adaptations that are associated with widespread health benefits. To uncover conserved protein phosphorylation events underlying this adaptive response, we performed mass spectrometry-based phosphoproteomic analyses of skeletal muscle from two widely used rodent models: treadmill running in mice and in situ muscle contraction in rats. We overlaid these phosphoproteomic signatures with cycling in humans to identify common cross-species phosphosite responses, as well as unique model-specific regulation. We identified > 22,000 phosphosites, revealing orthologous protein phosphorylation and overlapping signaling pathways regulated by exercise. This included two conserved phosphosites on stromal interaction molecule 1 (STIM1), which we validate as AMPK substrates. Furthermore, we demonstrate that AMPK-mediated phosphorylation of STIM1 negatively regulates store-operated calcium entry, and this is beneficial for exercise in Drosophila. This integrated cross-species resource of exercise-regulated signaling in human, mouse, and rat skeletal muscle has uncovered conserved networks and unraveled crosstalk between AMPK and intracellular calcium flux.

AIMS: To evaluate 1,5-anhydroglucitol (1,5-AG) according to clinical outcomes and assess the effects of glucose- and blood pressure-lowering interventions on change in 1,5-AG levels in people with type 2 diabetes. METHODS: We measured 1,5-AG in 6826 stored samples at baseline and in a random subsample of 684 participants at the 1-year follow-up visit in the ADVANCE trial. We examined baseline 1,5-AG [< 39.7, 39.7-66.2, ≥ 66.2 μmol/L (<6, 6-10, ≥10 μg/mL)] and microvascular and macrovascular events and mortality using Cox regression models during 5 years of follow-up. Using an intention-to-treat approach, we examined 1-year change in 1,5-AG (mean and percent) in response to the glucose- and blood pressure-lowering interventions in the subsample. RESULTS: Low 1,5-AG level [<39.7 μmol/L vs ≥ 66.2 μmol/L (<6 μg/mL vs ≥10 μg/mL)] was associated with microvascular events (hazard ratio 1.28, 95% confidence interval 1.03-1.60) after adjustment for risk factors and baseline glycated haemoglobin (HbA1c); however, the associations for macrovascular events and mortality were not independent of HbA1c. The glucose-lowering intervention was associated with a significant 1-year increase in 1,5-AG (vs standard control) of 6.69 μmol/L (SE 2.52) [1.01 μg/mL (SE 0.38)], corresponding to an 8.26% (SE 0.10%) increase from baseline. We also observed an increase in 1,5-AG of similar magnitude in response to the blood pressure intervention independent of the glucose-lowering effect. CONCLUSIONS: Our results suggest that 1,5-AG is a marker of risk in adults with type 2 diabetes, but only for microvascular events independently of HbA1c. We found that 1,5-AG was improved (increased) in response to an intensive glucose-lowering intervention, although the independent effect of the blood pressure-lowering intervention on 1,5-AG suggests potential non-glycaemic influences.

KEY POINTS: Linoleic acid consumption is increasing in Western populations. We investigated whether elevated linoleic acid in pregnancy was deleterious to mothers or offspring. Maternal and fetal body and organ weights were not affected by elevated linoleic acid consumption. Maternal lipids and leptin were altered following elevated linoleic acid consumption. Male offspring numbers were reduced following elevated linoleic acid consumption. ABSTRACT: Dietary intakes of linoleic acid (LA) have increased dramatically in Western populations, including in women of reproductive age. Pro-inflammatory effects of LA may have detrimental effects on maternal and offspring outcomes. We aimed to investigate whether consumption of a maternal diet with elevated LA altered maternal inflammatory or metabolic markers during pregnancy, fetal growth and/or the sex ratio of the offspring. Female Wistar Kyoto rats consumed a diet high in LA (HLA) (6.21% of energy) or a diet low in LA (LLA) (1.44% of energy) for 10 weeks prior to mating and during pregnancy. Pregnant rats were killed at embryonic day 20 (E20). There were no differences in maternal or fetal body weights or organ weights in the HLA group compared to the LLA group. There was no difference in maternal circulating cytokine concentrations between dietary groups. In the maternal liver, IL-1α concentrations were significantly lower, and TNF-α and IL-7 significantly higher in the HLA group. Total plasma cholesterol, LDL-cholesterol, HDL cholesterol and the total:HDL cholesterol ratio were lower in dams fed the HLA diet. mRNA expression of sterol regulatory element binding transcription factor 1 (SREBF-1) and leptin in maternal adipose tissue was lower in the HLA group, as were circulating leptin concentrations. The proportion of male fetuses was lower and circulating prostaglandin E metabolite concentrations were increased in the HLA group. In conclusion, consumption of a maternal diet high in linoleic acid alters cholesterol metabolism and prostaglandin E metabolite concentrations, which may contribute to the reduced proportion of male offspring.

KEY POINTS: Fetal growth is dependent on effective placental nutrient transportation, which is regulated by mammalian target of rapamycin (mTOR) complex 1 modulation of nutrient transporter expression. These transporters are dysregulated in pregnancies affected by uteroplacental insufficiency and maternal obesity. Nutrient transporters and mTOR were altered in placentae of mothers born growth restricted compared to normal birth weight dams, with maternal diet- and fetal sex-specific responses. Exercise initiated during pregnancy downregulated mTOR protein expression, despite an increase in mTOR activation in male associated placentae, and reduced nutrient transporter gene abundance, which was also dependent on maternal diet and fetal sex. Limited changes were characterized with exercise initiated before and continued throughout pregnancy in nutrient transporter and mTOR expression. Maternal exercise during pregnancy differentially regulated mTOR and nutrient transporters in a diet- and sex-specific manner, which likely aimed to improve late gestational placental growth and neonatal survival. ABSTRACT: Adequate transplacental nutrient delivery is essential for fetoplacental development. Intrauterine growth restriction and maternal obesity independently alter placental nutrient transporter expression. Although exercise is beneficial for maternal health, limited studies have characterized how the timing of exercise initiation influences placental nutrient transport. Therefore, this study investigated the impact of maternal exercise on placental mechanistic target of rapamycin (mTOR) and nutrient transporter expression in growth restricted mothers and whether these outcomes were dependent on maternal diet or fetal sex. Uteroplacental insufficiency or sham surgery was induced on embryonic day (E) 18 in Wistar-Kyoto rats. F1 offspring were fed a chow or high-fat diet from weaning and at 16 weeks were randomly allocated to an exercise protocol: sedentary, exercised prior to and during pregnancy, or exercised during pregnancy only. Females were mated with normal males (20 weeks) and F2 placentae collected at E20. Exercise during pregnancy only, reduced mTOR protein expression in all groups and increased mTOR activation in male associated placentae. Exercise during pregnancy only, decreased the expression of amino acid transporters in a diet- and sex-specific manner. Maternal growth restriction altered mTOR and system A amino acid transporter expression in a sex- and diet-specific manner. These data highlight that maternal exercise initiated during pregnancy alters placental mTOR expression, which may directly regulate amino acid transporter expression, to a greater extent than exercise initiated prior to and continued during pregnancy, in a diet- and fetal sex-dependent manner. These findings highlight that the timing of exercise initiation is important for optimal placental function.

AIMS: Patients with type 2 diabetes have a high risk of cardiovascular disease (CVD). Central obesity has been particularly associated with this risk relationship. We aimed to evaluate waist to height ratio (WHtR) as a predictor of risk in such patients. METHODS: WHtR was evaluated as a predictor of the risk of CVD and mortality amongst 11 125 participants with type 2 diabetes in the ADVANCE and ADVANCE-ON studies, and was compared with body mass index (BMI), waist circumference and waist hip ratio (WHR). Primary outcome was a composite of death from CVD, non-fatal myocardial infarction or non-fatal stroke. Secondary outcomes were myocardial infarction, stroke, cardiovascular death and death from any cause. Cox models were used, with bootstrapping to compare associations between anthropometric measures for the primary outcome. RESULTS: Median follow-up time was 9.0 years. There was a positive association between WHtR and adverse outcomes. The hazard ratio (HR) (confidence interval), per SD higher WHtR, was 1.16 (1.11-1.22) for the primary endpoint, with no heterogeneity by sex or region, but a stronger effect in individuals aged 66 years or older. The other 3 anthropometric measurements showed similar associations, although there was evidence that WHtR marginally outperformed BMI and WHR. Based on commonly used BMI cut-points, the equivalent WHtR cut-points were estimated to be 0.55 and 0.6, with no evidence of a difference across subgroups. CONCLUSIONS: In patients with diabetes, WHtR is a useful indicator of future adverse risk, with similar effects in different population subgroups.

AIM: To estimate the associations between risk factors and cognitive decline (CD)/dementia, and the sex differences in these risk factors in individuals with type 2 diabetes, while accounting for the competing risk of death. MATERIALS AND METHODS: The Action in Diabetes and Vascular Disease: Preterax and Diamicron Modified Release Controlled Evaluation (ADVANCE) trial of 11,140 individuals with type 2 diabetes was used to estimate the odds of CD/dementia using multinomial logistic regression. RESULTS: During a median 5-year follow-up, 1827 participants (43.2% women) had CD/dementia (1718 with CD only; 21 with dementia only; 88 with CD and dementia), and 929 (31.0% women) died without CD/dementia. Women had lower odds of CD/dementia than men (odds ratio [OR] [95% confidence interval], 0.88 [0.77, 1.00]); older age, higher total cholesterol, HbA1c, waist circumference, waist-to-height ratio, moderately increased albumin-creatinine ratio, stroke/transient ischaemic attack and retinal disease were each associated with greater odds of CD/dementia; higher years at education completion, baseline cognitive function, taller stature and current alcohol use were inversely associated. Higher waist circumference (women-to-men ratio of ORs [ROR], 1.05 [1.00, 1.10] per 5 cm) and presence of anxiety/depression (ROR, 1.28 [1.01, 1.63]) were associated with greater ORs for CD/dementia in women than men. CONCLUSIONS: Several risk factors were associated with CD/dementia. Higher waist circumference and mental health symptoms were more strongly associated with CD/dementia in women than men. Further studies should examine the mechanisms that underlie these sex differences.