Partial substitutions of animal with plant protein foods in Canadian diets have synergies and trade-offs among nutrition, health and climate outcomes.

Dietary guidelines emphasize the consumption of plant protein foods, but the implications of replacing animal with plant sources on a combination of diet sustainability dimensions are unknown. Using a combination of data from a national nutrition survey, greenhouse gas emissions from dataFIELD and relative risks from the Global Burden of Disease Study 2017, we assess the impact of partially substituting red and processed meat or dairy with plant protein foods in Canadian self-selected diets on nutrition, health and climate outcomes. The substitutions induced minor changes to the percentage of the population below requirements for nutrients of concern, but increased calcium inadequacy by up to 14% when dairy was replaced. Replacing red and processed meat or dairy increased life expectancy by up to 8.7 months or 7.6 months, respectively. Diet-related greenhouse gas emissions decreased by up to 25% for red and processed meat and by up to 5% for dairy replacements. Co-benefits of partially substituting red and processed meat with plant protein foods among nutrition, health and climate outcomes are relevant for reshaping consumer food choices in addressing human and planetary health.

In vivo genome-wide CRISPR screen reveals breast cancer vulnerabilities and synergistic mTOR/Hippo targeted combination therapy.

Triple negative breast cancer (TNBC) patients exhibit poor survival outcomes and lack effective targeted therapies. Using unbiased in vivo genome-wide CRISPR screening, we interrogated cancer vulnerabilities in TNBC and identified an interplay between oncogenic and tumor suppressor pathways. This study reveals tumor regulatory functions for essential components of the mTOR and Hippo pathways in TNBC. Using in vitro drug matrix synergy models and in vivo patient-derived xenografts, we further establish the therapeutic relevance of our findings and show that pharmacological inhibition of mTORC1/2 and oncoprotein YAP efficiently reduces tumorigenesis in TNBC. At the molecular level, we find that while verteporfin-induced YAP inhibition leads to apoptosis, torin1-mediated mTORC1/2 inhibition promotes macropinocytosis. Torin1-induced macropinocytosis further facilitates verteporfin uptake, thereby greatly enhancing its pro-apoptotic effects in cancer cells. Overall, our study underscores the power and robustness of in vivo CRISPR genome-wide screens in identifying clinically relevant and innovative therapeutic modalities in cancer.

Differential Regulation of Cancer Progression by CDK4/6 Plays a Central Role in DNA Replication and Repair Pathways.

Although the cyclin-dependent kinases CDK4 and CDK6 play fundamental roles in cancer, the specific pathways and downstream targets by which they exert their tumorigenic effects remain elusive. In this study, we uncover distinct and novel functions for these kinases in regulating tumor formation and metastatic colonization in various solid tumors, including those of the breast, prostate, and pancreas. Combining in vivo CRISPR-based CDK4 and CDK6 gene editing with pharmacologic inhibition approaches in orthotopic transplantation and patient-derived xenograft preclinical models, we defined clear functions for CDK4 and CDK6 in facilitating tumor growth and progression in metastatic cancers. Transcriptomic profiling of CDK4/6 CRISPR knockouts in breast cancer revealed these two kinases to regulate cancer progression through distinct mechanisms. CDK4 regulated prometastatic inflammatory cytokine signaling, whereas CDK6 mainly controlled DNA replication and repair processes. Inhibition of CDK6 but not CDK4 resulted in defective DNA repair and increased DNA damage. Multiple CDK6 DNA replication/repair genes were not only associated with cancer subtype, grades, and poor clinical outcomes, but also facilitated primary tumor growth and metastasis in vivo. CRISPR-based genomic deletion of CDK6 efficiently blocked tumor formation and progression in preestablished cell- and patient-derived xenograft preclinical models of breast cancer, providing a potential novel targeted therapy for these deadly tumors. SIGNIFICANCE: In-depth transcriptomic analysis identifies cyclin-dependent kinases CDK4 and CDK6 as regulators of metastasis through distinct signaling pathways and reveals the DNA replication/repair pathway as central in promoting these effects.

Protein consumption in Canadian habitual diets: usual intake, inadequacy, and the contribution of animal- and plant-based foods to nutrient intakes.

The 2019 Canada's Food Guide (CFG) emphasizes consumption of plant protein with implications for protein adequacy and nutrient intakes, yet a baseline with which to compare future dietary trends that may result from its adoption is not available. The objectives were to assess usual protein intake, inadequacy, and the contribution of animal- and plant-based foods to intake of protein, nutrients, and energy in Canada. Twenty-four-hour dietary recalls from the 2015 Canadian Community Health Survey - Nutrition were used to assess dietary intake among adults (n = 13 616). The National Cancer Institute method was used to estimate usual protein intake and inadequacy. Population ratios were used to determine the contribution of animal- and plant-based foods to intake of protein, nutrients, and energy. Usual protein intake averaged 79.47 ± 0.70 g/d; inadequacy was highest for females ≥71 y (9.76 ± 2.04%). Top protein contributors were red and processed meat (21.6 ± 0.55%), poultry and eggs (20.1 ± 0.81%), cereals, grains, and breads (19.5 ± 0.31%), and dairy (16.7 ± 0.38%). Dairy contributed most to calcium (53.4 ± 0.61%), vitamin D (38.7 ± 1.01%), but also saturated fat (40.6 ± 0.69%), whereas cereals, grains, and breads contributed most to iron (46.5 ± 0.57%) and vegetables and fruit to potassium (32 ± 0.45%). Given that animal sources contributed overwhelmingly to protein intake in 2015, dietary shifts towards plant protein needed to meet the 2019 CFG recommendations may pose a challenge, particularly for populations most at risk of inadequacy. Novelty: Older adults and females are most at risk of not meeting protein recommendations. Animal sources contribute two-thirds of the protein consumed by Canadian adults.

Acute hyperaminoacidemia does not suppress insulin-mediated glucose turnover in healthy young men.

Elevated circulating amino acids (AA) concentrations are purported to cause insulin resistance (IR) in humans. To quantify hyperaminoacidemia effects on insulin-mediated glucose turnover in healthy men, we performed 2-stage pancreatic clamps using octreotide with glucagon and growth hormone replacement. In the basal stage, insulin was infused to maintain euglycemia at postabsorptive levels. During the clamp stage, insulin was raised to postprandial levels, glycemia clamped at 5.5 mmol/L by glucose infusion, and branched-chain AA (BCAA) maintained at either postabsorptive (Hyper1; n = 8) or postprandial (Hyper2; n = 7) by AA infusion. Glucose turnover was measured by d-3-[3H]glucose dilution. Octreotide suppressed C-peptide; glucagon, growth hormone, and glycemia were maintained at postabsorptive levels throughout. Insulin did not differ at postabsorptive (72 ± 5 vs. 60 ± 5 pmol/L; Hyper1 vs. Hyper2) and increased to similar concentrations at basal (108 ± 11 vs. 106 ± 14) and clamp stages (551 ± 23 vs. 540 ± 25). Postabsorptive BCAA were maintained during Hyper1 and increased >2-fold (830 ± 26 µmol/L) during Hyper2. Endogenous glucose production was similarly suppressed (0.95 ± 0.16 vs. 1.37 ± 0.23 mg/kg lean body mass/min; Hyper1 vs. Hyper2) and basal glucose disposal (3.44 ± 0.12 vs. 3.67 ± 0.14) increased to similar levels (10.89 ± 0.56 vs. 11.11 ± 1.00) during the clamp. Thus, acute physiological elevation of AA for 3 h did not cause IR in healthy men. Novelty: A 2-step pancreatic clamp was used to quantify the effect of AA on insulin sensitivity in humans. Acute physiological elevation of circulating AA to postprandial levels does not cause IR in healthy men.

Role of Grb10 in mTORC1-dependent regulation of insulin signaling and action in human skeletal muscle cells.

Growth factor receptor-bound protein 10 (Grb10) is an adaptor protein that binds to the insulin receptor, upon which insulin signaling and action are thought to be inhibited. Grb10 is also a substrate for the mechanistic target of rapamycin complex 1 (mTORC1) that mediates its feedback inhibition on phosphatidylinositide 3-kinase (PI3K)/Akt signaling. To characterize the function of Grb10 and its regulation by mTORC1 in human muscle, primary skeletal muscle cells were isolated from healthy lean young men and then induced to differentiate into myotubes. Knockdown of Grb10 enhanced insulin-induced PI3K/Akt signaling and glucose uptake in myotubes, reinforcing the notion underlying its function as a negative regulator of insulin action in human muscle. The increased insulin responsiveness in Grb10-silenced myotubes was associated with a higher abundance of the insulin receptor. Furthermore, insulin and amino acids independently and additively stimulated phosphorylation of Grb10 at Ser476. However, acute inhibition of mTORC1 with rapamycin blocked Grb10 Ser476 phosphorylation and repressed a negative-feedback loop on PI3K/Akt signaling that increased myotube responsiveness to insulin. Chronic rapamycin treatment reduced Grb10 protein abundance in conjunction with increased insulin receptor protein levels. Based on these findings, we propose that mTORC1 controls PI3K/Akt signaling through modulation of insulin receptor abundance by Grb10. These findings have potential implications for obesity-linked insulin resistance, as well as clinical use of mTORC1 inhibitors.

Consumption of Milk and Alternatives and Their Contribution to Nutrient Intakes among Canadian Adults: Evidence from the 2015 Canadian Community Health Survey-Nutrition.

As a staple food and dense source of nutrients, milk and alternatives play an important role in nutrient adequacy. The aims of this study were to quantify the consumption of milk and alternatives within Canadian self-selected diets and determine their contribution to intakes of nutrients and energy. First, 24-h dietary recalls from the 2015 Canadian Community Health Survey-Nutrition were used to assess 1-d food and nutrient intakes among Canadian adults ≥19 y (n = 13,616). Foods were classified as milk and alternatives according to the 2007 Canada's Food Guide. Descriptive statistics were used to calculate daily servings of milk and alternatives by different age groups and demographic characteristics. Population ratios were used to discern their contribution to total intakes of nutrients and energy. Mean daily servings (±SE) were highest for milk (0.60 ± 0.02) and cheese (0.42 ± 0.01), intermediate for frozen dairy (0.16 ± 0.01) and yoghurt (0.14 ± 0.01), and lowest for soy and other dairy (<0.03). Intakes were lowest among Canadians 51+ y (1.3 ± 0.03), females (1.25 ± 0.03), non-Caucasians (1.06 ± 0.05), those with less than a secondary education (1.19 ± 0.05), and British Columbians (1.17 ± 0.05). Milk and alternatives contributed >20% to total intakes of calcium (52.62 ± 0.46%), vitamin D (38.53 ± 0.78%), saturated fat (28.84 ± 0.51%), vitamin B12 (27.73 ± 0.57%), vitamin A (26.16 ± 0.58%), phosphorus (24.76 ± 0.35%), and riboflavin (24.43 ± 0.37%), of which milk was the top source. Milk and alternatives contribute substantially to nutrient intakes and thus warrant further attention in terms of mitigating nutrient inadequacy among the Canadian population.

Insulin resistance of protein anabolism accompanies that of glucose metabolism in lean, glucose-tolerant offspring of persons with type 2 diabetes.

OBJECTIVE: To test whether protein anabolic resistance is an early defect in type 2 diabetes (T2D). RESEARCH DESIGN AND METHODS: Seven lean, normoglycemic T2D offspring (T2D-O) and eight matched participants without family history (controls; C) underwent a 3-hour hyperinsulinemic (40 mU/m2/min), euglycemic (5.5 mmol/L) and isoaminoacidemic clamp. Whole-body glucose and protein kinetics were measured with d-[3-3H]glucose and l-[l-13C]leucine, respectively. Plasma amino acids were measured by liquid chromatography-tandem mass spectrometry. RESULTS: Fasting glycemia and glucose kinetic variables did not differ between groups. Clamp decreases in glucose rate of appearance were not different, but rate of disappearance increased 29% less in T2D-O, to a significantly lower rate. Fasting leucine was higher in T2D-O, but kinetics did not differ. Clamp increases in leucine oxidation and decreases in endogenous rate of appearance (protein breakdown) were equal, but in T2D-O, non-oxidative rate of disappearance (protein synthesis) did not increase and net balance (synthesis-breakdown) did not become positive as in C. CONCLUSIONS: Resistance of whole-body protein anabolism (synthesis and net balance) accompanies resistance of glucose uptake in T2D-O. Mechanisms responsible, possible roles in the increased risk of developing diabetes, and its potential impact on long-term protein balance require definition.

CDK4 regulates cancer stemness and is a novel therapeutic target for triple-negative breast cancer.

Triple negative breast cancers exhibit very aggressive features and poor patient outcomes. These tumors are enriched in cancer stem cells and exhibit resistance to most treatments and chemotherapy. In this study, we found the cyclin-dependent kinase (CDK4) to act as a cancer stem cell regulator and novel prognostic marker in triple negative breast cancers. We found CDK4 to be highly expressed in these tumors and its expression to correlate with poor overall and relapse free survival outcomes, high tumor grade and poor prognostic features of triple negative breast cancer patients. Moreover, we found that blocking CDK4 expression or kinase activity, using a pharmacological inhibitor prevented breast cancer stem cell self-renewal. Interestingly, suppression of CDK4 expression or kinase activity reversed the basal-B TNBC mesenchymal phenotype to an epithelial- and luminal-like phenotype which correlates with better clinical prognosis. Finally, blocking CDK4 activity efficiently eliminated both normal and chemotherapy-resistant cancer cells in triple negative breast cancers, highlighting CDK4 as a promising novel therapeutic target for these aggressive breast tumors.

Protein and glucose metabolic responses to hyperinsulinemia, hyperglycemia, and hyperaminoacidemia in obese men.

OBJECTIVE: In insulin-resistant states, resistance of protein anabolism occurs concurrently with that of glucose, but can be compensated for by abundant amino acid (AA) provision. This effect and its mechanism were sought in obesity. METHODS: Pancreatic clamps were performed in 8 lean and 11 obese men, following 5-h postabsorptive, 3-h infusions of octreotide, basal glucagon, and growth hormone, with clamped postprandial-level insulin, glucose, and AA. Whole-body [1-(13) C]-leucine and [3-(3) H]-glucose kinetics, skeletal muscle protein ((2) H5 -phenylalanine) fractional synthesis rates, and insulin signaling were determined. RESULTS: Clamp Δ insulin and Δ branched-chain AA did not differ; fasting glucagon and growth hormone were maintained. Glucose uptake was 20% less in obese concurrent with less Akt(Ser473) , but also less IRS-1(Ser636/639) phosphorylation. Stimulation of whole-body, myofibrillar, and sarcoplasmic protein synthesis was similar. Whole-body protein catabolism suppression tended to be less (P=0.06), resulting in lesser net balance (1.09 ± 0.07 vs. 1.31 ± 0.08 µmol [kg FFM(-1) ] min(-1) , P=0.048). Increments in muscle S6K1(Thr389) phosphorylation were less in the obese, but 4E-BP1(Ser65) did not differ. CONCLUSIONS: Hyperaminoacidemia with hyperinsulinemia stimulated protein synthesis (possibly via nutrient signaling) normally in obesity, but suppression of proteolysis may be compromised. Whether long-term high protein intakes could compensate for the insulin resistance of protein anabolism remains to be determined.

Protein anabolic responses to a fed steady state in healthy aging.

BACKGROUND: Protein anabolism in response to feeding may be impaired with aging. To determine if this could contribute to muscle loss, we studied fed-state metabolic responses in healthy, non-sarcopenic elderly women. METHODS: Whole-body [(3)H]glucose and protein ([(13)C]leucine) kinetics were measured, and muscle protein fractional synthesis rate ([(2)H(5)]phenylalanine) and signaling events were assessed from vastus lateralis biopsies in eight elderly (73 ± 3 years) and eight young women (24 ± 1 years), using a simulated fed steady-state clamp. RESULTS: Both groups had similar muscle and lean body mass indices and activity level. During insulin, glucose (8 mmol/L), and amino acid (AA; 2× fasting) infusions, serum insulin was lower in the elderly women and C-peptide increased less. Glucose uptake was stimulated, and production suppressed similarly. Suppression of whole-body protein breakdown was less in the elderly women, leading to lower AA infusion rates, oxidation, and net positive protein balance, but differences were not present when adjusted for serum insulin. Whole-body protein synthesis and muscle protein fractional synthesis rate increased similarly. Similar increases in phosphorylated Akt(Ser473), PRAS40(Thr246), FoxO3a(Thr32), and rpS6(Ser240/244) indicated no alterations in insulin/nutrient signaling with aging. CONCLUSIONS: Both whole-body and muscle fed-state protein anabolic responses were preserved, as was insulin sensitivity of glucose metabolism, in active, healthy elderly women. This is consistent with other factors such as sedentarity, low protein intake, and concurrent diseases, being responsible for the sarcopenia of aging.